Application of Lactobacillus lentus

Abstract

The invention discloses an application of Lactobacillus farraginis, belonging to the field of biotechnology. The invention discloses an application of Lactobacillus farraginis in treating or improving mental illness. Lactobacillus farraginis can improve mental illness and alleviate the symptoms of mental illness related to the gut-brain axis.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to the application of Lactobacillus lentus. Background Art

[0002] In recent years, the incidence of mental illness has increased year by year. Mental illness is often accompanied by symptoms of behavioral disorders, such as autism, depression, bipolar disorder, etc. Mental illness has become a health problem that affects human life.

[0003] The intestinal flora can regulate the host's metabolism, immune and nervous systems, affect mental function, and play a vital role in regulating human health and disease. Dysbiosis can induce a variety of diseases, such as inflammatory bowel disease, anxiety or depression and other psychopathologies. Intestinal bacteria stimulate afferent neurons of the enteric nervous system and send signals to the brain through the vagus nerve. Through different mechanisms, intestinal microorganisms shape the structure of the stress response of the hypothalamic-pituitary-adrenal axis, which affects memory, mood and cognition. Probiotics are a type of bacteria that can improve the balance of the host's microecology and produce beneficial physiological effects on the host. They can prevent and treat a variety of diseases by regulating the balance of intestinal flora. It has been confirmed that probiotic therapy has considerable safety, acceptability and therapeutic potential in patients with mental illness, and probiotic-assisted therapy has shown good acceptability and tolerability in patients with mental illness.

[0004] Lactobacillus lentus is widely used in fermentation technology, but there is no research on the application of Lactobacillus lentus in improving mental illness. Summary of the invention

[0005] Based on this, the present invention proposes the application of Lactobacillus bran lentus in improving mental illness to fill the current research gap.

[0006] In order to achieve the above object, the present invention adopts the following technical solution: application of Lentilactobacillus farraginis in treating or improving mental illnesses.

[0007] Application of Lentilactobacillus farraginis in preparing products for preventing or treating mental illnesses.

[0008] Use of a culture of Lentilactobacillus farraginis in preparing a product for preventing or treating mental illness.

[0009] Furthermore, the above-mentioned products include medicines and bacterial agents.

[0010] Furthermore, the dosage forms of the above-mentioned medicine include oral dosage forms, external preparations, and suppositories.

[0011] Furthermore, the above-mentioned oral dosage forms include powders, granules, tablets, pills, capsules, suspensions, emulsions, syrups and aerosols.

[0012] Furthermore, the pharmaceutical composition of the above-mentioned medicine includes a carrier, an excipient and a diluent.

[0013] Furthermore, the above-mentioned medicine is administered to a mammal.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] Lentilactobacillus farraginis can improve mental illness, alleviate symptoms of mental illness related to the gut-brain axis, and have a positive effect on mental health. Changes in the intestinal microbial system that are beneficial to host health, the grooming time of mice gavaged with Lentilactobacillus farraginis was reduced compared with the control group, their stereotyped behavior was improved, their anxiety-like behavior was improved, their social skills were better, and their sense of novelty was improved, which has potential application value in the prevention and treatment of mental illness. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings are incorporated in and constitute a part of this specification and, together with the description, serve to explain the principles of the present invention.

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0018] Figure 1 This is a result diagram of feces water content of the experimental group and the control group of the present invention;

[0019] Figure 2 This is a graph showing the results of the repeated stereotyped behavior experiment of the present invention;

[0020] Figure 3 This is a result diagram of the central area residence time of the open field experiment of the present invention;

[0021] Figure 4 This is a result diagram of the total distance of movement in the open field experiment of the present invention;

[0022] Figure 5 This is a graph of the social ability results of the three-box social experiment;

[0023] Figure 6 This is a graph of the social novelty results for the three-box social experiment;

[0024] Figure 7 This is the trajectory result diagram of the three-box social experiment in sample one. DETAILED DESCRIPTION

[0025] The experimental methods in the following examples of the present invention, where no specific conditions are specified, are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0026] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0027] The Lactobacillus farraginis of the present invention was purchased from the China Industrial Microbiological Culture Collection Administration Center. 6131, the storage address is Building 6, No. 24, Jiuxianqiao Middle Road, Chaoyang District, Beijing.

[0028] The mice used in the following examples are inbred BTBR T~(+)tf / mice, which have been widely used in autism research. The behavioral phenotype of BTBR T~(+)tf / mice is consistent with the three core clinical symptoms of autism, showing low social behavior, abnormal ultrasonic vocalization behavior (language communication behavior), and highly repetitive self-grooming behavior (stereotyped behavior), and is considered to be a good animal model for studying the pathogenesis of autism and screening drug targets.

[0029] The culture medium used in the following examples is MRS broth purchased from Beijing Aoboxing Biotechnology Co., Ltd. with the product number 02-291.

[0030] Preparation Example: Cultivation of Lactobacillus lentus

[0031] The activated Lactobacillus lentus was inoculated into MRS liquid culture medium for anaerobic culture at 35°C for about 48 hours. The culture time could be adjusted according to the OD value, and the best OD value was between 0.6 and 0.8.

[0032] MRS liquid culture medium was prepared according to the bottle test instructions and then sterilized by high pressure at 121°C for 15 min, and then used after cooling;

[0033] Example 1: Testing the fecal water content of mice gavaged with Lactobacillus lentus

[0034] Step 1: Centrifuge 5 ml of Lactobacillus lentus with an OD value between 0.6 and 0.8 at 4000 rpm for 10 min, wash twice with dd water (double distilled water), resuspend with 5 ml dd water and then gavage BTBR mice.

[0035] Step 2: 20 BTBR mice of about 6 weeks old were divided into two groups, with 10 mice in each group, one group was treated with dd water as the control group, and the other group was treated with Lactobacillus lentus of grain bran as the experimental group. The intragastric dose was 1% of the body weight of the BTBR mice, and the intragastric administration lasted for 4 weeks.

[0036] Step 3: Collect fresh feces from each group of BTBR mice, weigh and record, dry the feces in a 56°C oven for 24 hours, weigh them, and then calculate the water content of the feces.

[0037] Experimental results: Figure 1 As shown, no abnormalities were found in the control group and the experimental group, which proves that the bacteria did not have an abnormal effect on the intestinal environment of BTBR mice and caused them to suffer from constipation or urination.

[0038] Example 2: Repeated stereotyped behavior experiment

[0039] Experimental preparation stage:

[0040] The BTBR mice were grouped, aged and gavaged in the same manner as described in Example 1, and stereotypic behavior tests were performed on them after 4 weeks.

[0041] Three days before the behavioral experiment, the experimenter needs to wear gloves and gently grasp the BTBR mice to allow the BTBR mice to adapt to them. Each BTBR mouse should be grasped for about 5 minutes, so that the BTBR mice can fully contact the experimenter's hands and no longer fear the subjects. During this process, the hands need to be cleaned with alcohol every time a mouse is changed. After the smell of alcohol dissipates, the next BTBR mouse can be touched. The contact with the BTBR mouse needs to be repeated until the behavioral experiment begins. And before the behavioral experiment begins, the BTBR mice need to be adapted in the preparation area of ​​the behavior room for half an hour.

[0042] Experimental stage:

[0043] Step 1: Prepare a grooming experiment observation box that has a color that is significantly different from the mouse's fur. Use a USB camera and adjust the position to ensure that the situation of the BTBR mice in the test box is fully recorded.

[0044] Step 2: Clean the observation box with 75% ethanol before placing each BTBR mouse, wipe the box dry with a clean paper towel, and after the smell of the box has dissipated, gently place the BTBR mouse in the grooming box with your palm, and record a 20-minute video of the BTBR mouse moving freely in the grooming box;

[0045] Step 3: The grooming behavior in the video was manually counted and analyzed in a double-blind manner. The first 10 minutes was the adaptation stage of the BTBR mice and no counting was performed. The behavior of the BTBR mice was counted and analyzed starting from the last 10 minutes.

[0046] Experimental results: Figure 2 As shown, the grooming time of BTBR mice gavaged with Lactobacillus lentus in grain bran was reduced, and their stereotyped behaviors were improved compared with those of the control group.

[0047] Example 3: Open field experiment

[0048] Experimental preparation stage:

[0049] The BTBR mice were grouped, aged, and gavage-administered in the same manner as described in Example 1, and stereotyped behavior tests were performed after 4 weeks. The adaptation phase of the mice before the behavior was consistent with the method described in Example 2; the device required a 40 cm × 40 cm × 30 cm hard plastic box with a white background, and the mice were placed in the behavior room adaptation area for 30 minutes before the experiment.

[0050] Experimental stage:

[0051] Step 1: Connect the camera to the center area at the intersection of 1 / 4 of the diagonal line at the bottom of the box, set up the Smart Video Tracking System (Smartv 3.0) software at the back end, test the recognition and ensure that the device can accurately recognize the mouse's movements.

[0052] Step 2: Gently place the mouse in the center of the open field, let it move freely for 30 minutes, and then replace it with the next mouse. Before replacing the mouse, clean up the feces and urine of the previous mouse, disinfect the box of the open field with 75% ethanol, wipe it with soft paper, and place the next mouse after the smell dissipates.

[0053] Step 3: Export the data from the software to analyze the time spent in the center area and the total distance traveled.

[0054] Experimental results: Figure 3-4 As shown in the figure, after gavage with Lactobacillus lentus, the mice in the experimental group stayed in the central area longer and moved more distance than those in the control group, which can reflect that the anxiety-like behavior of BTBR mice was improved.

[0055] Example 4: Three-box social experiment

[0056] Experimental preparation stage:

[0057] The three-box social experiment was the same as the BTBR mouse grouping, age and gavage method described in Example 1, and stereotyped behavior tests were performed after 4 weeks. BTBR mice need to be marked before the experiment, generally by painting the tail or ear tagging, and clipping the toes. The BTBR mice were gently placed on the hands and allowed to move freely, once a day, 3-5 minutes per BTBR mouse, for 5 days, so that the BTBR mice and the experimenter are fully familiar with each other.

[0058] The three-box experiment also requires the preparation of stimulus mice (mice used for social interaction), which can be selected as mice of the same age as the BTBR mice. Each small box of the three-box test box is 20cm×40cm×20cm, and the passage connecting the adjacent small boxes is 10cm wide. The cylindrical cage needs to be wiped clean with 75% alcohol. After the smell evaporates, the experiment begins. When replacing new experimental group mice, all items need to be wiped dry with alcohol. After the BTBR mice are moved to the adaptation area of ​​the behavior room for 1 hour, the experimental equipment is placed in advance as required, the camera is connected, the Smartv 3.0 software is set, and the parameters are debugged so that the Smartv 3.0 software automatically records the contact time between the BTBR mice and the social mice, ensuring a clear record of the situation in the three boxes.

[0059] Experimental stage:

[0060] Step 1: The experimenter who had sufficient contact with BTBR mice gently placed the BTBR mice in the central box of the three boxes, allowed the BTBR mice to freely explore the environment in the three boxes for 10 minutes, and then closed the two side channels connecting the central box with baffles to confine the BTBR mice in the central box;

[0061] Step 2: Place the stimulus mouse (MOUSE1) in the social cylinder cage, open the channels on both sides to allow the BTBR mice to move freely for 10 minutes, then restrict the BTBR mice in the central box, and then place a new stimulus mouse (MOUSE2) in the new object cage, and allow the BTBR mice to move freely for 10 minutes after opening the channels.

[0062] Step 3: Analyze the results based on software data.

[0063] Experimental results: Figure 5-7 The results show that BTBR mice are not good at socializing and are not sensitive enough to new things. However, after being treated with Lactobacillus lentus, the social trajectories of BTBR mice became more intensive, their social skills and social novelty showed better trends, and their sense of novelty to things also increased.

[0064] In addition, Lactobacillus lentus can be used in pharmaceutical compositions related to preventing or alleviating behavioral disorders of mental illnesses such as autism, depressive disorders, bipolar disorder, etc. In addition, the biosafety evaluation of the strain indicated that no inflammatory response occurred.

[0065] In addition, the present disclosure may provide a pharmaceutical composition comprising a Lactobacillus branus strain or a culture thereof, which is effective in preventing, improving or treating a variety of mental behavioral disorders.

[0066] The Lactobacillus lentus strain or its culture can be added to the pharmaceutical composition of the present disclosure in an amount of 0.001wt% to 100wt%. The pharmaceutical composition can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosol preparations, as well as external preparations and suppositories according to various typical methods.

[0067] Carriers, excipients and diluents in the pharmaceutical composition may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0068] Excipients: Preparations can usually be prepared using diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc.

[0069] Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc., and such solid preparations may be prepared by mixing the strain of the present disclosure or a culture thereof with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc., and in addition to simple excipients, lubricants may be used, such as magnesium stearate, talc, etc. Oral liquid preparations may include suspensions, solutions, emulsions, syrups, etc., and may contain not only simple diluents such as water or liquid paraffin, but also a variety of excipients such as wetting agents, sweeteners, aromatics, preservatives, etc.

[0070] Preparations for parenteral administration may include aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. Solid preparations for oral administration as non-aqueous solvents may include tablets, pills, powders, granules, capsules, etc., and such solid preparations may be prepared by mixing the strain of the present disclosure or its culture with at least one excipient, and the excipient may be selected from starch, calcium carbonate, sucrose, lactose, gelatin, etc.

[0071] In addition to simple excipients, lubricants such as magnesium stearate, talc, etc. can be used. Oral liquid preparations may include suspensions, solutions, emulsions, syrups, etc., which may contain not only simple diluents such as water or liquid paraffin, but also a variety of excipients such as wetting agents, sweeteners, flavoring agents, preservatives, etc.

[0072] Suspensions can use propylene glycol, polyethylene glycol, vegetable oil, injectable esters, etc., among which the vegetable oil can be olive oil and the injectable ester can be ethyl oleate; the base of the suppository can use witepsol polyglycol Tween 61, cocoa butter, lauryl ester, glycerol gelatin, etc.

[0073] The pharmaceutical composition of the present disclosure can be administered to mammals, such as mice, livestock, humans, etc., through various routes.

[0074] Since the strain of the present disclosure has low toxicity and minimal side effects, it can be used safely even when taken for a long time for preventive purposes. Since the strain of the present disclosure has low toxicity and minimal side effects, it is a drug that can be used safely even when taken for a long time for preventive purposes.

[0075] The above description is only a specific embodiment of the present invention, so that those skilled in the art can understand or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention.

[0076] It should be understood that the present invention is not limited to what has been described above and that various modifications and changes may be made without departing from its scope. The scope of the present invention is limited only by the appended claims.

Claims

1. Lactobacillus lentus Lentilactobacillus farraginis ) in the preparation of a medicine for treating autism, wherein the Lactobacillus lentus ( Lentilactobacillus farraginis ) is Lactobacillus lentus ( Lentilactobacillus farraginis )CICC6131.

2. The use according to claim 1, characterized in that: The dosage form of the drug includes an oral dosage form or an external preparation.

3. The use according to claim 2, characterized in that: The oral dosage forms include powders, granules, tablets, pills, capsules, suspensions, emulsions or syrups.

4. The use according to claim 1, characterized in that: The dosage form of the drug includes suppository.

5. The use according to claim 1, characterized in that: The pharmaceutical composition of the drug comprises a carrier, an excipient and a diluent.

6. The use according to claim 1, characterized in that: The medicament is administered to a mammal.

7. Lactobacillus lentus Lentilactobacillus farraginis ) in the preparation of a bacterial agent for treating autism, wherein the Lactobacillus lentus ( Lentilactobacillus farraginis ) is Lactobacillus lentus ( Lentilactobacillus farraginis )CICC6131.

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