Synthetic method of probiotic-artificial enzyme system

Abstract

The invention discloses a synthesis method of a probiotic-artificial enzyme system. The synthesis method comprises the following steps: dissolving L / D-GSH in deionized water; adding 84 [mu] L of a 476.3 mmol / L HAuCl4. 4H2O aqueous solution, then adding the HAuCl4. 4H2O aqueous solution into 36 mL of deionized water, and stirring for 30 min; slowly adding 2mL of a NaBH4 aqueous solution with the concentration of 0.88 mmol; performing ultrafiltration treatment in an ultrafilter; the method comprises the following steps: dispersing L-Au or D-Au in an aqueous solution to obtain an L-Au or D-Au aqueous solution; the preparation method comprises the following steps: dissolving PEG-NH2 in an L-Au or D-Au aqueous solution; 0.5 ml of a NaOH solution is added into the mixed solution; adding a bacterial suspension containing 1.15 mg of EDC (Ethylene Dichloride) and 1.3 mg of NHS (Hydroxysuccinimide); the artificial enzyme system can protect the probiotics from smoothly passing through the gastrointestinal tract and being colonized in the intestinal tract for a long time, and the chiral gold nano-enzyme can protect the probiotics from being damaged by ROS in the intestinal tract; the probiotic-artificial enzyme system can control the blood sugar level and the in-vivo inflammatory factor level of mice with non-alcoholic steatohepatitis, improve the intestinal flora distribution and treat non-alcoholic steatohepatitis.

Description

Technical Field

[0001] The invention relates to the technical field of artificial enzyme systems, and in particular to a method for synthesizing a probiotics-artificial enzyme system. Background Art

[0002] Chiral nanomaterials have received extensive attention in the biomedical field in recent years and play an important role in medical technology research; gold, as an inert precious metal with good biocompatibility, has a long history of research in the biomedical field; AuNP (gold nanoparticles) are the most widely studied nanoparticles among all precious metal nanoparticles because they have multiple surface functions and unique surface plasmon resonance, which can be used in many ways. Based on this, we chose gold as a raw material to synthesize chiral nanomaterials for the treatment of diseases. A large number of studies have shown that the performance of nanomaterials has an intricate relationship with factors such as their chirality; chiral inorganic nanomaterials can exhibit enzyme-like properties, and nanozymes overcome the shortcomings of natural enzymes such as recyclability, controllable activity, and low preparation cost; glutathione, as one of the most important antioxidants in the antioxidant system, can protect tissues from damage by reactive oxygen species; therefore, using the characteristics of the above materials to synthesize an artificial enzyme is an urgent problem that technicians in related fields need to solve. Summary of the invention

[0003] In order to solve the above technical problems, the technical solution provided by the present invention is: a method for synthesizing a probiotic-artificial enzyme system, comprising the following steps:

[0004] (1) Weigh 36.88 mg of L-GSH / D-GSH and dissolve it in 5 mL of deionized water;

[0005] (2) Add 84 μL of 476.3 mmol / L HAuCl to the solution in step (1). 4 ·4H 2 O aqueous solution, mix it with the solution in step (1) evenly, then add it into 36mL deionized water and stir for 30min;

[0006] (3) Slowly add 0.88 mmol of NaBH to the mixed solution of step (2) 4 2 mL of aqueous solution was added, and stirred rapidly to fully reduce the gold ions, and then stirred for 2 h until the solution turned brown;

[0007] (4) placing the product obtained in step (3) into an ultrafiltration machine for ultrafiltration treatment, and then washing the ultrafiltered product with deionized water three times to remove free GSH molecules to obtain L-Au or D-Au, i.e., chiral gold nanoparticles;

[0008] (5) dispersing the L-Au or D-Au obtained in step (4) in an aqueous solution to obtain an L-Au or D-Au aqueous solution;

[0009] (6) Take 10 ml of the L-Au or D-Au aqueous solution obtained in step (5) and 500 mg of PEG-NH 2 PEG-NH 2 Dissolved in L-Au or D-Au aqueous solution;

[0010] (7) Wait for the PEG-NH 2 After complete dissolution, add 0.5 ml of NaOH solution, react for 0.5 h, add 2 mL of AA solution, stir for 0.5 h, wash repeatedly with deionized water to obtain PEG-Au solution, and pour out for later use;

[0011] (8) Take 1 ml of the PEG-Au prepared in step (7), add the bacterial suspension containing 1.15 mg EDC and 1.3 mg NHS, stir for 0.5 h and then centrifuge to obtain a probiotic-artificial enzyme, which is then washed three times with PBS.

[0012] Preferably, the ultrafiltration treatment in step (4) is: ultrafiltration at a rotation speed of 10000 rpm for 15 min.

[0013] Preferably, the solubility of the NaOH solution in step (7) is 240 mg / mL.

[0014] Preferably, the preparation method of the bacterial suspension in step (8) comprises the following steps:

[0015] (8.1) Prepare modified YCFA liquid medium, activate the Faecalibacterium prausnitzii strain, and store it at 4 degrees Celsius for later use;

[0016] (8.2) The Faecalibacterium prausnitzii strain was inoculated into a modified YCFA liquid medium at a ratio of 5%, cultured at 37°C for 72 h, and the bacterial concentration was adjusted to 4 x 109 cfu / ml using a McFarland turbidimeter.

[0017] Preferably, the centrifugation in step (8) is: centrifugation at a rotation speed of 3000 rpm for 5 minutes.

[0018] The advantages of the present invention over the prior art are: (1) the artificial enzyme system of the present invention can protect probiotics from passing through the gastrointestinal tract smoothly and colonizing in the intestine for a long time, and the chiral gold nanozyme can protect probiotics from ROS damage in the intestine;

[0019] (2) The probiotic-artificial enzyme system of the present invention can control the blood sugar level and the level of inflammatory factors in the body of mice with non-alcoholic fatty liver disease, improve the distribution of intestinal flora, and treat non-alcoholic fatty liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a TEM electron microscope image of the chiral gold nanozyme of the present invention.

[0021] Figure 2 It is a TEM electron microscope picture of the probiotics-artificial enzyme system of the present invention.

[0022] Figure 3 It is a schematic diagram of the ability of the chiral gold nanoparticles of the present invention to scavenge ROS at the cellular level.

[0023] Figure 4 It is a schematic diagram of the chiral gold nanoparticles of the present invention verifying the ability to protect cells from protein, DNA, and lipid damage caused by ROS and the anti-inflammatory ability at the cellular level.

[0024] Figure 5 It is a schematic diagram of the colonization ability of the present invention in vivo 24 hours after administration.

[0025] Figure 6 It is a schematic diagram of the ability of the probiotic-artificial enzyme system of the present invention to reduce inflammation in hepatitis mice. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Generally, the components of the embodiments of the present invention described and shown in the drawings here can be arranged and designed in various different configurations.

[0027] Embodiment 1:

[0028] This embodiment discloses a method for synthesizing a probiotic-artificial enzyme system, comprising the following steps:

[0029] (1) Weigh 36.88 mg of L-GSH / D-GSH (oxidized glutathione / reduced glutathione) and dissolve it in 5 mL of deionized water;

[0030] (2) Add 84 μL of 476.3 mmol / L HAuCl to the solution in step (1). 4 ·4H 2 O (tetrachloroauric acid trihydrate) aqueous solution, after mixing it with the solution in step (1) evenly, add it into 36 mL of deionized water and stir for 30 min;

[0031] (3) Slowly add 0.88 mmol of NaBH to the mixed solution of step (2) 4 (Sodium borohydride) aqueous solution 2mL, stir rapidly to fully reduce the gold ions, and then stir for 2h until the solution turns brown;

[0032] (4) placing the product obtained in step (3) into an ultrafiltration machine for ultrafiltration treatment, and then washing the ultrafiltration product with deionized water for 3 times to remove free GSH molecules, thereby obtaining L-Au or D-Au, i.e., chiral nanogold particles; wherein the ultrafiltration treatment is: ultrafiltration at a rotation speed of 10,000 rpm for 15 min;

[0033] (5) dispersing the L-Au or D-Au obtained in step (4) in an aqueous solution to obtain a L-AU / D-AU aqueous solution;

[0034] (6) Take 10 ml of the L-Au or D-Au aqueous solution obtained in step (5) and 500 mg of PEG-NH 2 PEG-NH 2 (polyethylene glycol (PEG) derivatives with amino groups (-NH2) at both ends) dissolved in L-Au or D-Au aqueous solution;

[0035] (7) Wait for the PEG-NH 2 After complete dissolution, add 0.5 ml of NaOH (sodium hydroxide) solution, react for 0.5 h, then add 2 mL of AA (acrylic acid) solution, stir for 0.5 h, wash repeatedly with deionized water to obtain PEG-Au solution, pour out for later use; wherein the solubility of NaOH solution is 240 mg / mL;

[0036] (8) Take 1 ml of the PEG-Au prepared in step (7), add a bacterial suspension containing 1.15 mg of EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) and 1.3 mg of NHS (N-hydroxysuccinimide), stir for 0.5 h, and then centrifuge to obtain a probiotic-artificial enzyme, and wash it three times with PBS (phosphate buffered saline); the centrifugation treatment is: centrifuge at a speed of 3000 rpm for 5 min;

[0037] The preparation method of the bacterial suspension comprises the following steps:

[0038] (8.1) Prepare modified YCFA liquid medium, activate the Faecalibacterium prausnitzii strain, and store it at 4 degrees Celsius for later use;

[0039] (8.2) The Faecalibacterium prausnitzii strain was inoculated into a modified YCFA liquid culture medium at a ratio of 5%, cultured at 37°C for 72 h, and the bacterial concentration was adjusted to 4 x 109 cfu / ml using a McFarland turbidimeter.

[0040] 1. Experimental Verification

[0041] 1. Combined with Figure 1 The morphology of chiral gold nanoparticles was observed using a transmission electron microscope (TEM), and 40 L-Au and D-Au particles were measured to calculate the average particle size. The results showed that the average particle size of L-Au and D-Au was 3.4nm.

[0042] 2. The chiral properties of the nanomaterials were verified by circular dichroism (CD) spectroscopy. The results showed that the CD spectrum of L-Au showed a negative peak at 220nm, while D-Au showed a positive CD spectrum at this time, indicating that these gold nanoparticles have chiral properties; in addition, the UV-visible absorption spectrum of L-Au overlaps with that of D-Au; the zeta potential value of the synthesized L-Au is almost the same as that of D-Au; in summary, the synthesized gold nanoparticles have symmetrical CD spectra, almost the same particle size, UV absorption and surface charge, and it is easy to conclude that surface chirality is the only difference between L-Au and D-Au.

[0043] 3. Combined with attachment Figure 2 , TEM microscopy showed that Faecalibacterium prausnitzii was rod-shaped, about 1500-2000nm long, providing ample space for the attachment of gold nanoparticles; because chiral gold nanoparticles and Faecalibacterium prausnitzii are negatively charged and cannot be directly connected, the chiral gold nanoparticles were first purified with amino-containing polyethylene glycol and then bonded to the surface of Faecalibacterium prausnitzii through an amide condensation reaction. TEM showed that the probiotics were successfully connected to the chiral gold nanoparticles; further TEM element mapping and energy dispersive spectroscopy (EDS) analysis data jointly proved that the surfaces of these engineered bacteria were rich in AU, indicating that the chiral gold nanoparticles had been successfully anchored on the bacterial surface.

[0044] 4. Combine with attachment Figure 3-6 , verify the anti-ROS ability of the chiral L-Au (synthetic material containing glutathione) and D-Au (synthetic material containing glutathione) of this example:

[0045] The superoxide dismutase (SOD) and catalase (CAT) detection kits were used to detect the SOD and CAT activities of chiral gold nanoparticles at different concentrations. Catalase (CAT) has the ability to catalyze the oxidation of two molecules of H 2 O 2 Decomposition to generate O 2 and H 2 O's ability to prevent H 2 O 2Superoxide dismutase (SOD) is a key antioxidant enzyme against reactive oxygen species (ROS), catalyzing the oxidation of O 2 -·Converted to O 2 and H 2 O 2 The test results show that both chiral gold nanoparticles have anti-ROS ability. As the concentration increases, the chiral gold nanoparticles scavenge more ROS, but D-Au has a stronger ability to scavenge ROS.

[0046] In summary, chiral gold nanoparticles have therapeutic potential in inflammatory diseases and can protect probiotics from ROS damage in the gastrointestinal tract.

[0047] The present invention and its embodiments are described above, and such description is not restrictive. The drawings show only one embodiment of the present invention, and the actual structure is not limited thereto. In short, if ordinary technicians in the field are inspired by it, without departing from the purpose of the invention, they can design a structure and embodiment similar to the technical solution without creativity, which should belong to the protection scope of the present invention.

Claims

1. A method for synthesizing a probiotic-artificial enzyme system, characterized in that: The following steps are involved: (1) Weigh 36.88 mg of L-GSH / D-GSH and dissolve it in 5 mL of deionized water; (2) adding 84 μL of 476.3 mmol / L HAuCl4·4H2O aqueous solution to the solution of step (1), mixing the HAuCl4·4H2O aqueous solution with the solution of step (1) evenly, and then adding the mixture to 36 mL of deionized water and stirring for 30 min; (3) Slowly add 2 mL of 0.88 mmol of NaBH4 aqueous solution to the mixed solution of step (2), stir rapidly to fully reduce the gold ions, and stir for another 2 h until the solution turns brown; (4) placing the product obtained in step (3) into an ultrafiltration machine for ultrafiltration treatment, and then washing the ultrafiltered product with deionized water three times to remove free GSH molecules to obtain L-Au or D-Au, i.e., chiral gold nanoparticles; (5) dispersing the L-Au or D-Au obtained in step (4) in an aqueous solution to obtain a L-Au or D-Au solution; (6) Take 10 ml of the L-Au or D-Au aqueous solution obtained in step (5) and 500 mg of PEG-NH2, and dissolve the PEG-NH2 in the L-Au or D-Au aqueous solution; (7) After the PEG-NH2 in step (6) is completely dissolved, add 0.5 ml of NaOH solution thereto, react for 0.5 h, then add 2 mL of AA solution, stir for 0.5 h, and wash repeatedly with deionized water to obtain a PEG-Au solution, which is then poured out for later use; (8) Take 1 ml of the PEG-Au prepared in step (7), add the bacterial suspension containing 1.15 mg EDC and 1.3 mg NHS, stir for 0.5 h and then centrifuge to obtain a probiotic-artificial enzyme, which is then washed three times with PBS.

2. The method for synthesizing a probiotic-artificial enzyme system according to claim 1, characterized in that: The ultrafiltration treatment in step (4) is: ultrafiltration at a rotation speed of 10000 rpm for 15 minutes.

3. The method for synthesizing a probiotic-artificial enzyme system according to claim 1, characterized in that: The solubility of the NaOH solution in step (7) is 240 mg / mL.

4. The method for synthesizing a probiotic-artificial enzyme system according to claim 1, characterized in that: The preparation method of the bacterial suspension in step (8) comprises the following steps: (8.1) Prepare modified YCFA liquid medium, activate the Faecalibacterium prausnitzii strain, and store it at 4 degrees Celsius for later use; (8.2) The Faecalibacterium prausnitzii strain was inoculated into a modified YCFA liquid medium at a ratio of 5%, cultured at 37°C for 72 h, and the bacterial concentration was adjusted to 4 × 10 9 cfu / mI.

5. The method for synthesizing a probiotic-artificial enzyme system according to claim 1, characterized in that: The centrifugation in step (8) is: centrifugation at a speed of 3000 rpm for 5 minutes.

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