Application of ruminococcus torques and modified bacteria thereof in preparation of products for treating ulcerative colitis

Abstract

The present application relates to the technical field of probiotics, in particular to the application of a strain of Streptococcus ruminantium and a modified strain thereof in the preparation of a product for treating ulcerative colitis. The strain of Streptococcus ruminantium provided by the present application is a beneficial bacterium screened through differential analysis of clinical cohort stool samples, and is more in line with the intestinal flora characteristics of patients with ulcerative colitis. The present application modifies the strain of Streptococcus ruminantium through a manganese-iron bimetallic nanoscale enzyme, which can eliminate excess active oxygen in the intestinal environment and improve the treatment effect. In addition, the present application uses C18-PEG-PBA as a connecting agent, the hydrophobic end C18 of which can be inserted into the porous carbon skeleton of the manganese-iron bimetallic nanoscale enzyme through hydrophobic interaction, and the PBA end can undergo a mild esterification reaction with the polysaccharide on the surface of the probiotic bacteria, achieving stable connection without affecting the survival rate of the probiotic bacteria. The hydrophilic polyethylene glycol shell layer improves the biocompatibility, water solubility and stability of the product, and further improves the treatment effect of ulcerative colitis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of probiotics, in particular to the application of Ruminococcus torques and modified bacteria thereof in the preparation of products for treating ulcerative colitis. BACKGROUND

[0002] Ulcerative colitis is a chronic idiopathic inflammatory disease of the colon mainly involving the mucosa and submucosa of the rectum and colon. Although mesalazine, corticosteroids, immunosuppressants and biological agents have certain therapeutic effects, there is currently no cure, and the disease is prone to recurrence, which seriously affects the quality of life of patients and significantly increases the risk of colon cancer. Therefore, it is of great medical and social significance to further explore and find targeted clinical interventions for ulcerative colitis.

[0003] Previous studies have found that intestinal flora plays an important role in the development of ulcerative colitis, and many experiments and clinical studies have confirmed that probiotic-based therapy has high application prospects. However, existing probiotic preparations or products may not meet the intestinal flora characteristics of patients, and further screening of suitable individualized probiotics is still needed. SUMMARY

[0004] To solve the above problems, the present application provides the application of Ruminococcus torques and modified bacteria thereof in the preparation of products for treating ulcerative colitis. The Ruminococcus torques (ATCC 27756) provided by the present application is a beneficial bacteria selected by differential analysis of clinical cohort stool samples, which is more consistent with the intestinal flora characteristics of patients with ulcerative colitis and suitable for individualized treatment of patients with ulcerative colitis.

[0005] To achieve the above purpose, the present application provides the following technical solutions:

[0006] The present application provides the application of Ruminococcus torques and / or modified bacteria thereof in the preparation of products for treating ulcerative colitis, and the preservation number of the Ruminococcus torques is ATCC 27756.

[0007] Preferably, the product for treating ulcerative colitis includes a product for relieving one or more of weight loss, shortening of colon length and blood in stool and diarrhea caused by ulcerative colitis.

[0008] The application provides a nano-enzyme modified anaerobic rumen bacterium, comprising anaerobic rumen bacterium, manganese-iron bimetallic nano-enzyme and C18-PEG-PBA; C18 on the C18-PEG-PBA is connected with the manganese-iron bimetallic nano-enzyme through hydrophobic interaction, and PBA on the C18-PEG-PBA is connected with the anaerobic rumen bacterium through esterification reaction; the preservation number of the anaerobic rumen bacterium is ATCC 27756; and the preparation raw material of the manganese-iron bimetallic nano-enzyme comprises zinc nitrate hexahydrate, manganese chloride, iron trisacetylacetone and 2-methylimidazole.

[0009] Preferably, the preparation method of the manganese-iron bimetallic nano-enzyme comprises the following steps:

[0010] The zinc nitrate hexahydrate, manganese chloride, iron trisacetylacetone and 2-methylimidazole are dissolved in methanol to obtain a mixed solution;

[0011] The mixed solution is reacted at a temperature of 120 DEG C for 4 h, centrifuged, and the precipitate is collected;

[0012] The precipitate is calcined to obtain the manganese-iron bimetallic nano-enzyme.

[0013] Preferably, the mass ratio of the zinc nitrate hexahydrate, manganese chloride, iron trisacetylacetone and 2-methylimidazole is 1601:39:70:3700.

[0014] Preferably, the calcination condition comprises the following steps: increasing the temperature to 300 DEG C at a temperature increasing rate of 5 DEG C / min and keeping for 1 h, and then increasing the temperature to 900 DEG C at a temperature increasing rate of 5 DEG C / min and keeping for 4 h.

[0015] The application provides a preparation method of the nano-enzyme modified anaerobic rumen bacterium.

[0016] The C18-PEG-PBA solution and the manganese-iron bimetallic nano-enzyme are ultrasonically treated, the precipitate is collected, and the manganese-iron bimetallic nano-enzyme modified by the connecting agent is obtained;

[0017] The manganese-iron bimetallic nano-enzyme modified by the connecting agent and the anaerobic rumen bacterium liquid are mixed to obtain the nano-enzyme modified anaerobic rumen bacterium.

[0018] Preferably, the mass ratio of C18-PEG-PBA in the C18-PEG-PBA solution and the manganese-iron bimetallic nano-enzyme is 1:1; and the ratio of the manganese-iron bimetallic nano-enzyme modified by the connecting agent and the anaerobic rumen bacterium is 12.5 mu g-200 mu g:1x10 8 CFU-1x10 10 CFU.

[0019] Preferably, the ultrasonic treatment is ice bath ultrasonic treatment for 20 minutes; the reaction time of the manganese-iron bimetallic nanoenzyme modified Ruminococcus torques and the Ruminococcus torques bacterial solution after mixing is 20-30 minutes, and during the reaction, vortexing is performed for 5-10 seconds every 10 minutes.

[0020] The application provides application of the nanoenzyme modified Ruminococcus torques or the nanoenzyme modified Ruminococcus torques prepared by the preparation method in the above technical solution in preparation of a product for treating ulcerative colitis.

[0021] Beneficial effects:

[0022] The application provides application of Ruminococcus torques and / or modified bacteria thereof in preparation of a product for treating ulcerative colitis, and the Ruminococcus torques has a preservation number of ATCC 27756. The Ruminococcus torques (ATCC 27756) provided in the application is screened out through differential analysis of clinical cohort fecal samples, is more in line with the intestinal flora characteristics of patients with ulcerative colitis, and is suitable for individualized treatment of patients with ulcerative colitis.

[0023] Further, the nanoenzyme modified Ruminococcus torques provided in the application is modified by manganese-iron bimetallic nanoenzymes, and the superoxide dismutase and catalase activities of the manganese-iron bimetallic nanoenzymes can remove excess active oxygen in the intestinal environment, reduce the damage of active oxygen to the Ruminococcus torques, and improve the treatment effect on ulcerative colitis. In addition, C18-PEG-PBA is used as a connecting agent in the application, the hydrophobic end C18 of which can be inserted into the porous carbon skeleton of the manganese-iron bimetallic nanoenzyme through hydrophobic interaction; the PBA end can be subjected to a mild esterification reaction with polysaccharides on the surface of probiotics to realize stable connection without affecting the survival rate of the probiotics; the hydrophilic polyethylene glycol shell improves the biocompatibility, water solubility and stability of the product, and further improves the treatment effect on ulcerative colitis. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments.

[0025] Figure 1 Results of experiments on relieving ulcerative colitis in a dextran sulfate sodium (DSS)-induced mouse model by intestinal Ruminococcus torques; wherein, A is the change in the body weight of the mice during DSS induction, B is the change in the DAI score of the mice during DSS induction, C is a colon dissection diagram of the mice after DSS induction, and D is the difference in the colon length of the mice after DSS induction; * represents P<0.05, and ** represents P<0.01.

[0026] Figure 2 The morphology of the manganese-iron bimetallic nanozyme under a transmission electron microscope before etching;

[0027] Figure 3 The morphology of manganese-iron bimetallic nanozymes after etching under a transmission electron microscope;

[0028] Figure 4 The results show the detection results of catalase (CAT) and peroxidase (POD) activities of manganese-iron bimetallic nanozymes;

[0029] Figure 5 Experimental results demonstrating the ability of manganese-iron bimetallic nanozymes to scavenge reactive oxygen species; where A represents the scavenging ability of superoxide anion radicals (O₂). 2. - The experimental results for the ability of A to remove hydrogen peroxide (H2O2) are shown in Figure 1.

[0030] Figure 6 The experimental results show the binding ability of different concentrations of manganese-iron bimetallic nanozymes to probiotics.

[0031] Figure 7 The results show the effect of different concentrations of manganese-iron bimetallic nanozymes on the activity of probiotics;

[0032] Figure 8 Experimental results on the ability of probiotics to scavenge reactive oxygen species by modifying them with different concentrations of manganese-iron bimetallic nanozymes; where A represents the ability to scavenge superoxide anion radicals (O₂). 2. - The experimental results for A represent the ability to remove hydrogen peroxide (H2O2), and B represents the experimental results for the ability to remove hydrogen peroxide (H2O2). Detailed Implementation

[0033] This invention provides the use of *Ruminococcus torques* and / or its modified strains in the preparation of products for treating ulcerative colitis, wherein the *Ruminococcus torques* has the accession number ATCC27756. As one embodiment, the *Ruminococcus torques* with accession number ATCC 27756 was purchased from the American Tertiary Care Center (ATCC). As one embodiment, the product for treating ulcerative colitis includes products that alleviate one or more of the following symptoms caused by ulcerative colitis: weight loss, shortened colon length, and hematochezia / diarrhea. As one embodiment, the product can be a pharmaceutical product or a probiotic.

[0034] The present application provides a twisted chain coccus (ATCC 27756) which is a beneficial bacteria screened out through differential analysis of clinical cohort stool samples, and is more in line with the intestinal flora characteristics of patients with ulcerative colitis. The results of the examples show that the twisted chain coccus can alleviate the intestinal symptoms of mice caused by DSS induction, such as weight loss, colon length shortening, and hematochezia and diarrhea, and provides a new probiotic for treating ulcerative colitis, which can be suitable for individualized treatment of patients with ulcerative colitis.

[0035] Based on the above advantages, the present application provides a nano-enzyme modified twisted chain coccus, which comprises a twisted chain coccus, a manganese-iron bimetallic nano-enzyme and C18-PEG-PBA; the C18 on the C18-PEG-PBA is connected to the manganese-iron bimetallic nano-enzyme through hydrophobic interaction, and the PBA on the C18-PEG-PBA is connected to the twisted chain coccus through esterification; the preservation number of the twisted chain coccus is ATCC 27756; the raw materials for preparing the manganese-iron bimetallic nano-enzyme include zinc nitrate hexahydrate, manganese chloride, iron trisacetylacetone and 2-methylimidazole. The present application modifies the twisted chain coccus by the manganese-iron bimetallic nano-enzyme, utilizes the superoxide dismutase and catalase activity of the manganese-iron bimetallic nano-enzyme, can remove excess active oxygen in the intestinal environment, reduces the damage of active oxygen to the twisted chain coccus, and improves the treatment effect of ulcerative colitis; in addition, the present application uses C18 alkyl chain-polyethylene glycol-phenylboric acid (C18-PEG-PBA) as a linking agent, the hydrophobic end C18 of which can be inserted into the porous carbon skeleton of the manganese-iron bimetallic nano-enzyme through hydrophobic interaction; the PBA end can undergo a mild esterification reaction with the polysaccharide on the surface of the probiotic, realize stable connection, and does not affect the survival rate of the probiotic; the hydrophilic polyethylene glycol shell improves the biocompatibility, water solubility and stability of the product, and further improves the treatment effect of ulcerative colitis.

[0036] As an embodiment, the preparation method of the manganese-iron bimetallic nano-enzyme comprises:

[0037] Zinc nitrate hexahydrate, manganese chloride, iron trisacetylacetone and 2-methylimidazole are dissolved in methanol to obtain a mixed solution;

[0038] The mixed solution is reacted at a temperature of 120℃ for 4h, centrifuged, and the precipitate is collected;

[0039] The precipitate is calcined to obtain the manganese-iron bimetallic nano-enzyme.

[0040] As an embodiment, the present application dissolves zinc nitrate hexahydrate, manganese chloride and iron trisacetylacetone in methanol to obtain reagent A, dissolves 2-methylimidazole in methanol to obtain reagent B, adds reagent A to reagent B, and stirs vigorously at room temperature for at least 1h to obtain a mixed solution; the volume ratio of the reagent A to the reagent B is 1:1.

[0041] As an implementation form, the centrifugation conditions include: a rotation speed of 12000 rpm, a temperature of 4℃, and a centrifugation time of 10 min.

[0042] As an implementation form, before the precipitation calcination, the precipitate is washed 3-4 times with methanol and then dried.

[0043] As an implementation form, the mass ratio of the zinc nitrate hexahydrate, manganese chloride, iron trisacetylacetone and 2-methylimidazole is 1601:39:70:3700.

[0044] As an implementation form, the calcination conditions include: increasing the temperature to 300℃ at a temperature increasing rate of 5℃ / min and maintaining for 1 h, and then increasing the temperature to 900℃ at a temperature increasing rate of 5℃ / min and maintaining for 4 h.

[0045] The application provides a preparation method of the nano-enzyme modified Anaerococcus prevolii.

[0046] The C18-PEG-PBA solution and the manganese-iron bimetallic nano-enzyme are subjected to ultrasonic treatment, and the precipitate is collected to obtain the manganese-iron bimetallic nano-enzyme modified by the linker.

[0047] The manganese-iron bimetallic nano-enzyme modified by the linker and the Anaerococcus prevolii bacterial solution are mixed to obtain the nano-enzyme modified Anaerococcus prevolii.

[0048] As an implementation form, the mass ratio of the C18-PEG-PBA and the manganese-iron bimetallic nano-enzyme is 1:1; and the ratio of the manganese-iron bimetallic nano-enzyme modified by the linker and the Anaerococcus prevolii is 12.5 μg-200 μg:1×10 8 CFU-1×10 10 CFU. As another implementation form, the ratio of the manganese-iron bimetallic nano-enzyme modified by the linker and the Anaerococcus prevolii is 12.5 μg-200 μg:1×10 9 CFU; as another implementation form, the ratio of the manganese-iron bimetallic nano-enzyme modified by the linker and the Anaerococcus prevolii is 25 μg-100 μg:1×10 9 CFU; as another implementation form, the ratio of the manganese-iron bimetallic nano-enzyme modified by the linker and the Anaerococcus prevolii is 50 μg-100 μg:1×10 9 CFU.

[0049] As an embodiment, the ultrasonic treatment is ice bath ultrasonic treatment, and the time is 20 min. As an embodiment, the reaction time of the manganese-iron bimetallic nanoscale enzyme modified by the linker and the Ruminococcus torques bacterial solution after mixing is 20-30 min, and during the reaction, vortexing is performed every 10 min for 5-10 s. As another embodiment, the reaction time of the manganese-iron bimetallic nanoscale enzyme modified by the linker and the Ruminococcus torques bacterial solution after mixing is 25-30 min, and during the reaction, vortexing is performed every 10 min for 8-10 s.

[0050] Based on the above advantages, the application provides the application of the Ruminococcus torques modified by the nanoscale enzyme in the preparation of a product for treating ulcerative colitis. As an embodiment, the product for treating ulcerative colitis includes a product for relieving one or more of weight loss, colon length shortening, and bloody diarrhea caused by ulcerative colitis. As an embodiment, the product can be a drug or a probiotic.

[0051] In order to further illustrate the application, the application of the Ruminococcus torques and the modified bacteria thereof provided by the application in the preparation of a product for treating ulcerative colitis is described in detail below in combination with the accompanying drawings and examples, but they should not be understood as limiting the protection scope of the application.

[0052] Example 1: Intestinal Ruminococcus torques relieves dextran sodium sulfate (DSS)-induced mouse ulcerative colitis model

[0053] (1) Culture and preparation of intestinal Ruminococcus torques

[0054] Ruminococcus torques (ATCC 27756) was cultured in an anaerobic environment, and the culture medium used was ATCC Medium 1589, which was prepared as follows: 8 g of ATCC Medium 1589 was weighed into 100 mL of distilled water, 2 g of agarose was added, 0.05 g of L-cysteine hydrochloride, 0.5 mg of hemin chloride, and 0.1 mg of vitamin K1 were each added to one branch, 1 / 3 of the volume of the container was added with beef particles, and after mixing, high-pressure sterilization was performed at 121°C for 15 min. After sterilization was completed, it was placed in an anaerobic box composed of a mixture of 80% nitrogen, 10% hydrogen, and 10% carbon dioxide to remove oxygen overnight to obtain a deoxygenated liquid medium. The Ruminococcus torques freeze-dried powder was transferred to the deoxygenated liquid medium, and after 48 h of activation and culture, a flocculent precipitate was observed. After mixing by blowing, 200 μL of the culture was taken to a blood plate, and a L-shaped applicator stick was used to evenly spread it. Further culture was carried out in an anaerobic environment for 48 h, and the culture temperature was 37°C. Round transparent colonies appeared on the blood plate. A sterile cotton swab was used to scrape the colonies from the blood plate into a sterile tube of anaerobic PBS, and the concentration of the bacterial solution was adjusted to 1 x 10 9 CFU / mL to obtain a Ruminococcus torques bacterial solution.

[0055] (2) Treatment of mouse ulcerative colitis model by Ruminococcus torques

[0056] Eight-week-old C57BL / 6J male mice were divided into three groups after one week of pre-feeding: PBS group, DSS+PBS group, and DSS+R.torques group. The mice in the PBS group were given 200 μL / d of PBS by gavage for 17 days, and from the 10th day, the drinking water was changed to sterile water. The mice in the DSS+PBS group were given 200 μL / d of PBS by gavage for 17 days, and from the 10th day, the drinking water was changed to a dextran sulfate sodium (DSS) solution. The mice in the DSS+R.torques group were given 200 μL / d of Ruminococcus torques bacterial solution by gavage for 17 days, and from the 10th day, the drinking water was changed to a DSS solution. Body weight and DAI scores were recorded daily during the experiment. The method for DAI scoring is described in the literature

Wirtz S, Popp V, Kindermann M, et al. Chemically induced mouse models of acute and chronic intestinal inflammation. Nat Protoc. 2017; 12(7): 1295-1309.

[0057] The results show that the body weight of the DSS+R. torques group during the induction period is significantly higher than that of the DSS+PBS group, the DAI score is significantly lower than that of the DSS+PBS group, and the colon length of the DSS+R. torques group is significantly higher than that of the DSS+PBS group, indicating that Ruminococcus torques can alleviate the intestinal symptoms of mice caused by DSS induction, such as weight loss, colon length shortening, and bloody diarrhea, and provides a new probiotic for the treatment of ulcerative colitis.

[0058] Example 2 Preparation of manganese-iron bimetallic nanoscale enzyme and enzyme activity

[0059] (1) Preparation of manganese-iron bimetallic nanoscale enzyme

[0060] 1.601 g of zinc nitrate hexahydrate (Zn(NO3)2·6H2O), 39 mg of manganese chloride (MnCl2), and 70 mg of iron triacetylacetate (Fe(acac)3) were dissolved in 40 mL of methanol to obtain reagent A. 3.7 g of 2-methylimidazole (2-MI) was dissolved in 40 mL of methanol to obtain reagent B. Reagent A was added to reagent B, and the solution was stirred vigorously at room temperature for 1 h. The solution gradually changed from red transparent to orange. The stirred liquid was transferred to a high-pressure reaction kettle, and the reaction was carried out at 120℃ for 4 h. After cooling to room temperature, the precipitate was collected by centrifugation at 12000 rpm and 4℃ for 10 min. The precipitate was washed with methanol by centrifugation for 3-4 times under the same conditions. The washed precipitate was dried in a vacuum oven to obtain a micro-brown manganese-iron bimetallic framework (FeMnZIF-8). The FeMnZIF-8 was calcined in a tube furnace under the following conditions: the temperature was increased to 300℃ at a rate of 5℃ / min and maintained for 1 h, then increased to 900℃ at the same rate, and maintained for 4 h. After cooling to room temperature, a black manganese-iron bimetallic nanoscale enzyme was obtained. Under transmission electron microscopy, it was a rhombohedral dodecahedron with a particle size of about 200 nm, as shown in Figure 2 .

[0061] (2) As a comparison, the product after calcination of the FeMnZIF-8 prepared in step (1) was etched using the method described in the literature

Chu D, Zhao M, Rong S, et al. Dual-Atom Nanozyme Eye Drops Attenuate Inflammation and Break the Vicious Cycle in Dry Eye Disease. Nanomicro Lett. 2024; 16(1): 120. Published 2024 Feb 19.

[0062] (3) Hydrogen peroxidase (CAT) activity and peroxidase (POD) activity of manganese-iron bimetallic nanoscale enzyme

[0063] CAT activity: the manganese-iron bimetallic nanoscale enzyme prepared in step (1) was added to hydrogen peroxide (H2O2), and a large amount of bubbles was immediately generated Figure 4 A).

[0064] POD activity: 0.01 mL of color developing substrate TMB with a concentration of 10 mg / mL, 0.01 mL of 10M H2O2 and 0.05 mL of manganese-iron bimetallic nanoscale enzyme with a concentration of 1 mg / mL were added to 0.43 mL of 0.2M acetic acid-sodium acetate buffer (HAc / NaAc) with a pH of 5.5 and 0.43 mL of 0.2M acetic acid-sodium acetate buffer with a pH of 7.4, respectively. The control group used ultrapure water instead of manganese-iron bimetallic nanoscale enzyme. The reaction was carried out at room temperature for 5 min. POD can catalyze TMB to produce blue product, and by comparing with the control group, the POD activity of manganese-iron bimetallic nanoscale enzyme under different acid-base conditions can be reflected. It can be seen that under the condition of pH 7.4, the manganese-iron bimetallic nanoscale enzyme does not catalyze TMB to produce a large amount of blue product, indicating that the manganese-iron bimetallic nanoscale enzyme is acid-dependent and will not produce a large amount of active oxygen in the neutral-weak alkaline intestinal microenvironment Figure 4 B).

[0065] Example 3 Ability of manganese-iron bimetallic nanoscale enzyme to scavenge active oxygen

[0066] (1) Linker modified manganese-iron bimetallic nanoscale enzyme

[0067] 10 mg of C18-PEG-PBA was dissolved in 5 mL of water, and 10 mg of manganese-iron bimetallic nanoscale enzyme prepared in Example 2 was added thereto, and ultrasonic treatment was carried out in an ice bath for 20 min. Centrifugation was carried out at 12000 rpm and 4°C for 10 min, and the precipitate was collected. The precipitate was washed with water twice under the same centrifugation conditions. The precipitate after washing was dispersed with 5 mL of water to obtain linker modified manganese-iron bimetallic nanoscale enzyme (MnFe-PBA) with a concentration of 2 mg / mL.

[0068] (2) Ability to scavenge superoxide anion free radicals (O 2.- )·

[0069] Scavenging superoxide anion free radicals (O 2. -The ability of eliminating superoxide anion free radical can be reflected by the activity of superoxide dismutase (SOD). SOD can catalyze the dismutation of superoxide anion free radical to generate hydrogen peroxide (H2O2) and oxygen, and is an important antioxidant enzyme in organisms. The total SOD activity detection kit (WST-8 method) was used to detect the SOD enzyme activity. The MnFe-PBA stock solution with a concentration of 2 mg / mL was diluted with ultrapure water to a final concentration of 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, and 0 μg / mL. According to the kit instructions, the SOD enzyme activity of MnFe-PBA with different final concentrations was detected, and the results are shown in FIG. 2A. Figure 5 As shown in FIG. 2A, MnFe-PBA has good SOD enzyme activity.

[0070] (3) Ability of eliminating hydrogen peroxide (H2O2)

[0071] The ability of eliminating hydrogen peroxide (H2O2) can be reflected by the activity of catalase (CAT), which can catalyze hydrogen peroxide to generate water and oxygen. The catalase detection kit was used to detect the CAT enzyme activity. The MnFe-PBA stock solution with a concentration of 2 mg / mL was diluted with ultrapure water to a final concentration of 250 μg / mL, 200 μg / mL, 150 μg / mL, 100 μg / mL, and 50 μg / mL. According to the kit instructions, the CAT enzyme activity of MnFe-PBA with different final concentrations was detected, and the results are shown in FIG. 2B. Figure 5 As shown in FIG. 2B, MnFe-PBA has good CAT enzyme activity.

[0072] Example 4: Ability of combination of different concentrations of manganese-iron bimetallic nanoscale enzyme and probiotics

[0073] The intestinal Treponema succinifaciens was cultured in a liquid medium to 1×10 8 CFU / mL, 2 mL was taken into a centrifugal tube, centrifuged at 3000 rpm and 4°C for 5 min, the precipitate was collected and washed with phosphate buffered saline (PBS) twice under the same centrifugal condition, and the washed precipitate was resuspended with 1 mL of PBS to obtain a resuspended bacterial solution (denoted as Ruminococcus torques) with a concentration of 2×10 8 CFU / mL.

[0074] The MnFe-PBA prepared in Example 3 was diluted to 200 μg / 100 μL, 40 μg / 100 μL, and 4 μg / 100 μL, and 100 μL of each was uniformly mixed with 100 μL of the bacterial solution, respectively, and reacted for 30 min, during which the mixture was vortexed for 10 s every 10 min to obtain reaction solutions corresponding to different concentrations of MnFe-PBA. The bacterial solution and different reaction solutions were observed under a transmission electron microscope, and the results are shown in FIG. 3. Figure 6It can be seen that different concentrations of manganese-iron bimetallic nanoscale enzymes can successfully modify intestinal torsional Ruminococcus and will not damage the activity and structure of the bacteria.

[0075] Example 5 Influence of different concentrations of manganese-iron bimetallic nanoscale enzymes on the activity of probiotics

[0076] Ruminococcus torques (Rt) was cultured in an anaerobic environment for 48 h, and flocculent precipitates were visible in the liquid medium. After mixing by blowing, 200 μL of the culture was taken to a blood plate and evenly smeared with an L-type smear rod. The blood plate was then cultured in an anaerobic environment for 48 h, and round transparent colonies appeared on the blood plate. The colonies were scraped from the blood plate with a sterile cotton swab into a sterile tube containing anaerobic PBS, and the concentration of the bacterial solution was adjusted to 2×10 9 CFU / mL.

[0077] The MnFe-PBA prepared in Example 3 was diluted to 200 μg / mL, 100 μg / mL, and 50 μg / mL, and 500 μL of each was mixed with 500 μL of the bacterial solution. After 30 min of reaction, the mixture was vortexed for 10 s every 10 min. This resulted in Ruminococcus torques modified by manganese-iron bimetallic nanoscale enzymes at different concentrations: Rt@MnFe-PBA 100 (1×10 9 CFU Rt / 100 μg MnFe-PBA), Rt@MnFe-PBA 50 (1×10 9 CFU Rt / 50 μg MnFe-PBA), Rt@MnFe-PBA 25 (1×10 9 CFU Rt / 25 μg MnFe-PBA). 100 μL of each of the modified bacterial solutions was resuspended in a new liquid culture medium, and the activity of the bacteria was observed after 24 h of culture in an anaerobic environment. A new liquid culture medium was used as a blank control, and 100 μL of Ruminococcus torques bacterial solution with a concentration of 1×10 9 CFU / mL was resuspended in a new liquid culture medium as a positive control. The results are shown in Figure 7 It can be seen that different concentrations of MnFe-PBA combined with bacteria do not affect the activity of the bacteria.

[0078] Example 6 Enhancement of the ability of probiotics to scavenge reactive oxygen species by modification with different concentrations of manganese-iron bimetallic nanoscale enzymes

[0079] The method of Example 5 was used to prepare Ruminococcus torques modified by manganese-iron bimetallic nanoscale enzymes at different concentrations: Rt@MnFe-PBA 100 (1×109 CFU Rt / 100μg MnFe-PBA), Rt@MnFe-PBA 50 (1×10 9 CFU Rt / 50μg MnFe-PBA), Rt@MnFe-PBA 25 (1×10 9 CFU Rt / 25μg MnFe-PBA).

[0080] SOD and CAT activity of Ruminococcus torques at 1×10 9 CFU / mL were determined by the same principle and method as in Example 3. The results are shown in Table 5. Figure 8 It can be seen that the manganese-iron bimetallic nanoscale enzyme modified at different concentrations can improve the ability of probiotics to scavenge reactive oxygen species.

[0081] Although the above examples have made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which belong to the protection scope of the present application.

Claims

1. A nano-enzyme modified Fusicatenibacter succinatens, characterized in that, The C18 on the C18-PEG-PBA is connected with the manganese-iron bimetallic nanoscale enzyme through hydrophobic interaction, and the PBA on the C18-PEG-PBA is connected with the twisted chain rumen bacterium through esterification reaction; the preservation number of the twisted chain rumen bacterium is ATCC 27756; the preparation raw materials of the manganese-iron bimetallic nanoscale enzyme include: zinc nitrate hexahydrate, manganese chloride, iron trisacetylacetone and 2-methylimidazole; The preparation method of the manganese-iron bimetallic nanoscale enzyme comprises: The zinc nitrate hexahydrate, manganese chloride, iron trisacetylacetone and 2-methylimidazole are dissolved in methanol to obtain a mixed solution; The mixed solution is reacted at a temperature of 120 DEG C for 4h, centrifuged, and the precipitate is collected; The precipitate is calcined to obtain the manganese-iron bimetallic nanoscale enzyme.

2. The nanoenzyme-modified Z. torasil of claim 1, wherein, The mass ratio of the zinc nitrate hexahydrate, manganese chloride, iron trisacetylacetone and 2-methylimidazole is 1601:39:70:3700.

3. The nanoenzyme-modified Z. torasil of claim 1, wherein, The calcination conditions include: increasing the temperature to 300 DEG C at a temperature increasing rate of 5 DEG C / min for 1h, and then increasing the temperature to 900 DEG C at a temperature increasing rate of 5 DEG C / min for 4h.

4. Process for the preparation of a nano-enzyme modified Ruminococcus toruosus according to any one of claims 1 to 3, characterized in that, The method comprises the following steps: The C18-PEG-PBA solution and the manganese-iron bimetallic nanoscale enzyme are subjected to ultrasonic treatment, the precipitate is collected, and the manganese-iron bimetallic nanoscale enzyme modified by the connecting agent is obtained; The manganese-iron bimetallic nanoscale enzyme modified by the connecting agent and the twisted chain rumen bacterium solution are mixed to obtain the nanoscale enzyme modified twisted chain rumen bacterium.

5. The preparation method according to claim 4, characterized in that, The mass ratio of C18-PEG-PBA and manganese-iron bimetal nanoscale enzyme in the C18-PEG-PBA solution is 1:1; the ratio of the manganese-iron bimetal nanoscale enzyme modified by the linker and the T. praeacidoactylus is 12.5 μg-200 μg:1×10 8 CFU-1×10 10 CFU.

6. The preparation method according to claim 4, characterized in that, The ultrasonic treatment is ice bath ultrasonic treatment, and the time is 20min; the reaction time of the manganese-iron bimetallic nanoscale enzyme modified by the connecting agent and the twisted chain rumen bacterium solution after mixing is 20-30min, and during the reaction, every 10min is vortexed for 5-10s.

7. The use of the nanoscale enzyme modified twisted chain rumen bacterium of any one of claims 1-3 or the nanoscale enzyme modified twisted chain rumen bacterium prepared by the preparation method of any one of claims 4-6 in the preparation of a product for treating ulcerative colitis.

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