Bacillus licheniformis F0726-coated Fe-TA-coated GN compound as well as preparation method and application thereof in AD (Alzheimer's disease)

Abstract

The invention provides a bacillus licheniformis F0726 (at) Fe-TA (at) GN compound as well as a preparation method and application thereof in AD (Alzheimer's disease). Relates to the microbial field. Experiments prove that after the bacillus licheniformis F0726 is embedded by iron / tannic acid / glucan, the resistance of the bacillus licheniformis F0726 in gastric acid and intestinal juice is greatly improved. When the F0726-coated Fe-TA-coated GN is applied to an Alzheimer's disease model mouse induced by brain injection of A beta oligomer, the spatial learning and memory disorder of the AD model mouse can be better improved, the inflammation level of the AD mouse is reduced, and the SCFAs content is increased. The invention provides a new way for developing drugs related to prevention and treatment of Alzheimer's disease, and provides a new idea for probiotic storage and synergistic medication.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms, and specifically relates to a Bacillus licheniformis F0726@Fe-TA@GN complex, a preparation method thereof, and an application thereof in AD. Background Art

[0002] Bacillus licheniformis is an aerobic Gram-positive bacterium widely found in various ecological environments such as water bodies and soil. It is a common endophytic bacterium in plants, especially in the roots and stems of plants. Bacillus licheniformis competes with pathogens for nutrients and living space, produces metabolites that inhibit the growth of pathogens, secretes lytic substances that dissolve the cell walls or cell membranes of pathogens, and even promotes plant growth and induces plant resistance, thereby playing its role in preventing and curing diseases. Bacillus licheniformis does not pollute the environment, is harmless to humans and animals, has strong environmental adaptability, can produce spores with strong stress resistance, and is extremely easy to isolate and culture. It is considered to be an ideal plant disease biocontrol bacterium.

[0003] Alzheimer's disease (AD) is the most common type of dementia. It is a degenerative disease of the central nervous system characterized by progressive cognitive dysfunction and behavioral impairment. In recent years, there have been more and more studies on the treatment of AD, but the treatment of AD is still difficult. AD patients generally have symptoms such as cognitive impairment, behavioral disorders, memory loss, language disorders, and personality changes. Studies have found that the pathological characteristics of patients with Alzheimer's disease include senile plaques formed by Aβ deposition, neurofibrillary tangles produced by hyperphosphorylation of tau protein, and neuronal loss mainly in the hippocampal cortex. At present, the main treatment drugs for Alzheimer's disease are acetylcholinesterase inhibitors (tacrine, donepezil, capracin, galantamine, etc.) and aspartate receptor antagonists (memantine), but these drugs will more or less produce some side effects, such as strong hepatotoxicity, nausea and vomiting, etc.

[0004] However, probiotic lactobacilli are sensitive to extreme environmental conditions, have poor stability during food processing and storage, and have low bioavailability when exposed to digestive tract conditions. Therefore, when probiotics are processed, stored, and in the host's gastrointestinal tract, they will inevitably be subjected to various stresses from the external environment, which will in turn affect their stability and bioavailability, which greatly limits their probiotic efficacy and application in the food industry. At present, many encapsulation methods involve the use of high / low temperatures and organic solvents, which can easily cause loss of vitality and safety risks; in view of this, we should further deepen basic research and strengthen technological innovation, combine feasible technologies such as nanoprecipitation to synergize prebiotics, protect probiotics from various environmental stresses while ensuring their probiotic efficacy, and solve the key problem of probiotic stability and functionality under environmental stress. Summary of the invention

[0005] To solve the above technical problems, the present invention aims to provide a Bacillus licheniformis F0726@Fe-TA@GN complex, a preparation method thereof, and an application in AD. The Bacillus licheniformis F0726@Fe-TA@GN complex, namely F0726@Fe-TA@GN, can improve the spatial learning ability and memory impairment of AD mice, improve the levels of inflammatory factors in the brains of AD mice, and regulate the content of SCFAs in the cecal contents of AD mice.

[0006] The present invention provides a preparation method of a Bacillus licheniformis F0726@Fe-TA@GN complex. Bacillus licheniformis F0726 is encapsulated in a metal network phenolic structure composed of iron and tannic acid (Fe-TA), and a prebiotic barrier composed of dextran (GN) is encapsulated on its outer layer to resist adverse environments such as gastric acid and intestinal juice.

[0007] Furthermore, Bacillus licheniformis F0726 was deposited at the Guangdong Provincial Microbial Culture Collection Center on March 18, 2024; the deposit address is the 5th floor of Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences; the deposit number is GDMCC No. 64432.

[0008] The embedding method of the above F0726@Fe-TA@GN is as follows:

[0009] a1. Culturing the Bacillus licheniformis F0726 bacterial solution: Inoculate the Bacillus licheniformis F0726 into a 5 ml LB liquid test tube and culture it at 37°C and 200 r / min for 12 hours;

[0010] a2. Centrifuge the bacterial solution obtained in a1 at 8000 rpm for 5 min, wash it with sterile PBS buffer, and then resuspend it in sterile PBS buffer to make the F0726 bacterial concentration 10 8 ~10 10 CFU / mL;

[0011] a3. Add 100 μL of the F0726 bacterial suspension prepared in a2, 10 μL of a 40 mg / mL TA solution, 10 μL of the TA solution, and 10 μL of a 10 mg / mL FeCl3 solution to 880 μL of PBS buffer, and vortex and mix well to obtain F0726@Fe-TA;

[0012] a4. Add GN solution to the F0726@Fe-TA prepared in a3 to make the final concentration of GN in the system 1.25 mg / mL, and gently mix well to finally obtain the Bacillus licheniformis F0726@Fe-TA@GN complex; furthermore, F0726@Fe-TA@GN is used to improve the resistance of Bacillus licheniformis F0726 in gastric juice and intestinal juice.

[0013] Furthermore, F0726@Fe-TA@GN is used to improve the spatial learning and memory impairment in Alzheimer's disease mice.

[0014] Furthermore, F0726@Fe-TA@GN is used to reduce the levels of inflammatory factors in the brains of Alzheimer's disease mice.

[0015] Furthermore, F0726@Fe-TA@GN is used to regulate the content of SCFAs in the cecal contents of Alzheimer's disease mice.

[0016] The present invention formulates F0726@Fe-TA@GN into the dosage forms of drugs, health products or foods, and the viable count of Bacillus licheniformis F0726 in the drugs, health products or foods is 1×10 8 CFU / mL to 1×10 10 CFU / mL.

[0017] The technical solution of the present invention has the following advantages:

[0018] The present invention proves through experiments that F0726@Fe-TA@GN can improve the resistance of Bacillus licheniformis F0726 in gastric juice and intestinal juice. In Alzheimer's disease model mice induced by intracerebral injection of Aβ oligomers, it can improve the spatial learning and memory impairment of AD model mice, reduce the levels of inflammatory factors in the brains of AD mice, and improve the content of SCFAs in the cecal contents of AD mice, providing a new approach for the development of drugs related to the prevention and treatment of Alzheimer's disease, as well as potential probiotic resources. Description of the Drawings

[0019] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0020] Figure 1 Zeta potential (a) and particle size distribution diagram (b) of F0726@Fe-TA@GN;

[0021] Figure 2 Scanning diagram of F0726@Fe-TA@GN under TEM. (a1) Scanning diagram of F0726 under TEM at 5 μm, (a2) Scanning diagram of F0726 under TEM at 1 μm, (b1) Scanning diagram of F0726@Fe-TA@GN under TEM at 5 μm, (b2) Scanning diagram of F0726@Fe-TA@GN under TEM at 1 μm;

[0022] Figure 3 The survival rates of F0726@Fe-TA@GN in gastric juice (a) and intestinal juice (b);

[0023] Figure 4 The activity path diagrams of the water maze experiment for AD mice after intervention treatment with F0726@Fe-TA@GN; (a) is the activity path diagram of the Control group, (b) is the activity path diagram of the Model group, (c) is the activity path diagram of the F0726 group, and (d) is the activity path diagram of the F0726@Fe-TA@GN group;

[0024] Figure 5 The statistical results of the number of times of crossing the platform in the water maze experiment for AD mice after intervention treatment with F0726@Fe-TA@GN;

[0025] Figure 6 For the TLR4 / MYD 88 / NF-κB / p NF-κB

[0026] WB results of AD mice after intervention treatment with F0726@Fe-TA@GN;

[0027] Figure 7 The statistical results of the SCFAs content in the cecal contents of AD mice after intervention treatment with F0726@Fe-TA@GN. (a) is the statistical result of the 2-methylbutyric acid content, and (b) is the statistical result of the isobutyric acid content. Detailed implementation manners

[0028] The following embodiments are provided to better further understand the present invention. It is not limited to the described optimal implementation manner, and does not limit the content and protection scope of the present invention. Any product that is the same as or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other existing technologies falls within the protection scope of the present invention.

[0029] For those not specifying specific experimental steps or conditions in the embodiments, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. The raw materials or instruments used are all conventional products that can be obtained through commercial purchase, including but not limited to the raw materials or instruments used in the embodiments of the present application.

[0030] Research shows that the increase in Aβ protein is the main inducement for the occurrence of AD. Therefore, currently, injecting Aβ42 oligomers into the brain can induce AD symptoms in mice. This injection method can simulate the changes at the molecular level and behavioral level, such as the weakening of learning and memory abilities. This modeling method has a short modeling time, but there are large individual differences, and the injection method is quite skillful. The increase in the Aβ protein content in the mouse brain can trigger a series of other symptoms, such as oxidative stress, inflammatory responses, etc., ultimately leading to cell death.

[0031] The following will specifically describe a Bacillus licheniformis F0726@Fe-TA@GN complex, its preparation method, and its application in AD in combination with specific experiments.

[0032] Example 1

[0033] I. Experimental materials and instruments

[0034] 1. Experimental animals

[0035] Male C57BL / 6 mice, 8 weeks old, weighing 20 - 25 g, were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd. Six mice were housed in each cage and were all raised in an environment with a temperature of 22 - 25 °C, a 12-hour day-night rhythm, and free access to food and water.

[0036] 2. Drugs and main reagents

[0037] Bacillus licheniformis F0726 has been deposited in the Guangdong Provincial Microbial Culture Collection Center, with the deposit number: GDMCC No. 64432; the Aβ42 protein used for modeling was purchased from Shanghai Gil Biochemical Co., Ltd. FeCl3 was purchased from Macklin, and dextran and tannic acid were purchased from Bio-Thera. In addition, other information about Bacillus licheniformis F0726, including isolation, identification, deposit, etc., can refer to the Chinese patent with the application number 202410732987.X (the invention name is a Bacillus licheniformis F0726 for improving neuroinflammation in Alzheimer's disease and its application).

[0038] II. Experimental methods

[0039] 1. Embedding method of F0726@Fe-TA@GN

[0040] a1. Culturing the Bacillus licheniformis F0726 bacterial solution: Inoculate the Bacillus licheniformis F0726 into a 5 ml LB liquid test tube and culture it at 37 °C and 200 r / min for 12 hours;

[0041] a2. Centrifuge the bacterial solution obtained in a1 at 8000 rpm for 5 min, wash it with sterile PBS buffer, and then resuspend it in sterile PBS buffer to make the F0726 bacterial concentration 10 8 ~

[0042] 10 10 CFU / mL;

[0043] a3. Add 100 μL of the F0726 bacterial suspension prepared in a2, 10 μL of a 40 mg / mL TA solution, and 10 μL of a 10 mg / mL FeCl3 solution to 880 μL of PBS buffer, and vortex to mix evenly to obtain F0726@Fe-TA;

[0044] a4. Add the GN solution to the F0726@Fe-TA prepared in a3 so that the final concentration of GN in the system is 1.25 mg / mL, and gently mix to finally obtain the Bacillus licheniformis F0726@Fe-TA@GN complex.

[0045] 2. Zeta potential and TEM scanning

[0046] For the F0726@Fe-TA@GN obtained in a4, first fix the sample with 2.5% glutaraldehyde, and then rinse it 3 times with 0.1 M phosphate buffer at pH 7.0. Then dehydrate the sample and dry it in a critical point dryer before observation. Carefully deposit a drop of the bacterial suspension on a Formvar / carbon 200-mesh grid and dry it in air before TEM observation. Subsequently, use a Zeta potential analyzer to detect the potential and particle size changes of F0726@Fe-TA@GN.

[0047] 3. Gastrointestinal simulation experiment

[0048] Incubate F0726 and F0726@Fe-TA@GN separately in simulated intestinal fluid (SIF, pH 7.4 phosphate buffer supplemented with 1.60 mg / mL trypsin) for 1 hour and in simulated gastric fluid (SGF, pH 2.0 HCl solution supplemented with 3.2 mg / mL pepsin) for 30 min. Next, incubate the solution at 37 °C and shake it at 200 rpm. Take 300 μL aliquots of the reaction solution at predetermined time points, centrifuge to obtain the precipitate, and wash it with PBS. Dilute the obtained bacterial suspension series and spread it on solid LB plates. After incubating at 37 °C for 24 hours, perform colony counting.

[0049] 4. Establishment of an AD mouse model by intracerebral injection of Aβ42 and experimental grouping

[0050] Randomly divide the mice into a normal group, a model group, a Bacillus licheniformis F0726 group, and an F0726@Fe-TA@GN group, with 8 mice in each group. Mice in the AD model group and the drug administration group are intracerebrally injected with Aβ42 oligomeric protein to establish an AD mouse model; mice in the normal group only receive intracerebral injection of the same volume of normal saline.

[0051] Mice in the drug administration group start intragastric administration with Bacillus licheniformis F0726 and F0726@Fe-TA@GN one week before intracerebral injection, with a concentration of 1×109 CFU / mL, with a dosage of 0.2 ml / d / rat, and intragastric administration was carried out continuously for 21 days. The mice in the model group and the normal group were intragastrically administered the same volume of normal saline daily.

[0052] 5. Morris water maze experiment

[0053] The Morris water maze system mainly includes a water maze device, an automatic water maze image acquisition system, and a software analysis system. The pool is divided into four areas, namely the first quadrant (northeast), the second quadrant (southeast), the third quadrant (southwest), and the fourth quadrant (northwest). Before the experiment, a certain amount of water was injected into the pool, and the heating device was turned on to control the water temperature at 25 ± 1 °C. On the first day, the platform was placed at a position 1.5 cm above the water surface in the middle of the fourth quadrant. The mice were placed into the pool facing the pool wall in the second quadrant, and the mice were allowed to search for the platform in the pool. If the platform was not found, the mice were manually guided to stay on the platform for 10 s. On the second to sixth days, the platform was placed 1.5 cm underwater, and the operation on the first day was repeated. The time required for the mice to find the hidden platform and stay on it for 5 s was recorded, which was denoted as the latency. Each mouse was trained four times a day, and the interval between each training was 15 - 20 min to ensure that the mice had enough rest time.

[0054] 24 h after the last positioning navigation experiment of the hidden platform. Keeping the external conditions such as the control object, water depth, light, temperature, humidity, etc. unchanged, only the water maze platform was removed. The mice were gently placed into the water facing the pool wall of the second quadrant of the water maze. The swimming trajectories of the mice in the water within 90 s, the percentage of the distance in the fourth quadrant, and the number of times of crossing the platform position were recorded.

[0055] Sampling of mouse samples: The brains and cecal contents of the mice were collected after euthanasia and stored in a -80 °C refrigerator.

[0056] 6. WB experiment

[0057] Take 100 mg of mouse brain, mix it with RAPi and protease inhibitor at a ratio of 1:10, grind it, and centrifuge at 4 °C and 12000 rpm for 10 min. The supernatant is the protein stock solution. Add 1 / 4 of the total volume of protein loading buffer and heat it at 95 °C for 10 min. Separate the proteins by 10% SDS PAGE and transfer them to PVDF. The transferred PVDF membrane was placed in 5% skim milk powder for blocking, then incubated with the primary antibody overnight, incubated with the secondary antibody, and washed the membrane. Use a gel imaging system to take pictures, and use Image J software to perform gray scale analysis on the pictures.

[0058] 7. Detection of SCFAs content

[0059] The feces were immediately stored in a -80°C refrigerator after collection, resuspended with 500 μL of saturated NaCl solution, and acidified with 20 μL of 10% H2SO4; 800 μL of ether was added, shaken and mixed evenly to extract fatty acids, and then centrifuged at 18,000 g for 15 min; the upper ether phase was taken, and 0.25 g of anhydrous Na2SO4 was added for drying; after standing for 30 min, centrifuged at 18,000 g for 5 min, the upper ether phase was taken, and the content of short-chain fatty acids (acetic acid, propionic acid, isobutyric acid, butyric acid, and isovaleric acid) was analyzed by GC-MS.

[0060] Statistical analysis

[0061] The experimental data were processed using Graphpad Prism 8.0 software. All data were expressed as mean ± standard deviation. The comparison between two groups of data was performed using a t-test. ANOVA was used for multiple groups of data, and the Q-test was used when there was a significant difference. P < 0.05 was defined as a significant difference.

[0062] III. Experimental results

[0063] 1. In vitro characterization results

[0064] As Figure 1 shown, the changes in ζ potential and size confirmed the successful step-by-step coating of Fe-TA and GN on F0726. To observe the embedding situation more intuitively, a biological scanning electron microscope was used for observation. According to Figure 2 the display, the surface of the naked short one is relatively smooth, while the surface of the embedded Bacillus licheniformis can clearly observe an irregular embedding layer, further verifying the feasibility of this method, indicating that our embedding method can firmly bind to the surface of Bacillus licheniformis F0726.

[0065] To further prove that the encapsulated Bacillus licheniformis can resist the high-acid and high-salt environment in the gastrointestinal tract and the protective effect of the "armored coat" on F0726 of lichen, we simulated the environments of gastric juice (SGF) and intestinal juice (SIF) in vitro ( Figure 3 ). The results showed that the "armored coat" had a good protective effect on Bacillus licheniformis, and 35.68% of F0726 still survived, greatly improving the tolerance of Bacillus licheniformis to intestinal juice and strong acid environment.

[0066] 2. Results of the water maze experiment

[0067] The activity paths of each experimental group on the 6th day are shown in the experiment as Figure 4 shown. From Figure 5The experimental results show that after 6 days of training, the difference between the model group and the wild group becomes more and more obvious, indicating that the model of intracerebral injection of Aβ42 oligomers is successfully established. The latency time of the model group mice is significantly higher than that of the drug administration group, and the latency of each drug administration treatment group shortens with the increase of the training days. And compared with the control group, the drug administration group showed significant differences on the 5th and 6th days. It shows that F0726@Fe-TA@GN provided by the present invention has a better improvement effect on the memory behavior ability of mice. After removing the platform, compared with the model group, the number of times the drug administration group mice crossed the platform in the Morris water maze increased significantly.

[0068] 3. WB detection results

[0069] Figure 6 It is the statistical result of the WB content of TLR4 / MYD 88 / NF-κB / p NF-κB in the mouse brain. Compared with the wild group, the TLR4 / MYD 88 / NF-κB / p NF-κB pathway in the model group mice is promoted, indicating that intracerebral injection of Aβ oligomers can induce AD symptoms, while F0726@Fe-TA@GN and Bacillus licheniformis F0726 inhibited the TLR4 / MYD 88 / NF-κB / p NF-κB pathway in the mouse serum, and the inhibitory effect of F0726@Fe-TA@GN is more obvious. This shows that F0726@Fe-TA@GN can better inhibit the inflammatory pathway of TLR4 / MYD 88 / NF-κB / p NF-κB in AD model mice and relieve the neuroinflammation caused by Aβ oligomers.

[0070] 4. SCFAs content detection results

[0071] Figure 7 It is the statistical result of the short-chain fatty acids (SCFAs) of the intestinal flora in the mouse feces. After intervention with F0726@Fe-TA@GN, the contents of 2-methylbutyric acid (2-BA) and isobutyric acid (IBA) are significantly higher than those in the Model group and almost reach the wild group level. This is because the combination of GN in F0726@Fe-TA@GN has the behavior of regulating the intestine, thus affecting the composition and metabolism of the intestinal flora and resulting in the production of more short-chain fatty acids. That is, GN in F0726@Fe-TA@GN will act synergistically with F0726 to effectively relieve Alzheimer's disease.

[0072] In summary, F0726@Fe-TA@GN enhances the resistance of Bacillus licheniformis F0726 in gastric juice and intestinal juice. In a mouse model of Alzheimer's disease induced by intracerebral injection of Aβ oligomers, it can improve the spatial learning and memory impairment of AD model mice, reduce the levels of inflammatory factors in the brains of AD mice, and improve the content of SCFAs in the cecal contents of AD mice, providing a new approach for the development of drugs related to the prevention and treatment of Alzheimer's disease, as well as potential probiotic resources.

[0073] Obviously, the above examples are only for illustration purposes and are not limitations on the implementation. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementations here. The obvious changes or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. A method for preparing a Bacillus licheniformis F0726@Fe-TA@GN complex, characterized in that: The preparation method is: a1. Culture of Bacillus licheniformis F0726: The Bacillus licheniformis F0726 was inoculated in a 5 ml LB liquid test tube and cultured at 37 ° C, 200 r / min for 12 hours; a2. The bacterial solution in a1 was centrifuged at 8000 rpm for 5 min, washed with sterile PBS buffer and resuspended in sterile PBS buffer to a concentration of 10 F0726 bacteria. 8 ~10 10 CFU / mL; a3. Add 100 μL of F0726 bacterial suspension prepared in a2, 10 μL of 40 mg / mL TA solution and 10 μL of 10 mg / mL FeCl3 solution to 880 μL PBS buffer and vortex to mix to obtain F0726@Fe-TA; a4. Add GN solution to the F0726@Fe-TA prepared in a3 to make the final concentration of GN in the system 1.25 mg / mL, mix gently, and finally obtain Bacillus licheniformis F0726@Fe-TA@GN complex; The Bacillus licheniformis F0726 has been deposited in Guangdong Microbiological Culture Collection Center with the deposit number: GDMCC No.64432.

2. The Bacillus licheniformis F0726@Fe-TA@GN complex prepared by the preparation method according to claim 1, characterized in that: Bacillus licheniformis F0726 was first encapsulated in a metal mesh phenol structure composed of iron and tannic acid, and then β-glucan was encapsulated in its outermost layer.

3. The use of the Bacillus licheniformis F0726@Fe-TA@GN complex according to claim 2 in the preparation of a drug, characterized in that: Use of the Bacillus licheniformis F0726@Fe-TA@GN complex in the preparation of a drug for preventing and treating Alzheimer's disease, wherein the number of viable bacteria of F0726@Fe-TA@GN in the drug is 1×10 8 CFU / mL~1×10 10 CFU / mL.

4. The use according to claim 3, characterized in that: The Bacillus licheniformis F0726@Fe-TA@GN complex is used in the preparation of a drug for improving spatial learning and memory disorders in Alzheimer's disease.

5. The use according to claim 3, characterized in that: The Bacillus licheniformis F0726@Fe-TA@GN complex is used in the preparation of a drug for improving the level of inflammatory factors in the brain of Alzheimer's disease.

6. The use according to claim 3, characterized in that: The Bacillus licheniformis F0726@Fe-TA@GN complex is used in the preparation of a drug for regulating the SCFAs content in the cecal contents of Alzheimer's disease.

7. The use of the Bacillus licheniformis F0726@Fe-TA@GN complex according to claim 2, characterized in that: The Bacillus licheniformis F0726@Fe-TA@GN complex is used in the preparation of medicines and health products for improving learning and memory ability or regulating intestinal flora.

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