Silver nano probiotic preparation as well as preparation method and application thereof

Abstract

The invention relates to a silver nano probiotic preparation and a preparation method and application thereof, which can effectively reduce the toxicity of silver nanoparticles and enhance the treatment effect of the silver nanoparticles, and adopts the technical scheme that the silver nano probiotic preparation is characterized in that the silver nanoparticles are loaded on the surface of bacillus subtilis, the particle size of the silver nanoparticles is 20-100 nm, and the particle size of the bacillus subtilis is 20-100 nm. The mass ratio of the silver nanoparticles to the bacillus subtilis is (4-10): 100, the prepared silver nano probiotic preparation does not affect proliferation of probiotics, can prolong the residence time of the silver nanoparticles in intestinal tracts and reduce the potential toxic and side effects of the silver nanoparticles, has good biocompatibility and safety, is wide in application range, and can be widely applied to the field of biological medicines. The compound can be effectively used for preparing medicines for treating colitis, bacterial infection, inflammatory bowel diseases, irritable bowel syndromes and the like and preparing oral preparations for treating diseases, and new ways of the medicines for treating colitis, bacterial infection, inflammatory bowel diseases and irritable bowel syndromes and the oral preparations thereof are developed.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and particularly to a silver nanoparticle probiotic preparation, a preparation method thereof and an application thereof. Background Art

[0002] Silver nanoparticles refer to silver particles with a size in the range of 1 - 100 nm in at least one dimension. As a highly potential nanomaterial, silver nanoparticles exhibit diverse biological activities due to their unique physical and chemical characteristics. Silver nanoparticles can not only release Ag + , bind to thiol groups on the cell membrane, disrupt the integrity of the cell membrane, interfere with cell functions, but also induce the generation of intracellular reactive oxygen species, trigger oxidative stress, resulting in lipid peroxidation and mitochondrial dysfunction. In addition, silver nanoparticles can also interact with DNA, proteins and enzymes, inhibit gene expression, protein synthesis and enzyme activity, and thus affect the regulation of cell signaling pathways. Therefore, silver nanoparticles have been widely used in the biomedical field and shown important value in aspects such as antibacterial, anti-inflammatory, anti-cancer, antiviral, anti-diabetic, drug delivery, promoting wound repair and bone healing, enhancing vaccine immunogenicity, preventing cardiovascular diseases, developing biosensors and as additives for dental materials. However, their potential toxicity problems have also attracted the general attention of researchers. For example, the transformation of silver nanoparticles in the body after oral exposure can cause damage such as abnormal microvilli and intestinal glands of intestinal epithelial cells. Silver nanoparticles in the intestinal lumen can be absorbed into the systemic circulation through the gastrointestinal tract, accumulate in organ tissues across various biological barriers, and thus induce potential organ tissue toxicity, bringing unpredictable health effects. Therefore, how to reduce the potential toxicity of silver nanoparticles and prolong the intestinal retention of silver nanoparticles is a key technical problem to be solved urgently.

[0003] In recent years, probiotics have attracted much attention due to their unique advantages and have been widely explored as an efficient platform for oral delivery of nano-drugs. Bacillus subtilis is one of the most representative probiotic drug delivery carriers. First of all, Bacillus subtilis has been approved by the FDA as a safe food supplement and additive, and has various applications in the biomedical field, especially in the treatment of intestinal diseases, playing important roles such as reshaping the intestinal flora, balancing the immune system and repairing the intestinal barrier. More importantly, Bacillus subtilis can form spores under specific conditions, and its surface peptidoglycan and proteins are rich in various functional groups such as amino, carboxyl, hydroxyl and mercapto groups, which provides the possibility for loading molecules such as drugs. Some researchers have utilized Bacillus subtilis to achieve targeted delivery of various drugs such as monoclonal antibodies and hydrophobic chemical molecules in the intestine. Based on this, constructing a novel silver nanoparticle oral preparation by loading silver nanoparticles with Bacillus subtilis is expected to become a good drug delivery system for prolonging the retention of silver nanoparticles in the intestine, reducing their toxicity and enhancing their activity, but there has been no public report so far. Summary of the Invention

[0004] In view of the above situation, to solve the defects of the prior art, the object of the present invention is to provide a silver nano-probiotic preparation, its preparation method and application, which can effectively reduce the toxicity of silver nanoparticles and enhance its therapeutic effect.

[0005] One of the technical solutions provided by the present invention is to provide a silver nano-probiotic preparation, which is obtained by loading silver nanoparticles on the surface of Bacillus subtilis, wherein the particle size of the silver nanoparticles is 20-100 nm, and the mass ratio of the silver nanoparticles to Bacillus subtilis is 4-10:100.

[0006] Another technical solution provided by the present invention is to provide a preparation method of a silver nano-probiotic preparation. Specifically, Bacillus subtilis forms spores under certain transformation conditions, and then silver nanoparticles are synthesized by a chemical reducing reagent or a traditional Chinese medicine extract and loaded on the surface of Bacillus subtilis spores. The transformation conditions are high-temperature heating or transfer to a sporulation medium containing manganese sulfate; the chemical reducing reagent is one of sodium citrate, sodium borohydride, ascorbic acid, ethylene glycol and N, N-dimethylformamide; the traditional Chinese medicine extract is one of the extracts of traditional Chinese medicines such as Coptis chinensis, Glycyrrhiza uralensis, Astragalus membranaceus, Cornus officinalis or Astragalus membranaceus.

[0007] Preferably, the chemical reducing reagent is sodium citrate or sodium borohydride.

[0008] Preferably, the traditional Chinese medicine extract is the extract of Coptis chinensis.

[0009] Preferably, the preparation method of the silver nano-probiotic preparation is as follows: Bacillus subtilis is transferred to a sporulation medium containing 50 mg / L manganese sulfate, cultured at 37 °C and 250 r / min for 48 h, centrifuged at 5×10 3 r / min for 10 min to obtain Bacillus subtilis spores; after heating 100 mL of 0.001 mol / L silver nitrate solution to 100 °C, 5 mL of 1% sodium citrate solution is added dropwise, reacted for 90 min, then 10 mL of Bacillus subtilis spore suspension is added, and vigorously stirred for 120 min, followed by centrifugation and washing with ultrapure water to obtain the silver nano-probiotic preparation.

[0010] Preferably, the preparation method of the silver nano-probiotic preparation is as follows: a suspension of Bacillus subtilis growing to the stationary phase is heated at 90 °C for 10 min, then 10 mL of 0.002 mol / L silver nitrate solution is added, and 10 mL of 0.01% sodium borohydride aqueous solution is slowly added dropwise while stirring, reacted for 120 min, followed by centrifugation and washing with ultrapure water to obtain the silver nano-probiotic preparation.

[0011] Preferably, the preparation method of the silver nanometer probiotic preparation is as follows: Bacillus subtilis is transferred to a sporulation medium containing 30 mg / L manganese sulfate, cultured at 30 °C and 350 r / min for 36 h, and centrifuged at 5×10 3 r / min for 10 min to obtain Bacillus subtilis spores; take 60 mL of 0.002 mol / L silver nitrate solution, and successively add 10 mL of Bacillus subtilis spore suspension and 1.5 mL of 0.01 mol / L ascorbic acid under stirring, react for 10 h, and after centrifugation and washing with ultrapure water, the silver nanometer probiotic preparation is obtained.

[0012] Preferably, the preparation method of the silver nanometer probiotic preparation is as follows: the Bacillus subtilis is a Bacillus subtilis suspension grown to the plateau phase, heated at 80 °C for 30 min, then 20 mL of 0.01 mol / L silver nitrate solution is added, 5 mL of 10% Coptis chinensis extract is added under stirring, boiled for 120 min, naturally cooled, and after centrifugation and washing with ultrapure water, the silver nanometer probiotic is obtained; the Coptis chinensis extract is obtained by placing 8 g of Coptis chinensis powder in 100 mL of ultrapure water, heating and stirring at 100 °C for 45 min, filtering, centrifuging the filtrate, and collecting the supernatant.

[0013] Preferably, the preparation method of the silver nanometer probiotic preparation is as follows: Bacillus subtilis is transferred to a sporulation medium containing 40 mg / L manganese sulfate, cultured at 37 °C and 200 r / min for 60 h, and centrifuged at 5×10 3 r / min for 10 min to obtain Bacillus subtilis spores; take 50 mL of 0.001 mol / L silver nitrate solution, and successively add 10 mL of Bacillus subtilis spore suspension and 10 mL of 20% Glycyrrhiza uralensis extract under stirring, stir in the dark at room temperature for 60 min, and after centrifugation and washing with ultrapure water, the silver nanometer probiotic is obtained; the Glycyrrhiza uralensis extract is obtained by taking 200 g of Glycyrrhiza uralensis rhizome powder, extracting three times with 95% ethanol, each time with 10 times the volume, combining the extracts, concentrating, precipitating with 95% ethanol, and centrifuging to collect the supernatant.

[0014] Preferably, the preparation method of the silver nanometer probiotic preparation is as follows: the Bacillus subtilis is a Bacillus subtilis suspension grown to the plateau phase, heated at 90 °C for 10 min, then 100 mL of 0.002 mol / L silver nitrate solution is added, 15 mL of 5% Scutellaria baicalensis aqueous solution is added under stirring, stirred in the dark at room temperature for 30 min and then ultrasonically treated for 30 min, repeat this step three times, after the reaction is completed, and after centrifugation and washing with ultrapure water, the silver nanometer probiotic is obtained; the Scutellaria baicalensis aqueous solution is obtained by taking 5 g of Scutellaria baicalensis leaves, extracting with 100 mL of distilled water at 90 °C for 30 min, cooling to room temperature, filtering, centrifuging, and collecting the supernatant.

[0015] The third technical solution provided by the present invention is the application of the silver nanoparticle probiotic preparation in the preparation of drugs for treating colitis, bacterial infection, inflammatory bowel disease, and irritable bowel syndrome.

[0016] The fourth technical solution provided by the present invention is the application of the silver nanoparticle probiotic preparation in the preparation of an oral dosage form for treating gastrointestinal diseases.

[0017] The beneficial technical effects of the present invention: 1. The silver nanoparticle probiotic preparation prepared by the present invention does not affect the proliferation of probiotics, can prolong the residence time of silver nanoparticles in the intestine, reduce the potential toxic and side effects of silver nanoparticles. In the experiment, no obvious adverse reactions were found, and it has a good anti-inflammatory effect. The co-administration enhanced the anti-inflammatory effect of silver nanoparticles; 2. It has good biocompatibility and safety, a wide range of applications, can be effectively used in the preparation of drugs for treating colitis, bacterial infection, inflammatory bowel disease, irritable bowel syndrome, etc. and the preparation of oral preparations for treating diseases, opening up new ways for the treatment of colitis, bacterial infection, inflammatory bowel disease, irritable bowel syndrome drugs and their oral preparations. Specific embodiments

[0018] The following detailed description of the specific embodiments of the present invention is given in conjunction with the examples. Examples

[0019] In the specific implementation of the present invention, a preparation method of a silver nanoparticle probiotic preparation includes the following steps: Inoculate the Bacillus subtilis strain preserved in glycerol into LB liquid medium at an inoculation amount of 1%, and shake and activate it in a constant temperature shaking incubator at 37°C and 200 r / min for 12 h to obtain a seed solution. Transfer the seed solution to a spore-producing medium containing manganese sulfate (peptone 5.0 g / L, beef extract 3.0 g / L, NaCl 5.0 g / L, manganese sulfate 50 mg / L, pH 7.0, 121°C, conventional steam sterilization for 20 min) at an inoculation amount of 10%, and culture it at 37°C and 250 r / min for 48 h. Stain with malachite green and examine under a microscope. When the spore yield reaches more than 95%, centrifuge the crude spore product at 5×10 3 r / min and 4°C for 10 min, and resuspend it with sterile water to obtain a spore suspension; Take 100 mL of 0.001 mol / L silver nitrate solution, heat it to 100°C with stirring at 2.5×10 3 r / min, dropwise add 5 mL of 1% sodium citrate solution, boil for 90 min, and add 10 mL of 10 9CFU / mL Bacillus subtilis spore suspension, continue stirring for 120 min. When the reaction is completed, centrifuge the reaction solution at 5×10 3 r / min for 10 min, discard the supernatant, resuspend the precipitate with ultrapure water, wash 3 times, collect the silver nanoprobeiotic solution, put it into a brown reagent bottle, and store it at 4℃ to obtain. Example

[0020] In a specific implementation of the present invention, a preparation method of a silver nanoprobeiotic preparation includes the following steps: Transfer Bacillus subtilis in the exponential growth phase to an LB expansion medium at an inoculation amount of 30%, culture at 48℃ and 150 r / min until the stationary phase, heat at 90℃ for 10 min. When the spore yield reaches more than 95%, filter the precipitate through gauze, wash 3 times with sterile water, and obtain a spore suspension with sterile ultrapure water; add a 0.02 mol / L silver nitrate solution according to a volume ratio of 10:1, place it on a magnetic stirrer, and heat to 90℃ under stirring at 3×10 3 r / min, gradually add 10 mL of 1% sodium citrate solution dropwise, boil for 90 min. When the color of the reaction system gradually changes from the initial white to yellowish brown, terminate the reaction, naturally cool to room temperature, and centrifuge the synthesized reaction solution at 5×10 3 r / min for 10 min, discard the supernatant, resuspend the precipitate with ultrapure water to obtain a silver nanoprobeiotic solution. Example

[0021] In a specific implementation of the present invention, a preparation method of a silver nanoprobeiotic preparation includes the following steps: Transfer the seed liquid of Bacillus subtilis strain in the exponential growth phase to an LB expansion medium at an inoculation amount of 50%, culture at 30℃ and 220 r / min until the stationary phase, heat at 85℃ for 20 min. When the spore yield reaches more than 95%, centrifuge the crude spore product at 5×10 3 r / min at 4℃ for 10 min, resuspend the precipitate with sterile water to obtain a spore suspension; Take 10 mL of freshly prepared 0.02 mol / L silver nitrate solution, place it on a magnetic stirrer, and slowly add 10 mL of 0.01% sodium borohydride aqueous solution dropwise while stirring at 3.5×10 3 r / min for 120 min, add 10 mL of the above-mentioned Bacillus subtilis spore suspension, and stir strongly for 100 min. The whole experimental process is carried out in a light-proof environment. When the reaction is completed, centrifuge the reaction solution at 5×10 3 r / min for 10 min, discard the supernatant, resuspend the precipitate with ultrapure water to obtain a silver nanoprobeiotic solution. Example

[0022] In a specific implementation of the present invention, a preparation method of a silver nano-probiotic preparation comprises the following steps: Inoculate the Bacillus subtilis strain preserved with glycerol into the LB liquid medium at an inoculation amount of 1%, and shake and activate it in a constant temperature shaking incubator at 37°C and 200 r / min for 12 h to obtain a seed solution. Transfer the seed solution to a sporulation medium containing manganese sulfate (peptone 5.0 g / L, beef extract 3.0 g / L, NaCl 5.0 g / L, manganese sulfate 30 mg / L, pH 7.0, 134°C, high-temperature short-time sterilization for 2 min) at an inoculation amount of 10%, and culture it at 30°C and 350 r / min for 36 h. When the spore yield reaches more than 95%, filter and collect the precipitate with a gauze, and wash it 3 times with sterile water to obtain a spore suspension; Take 10 mL of freshly prepared 0.02 mol / L silver nitrate solution, add 10 mL of the Bacillus subtilis spore suspension, place it on a magnetic stirrer and stir at 2.5×10 3 r / min, and while stirring, dropwise add 10 mL of 0.01% sodium borohydride aqueous solution, and react for 120 min. The whole experimental process is carried out in a light-proof environment. When the color of the reaction system gradually changes from the initial white to yellow and finally stabilizes to brownish-yellow, terminate the reaction, centrifuge the synthesized reaction solution at 5×10 3 r / min for 10 min, discard the supernatant, resuspend the precipitate with ultrapure water and wash it 3 times, collect the silver nano-probiotic solution, put it into a brown reagent bottle, and store it at 4°C. Example

[0023] In a specific implementation of the present invention, a preparation method of a silver nano-probiotic preparation comprises the following steps: Inoculate the Bacillus subtilis strain preserved with glycerol into the LB liquid medium, and shake and activate it in a constant temperature shaking incubator at 30°C and 300 r / min for 8 h to obtain a seed solution. Transfer the seed solution to a sporulation medium containing manganese sulfate (peptone 5.0 g / L, beef extract 3.0 g / L, NaCl 5.0 g / L, manganese sulfate 70 mg / L, pH 7.0, 172°C, steam explosion treatment for 2 min) at an inoculation amount of 30%, and culture it at 48°C and 150 r / min for 24 h. When the spore yield reaches more than 95%, filter and discard the liquid, and wash the precipitate with sterile water to obtain a spore suspension; Under stirring conditions, 20 mL of freshly prepared 0.01 mol / L silver nitrate solution was successively added to the spore suspension, followed by 5 mL of 10% Coptis chinensis extract (prepared by placing 8 g of Coptis chinensis powder in 100 mL of ultrapure water, heating and stirring at 100 °C for 45 min, filtering, centrifuging the filtrate, and collecting the supernatant). The mixture was boiled for 120 min, and then 10 mL of the solution from step 1) was added, and it was boiled for another 120 min under dark conditions. When the reaction was completed, the reaction solution was centrifuged at 5×10 3 r / min for 10 min. The supernatant was discarded, the precipitate was resuspended with ultrapure water and washed 3 times, and the silver nanoprobeiotic solution was collected and placed in a brown reagent bottle for storage at 4 °C. Example

[0024] In a specific implementation of the present invention, a method for preparing a silver nanoprobeiotic preparation includes the following steps: The Bacillus subtilis strain preserved with glycerol was inoculated into LB liquid medium at an inoculation amount of 3%, and cultured with shaking and activation at 48 °C and 150 r / min in a constant temperature shaking incubator for 24 h to obtain a seed solution. The seed solution was transferred to an LB expansion medium at an inoculation amount of 50%, and cultured at 30 °C and 220 r / min until the stationary phase. Then it was heated at 80 °C for 30 min. When the spore yield reached more than 95%, the crude spore product was centrifuged at 5×10 3 r / min at 4 °C for 10 min. The gray bacterial cells and culture medium impurities on the top layer of the precipitate were carefully scraped off, and the white spores at the bottom of the centrifuge tube were resuspended and washed 3 times with sterile water to obtain a spore suspension; 20 mL of freshly prepared 0.01 mol / L silver nitrate solution was added to the above spore suspension, and it was stirred at 3.5×10 3 r / min on a magnetic stirrer. While stirring, 10 mL of 10% Scutellaria baicalensis extract (prepared by placing 5 g of Scutellaria baicalensis root powder in 100 mL of 98% ethanol, treating at room temperature for 24 h, filtering, centrifuging the filtrate, and collecting the supernatant) was added. The mixture was boiled for 90 min. After the reaction ended, it was centrifuged, washed with ultrapure water, and the precipitate was resuspended to obtain a silver nanoprobeiotic solution.

[0025] The silver nanoprobeiotic preparation prepared by the method described in Examples 1-6 is effectively used for preparing drugs for treating colitis, bacterial infection, inflammatory bowel disease, and irritable bowel syndrome, as well as for use in preparing an oral administration preparation for treating gastrointestinal diseases, to realize the application of this novel silver nanoparticle oral preparation in preparing drugs for treating colitis, bacterial infection, inflammatory bowel disease, and irritable bowel syndrome, and for use in preparing an oral administration preparation.

[0026] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those skilled in the art, additional modifications can be easily achieved. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to specific details.

[0027] The present invention uses Bacillus subtilis with probiotic benefits to prepare a novel micro-nano probiotic silver nanoparticle preparation based on the combination of Bacillus subtilis and silver nanoparticles. This drug has the following characteristics: 1) The proliferation ability of Bacillus subtilis loaded with silver nanoparticles is not affected; 2) The prepared micro-nano drug delivery system can prolong the retention of silver nanoparticles in the intestine and reduce the toxic and side effects of silver nanoparticles; 3) The prepared micro-nano drug delivery system has good anti-inflammatory activity and can enhance the effect of drug treatment for colitis.

[0028] The present invention relates to the construction of a novel silver nanoparticle preparation. First, taking advantage of the property that Bacillus subtilis can produce spores under the condition of high cell density or in the presence of manganese ions, Bacillus subtilis is inoculated in different culture media and cultured under different culture conditions to obtain a spore suspension. Then, taking advantage of the property that the active groups on the spore surface can physically adsorb silver nanoparticles, the spores and silver nanoparticles are mixed and stirred, or the spore suspension is added during the synthesis of silver nanoparticles to prepare silver nano-probiotics. In this process, Bacillus subtilis provides reaction sites for the attachment of silver nanoparticles on its surface, forming silver nanoparticles with uniform size. Silver nanoparticles and the antibacterial substances secreted by Bacillus subtilis play synergistic roles in anti-inflammatory, antibacterial, etc.

[0029] The silver nanoparticles used in the present invention are metal nanoparticles with anti-inflammatory and antibacterial activities prepared by chemical reduction method or biological reduction method from silver nitrate. As an important metal, silver has been widely used for treating infections, preventing bacterial growth, accelerating wound healing, etc. since ancient times, and it is a good anti-inflammatory and antibacterial material. Silver nanoparticles can slowly release Ag + ⁺, generate reactive oxygen species, and have excellent biological activity. The prepared micro-nano drug delivery system produces a synergistic effect without affecting the proliferation of probiotics, prolongs the intestinal retention of silver nanoparticles, reduces the toxicity of silver nanoparticles, and enhances the effect of silver nanoparticles in treating colitis. The relevant experimental data are as follows: Experiment 1: Particle size and zeta potential of silver nano-probiotics The silver nanoparticles and silver nano-probiotics were diluted to appropriate concentrations with ultrapure water. 1 mL of each sample was taken and placed in a cuvette, and the average particle size and Zeta potential of the samples were measured at 25 °C using a nanoparticle size and zeta potential analyzer. The experimental results showed that the average particle size of the silver nanoparticles was (94.69 ± 2.24) nm, and the potential was (-20.47 ± 0.38) mV. The particle size and potential of the silver nano-probiotics were (1457.39 ± 39.97) nm and (-29.67 ± 0.25) mV, respectively.

[0030] Experiment 2: Changes in cell proliferation before and after probiotics loaded with silver nanoparticles The dilution plating method was used to investigate the changes in cell proliferation of Bacillus subtilis after loading with silver nanoparticles. The experiment was divided into three groups: the Bacillus subtilis group; the mixed group of silver nanoparticles and Bacillus subtilis; the silver nano-probiotic group. The initial number of bacteria in each group was kept the same. After gradient dilution with sterile water, they were spread on LB solid medium and incubated in an incubator at 37 °C for 12 h, and the colony growth was observed. The experimental results showed that there were no significant differences in colony morphology or CFU count between the silver nano-probiotic group and the Bacillus subtilis group and the mixed group of silver nanoparticles and Bacillus subtilis, indicating that loading with silver nanoparticles had no significant effect on the germination and proliferation of Bacillus subtilis.

[0031] Experiment 3: Determination of fecal elimination after single-dose administration to explore the intestinal retention of silver nanoparticles After single-dose administration, mouse feces were collected at different time points, and the silver content in the feces was detected by ICP-MS to investigate the intestinal retention of silver nanoparticles. Normal (healthy) BABL / c mice were randomly divided into two groups, with 6 mice in each group and three replicates, and the average value was taken: 1) the silver nanoparticle group; 2) the silver nano-probiotic group. They were orally administered once by gavage. The dosage of silver nanoparticles was 5 mg / kg. The bedding of each group of mice was changed at 0 h, 1 h, 2 h, 3 h, 6 h, 9 h, 12 h, 18 h, and 24 h of the experiment, and all fecal samples in the bedding at different time points were collected. The silver content in the fecal samples of each group was detected by ICP-MS. The results showed that among the detected time points, the fecal silver concentration peak of the silver nanoparticle group reached at 3 h. Compared with the silver nanoparticle group, the fecal silver concentration peak of the silver nano-probiotic group reached at 6 h, indicating that the anchoring of Bacillus subtilis prolonged the intestinal retention time of silver nanoparticles.

[0032] Experiment 4: Effects of silver nano-probiotics on the liver function of healthy mice The liver is the main storage site of silver nanoparticles in the body, and liver function indicators are also important indicators reflecting the toxicity of silver nanoparticles. Twenty-four healthy BABL / c mice were randomly divided into three groups (8 mice in each group), namely: 1) normal saline control group, 2) silver nanoparticle group, and 3) silver nanoprobeiotic group. The dosage of silver nanoparticles was 2 mg / kg, and the dosage of Bacillus subtilis was 5×10 10 CFU / kg. They were administered orally every day, and blood was collected from the orbital cavity after 21 days. Part of the collected blood samples was stored in centrifuge tubes without anticoagulant to detect the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum. The experimental results showed that compared with the normal saline control group, the levels of serum ALT and AST in the silver nanoparticle group were significantly increased (2.22 times and 2.30 times that of the normal saline control group, respectively); compared with the silver nanoparticle group, the levels of serum ALT and AST in the silver nanoprobeiotic group were significantly decreased (0.58 times and 0.52 times that of the silver nanoparticle group, respectively), indicating that the compounding of Bacillus subtilis could effectively reduce the liver injury caused by the transport of free silver nanoparticles and alleviate the liver toxicity side effects.

[0033] Experiment 5: Therapeutic activity of silver nanoprobeiotics on murine colitis A chronic colitis model was established in male BALB / c mice and randomly divided into 4 groups, with 10 mice in each group: 1) normal saline control group, 2) colitis model group, 3) silver nanoparticle group, and 4) silver nanoprobeiotic group. The most widely used dextran sulfate sodium induction method was used to construct a murine chronic colitis model. During the model establishment, drug treatment was carried out simultaneously. The normal saline control group and the colitis model group were given an equal volume of normal saline, and the other groups were intragastrically administered silver nanoparticles and silver nanoprobeiotics, respectively. The dosage of silver nanoparticles was 2 mg / kg, and the dosage of Bacillus subtilis was 5×10 10 CFU / kg. They were administered once a day for 28 consecutive days. The body weights of the mice in each group were regularly recorded every two days, and the disease activity index (DAI) of the mice was calculated. After the last administration, all mice were fasted but not water-deprived for 12 h, and then sacrificed by cervical dislocation. The whole colon tissue was dissected from the abdominal cavity of the mice, rinsed with normal saline and blotted dry with filter paper, and then the intestinal segment was straightened to measure the colorectal length. The experimental results showed that compared with the normal saline control group, the body weight of the mice in the colitis model group decreased significantly periodically, the DAI score increased significantly periodically, and the final colon length was significantly shortened. Compared with the colitis model group, treatment with silver nanoprobeiotics could significantly restore the weight loss of colitis mice, reduce the DAI score of colitis mice, and relieve the colon shortening of colitis mice, showing an obvious therapeutic effect on colitis.

[0034] Experiment 6: Effects of silver nanoprobeiotics on murine intestinal flora A mouse model of chronic colitis was constructed by dextran sulfate sodium induction method. Silver nanometer probiotics were fed orally, and mouse fecal DNA was extracted for metagenomic detection. The experimental results showed that silver nanometer probiotics could regulate the changes of intestinal flora, inhibit the proliferation of harmful bacteria and promote the proliferation of beneficial bacteria at the same time. For example, the treatment with nanometer probiotics would increase Lactobacillus, Paramuribaculum, and Alistipes the relative abundance of Prevotella and decrease the relative abundance of Lactobacillus It can regulate the intestinal flora and maintain the homeostasis of intestinal barrier through extracellular vesicles, thereby improving acute colitis. Intestinal symbiont Alistipes can reduce intestinal epithelial damage and cytokine secretion, and improve experimental colitis in mice. Prevotella The severity of DSS-induced colitis can be aggravated by reducing the expression of ATF4 and disturbing the intestinal microbiota. The results of KEGG pathway analysis showed that silver nanometer probiotics could reverse extracellular biosynthesis and DNA replication induced by colitis, which was beneficial to restore the normal physiological functions of the intestinal flora.

[0035] In summary, the preparation method of the present invention is simple, with rich raw materials and easy to produce and prepare. The prepared silver nanometer probiotic delivery system does not affect the proliferation of probiotics, can prolong the retention of silver nanoparticles in the intestine, reduce the potential toxicity of silver nanoparticles, and has good anti-inflammatory effects. The co-administration enhances the anti-inflammatory effect of silver nanoparticles, has good biocompatibility and safety, a wide range of applications, and can be effectively used for the preparation of drugs for treating colitis, bacterial infections, inflammatory bowel disease, irritable bowel syndrome, etc. and the preparation of oral preparations for treating diseases, opening up new ways for the treatment of colitis, bacterial infections, inflammatory bowel disease, irritable bowel syndrome drugs and their oral preparations, and having practical clinical significance and popularization and application value.

Claims

1. A silver nano probiotic preparation, characterized in that, The silver nano-probiotic preparation is to load silver nanoparticles on the surface of Bacillus subtilis, wherein the particle size of the silver nanoparticles is 20-100 nm, and the mass ratio of the silver nanoparticles to Bacillus subtilis is 4-10:

100.

2. The preparation method of the silver nano probiotic preparation according to claim 1, characterized in that, Bacillus subtilis forms spores under certain transformation conditions, and then silver nanoparticles are synthesized and loaded on the surface of Bacillus subtilis by using a chemical reducing reagent or a traditional Chinese medicine extract; the transformation conditions are high-temperature heating or transfer to a sporulation medium containing manganese sulfate; the chemical reducing reagent is one of sodium citrate, sodium borohydride, ascorbic acid, ethylene glycol or N, N-dimethylformamide, and the traditional Chinese medicine extract is one of the traditional Chinese medicine extracts of Coptis chinensis, Glycyrrhiza uralensis, Astragalus membranaceus, Cornus officinalis or Astragalus membranaceus.

3. The preparation method of the silver nano-probiotic preparation according to claim 2, characterized in that, The described Bacillus subtilis is transferred to a sporulation medium containing 50 mg / L manganese sulfate and cultured at 37°C and 250 r / min for 48 h, then centrifuged at 5×10 3 r / min for 10 min to obtain Bacillus subtilis spores; after heating 100 mL of 0.001 mol / L silver nitrate solution to 100°C, 5 mL of 1% sodium citrate solution is added dropwise, reacted for 90 min, then 10 mL of Bacillus subtilis spore suspension is added, and strongly stirred for 120 min. After centrifugation and washing with ultrapure water, the silver nanoprobeiotic preparation is obtained.

4. The preparation method of the silver nano probiotic preparation according to claim 2, characterized in that, The Bacillus subtilis is a suspension of Bacillus subtilis grown to the stationary phase, heated at 90 °C for 10 min, then 10 mL of 0.002 mol / L silver nitrate solution is added, and 10 mL of 0.01% sodium borohydride aqueous solution is slowly added dropwise while stirring. After reacting for 120 min, it is centrifuged and washed with ultrapure water to obtain the silver nano-probiotic preparation.

5. The preparation method of the silver nano probiotic preparation according to claim 2, characterized in that, The described Bacillus subtilis is transferred to a sporulation medium containing 30 mg / L manganese sulfate and cultured at 30 °C and 350 r / min for 36 h, and then centrifuged at 5×10 3 r / min for 10 min to obtain Bacillus subtilis spores; 60 mL of 0.002 mol / L silver nitrate solution is taken, and 10 mL of the Bacillus subtilis spore suspension and 1.5 mL of 0.01 mol / L ascorbic acid are successively added under stirring, and the reaction is carried out for 10 h. After centrifugation and washing with ultrapure water, the silver nanoparticle probiotic preparation is obtained.

6. The preparation method of the silver nano probiotic preparation according to claim 2, characterized in that, The Bacillus subtilis is a suspension of Bacillus subtilis grown to the stationary phase, heated at 80 °C for 30 min, then 20 mL of 0.01 mol / L silver nitrate solution is added, and 5 mL of 10% Coptis chinensis extract is added under stirring, and boiled for 120 min, and then naturally cooled. After centrifugation and washing with ultrapure water, the silver nano-probiotic is obtained; the Coptis chinensis extract is prepared by placing 8 g of Coptis chinensis powder in 100 mL of ultrapure water, heating and stirring at 100 °C for 45 min, filtering, centrifuging the filtrate, and collecting the supernatant.

7. The preparation method of the silver nano-probiotic preparation according to claim 2, characterized in that, The described Bacillus subtilis is transferred to a sporulation medium containing 40 mg / L manganese sulfate and cultured at 37°C and 200 r / min for 60 h, and then centrifuged at 5×10 3 r / min for 10 min to obtain Bacillus subtilis spores; take 50 mL of 0.001 mol / L silver nitrate solution, and successively add 10 mL of Bacillus subtilis spore suspension and 10 mL of 20% licorice extract under stirring, and stir in the dark at room temperature for 60 min, and then centrifuge and wash with ultrapure water to obtain silver nano-probiotics; the licorice extract is obtained by taking 200 g of licorice root powder, extracting it three times with 95% ethanol, each time with 10 times the volume, combining the extracts, concentrating, precipitating with 95% ethanol, and centrifuging to collect the supernatant.

8. The preparation method of the silver nano probiotic preparation according to claim 2, characterized in that, The Bacillus subtilis is a suspension of Bacillus subtilis grown to the stationary phase, heated at 90 °C for 10 min, then 100 mL of 0.002 mol / L silver nitrate solution is added, and 15 mL of 5% Scutellaria baicalensis aqueous solution is added under stirring. After stirring in the dark at room temperature for 30 min, it is ultrasonically treated for 30 min, and this step is repeated three times. After the reaction is completed, it is centrifuged and washed with ultrapure water to obtain the silver nano-probiotic; the Scutellaria baicalensis aqueous solution is prepared by taking 5 g of Scutellaria baicalensis leaves, extracting with 100 mL of distilled water at 90 °C for 30 min, cooling to room temperature, filtering, centrifuging, and collecting the supernatant.

9. Use of the silver nano-probiotic preparation according to claim 1 in the preparation of drugs for treating colitis, bacterial infection, inflammatory bowel disease, and irritable bowel syndrome.

10. Use of the silver nano-probiotic preparation according to claim 1 in the preparation of an oral preparation for treating gastrointestinal diseases.