Ruminococcus Torque and application of modified bacteria of ruminococcus Torque in preparation of products for treating ulcerative colitis

Abstract

The invention relates to the technical field of probiotics, in particular to ruminococcus Torque and application of modified bacteria of the ruminococcus Torque in preparation of products for treating ulcerative colitis. The ruminococcus torsionosus provided by the invention is beneficial bacteria screened out through difference analysis of excrement samples in a clinical queue, and is more in line with intestinal flora characteristics of patients with ulcerative colitis. According to the invention, the ruminococcus tornadus is modified by the ferromanganese bimetallic nano-enzyme, so that excessive active oxygen in the intestinal environment can be removed, and the treatment effect is improved; besides, C18-PEG-PBA is used as a connecting agent, the hydrophobic end C18 of the C18 can be inserted into a porous carbon skeleton of the ferromanganese bimetallic nano-enzyme through hydrophobic interaction, the PBA end of the C18 can be subjected to mild esterification reaction with polysaccharide on the surfaces of the probiotics, stable connection is achieved, the survival rate of the probiotics is not affected, and the preparation method is simple and convenient to operate. Due to the hydrophilic polyethylene glycol shell layer, the biocompatibility, the water solubility and the stability of the product are improved, and the treatment effect on ulcerative colitis is further improved.

Description

Technical Field

[0001] The invention relates to the technical field of probiotics, and in particular to application of Ruminococcus contortus and modified bacteria thereof in preparing products for treating ulcerative colitis. Background Art

[0002] Ulcerative colitis is a chronic, idiopathic inflammatory disease of the colon that primarily affects the rectum, colon mucosa, and submucosa. 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 patient's quality of life and significantly increases the risk of colon cancer. Therefore, in-depth exploration and search for targeted clinical intervention measures for ulcerative colitis have important medical and social significance.

[0003] Previous studies have found that intestinal flora plays an important role in the occurrence and development of ulcerative colitis, and many experimental and clinical studies have also confirmed that probiotic-based therapies have a high application prospect. However, existing probiotic preparations or products may not meet the characteristics of the patient's intestinal flora, and further screening of appropriate and individualized probiotics is still needed. Summary of the invention

[0004] In order to solve the above problems, the present invention provides the use of Ruminococcus contortus and its modified bacteria in the preparation of products for treating ulcerative colitis. The Ruminococcus contortus (ATCC 27756) provided by the present invention is a beneficial bacteria screened out through differential analysis of fecal samples of a clinical cohort, which 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.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides the use of Ruminococcus torques and / or its modified bacteria in preparing a product for treating ulcerative colitis. The deposit number of the Ruminococcus torques is ATCC 27756.

[0007] Preferably, the product for treating ulcerative colitis includes a product that alleviates one or more of weight loss, shortened colon length and bloody diarrhea caused by ulcerative colitis.

[0008] The invention provides a nanozyme-modified Ruminococcus twisted, comprising Ruminococcus twisted, manganese-iron bimetallic nanozyme and C18-PEG-PBA; C18 on the C18-PEG-PBA is connected to the manganese-iron bimetallic nanozyme through hydrophobic interaction, and PBA on the C18-PEG-PBA is connected to the Ruminococcus twisted through an esterification reaction; the preservation number of the Ruminococcus twisted is ATCC 27756; the raw materials for preparing the manganese-iron bimetallic nanozyme include: zinc nitrate hexahydrate, manganese chloride, ferric triacetylacetonate and 2-methylimidazole.

[0009] Preferably, the preparation method of the manganese-iron bimetallic nanozyme comprises:

[0010] Dissolving zinc nitrate hexahydrate, manganese chloride, ferric triacetylacetonate and 2-methylimidazole in methanol to obtain a mixed solution;

[0011] The mixed solution was reacted at 120° C. for 4 h, centrifuged, and the precipitate was collected;

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

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

[0014] Preferably, the calcination conditions include: heating to 300° C. at a heating rate of 5° C. / min and maintaining for 1 hour, then heating to 900° C. at a heating rate of 5° C. / min and maintaining for 4 hours.

[0015] The present invention provides a method for preparing the nanozyme-modified Ruminococcus twisted chain described in the above technical scheme, comprising the following steps:

[0016] The C18-PEG-PBA solution and the manganese-iron bimetallic nanozyme are subjected to ultrasonic treatment, and the precipitate is collected to obtain the linker-modified manganese-iron bimetallic nanozyme;

[0017] The linker-modified manganese-iron bimetallic nanozyme and the bacterial solution of Ruminococcus contortus are mixed to obtain the nanozyme-modified Ruminococcus contortus.

[0018] Preferably, the mass ratio of C18-PEG-PBA to manganese-iron bimetallic nanozyme in the C18-PEG-PBA solution is 1:1; the ratio of manganese-iron bimetallic nanozyme modified by the linker to Ruminococcus twisted is 12.5 μg to 200 μg: 1×10 8 CFU~1×10 10 CFU.

[0019] Preferably, the ultrasonic treatment is ice bath ultrasonication for 20 minutes; the reaction time after mixing the linker-modified manganese-iron bimetallic nanozyme and the twisted chain Ruminococcus bacterial liquid is 20 to 30 minutes, and during the reaction, vortexing is performed for 5 to 10 seconds every 10 minutes.

[0020] The present invention provides the use of the nanozyme-modified Ruminococcus twisted chain described in the above technical scheme or the nanozyme-modified Ruminococcus twisted chain prepared by the preparation method described in the above technical scheme in the preparation of products for treating ulcerative colitis.

[0021] Beneficial effects:

[0022] The present invention provides the use of Ruminococcus torques and / or its modified bacteria in preparing a product for treating ulcerative colitis, wherein the deposit number of the Ruminococcus torques is ATCC 27756. The Ruminococcus torques (ATCC 27756) provided by the present invention is a beneficial bacterium screened out through differential analysis of fecal samples of a clinical cohort, is more consistent with the intestinal flora characteristics of patients with ulcerative colitis, and is suitable for individualized treatment of patients with ulcerative colitis.

[0023] Furthermore, the nanozyme-modified Ruminococcus twisted provided by the present invention modifies the Ruminococcus twisted by manganese-iron bimetallic nanozyme, and utilizes the superoxide dismutase and catalase activities of the manganese-iron bimetallic nanozyme to remove excess reactive oxygen in the intestinal environment, reduce the damage of reactive oxygen to the Ruminococcus twisted, and improve the therapeutic effect of ulcerative colitis; in addition, the present invention utilizes carbon 18 alkyl chain-polyethylene glycol-phenylboronic acid (C18-PEG-PBA) as a connector, and its hydrophobic end C18 can be inserted into the porous carbon skeleton of the manganese-iron bimetallic nanozyme through hydrophobic interaction; the PBA end can undergo a mild esterification reaction with the polysaccharide on the surface of the probiotic to achieve a stable connection without affecting the survival rate of the probiotic; the hydrophilic polyethylene glycol shell improves the biocompatibility, water solubility and stability of the product, further improving the therapeutic effect of ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0025] Figure 1 The experimental results of intestinal Ruminococcus torsion alleviating the mouse ulcerative colitis model induced by dextran sodium sulfate (DSS); A is the change of mouse body weight during DSS induction, B is the change of mouse DAI score during DSS induction, C is the anatomical diagram of mouse colon after DSS induction, and D is the difference of mouse colon length after DSS induction; * is P < 0.05, ** is P < 0.01;

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

[0027] Figure 3 This is the morphology of the manganese-iron bimetallic nanozyme after etching under a transmission electron microscope;

[0028] Figure 4 The results of the catalase (CAT) activity and peroxidase (POD) activity testing of the manganese-iron bimetallic nanozyme;

[0029] Figure 5 is the experimental result of the ability of manganese-iron bimetallic nanozyme to scavenge active oxygen; wherein A is the superoxide anion free radical (O 2. - ) ability, B is the ability to remove hydrogen peroxide (H 2 O 2 )’s ability to test results;

[0030] Figure 6 The experimental results of the binding ability of manganese-iron bimetallic nanozymes with probiotics at different concentrations;

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

[0032] Figure 8 The experimental results of different concentrations of manganese-iron bimetallic nanozymes to improve the ability of probiotics to scavenge reactive oxygen species; A is the superoxide anion free radical (O 2. - ) ability, B is the ability to remove hydrogen peroxide (H 2 O 2 )’s ability to perform experiments. DETAILED DESCRIPTION

[0033] The present invention provides the use of Ruminococcus torques and / or its modified bacteria in the preparation of products for treating ulcerative colitis, wherein the Ruminococcus torques has a deposit number of ATCC27756. As an embodiment, the Ruminococcus torques with a deposit number of ATCC 27756 is purchased from ATCC in the United States. As an embodiment, the product for treating ulcerative colitis includes a product for relieving one or more of weight loss, shortened colon length, and bloody diarrhea caused by ulcerative colitis. As an embodiment, the product can be a medicine or a probiotic.

[0034] The Ruminococcus contortus (ATCC 27756) provided by the present invention is a beneficial bacteria screened out through differential analysis of fecal samples of a clinical cohort, and is more consistent with the intestinal flora characteristics of patients with ulcerative colitis. The results of the embodiment show that the Ruminococcus contortus can alleviate the intestinal symptoms of weight loss, shortened colon length, and bloody diarrhea in mice induced by DSS, providing a new probiotic for the treatment of ulcerative colitis, and can be suitable for individualized treatment of patients with ulcerative colitis.

[0035] Based on the above advantages, the present invention provides a nanozyme-modified Ruminococcus twisted, including Ruminococcus twisted, manganese-iron bimetallic nanozyme and C18-PEG-PBA; the C18 on the C18-PEG-PBA is connected to the manganese-iron bimetallic nanozyme through hydrophobic interaction, and the PBA on the C18-PEG-PBA is connected to the Ruminococcus twisted through an esterification reaction; the preservation number of the Ruminococcus twisted is ATCC 27756; the raw materials for preparing the manganese-iron bimetallic nanozyme include: zinc nitrate hexahydrate, manganese chloride, triacetylacetonate iron and 2-methylimidazole. The present invention modifies the twisted chain Ruminococcus by manganese-iron bimetallic nanozyme, and utilizes the superoxide dismutase and catalase activities of the manganese-iron bimetallic nanozyme to remove excess reactive oxygen in the intestinal environment, reduce the damage of reactive oxygen to the twisted chain Ruminococcus, and improve the therapeutic effect of ulcerative colitis; in addition, the present invention utilizes carbon 18 alkyl chain-polyethylene glycol-phenylboronic acid (C18-PEG-PBA) as a linker, and its hydrophobic end C18 can be inserted into the porous carbon skeleton of the manganese-iron bimetallic nanozyme through hydrophobic interaction; the PBA end can undergo a mild esterification reaction with the polysaccharide on the surface of the probiotic to achieve a stable connection without affecting the survival rate of the probiotic; the hydrophilic polyethylene glycol shell layer improves the biocompatibility, water solubility and stability of the product, and further improves the therapeutic effect of ulcerative colitis.

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

[0037] Dissolving zinc nitrate hexahydrate, manganese chloride, ferric triacetylacetonate and 2-methylimidazole in methanol to obtain a mixed solution;

[0038] The mixed solution was reacted at 120° C. for 4 h, centrifuged, and the precipitate was collected;

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

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

[0041] As an embodiment, the centrifugal conditions include: a rotation speed of 12000 rpm, a temperature of 4°C, and a centrifugal time of 10 min.

[0042] As an embodiment, before the precipitate is calcined, the precipitate is washed 3 to 4 times with methanol and then dried.

[0043] As an embodiment, the mass ratio of zinc nitrate hexahydrate, manganese chloride, ferric triacetylacetonate and 2-methylimidazole is 1601:39:70:3700.

[0044] As an embodiment, the calcination conditions include: heating to 300° C. at a heating rate of 5° C. / min and maintaining for 1 hour, then heating to 900° C. at a heating rate of 5° C. / min and maintaining for 4 hours.

[0045] The present invention provides a method for preparing the nanozyme-modified Ruminococcus twisted chain described in the above technical scheme, comprising the following steps:

[0046] Ultrasonic treatment is performed on the C18-PEG-PBA solution and the manganese-iron bimetallic nanozyme, and the precipitate is collected to obtain the linker-modified manganese-iron bimetallic nanozyme;

[0047] The linker-modified manganese-iron bimetallic nanozyme and the bacterial solution of Ruminococcus contortus are mixed to obtain the nanozyme-modified Ruminococcus contortus.

[0048] As an embodiment, the mass ratio of the C18-PEG-PBA to the manganese-iron bimetallic nanozyme is 1:1; the ratio of the manganese-iron bimetallic nanozyme modified by the linker to the twisted chain Ruminococcus is 12.5 μg to 200 μg: 1×10 8 CFU~1×10 10 As another embodiment, the ratio of the linker-modified manganese-iron bimetallic nanozyme to the twisted Ruminococcus is 12.5 μg to 200 μg: 1×10 9 CFU; As another embodiment, the ratio of the linker-modified manganese-iron bimetallic nanozyme to the twisted chain Ruminococcus is 25 μg to 100 μg: 1×10 9 CFU; As another embodiment, the ratio of the linker-modified manganese-iron bimetallic nanozyme to the twisted chain Ruminococcus is 50 μg to 100 μg: 1×10 9 CFU.

[0049] As an embodiment, the ultrasonic treatment is ice bath ultrasonic treatment for 20 minutes. As an embodiment, the reaction time of the linker-modified manganese iron bimetallic nanozyme and the twisted chain Ruminococcus bacterial solution after mixing is 20 to 30 minutes, and during the reaction, vortexing is performed for 5 to 10 seconds every 10 minutes; as another embodiment, the reaction time of the linker-modified manganese iron bimetallic nanozyme and the twisted chain Ruminococcus bacterial solution after mixing is 25 to 30 minutes, and during the reaction, vortexing is performed for 8 to 10 seconds every 10 minutes.

[0050] Based on the above advantages, the present invention provides the use of the nanozyme-modified twisted Ruminococcus described in the above technical solution or the nanozyme-modified twisted Ruminococcus prepared by the preparation method described in the above technical solution in the preparation of products for treating ulcerative colitis. As an embodiment, the product for treating ulcerative colitis includes a product that relieves one or more of weight loss, shortened colon length, 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 present invention, the application of the twisted Ruminococcus and its modified bacteria provided by the present invention in the preparation of products for treating ulcerative colitis is described in detail below in combination with the drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0052] Example 1: Intestinal Ruminococcus contortus alleviates the dextran sulfate sodium (DSS)-induced ulcerative colitis model in mice

[0053] (1) Cultivation and preparation of intestinal Ruminococcus contortus

[0054] Ruminococcus torques (ATCC 27756) was cultured in an anaerobic environment. The culture medium used was ATCC Medium 1589. The preparation method was as follows: 8 g ATCC Medium 1589 was weighed into 100 mL of distilled water, 2 g agarose was added, 0.05 g L-cysteine ​​hydrochloride, 0.5 mg hemin chloride, 0.1 mg vitamin K1, 1 / 3 of the volume of the container was added with diced beef, mixed well, and sterilized at 121° C. for 15 min. After sterilization, it was placed in an anaerobic box composed of a mixed gas of 80% nitrogen, 10% hydrogen, and 10% carbon dioxide for overnight deoxygenation to obtain a deoxygenated liquid culture medium. Transfer the lyophilized powder of Ruminococcus twisted to the deoxygenated liquid culture medium, fully activate it, and culture it for 48 hours until flocculent precipitates are visible. After blowing and mixing, take 200 μL of the culture to the blood plate, spread it evenly with an L-shaped coating stick, and continue to culture it in an anaerobic environment for 48 hours at a temperature of 37°C. Circular transparent colonies can be seen on the blood plate. Use a sterile cotton swab to scrape the colonies from the blood plate into a sterile tube of anaerobic PBS, and adjust the bacterial solution concentration to 1×10 9 CFU / mL, and obtain Ruminococcus torques bacterial solution.

[0055] (2) Treatment of mouse ulcerative colitis model with intestinal Ruminococcus contortus

[0056] Thirty 8-week-old C57BL / 6J male mice were pre-fed for one week and then divided into three groups: PBS group, DSS+PBS group, and DSS+R.torques group. Mice in the PBS group were treated with 200 μL / d PBS by gavage for 17 days, and the drinking water was changed to sterile water from the 10th day. Mice in the DSS+PBS group were treated with 200 μL / d PBS by gavage for 17 days, and the drinking water was changed to dextran sulfate sodium (DSS) solution from the 10th day. Mice in the DSS+R.torques group were treated with 200 μL / d Ruminococcustorques bacterial solution by gavage for 17 days, and the drinking water was changed to DSS solution from the 10th day. During the experiment, body weight was recorded and DAI scores were performed every day. The DAI scoring method can be found 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.]. After the experiment, anatomical sampling was performed to measure the colon length of the mice in their natural state. The results are shown in Figure 1 .

[0057] The results showed that during the induction period, the body weight of the DSS+R.torques group was significantly higher than that of the DSS+PBS group, the DAI score was significantly lower than that of the DSS+PBS group, and the colon length of the DSS+R.torques group was significantly higher than that of the DSS+PBS group, indicating that Ruminococcus torques can alleviate the intestinal symptoms of weight loss, shortened colon length, and bloody diarrhea in mice induced by DSS, providing a new probiotic for the treatment of ulcerative colitis.

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

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

[0060] 1.601 g of zinc nitrate hexahydrate (Zn(NO 3 ) 2 6H 2 O), 39 mg manganese chloride (MnCl 2 ) and 70 mg ferric triacetylacetonate (Fe(acac) 3 ) was 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 stirred vigorously at room temperature for 1 hour. The solution gradually changed from red and transparent to orange. The stirred liquid was transferred to a high-pressure reactor and reacted at 120°C for 4 hours. After cooling to room temperature, centrifuged at 12000 rpm and 4°C for 10 minutes, and the precipitate was collected. The precipitate was centrifuged and washed 3 to 4 times with methanol under the same centrifugal conditions as before. The washed precipitate was dried in a vacuum oven to obtain a slightly brown manganese-iron bimetallic skeleton (FeMnZIF-8). FeMnZIF-8 was calcined in a tubular furnace under the following calcination conditions: the temperature was increased to 300°C at a rate of 5°C / min and maintained for 1 hour, and then the temperature was increased to 900°C at the same rate and maintained for 4 hours. After cooling to room temperature, a black manganese-iron bimetallic nanozyme was obtained. Under a transmission electron microscope, it appeared as a rhombic dodecahedron with a particle size of about 200 nm. Figure 2 .

[0061] (2) As a comparison, the present invention adopts 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 DryEye Disease. Nanomicro Lett. 2024; 16(1): 120. Published 2024 Feb 19.] to etch the product of FeMnZIF-8 calcined in step (1): washing with 12% hydrogen fluoride, 1M hydrochloric acid, water and methanol for 3 times, and vacuum drying at 60°C. The transmission electron microscopy morphology is as follows Figure 3 It can be seen that the performance after etching is worse than before etching, so etching is chosen not to be performed in subsequent applications.

[0062] (3) Catalase (CAT) and peroxidase (POD) activities of manganese-iron bimetallic nanozymes

[0063] CAT activity: The manganese-iron bimetallic nanozyme prepared in step (1) was added with hydrogen peroxide (H 2 O 2 ) can immediately generate a large number of bubbles ( Figure 4 (A).

[0064] POD activity: 0.01 mL of 10 mg / mL chromogenic substrate TMB, 0.01 mL of 10 M HAc, and 0.43 mL of 0.2 M acetic acid-sodium acetate buffer (HAc / NaAc) at pH 5.5 and 0.43 mL of 0.2 M acetic acid-sodium acetate buffer at pH 7.4 were added respectively. 2 O 2 and 0.05mL of 1mg / mL manganese-iron bimetallic nanozyme. Ultrapure water was used in the control group instead of manganese-iron bimetallic nanozyme. The reaction was carried out at room temperature for 5 minutes. POD catalyzes TMB to produce a blue product. The colorimetric analysis with the control group reflects the POD activity of the manganese-iron bimetallic nanozyme under different acid-base conditions. It can be seen that under pH 7.4, the manganese-iron bimetallic nanozyme did not catalyze TMB to produce a large amount of blue products, indicating that the manganese-iron bimetallic nanozyme is acid-dependent and does not produce a large amount of reactive oxygen species in the neutral-weakly alkaline intestinal microenvironment ( Figure 4 (middle B).

[0065] Example 3 Ability of manganese-iron bimetallic nanozymes to scavenge reactive oxygen species

[0066] (1) Linker modification of manganese-iron bimetallic nanozymes

[0067] 10 mg of C18-PEG-PBA was dissolved in 5 mL of water, and 10 mg of the manganese-iron bimetallic nanozyme prepared in Example 2 was added thereto, and the mixture was ultrasonicated in an ice bath for 20 min. Centrifuged at 12000 rpm and 4 ° C for 10 min, the precipitate was collected, and the precipitate was washed twice by centrifugation with water under the same centrifugation conditions as before. The washed precipitate was ultrasonically dispersed with 5 mL of water to obtain the linker-modified manganese-iron bimetallic nanozyme (MnFe-PBA) at a concentration of 2 mg / mL.

[0068] (2) Remove superoxide anion free radicals (O 2.- )

[0069] Scavenges superoxide anion free radicals (O 2. - ) can be reflected by the activity of superoxide dismutase (SOD). SOD can catalyze the dismutation of superoxide anion free radicals to generate hydrogen peroxide (H 2 O 2 ) and oxygen, and is an important antioxidant enzyme in organisms. Use the total SOD activity detection kit (WST-8 method) to detect SOD enzyme activity. Dilute the 2 mg / mL MnFe-PBA stock solution 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. Refer to the kit instructions to detect the SOD enzyme activity of different final concentrations of MnFe-PBA. The results are as follows: Figure 5 In Figure A, it can be seen that MnFe-PBA has good SOD enzyme activity.

[0070] (3) Removal of hydrogen peroxide (H 2 O 2 )

[0071] Removal of hydrogen peroxide (H 2 O 2 ) can be reflected by the activity of catalase (CAT), which can catalyze hydrogen peroxide to produce water and oxygen. Use a catalase detection kit to detect CAT enzyme activity. Dilute the 2 mg / mL MnFe-PBA stock solution with ultrapure water to a final concentration of 250 μg / mL, 200 μg / mL, 150 μg / mL, 100 μg / mL, and 50 μg / mL. Refer to the kit instructions to detect the CAT enzyme activity of MnFe-PBA at different final concentrations. The results are as follows: Figure 5 In B, it can be seen that MnFe-PBA has good CAT enzyme activity.

[0072] Example 4 Binding ability of manganese-iron bimetallic nanozymes with probiotics at different concentrations

[0073] Ruminococcus spp. was cultured in liquid medium until 1×10 8 CFU / mL, take 2mL into a centrifuge tube, centrifuge at 3000rpm, 4℃ for 5min, collect the precipitate, wash the precipitate twice with phosphate buffered saline (PBS) by centrifugation, centrifugation conditions are the same as before, resuspend the washed precipitate with 1mL PBS to obtain the resuspended bacterial solution (referred as Ruminococcus torques), the concentration is 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 taken and evenly mixed with 100 μL of bacterial solution, respectively, and reacted for 30 minutes, vortexed for 10 seconds every 10 minutes, and reaction solutions corresponding to different MnFe-PBA concentrations were obtained. The bacterial solution and different reaction solutions were observed under a transmission electron microscope, and the results are shown in FIG. Figure 6 It can be seen that different concentrations of manganese-iron bimetallic nanozymes can successfully modify intestinal Ruminococcus contortus without destroying the activity and structure of the bacteria.

[0075] Example 5 Effects of different concentrations of manganese-iron bimetallic nanozymes on the activity of probiotics

[0076] Ruminococcus torques (Rt) was cultured in an anaerobic environment for 48 hours. Flocculent precipitates were visible in the liquid culture medium. After mixing by blowing, 200 μL of the culture was taken to a blood plate and evenly spread with an L-shaped coating stick. The culture was continued in an anaerobic environment for 48 hours. Round transparent colonies were visible on the blood plate. The colonies were scraped from the blood plate with a sterile cotton swab and placed in a sterile tube of anaerobic PBS. 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 taken and evenly mixed with 500 μL of bacterial solution, respectively, and reacted for 30 min, during which vortexing was performed for 10 s every 10 min. The Ruminococcus torques modified with manganese-iron bimetallic nanozymes of different concentrations were obtained: Rt@MnFe-PBA 100 (1×10 9 CFU Rt / 100μg MnFe-PBA), Rt@MnFe-PBA 50 (1×10 9 CFU Rt / 50μg MnFe-PBA), Rt@MnFe-PBA 25 (1×10 9CFU Rt / 25μg MnFe-PBA). Take 100μL of each of the modified bacterial solutions and resuspend them in new liquid culture medium. After culturing in an anaerobic environment for 24h, count the cells to observe whether MnFe-PBA has an effect on the activity of the bacteria. Use the new liquid culture medium as a blank control, and take 100μL of 1×10 9 CFU / mL of Ruminococcus torques bacteria were resuspended in new liquid culture medium as a positive control. Figure 7 It can be seen that different concentrations of MnFe-PBA do not affect the activity of bacteria after combining with bacteria.

[0078] Example 6 Modification of manganese-iron bimetallic nanozymes with different concentrations to enhance the ability of probiotics to scavenge reactive oxygen species

[0079] The method of Example 5 was used to prepare Ruminococcustorques modified with manganese-iron bimetallic nanozymes of different concentrations: Rt@MnFe-PBA 100 (1×10 9 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] Use 1×10 9 The SOD and CAT enzyme activities were determined in the Ruminococcus torques bacterial solution with CFU / mL and the modified bacterial solution, and the principle and method were the same as in Example 3. The results are shown in Figure 8 It can be seen that different concentrations of manganese-iron bimetallic nanozyme modification have enhanced the ability of probiotics to eliminate reactive oxygen species.

[0081] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of Ruminococcus torques and / or its modified bacteria in the preparation of products for treating ulcerative colitis, wherein the deposit number of Ruminococcus torques is ATCC 27756.

2. The use according to claim 1, characterized in that: The product for treating ulcerative colitis includes a product for alleviating one or more of weight loss, shortening of colon length and bloody diarrhea caused by ulcerative colitis.

3. A nanozyme-modified Ruminococcus contortus, characterized in that: It includes Ruminococcus twisted, manganese-iron bimetallic nanozyme and C18-PEG-PBA; C18 on the C18-PEG-PBA is connected to the manganese-iron bimetallic nanozyme through hydrophobic interaction, and PBA on the C18-PEG-PBA is connected to the Ruminococcus twisted through esterification reaction; the preservation number of Ruminococcus twisted is ATCC 27756; the raw materials for preparing the manganese-iron bimetallic nanozyme include: zinc nitrate hexahydrate, manganese chloride, triacetylacetonate iron and 2-methylimidazole.

4. The nanozyme-modified Ruminococcus contortus according to claim 3, characterized in that: The preparation method of the manganese-iron bimetallic nanozyme comprises: Dissolving zinc nitrate hexahydrate, manganese chloride, ferric triacetylacetonate and 2-methylimidazole in methanol to obtain a mixed solution; The mixed solution was reacted at 120° C. for 4 h, centrifuged, and the precipitate was collected; The precipitate is calcined to obtain the manganese-iron bimetallic nanozyme.

5. The nanozyme-modified Ruminococcus contortus according to claim 4, characterized in that: The mass ratio of the zinc nitrate hexahydrate, manganese chloride, ferric triacetylacetonate and 2-methylimidazole is 1601:39:70:3700.

6. The nanozyme-modified Ruminococcus contortus according to claim 4, characterized in that: The calcination conditions include: heating to 300° C. at a heating rate of 5° C. / min and maintaining for 1 hour, then heating to 900° C. at a heating rate of 5° C. / min and maintaining for 4 hours.

7. The method for preparing the nanozyme-modified Ruminococcus twisted chain according to any one of claims 3 to 6, characterized in that: The following steps are involved: The C18-PEG-PBA solution and the manganese-iron bimetallic nanozyme are subjected to ultrasonic treatment, and the precipitate is collected to obtain the linker-modified manganese-iron bimetallic nanozyme; The linker-modified manganese-iron bimetallic nanozyme and the bacterial solution of Ruminococcus contortus are mixed to obtain the nanozyme-modified Ruminococcus contortus.

8. The preparation method according to claim 7, characterized in that: The mass ratio of C18-PEG-PBA and manganese-iron bimetallic nanozyme in the C18-PEG-PBA solution is 1:1; the ratio of manganese-iron bimetallic nanozyme modified by the linker to Ruminococcus twisted is 12.5 μg to 200 μg: 1×10 8 CFU~1×10 10 CFU.

9. The preparation method according to claim 7, characterized in that: The ultrasonic treatment is ice bath ultrasonic treatment for 20 minutes; the reaction time after the linker-modified manganese-iron bimetallic nanozyme and the twisted chain Ruminococcus bacterial liquid are mixed is 20 to 30 minutes, and during the reaction, vortexing is performed for 5 to 10 seconds every 10 minutes.

10. Use of the nanozyme-modified Ruminococcus twisted as described in any one of claims 3 to 6 or the nanozyme-modified Ruminococcus twisted prepared by the preparation method described in any one of claims 7 to 9 in the preparation of a product for treating ulcerative colitis.

Images