Application of giant coccus Eschersoni KCTC5187 in preparation of medicines for improving learning and memory, protecting nerves, resisting oxidation, reducing fat and protecting intestinal barrier

Abstract

The invention discloses application of giant coccus Eschersoni KCTC5187 in preparation of medicines for improving learning and memory, protecting nerves, resisting oxidation, reducing fat and protecting intestinal barriers, and belongs to the technical field of biology. The invention provides a scheme for people with high calorie and high fat diet, and the giant coccus Echeli KCTC5187 or a microbial agent thereof is used for regulating lipid deposition of the people with high calorie and high fat diet, enhancing the activity of neurons, resisting oxidation and effectively improving the learning and memory ability of the people with high calorie and high fat diet; related products are effectively developed based on the strain, and an innovative and effective solution is provided for solving health challenges confronted by people at present.

Description

Technical Field

[0001] The invention specifically relates to the application of Megasphaera elsdenii KCTC5187 in the preparation of drugs for improving learning and memory, neuroprotection, anti-oxidation, fat reduction and intestinal barrier protection, and belongs to the field of biotechnology. Background Art

[0002] In recent years, with the change of residents' dietary structure, the intake of high-fat food has increased significantly. The lipid metabolism disorder caused by high-fat diet, characterized by hyperlipidemia, excessive accumulation of visceral fat and obesity, has been increasing year by year, becoming one of the public health problems that need to be solved urgently worldwide. Long-term high-fat diet causes energy metabolism imbalance, induces lipid metabolism and sugar metabolism disorders, and increases the risk of obesity, diabetes, gastrointestinal dysfunction, hypertension, myocardial infarction, stroke, dementia, obstructive sleep apnea and cancer. This trend is also particularly evident in the adolescent group. Due to the accelerated pace of life, the prevalence of fast food culture and the formation of bad eating habits, many adolescents have too high a proportion of high-fat and high-calorie foods in their daily diet, resulting in energy intake far exceeding the body's needs, which in turn causes a series of health problems.

[0003] It is worth noting that long-term high-fat, high-calorie diets not only increase the risk of chronic diseases such as obesity, diabetes, and cardiovascular disease, but also have a significant negative impact on the development of the nervous system and learning and memory abilities (Ma Weiwei, Yuan Linhong, Jiang Lizhen, Ma Mengwei, Li Haibin, Jia Yichen, Xiao Rong. Effects of high-fat diet on learning and memory abilities of obese rats [J]. Chinese Public Health, 2013, 29(8): 1179-1181.). Studies have shown that lipid metabolism disorders are one of the key factors leading to this problem. Under abnormal lipid metabolism, excessive lipid accumulation in the body not only affects the normal function of cells, but may also directly damage learning and memory abilities by interfering with the synthesis and release of neurotransmitters and affecting information transmission between neurons.

[0004] The threat of lipid metabolism disorders to learning and memory abilities is mainly manifested in the following aspects (Zhao Meiqi, Zhang Peng, Guo Yanmin, et al. Research progress on the effects of high-fat diet on cognitive functions such as learning and memory [J]. Food Science, 2017, 38(9): 258-264.): A high-fat and high-sugar diet will lead to excessive intake of fat and sugar in the body, which in turn leads to health problems such as obesity and diabetes. These diseases not only affect the cardiovascular system, but also damage the normal function of the brain, leading to problems such as memory loss and lack of concentration; a high-fat and high-sugar diet can easily lead to chronic inflammatory reactions in the body, which can also spread to the brain and cause neuroinflammation. Neuroinflammation interferes with the normal communication between neurons, affects the storage and retrieval process of memory, and thus reduces the level of memory; in addition, free radicals and other oxidative stress products produced during lipid metabolism may also cause damage to brain cells; a high-fat and high-sugar diet may interfere with the balance of neurotransmitters and affect the normal activity of neurons in the brain.

[0005] As microbiome research deepens, people are increasingly aware of the important role of intestinal microbiota in health and disease. Studies have shown that a high-fat or high-sugar and high-fat diet causes changes in the intestinal microbiome, enlargement of white adipose tissue and metabolic dysfunction, leading to learning and memory impairment and increased risk of dementia. Therefore, regulating the intestinal microbiota by supplementing probiotics may help improve lipid metabolism and its related cognitive impairment.

[0006] In summary, the use of probiotics to prepare drugs to improve diet-induced learning and memory impairment, neurodegeneration, lipid metabolism disorders, etc. is of great significance for improving the quality of life and preventing diseases. In view of the severity and urgency of the above problems, it is particularly important to develop products or methods that can effectively improve the decline in learning and memory ability caused by a high-calorie, high-fat diet. Summary of the invention

[0007] Megasphaera elsdenii is a Gram-negative bacterium belonging to the genus Megasphaera, with a relative abundance of up to 2.4×10 8 CFU / g. In order to solve the shortcomings of the prior art, the present invention aims to provide a solution for people with high-calorie, high-fat diets, using Megasphaera elsdenii KCTC5187 or its bacterial agent to effectively improve their learning and memory abilities by regulating lipid deposition, enhancing neuronal vitality and anti-oxidation mechanisms, and providing an innovative and effective solution to the health challenges currently faced by people.

[0008] The first object of the present invention is to provide the use of Megasphaera elsdenii KCTC5187 or its microbial agent in the preparation of products for improving learning and memory.

[0009] In one embodiment of the present invention, the improvement of learning and memory is to improve the learning and memory ability of people with high fat content or patients with lipid metabolism disorders.

[0010] In one embodiment of the present invention, the microbial agent uses Megasphaera elsdenii KCTC5187 as a main microorganism.

[0011] In one embodiment of the present invention, the viable count of Megasphaera elsdenii KCTC5187 in the bacterial agent is not less than 1×10 8 CFU / mL or 1×10 8 CFU / g.

[0012] In one embodiment of the present invention, the bacterial agent is obtained by preparing the seed solution of Megasphaera elsdenii KCTC5187 and then expanding the culture.

[0013] In one embodiment of the present invention, the microbial agent contains living cells of the KCTC5187 strain of the present invention, dry cells of the KCTC5187 strain obtained by freeze-drying, immobilized cells of the KCTC5187 strain, liquid agent of the KCTC5187 strain, solid agent of the KCTC5187 strain, or the KCTC5187 strain in any other form.

[0014] In one embodiment of the present invention, the bacterial agent further contains other applicable strains of any species, including but not limited to Bacillus licheniformis, Saccharomyces cerevisiae, Bacillus subtilis, and Lactobacillus.

[0015] In one embodiment of the present invention, the product includes but is not limited to medicines, foods, food additives, feeds, feed additives, cosmetics, and health products.

[0016] In one embodiment of the present invention, the drug contains the above-mentioned Megasphaera elsdenii KCTC5187 or its bacterial agent, and pharmaceutically acceptable excipients.

[0017] In one embodiment of the present invention, the auxiliary materials include any one of carriers, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, antibacterial agents or buffers, or a combination of at least two of them.

[0018] In one embodiment of the present invention, the carrier includes but is not limited to liposomes and nanoparticles.

[0019] In one embodiment of the present invention, the excipient is one or more of mannitol, lactose, fatty acid, and polyethylene glycol.

[0020] In one embodiment of the present invention, the dosage form of the drug includes any one of suspension, granules, capsules, powders, tablets, emulsions, solutions, pills, injections, suppositories, enemas, aerosols, patches or drops.

[0021] In one embodiment of the present invention, the administration route of the drug includes oral administration, sublingual administration, skin mucosal administration, inhalation administration or injection administration.

[0022] In one embodiment of the present invention, the food is solid food, liquid food or semi-solid food.

[0023] In one embodiment of the present invention, the content of Megasphaera elsdenii KCTC5187 in the product is not less than 1×10 8 CFU / g or 1×10 8 CFU / mL.

[0024] In one embodiment of the present invention, the product has at least the function of improving learning and memory, and may also have one or more of the following functions (a) to (d):

[0025] (a) Neuroprotection;

[0026] (b) Antioxidant;

[0027] (c) fat loss;

[0028] (d) Protect the intestinal barrier.

[0029] In one embodiment of the present invention, the neuroprotection is to increase the strength of dopamine neurons, glutamate neurons, γ-aminobutyric acid neurons and / or serotonergic neurons.

[0030] In one embodiment of the present invention, the antioxidant is to reduce ROS and O2 - level.

[0031] In one embodiment of the present invention, the fat reduction is to reduce lipid deposition.

[0032] In one embodiment of the present invention, the protection of the intestinal barrier is to reduce intestinal leakage and alleviate intestinal barrier damage.

[0033] The second object of the present invention is to provide the use of Megasphaera elsdenii KCTC5187 or its microbial agent in the preparation of products that have the effects of improving learning and memory, neuroprotection, anti-oxidation, fat reduction, and protecting the intestinal barrier.

[0034] In one embodiment of the present invention, the improvement of learning and memory is to improve the learning and memory ability of people with high fat content or patients with lipid metabolism disorders.

[0035] In one embodiment of the present invention, the neuroprotection is to increase the strength of dopamine neurons, glutamate neurons, γ-aminobutyric acid neurons and / or serotonergic neurons.

[0036] In one embodiment of the present invention, the antioxidant is to reduce ROS and O2 - level.

[0037] In one embodiment of the present invention, the fat reduction is to reduce lipid deposition.

[0038] In one embodiment of the present invention, the protection of the intestinal barrier is to reduce intestinal leakage and alleviate intestinal barrier damage.

[0039] In one embodiment of the present invention, the microbial agent uses Megasphaera elsdenii KCTC5187 as a main microorganism.

[0040] In one embodiment of the present invention, the viable count of Megasphaera elsdenii KCTC5187 in the bacterial agent is not less than 1×10 8 CFU / mL or 1×10 8 CFU / g.

[0041] In one embodiment of the present invention, the bacterial agent is obtained by preparing the seed solution of Megasphaera elsdenii KCTC5187 and then expanding the culture.

[0042] In one embodiment of the present invention, the microbial agent contains living cells of the KCTC5187 strain of the present invention, dry cells of the KCTC5187 strain obtained by freeze-drying, immobilized cells of the KCTC5187 strain, liquid agent of the KCTC5187 strain, solid agent of the KCTC5187 strain, or the KCTC5187 strain in any other form.

[0043] In one embodiment of the present invention, the bacterial agent further contains other applicable strains of any species, including but not limited to Bacillus licheniformis, Saccharomyces cerevisiae, Bacillus subtilis, and Lactobacillus.

[0044] In one embodiment of the present invention, the product includes but is not limited to medicines, foods, food additives, feeds, feed additives, cosmetics, and health products.

[0045] In one embodiment of the present invention, the drug contains the above-mentioned Megasphaera elsdenii KCTC5187 or its bacterial agent, and pharmaceutically acceptable excipients.

[0046] In one embodiment of the present invention, the auxiliary materials include any one of carriers, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, antibacterial agents or buffers, or a combination of at least two of them.

[0047] In one embodiment of the present invention, the carrier includes but is not limited to liposomes and nanoparticles.

[0048] In one embodiment of the present invention, the excipient is one or more of mannitol, lactose, fatty acid, and polyethylene glycol.

[0049] In one embodiment of the present invention, the dosage form of the drug includes any one of suspension, granules, capsules, powders, tablets, emulsions, solutions, pills, injections, suppositories, enemas, aerosols, patches or drops.

[0050] In one embodiment of the present invention, the administration route of the drug includes oral administration, sublingual administration, skin mucosal administration, inhalation administration or injection administration.

[0051] In one embodiment of the present invention, the food is solid food, liquid food or semi-solid food.

[0052] In one embodiment of the present invention, the content of Megasphaera elsdenii KCTC5187 in the product is not less than 1×10 8 CFU / g or 1×10 8 CFU / mL.

[0053] The third object of the present invention is to provide a drug with the function of improving learning and memory, wherein the drug contains the above-mentioned Megasphaera elsdenii KCTC5187 or its microbial agent, and pharmaceutically acceptable excipients.

[0054] In one embodiment of the present invention, the improvement of learning and memory function is to improve the learning and memory ability of people with high fat content or patients with lipid metabolism disorders.

[0055] In one embodiment of the present invention, the bacterial agent contains living cells of the KCTC5187 strain of the present invention, dry bacteria of the KCTC5187 strain obtained by freeze-drying, immobilized cells of the KCTC5187 strain, liquid bacterial agent of the KCTC5187 strain, solid bacterial agent of the KCTC5187 strain, or the KCTC5187 strain in any other form.

[0056] In one embodiment of the present invention, the auxiliary materials include any one of carriers, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, antibacterial agents or buffers, or a combination of at least two of them.

[0057] In one embodiment of the present invention, the carrier includes but is not limited to liposomes and nanoparticles.

[0058] In one embodiment of the present invention, the excipient is one or more of mannitol, lactose, fatty acid, and polyethylene glycol.

[0059] In one embodiment of the present invention, the dosage form of the drug includes any one of suspension, granules, capsules, powders, tablets, emulsions, solutions, pills, injections, suppositories, enemas, aerosols, patches or drops.

[0060] In one embodiment of the present invention, the administration route of the drug includes oral administration, sublingual administration, skin mucosal administration, inhalation administration or injection administration.

[0061] In one embodiment of the present invention, the medicine further contains strains of any genus that can be used in medicine, including but not limited to Bacillus licheniformis, Saccharomyces cerevisiae, Bacillus subtilis, and Lactobacillus.

[0062] In one embodiment of the present invention, the number of viable bacteria of Megasphaera elsdenii KCTC5187 in the drug is not less than 1×10 8 CFU / mL or 1×10 8 CFU / g.

[0063] The fourth object of the present invention is to provide the use of Megasphaera elsdenii KCTC5187 or its microbial agent in the preparation of a product for alleviating the decline in learning and memory ability caused by a high-fat diet.

[0064] In one embodiment of the present invention, the microbial agent uses Megasphaera elsdenii KCTC5187 as a main microorganism.

[0065] In one embodiment of the present invention, the viable count of Megasphaera elsdenii KCTC5187 in the bacterial agent is not less than 1×10 8 CFU / mL or 1×10 8 CFU / g.

[0066] In one embodiment of the present invention, the bacterial agent is obtained by preparing the seed solution of Megasphaera elsdenii KCTC5187 and then expanding the culture.

[0067] In one embodiment of the present invention, the microbial agent contains living cells of the KCTC5187 strain of the present invention, dry cells of the KCTC5187 strain obtained by freeze-drying, immobilized cells of the KCTC5187 strain, liquid agent of the KCTC5187 strain, solid agent of the KCTC5187 strain, or the KCTC5187 strain in any other form.

[0068] In one embodiment of the present invention, the bacterial agent further contains other applicable strains of any species, including but not limited to Bacillus licheniformis, Saccharomyces cerevisiae, Bacillus subtilis, and Lactobacillus.

[0069] In one embodiment of the present invention, the product includes but is not limited to medicines, foods, food additives, and health products.

[0070] In one embodiment of the present invention, the drug contains the above-mentioned Megasphaera elsdenii KCTC5187 or its bacterial agent, and pharmaceutically acceptable excipients.

[0071] In one embodiment of the present invention, the auxiliary materials include any one of carriers, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, antibacterial agents or buffers, or a combination of at least two of them.

[0072] In one embodiment of the present invention, the carrier includes but is not limited to liposomes and nanoparticles.

[0073] In one embodiment of the present invention, the excipient is one or more of mannitol, lactose, fatty acid, and polyethylene glycol.

[0074] In one embodiment of the present invention, the dosage form of the drug includes any one of suspension, granules, capsules, powders, tablets, emulsions, solutions, pills, injections, suppositories, enemas, aerosols, patches or drops.

[0075] In one embodiment of the present invention, the administration route of the drug includes oral administration, sublingual administration, skin mucosal administration, inhalation administration or injection administration.

[0076] The fifth object of the present invention is to provide the use of Megasphaera elsdenii KCTC5187 or its microbial agent in the preparation of a product for alleviating the decline in learning and memory ability of adolescents caused by a high-fat diet.

[0077] In one embodiment of the present invention, the microbial agent uses Megasphaera elsdenii KCTC5187 as a main microorganism.

[0078] In one embodiment of the present invention, the viable count of Megasphaera elsdenii KCTC5187 in the bacterial agent is not less than 1×10 8 CFU / mL or 1×10 8 CFU / g.

[0079] In one embodiment of the present invention, the bacterial agent is obtained by preparing the seed solution of Megasphaera elsdenii KCTC5187 and then expanding the culture.

[0080] In one embodiment of the present invention, the microbial agent contains living cells of the KCTC5187 strain of the present invention, dry cells of the KCTC5187 strain obtained by freeze-drying, immobilized cells of the KCTC5187 strain, liquid agent of the KCTC5187 strain, solid agent of the KCTC5187 strain, or the KCTC5187 strain in any other form.

[0081] In one embodiment of the present invention, the bacterial agent further contains other applicable strains of any species, including but not limited to Bacillus licheniformis, Saccharomyces cerevisiae, Bacillus subtilis, and Lactobacillus.

[0082] In one embodiment of the present invention, the product includes but is not limited to medicines, foods, food additives, and health products.

[0083] In one embodiment of the present invention, the drug contains the above-mentioned Megasphaera elsdenii KCTC5187 or its bacterial agent, and pharmaceutically acceptable excipients.

[0084] In one embodiment of the present invention, the auxiliary materials include any one of carriers, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, antibacterial agents or buffers, or a combination of at least two of them.

[0085] In one embodiment of the present invention, the carrier includes but is not limited to liposomes and nanoparticles.

[0086] In one embodiment of the present invention, the excipient is one or more of mannitol, lactose, fatty acid, and polyethylene glycol.

[0087] In one embodiment of the present invention, the dosage form of the drug includes any one of suspension, granules, capsules, powders, tablets, emulsions, solutions, pills, injections, suppositories, enemas, aerosols, patches or drops.

[0088] In one embodiment of the present invention, the administration route of the drug includes oral administration, sublingual administration, skin mucosal administration, inhalation administration or injection administration.

[0089] Beneficial effects:

[0090] The present invention confirms that Megasphaera elsdenii KCTC5187 has the effects of improving learning and memory, neuroprotection, anti-oxidation, fat reduction, and protecting the intestinal barrier, which are specifically embodied in:

[0091] (1) Behavioral experiments confirmed that Megasphaera elsdenii KCTC5187 can significantly improve learning and memory in mice and Caenorhabditis elegans;

[0092] (2) Neuronal fluorescence imaging analysis confirmed that Megasphaera elsdenii KCTC5187 can significantly improve the degeneration of dopamine, glutamate, γ-aminobutyric acid, and serotonin neurons in C. elegans;

[0093] (3) By detecting ROS and superoxide anions (O2 - ) levels confirmed that Megasphaera elsdenii KCTC5187 could reduce ROS and superoxide anions (O2 - ) level, exerting an antioxidant effect;

[0094] (4) Nile red staining experiments confirmed that Megasphaera elsdenii KCTC5187 could reduce lipid deposition;

[0095] (5) Brilliant blue staining experiments confirmed that Megasphaera elsdenii KCTC5187 could significantly improve intestinal barrier damage.

[0096] Therefore, Megasphaera elsdenii KCTC5187 has broad application prospects and promotion value in the preparation of drugs for improving learning and memory, neuroprotection, anti-oxidation, fat reduction, and protecting the intestinal barrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] Figure 1This is a diagram showing the effect of Giant Coccus elsdenii KCTC5187 on the learning and memory ability of Caenorhabditis elegans.

[0098] Figure 2 Representative graphs showing the effects of Megasphaera elsdenii KCTC5187 on dopamine, glutamate, GABA, and serotonergic neurons in Caenorhabditis elegans.

[0099] Figure 3 This is a fluorescence quantitative analysis of the effects of Megasphaera elsdenii KCTC5187 on the levels of dopamine, glutamate, γ-aminobutyric acid and serotonergic neurons in Caenorhabditis elegans.

[0100] Figure 4 Effects of Megasphaera elsdenii KCTC5187 on ROS and O2 in Caenorhabditis elegans - Representative plots of horizontal effects.

[0101] Figure 5 Effects of Megasphaera elsdenii KCTC5187 on ROS and O2 in Caenorhabditis elegans - Quantitative analysis of levels.

[0102] Figure 6 Representative images of the effects of Megasphaera elsdenii KCTC5187 on lipid deposition in Caenorhabditis elegans.

[0103] Figure 7 This is a quantitative analysis of lipid deposition in Caenorhabditis elegans by Megasphaera elsdenii KCTC5187.

[0104] Figure 8 Representative images of the effects of Megasphaera elsdenii KCTC5187 on the intestinal barrier of Caenorhabditis elegans.

[0105] Fig. 9 This is a quantitative analysis of the effect of Megasphaera elsdenii KCTC5187 on the intestinal leaky area of ​​Caenorhabditis elegans. DETAILED DESCRIPTION

[0106] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0107] The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples were repeated three times, and the results were averaged.

[0108] In the following examples, unless otherwise specified, all solutions mentioned use water as the solvent.

[0109] The reagents and materials involved in the following examples are as follows:

[0110] Cholesterol (Cat. No.: 69008216), anhydrous ethanol (Cat. No.: 40021279), sodium hypochlorite (Cat. No.: 40021279), sodium chloride (Cat. No.: 10019360), agar powder (Cat. No.: 10000582), tryptone (Cat. No.: 69024138), yeast powder (Cat. No.: 5501460B), sodium hydroxide (Cat. No.: G19852BYd), glucose (Cat. No.: T0887), soluble starch (Cat. No.: 10021328), sodium acetate (Cat. No.: 10018818), cysteine ​​(Cat. No.: 62007534), and levamisole (Cat. No.: L0438) were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0111] Beef extract (catalog number: G8270) and brilliant blue (catalog number: E8500) were purchased from Beijing Solebow Technology Co., Ltd.;

[0112] DCFH-DA (Cat. No.: S0033S), DHE (Cat. No.: S0063), and lipid droplet red fluorescence detection kit (Cat. No.: C2051S) were purchased from Biotechnologies;

[0113] N2 wild-type, BZ555, DA1240, EG1285, and GR1366 Caenorhabditis elegans were provided by the CGC American Caenorhabditis Center (University of Minnesota, MN, USA);

[0114] Megasphaera elsdenii KCTC5187 was purchased from Beijing Biobo Biotechnology Co., Ltd.

[0115] The solutions involved in the following examples are as follows:

[0116] Stock solution preparation: 1M phosphate buffer (35.6g K2HPO4, 108.3g KH2PO4, pH 6.0, constant volume 1L); 1M CaCl2 solution; 1M MgSO4 solution; 5mg / mL cholesterol solution (prepared with anhydrous ethanol).

[0117] M9 buffer: 3g KH2PO4, 6g Na2HPO4, 5g NaCl and 1mL 1M MgSO4 solution, 1L deionized water, mix well, and autoclave at 121℃ for 20min.

[0118] Lysis solution: NaClO 5mL (5%), NaOH (10mol / L) 1.25mL, and sterile water 18.75mL, prepared for use immediately.

[0119] Brilliant blue staining solution: Dissolve 5 g of brilliant blue in 95 mL of sterile water to prepare 5% brilliant blue staining solution.

[0120] 2% glucose solution: Dissolve 2 g glucose in 98 mL sterile water.

[0121] Nematode anesthetic levamisole: 1 M levamisole was prepared using M9 buffer for in vivo observation of nematodes.

[0122] Agarose pad: Prepare 2% agarose solution, heat and melt it, then take 800 μL and pour it into a small square grid made of six slides (two overlapping slides on each side and two cross slides in the middle). Use it as soon as possible after the agar solidifies.

[0123] The culture medium involved in the following examples is as follows:

[0124] RCM solid medium (1 L): 10 g tryptone, 3 g yeast extract, 5 g glucose, 0.5 g cysteine, 3 g sodium acetate, 5 g sodium chloride, 15 g agar, 1 g soluble starch, autoclave at 115 °C for 20 min.

[0125] RCM liquid medium (1L): 10 g tryptone, 3 g yeast extract, 5 g glucose, 0.5 g cysteine, 3 g sodium acetate, 5 g sodium chloride, 1 g soluble starch, autoclaved at 115°C for 20 min.

[0126] LB liquid medium: 10 g of tryptone, 5 g of yeast powder, 10 g of NaCl, add deionized water to 1 L, adjust the pH to 7.0, and sterilize at 121°C for 20 min.

[0127] NGM medium preparation (1L): 3g NaCl, 2.5g peptone, 20g agar, 25mL 1M phosphate buffer, 1mL 1M CaCl2 solution, 1mL 1M MgSO4 solution, 973mL H2O. Autoclave at 121℃ for 20min. Cool to room temperature, add 1mL 5mg / mL cholesterol solution, mix well, and add to the culture dish.

[0128] The experimental methods involved in the following examples are as follows:

[0129] 1. Preparation of bacterial suspension:

[0130] (1) Strain activation:

[0131] Take a glycerol storage tube of Megasphaera elsdenii KCTC5187, inoculate it into RCM liquid medium, and culture it in an anaerobic workstation at 37°C for 18 hours to activate the strain;

[0132] The glycerol stock tube of Escherichia coli OP50 was inoculated into LB liquid medium and cultured in a shaking incubator (180 rpm) at 37°C for 18 h.

[0133] (2) Subculture of strains:

[0134] Take the activated culture fluid of Megasphaera elsdenii KCTC5187 strain, inoculate it into new RCM liquid medium at an inoculum volume of 2% (v / v), and culture it in an anaerobic workstation at 37°C for 18 hours. Repeat this step to pass the strain three times, and repeat this step to expand the culture;

[0135] The activated culture fluid of Escherichia coli OP50 was inoculated into LB liquid medium in the same manner as above, and cultured in a 37°C shaking incubator (180 rpm).

[0136] (3) Calculation of viable bacteria:

[0137] The KCTC5187 bacterial solution and OP50 bacterial solution were diluted to 10 5 , 10 6 and 10 7 The diluted KCTC5187 bacterial solution and OP50 bacterial solution were spread on the surface of RCM solid culture medium and LB liquid culture medium respectively, and counted after 48 hours of culture.

[0138] (4) Preparation of bacteria:

[0139] The number of viable bacteria was 1×10 8 The KCTC5187 and OP50 bacterial suspensions with CFU / mL were centrifuged at 5000 rpm for 5 min to remove the culture medium, and then washed with sterile water and centrifuged (5000 rpm for 5 min) to obtain bacterial precipitates.

[0140] 2. Preparation of nematodes:

[0141] (1) Nematode culture

[0142] In the present invention, N2 wild type, BZ555, DA1240, EG1285 and GR1366 Caenorhabditis elegans were cultured in a medium containing 1×10 8 The cells were cultured in NGM culture dishes with OP50 bacterial suspensions of 10.3 CFU / mL and maintained and tested in a constant temperature incubator at 20°C using standard methods.

[0143] (2) Nematode passaging

[0144] Under laboratory conditions, nematodes reproduce quickly. To prevent the nematodes from starving and affecting the experiment, subculture inoculation is required. Use a razor blade to cut the old culture medium containing nematodes into a suitable size and transfer it to a new NGM culture dish.

[0145] (3) Synchronization of nematodes

[0146] When there are enough eggs and reproductive nematodes on the NGM culture dish, rinse them with M9 buffer into a 15 mL centrifuge tube, repeat the wash twice, discard the supernatant and transfer to a 1.5 mL centrifuge tube, add 1 mL of lysis buffer, rotate on a rotary mixer for 10 min, centrifuge at 5000 × g for 1 min, wash three times with M9 buffer, add M9 buffer and culture in a 20°C incubator overnight. When the eggs hatch, you can obtain N2 wild type, BZ555, DA1240, EG1285 or GR1366 larvae of the same L1 period.

[0147] Example 1: Effect of Megasphaera elsdenii KCTC5187 on the learning and memory ability of the high-fat nematode model

[0148] The effect of Megasphaera elsdenii KCTC5187 on the learning and memory ability of the high-fat Caenorhabditis elegans model was tested. The specific experiment is as follows:

[0149] 1. Animal experiment grouping:

[0150] The synchronized L1 stage N2 wild type, BZ555, DA1240, EG1285, and GR1366 nematodes were divided into three groups: M. elsdenii + Glu group, control group, and Glu group (a high-fat nematode model).

[0151] M. elsdenii + Glu group: 0.5 mL of the bacterial concentration was 1 × 10 8CFU / mL of KCTC5187 bacterial suspension (obtained by resuspending the above-cultured KCTC5187 bacteria in 2% glucose solution) was spread on a 60 mm NGM culture dish, and equal amounts of synchronized L1-stage N2 wild-type, BZ555, DA1240, EG1285, and GR1366 nematodes were transferred to the above-mentioned culture dishes and placed in a constant temperature incubator at 20°C for culture.

[0152] Control group: 0.5 mL of bacterial concentration was 1×10 8 CFU / mL of OP50 bacterial suspension (obtained by resuspending the above-cultured OP50 bacteria with sterile water) was spread on a 60 mm NGM culture dish, and equal amounts of synchronized L1-stage N2 wild-type, BZ555, DA1240, EG1285, and GR1366 nematodes were transferred to the above culture dishes as the control group and cultured in a constant temperature incubator at 20°C.

[0153] Glu group (high lipid nematode model): 0.5 mL of bacterial concentration was 1×10 8 CFU / mL of OP50 bacterial suspension (obtained by resuspending the above-cultured OP50 bacteria in 2% glucose solution) was spread on a 60 mm NGM culture dish, and equal amounts of synchronized L1-stage N2 wild-type, BZ555, DA1240, EG1285, and GR1366 nematodes were transferred to the above-mentioned culture dishes, respectively, as the Glu group, and cultured in a constant temperature incubator at 20°C.

[0154] 2. Evaluate the learning and memory of the N2 wild-type nematodes, and calculate the learning and memory chemotaxis index of the N2 wild-type nematodes in the above three groups. The specific method is as follows:

[0155] On the fifth day of culture, the N2 wild-type nematodes of the above three groups were transferred to a blank culture dish without food (i.e., without bacterial suspension) and starved for 1 hour. Then, the nematodes were transferred to the corresponding NGM culture dish coated with food, and 10% butanone was dripped on the lid of the culture dish. The nematodes were fed for 1 hour and then starved for 1 hour. Then, the nematodes were transferred to the chemotaxis plate to evaluate learning and memory, and the chemotaxis of the nematodes was measured at 0, 1 and 2 hours after the nematodes were transferred to the chemotaxis plate.

[0156] The food in each group is:

[0157] M. elsdenii + Glu group: 0.5 mL of bacterial concentration is 1 × 10 8 CFU / mL of KCTC5187 bacterial suspension (resuspend the KCTC5187 cells cultured above in 2% glucose solution); control group: 0.5 mL of bacterial concentration of 1×10 8CFU / mL of OP50 bacterial suspension (use sterile water to resuspend the above cultured OP50 bacteria); Glu group: 0.5mL bacterial concentration of 1×10 8 CFU / mL of OP50 bacterial suspension (2% glucose solution was used to resuspend the above cultured OP50 bacteria);

[0158] The nematodes were placed in the starting area of ​​the test plate. The NGM plate had two test areas, the butanone area (1 μl butanone: ethanol (1:10) + 1 μl anesthetic) and the ethanol control area (1 μl ethanol + 1 μl anesthetic). After one hour, the number of nematodes in the butanone area and the ethanol control area was counted. Learning and memory chemotaxis index = (the number of nematodes treated with butanone - the number of nematodes in the ethanol control) / (the total number of nematodes).

[0159] Learning and memory chemotaxis index Figure 1 shown.

[0160] The results show:

[0161] (1) The average chemotaxis index of the control group at 0 h was 0.4815. Under glucose induction, the chemotaxis index of the Glu group (model group) at 0 h decreased, with an average value of 0.3222, a decrease of 33.08% compared with the control group. The intervention of Megasphaera elsdenii KCTC5187 significantly increased the chemotaxis index of nematodes (P < 0.01). The average chemotaxis index of the M. elsdenii + Glu group at 0 h was 0.6407.

[0162] (2) The average chemotaxis index of the control group was 0.4933 at 1 h. Under glucose induction, the chemotaxis index of the Glu group (model group) was significantly decreased compared with the control group (P < 0.05), with an average value of 0.1917, a decrease of 61.13% compared with the control group. The intervention of Megasphaera elsdenii KCTC5187 significantly increased the chemotaxis index of nematodes (P < 0.05). The average chemotaxis index of M. elsdenii + Glu group was 0.4333 at 1 h.

[0163] (3) The average 2h learning and memory chemotaxis index of the control group was 0.1367. Under glucose induction, the 2h learning and memory chemotaxis index of the Glu group (model group) decreased compared with the control group, with an average value of 0.0444, a decrease of 67.52% compared with the control group. The intervention of Megasphaera elsdenii KCTC5187 increased the nematode learning and memory chemotaxis index, and the average 2h learning and memory chemotaxis index of the M. elsdenii + Glu group was 0.3533.

[0164] Example 2: Effect of Megasphaera elsdenii KCTC5187 on neuroprotection in a high-fat nematode model

[0165] According to the method of Example 1, M. elsdenii + Glu group, control group and Glu group (high-fat nematode model) of BZ555, DA1240, EG1285 and GR1366 nematodes were obtained.

[0166] The fluorescence signal intensity of dopamine neurons of BZ555 nematodes, the fluorescence signal intensity of glutamate neurons of DA1240 nematodes, the fluorescence signal intensity of γ-aminobutyric acid neurons of EG1285 nematodes, and the fluorescence signal intensity of serotonergic neurons of GR1366 nematodes in the above three groups were measured to evaluate the neuronal development of nematodes. The specific method is as follows:

[0167] The BZ555, DA1240, EG1285 and GR1366 Caenorhabditis elegans of the above three groups cultured to day 8 were washed 2-3 times, and the Caenorhabditis elegans were aspirated onto a glass slide and anesthetized with 25 mM levamisole. Fluorescence imaging was performed using a laser confocal scanning microscope (Olympus FV1200, Japan) under excitation light (488-519 nm), and fluorescence quantification was performed using Image J (National Institutes of Health, Bethesda, MD, USA). Twenty Caenorhabditis elegans were observed in each group, and three parallel experiments were performed.

[0168] Representative images and quantitative analysis results of fluorescent signals of dopamine, glutamate, γ-aminobutyric acid, and serotonergic neurons in Caenorhabditis elegans Figure 2 and Figure 3 shown.

[0169] The results show:

[0170] (1) After 8 days of intervention with Megasphaera elsdenii KCTC5187, the average fluorescence area of ​​dopamine neurons in the control group was 6.1124. Under glucose induction, compared with the control group, the average fluorescence area of ​​dopamine neurons in the model group Glu group decreased significantly by 44.37% (P < 0.001), and the average fluorescence area was 3.4002. After intervention with Megasphaera elsdenii KCTC5187, the average fluorescence area of ​​dopamine neurons in nematodes increased significantly by 27.92% (P < 0.05), and the average value was 4.4175.

[0171] (2) The average fluorescence intensity of glutamate neurons in the control group was 40.2865. Under glucose induction, the average fluorescence intensity of glutamate neurons in the model group Glu group decreased significantly by 7.58% (P < 0.001) compared with the control group, and the average fluorescence intensity was 37.2312. After the intervention of Megasphaera elsdenii KCTC5187, the average fluorescence intensity of glutamate neurons in C. elegans increased significantly by 16.6% (P < 0.001), and the average value was 43.4122.

[0172] (3) The average fluorescence intensity of γ-aminobutyric acid neurons in the control group was 41.1424. Under glucose induction, the average fluorescence intensity of γ-aminobutyric acid neurons in the model group Glu group was 41.2645, which was not significantly different from the control group. After intervention with Megasphaera elsdenii KCTC5187, the average fluorescence intensity of γ-aminobutyric acid neurons in C. elegans increased significantly by 10.82% (P < 0.01), with an average value of 45.7328.

[0173] (4) The average fluorescence area of ​​serotonin neurons in the control group was 2.2351. Under glucose induction, compared with the control group, the average fluorescence area of ​​serotonin neurons in the model group Glu group decreased by 21.22%, and the average fluorescence intensity was 1.7608. After the intervention of Megasphaera elsdenii KCTC5187, the average fluorescence area of ​​serotonin neurons in C. elegans increased significantly by 56.73% (P < 0.001), and the average value was 4.0701.

[0174] Example 3: Effect of Megasphaera elsdenii KCTC5187 on Antioxidant Effects in a High-fat Nematode Model

[0175] According to the method of Example 1, the M. elsdenii + Glu group, the control group and the Glu group (a high-fat nematode model) of N2 wild-type nematodes were obtained.

[0176] 2,7-dichlorofluorescein diacetate (DCFH-DA) and dihydroethylammonium (DHE) probes were used to measure ROS and O2 in the N2 wild-type nematodes in the above three groups. - Level, the specific method is as follows:

[0177] The N2 wild-type Caenorhabditis elegans of the three groups cultured to the eighth day were washed 2-3 times and incubated with 10 μM DCFH-DA and 10 μM DHE probes at 37°C in the dark for 60 minutes. After the dye was fully washed off with M9 buffer, the Caenorhabditis elegans were aspirated onto a glass slide and anesthetized with 25 mM levamisole. Fluorescence imaging was performed using a laser confocal scanning microscope (Olympus FV1200, Japan) at excitation light (488-519 nm) and (488-518 nm), and fluorescence quantification was performed using Image J (National Institutes of Health, Bethesda, MD, USA). At least 20 Caenorhabditis elegans were observed in each group, and three parallel experiments were performed.

[0178] ROS and O2 in Caenorhabditis elegans - Representative images and quantitative analysis results are shown in Figure 4 and Figure 5 shown.

[0179] The results show:

[0180] (1) After 8 days of intervention with Megasphaera elsdenii KCTC5187, the average fluorescence intensity of ROS in the control group was 43.9941. Under glucose induction, the average fluorescence intensity of ROS in the model group Glu group was significantly increased (P < 0.01) compared with the control group, with an average fluorescence intensity of 48.1649, and the average fluorescence intensity of the KCTC5187 group was 34.3893, with a significant decrease of 28.60%;

[0181] (2) O2 in the control group - The average fluorescence intensity was 17.5948. Compared with the control group, the model group Glu group O2 - The mean fluorescence intensity was significantly increased (P < 0.01), with a mean fluorescence intensity of 19.5834, while that of the KCTC5187 group was 17.4922, and the fluorescence intensity decreased by 10.67%, that is, the ROS and O2 - The fluorescence intensity was significantly lower than that of the model group.

[0182] It can be seen that Megasphaera elsdenii KCTC5187 can significantly improve the antioxidant level of nematodes.

[0183] Example 4: Effect of Megasphaera elsdenii KCTC5187 on lipid deposition in a high-fat nematode model

[0184] According to the method of Example 1, the M. elsdenii + Glu group, the control group and the Glu group (a high-fat nematode model) of N2 wild-type nematodes were obtained.

[0185] The lipid droplet red fluorescence detection kit (fluorescent probe Nile Red) was used to measure the lipid level in N2 wild-type nematodes. The specific method is as follows:

[0186] The N2 wild-type Caenorhabditis elegans of the three groups cultured to the eighth day were washed 2-3 times and incubated in the dark at 37°C for 60 minutes using the Nile Red probe. After the dye was fully washed off with M9 buffer, the Caenorhabditis elegans were aspirated onto a glass slide and anesthetized with 25 mM levamisole. Fluorescence imaging was performed using a laser confocal scanning microscope under excitation light (552-636 nm), and fluorescence quantification was performed using Image J. Twenty Caenorhabditis elegans were observed in each group, and three parallel experiments were performed.

[0187] Representative images of C. elegans Nile red staining and quantitative analysis results are shown in Figure 6 and Figure 7 shown.

[0188] The results show:

[0189] After 8-day intervention with Megasphaera elsdenii KCTC5187, the average fluorescence intensity of lipids in the control group was 83.6291. Under glucose induction, compared with the control group, the average fluorescence intensity of lipids in the Glu group of the model group was significantly increased (P < 0.0001), with an average fluorescence intensity of 121.0896. The average fluorescence intensity of the KCTC5187 group was 82.9663, and the fluorescence intensity was significantly decreased by 31.48%. Megasphaera elsdenii KCTC5187 can significantly reduce lipid deposition (P < 0.001).

[0190] Example 5: Effects of Megasphaera elsdenii KCTC5187 on the intestinal barrier in a high-fat nematode model

[0191] According to the method of Example 1, the M. elsdenii + Glu group, the control group and the Glu group (a high-fat nematode model) of N2 wild-type nematodes were obtained.

[0192] 0.5 mL of 5% brilliant blue staining solution was added to the culture dishes of the three groups of N2 wild-type Caenorhabditis elegans cultured to the eighth day. The N2 wild-type nematodes were fed with the 5% brilliant blue staining solution, and the leakage of brilliant blue in the nematode intestine was observed under a microscope to determine the degree of intestinal damage in the nematodes.

[0193] The N2 wild-type nematodes of the three groups on the eighth day of the test were washed three times with M9 buffer, then fixed on a 2% agarose pad with 25 mM levamisole and imaged using a Nikon upright fluorescence microscope. The blue dye leakage area of ​​the nematodes was analyzed using Image J software.

[0194] Representative images and quantitative analysis of intestinal permeability Figure 8 and Fig. 9 shown.

[0195] The results show:

[0196] After 8 days of intervention with Megasphaera elsdenii KCTC5187, the average leakage area of ​​brilliant blue in the control group was 46.7458%. Under glucose induction, the average leakage area of ​​the Glu group in the model group was significantly increased compared with the control group (P < 0.05), with an average leakage area of ​​63.1129%, and the average leakage area of ​​the KCTC5187 group was 11.7711%.

[0197] The above shows that Megasphaera elsdenii KCTC5187 can alleviate intestinal barrier damage.

[0198] In summary, the Megasphaera elsdenii KCTC5187 in the present invention has the effects of improving learning and memory, neuroprotection, anti-oxidation, fat reduction, and protection of the intestinal barrier, and can be used to prepare drugs for improving learning and memory, neuroprotection, anti-oxidation, fat reduction, and protection of the intestinal barrier. Those skilled in the art can refer to the content of this article, and appropriately improving the process parameters is within the scope of protection of the present invention.

[0199] It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The products of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the products described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

Claims

1. Use of Megasphaera elsdenii KCTC5187 or its microbial agent in the preparation of products for improving learning and memory.

2. The use according to claim 1, characterized in that: The improving learning and memory is to improve the learning and memory abilities of patients with high fat content or lipid metabolism disorders.

3. The use according to claim 1 or 2, characterized in that: The microbial agent uses Megasphaera elsdenii KCTC5187 as the main microorganism; Optionally, the microbial agent contains living cells of the KCTC5187 strain of the present invention, dry cells of the KCTC5187 strain obtained by freeze-drying, immobilized cells of the KCTC5187 strain, liquid agent of the KCTC5187 strain, solid agent of the KCTC5187 strain, or the KCTC5187 strain in any other form; Optionally, the bacterial agent also contains other applicable strains of any species, including but not limited to Bacillus licheniformis, Saccharomyces cerevisiae, Bacillus subtilis, and Lactobacillus.

4. The use according to any one of claims 1 to 3, characterized in that: The products include but are not limited to medicines, foods, food additives, feeds, feed additives, cosmetics, and health products; Optionally, the drug contains the above-mentioned Megasphaera elsdenii KCTC5187 or its bacterial agent, and pharmaceutically acceptable excipients; Optionally, the dosage form of the drug includes any one of suspension, granules, capsules, powders, tablets, emulsions, solutions, pills, injections, suppositories, enemas, aerosols, patches or drops; Optionally, the administration route of the drug includes oral administration, sublingual administration, skin mucosal administration, inhalation administration or injection; Optionally, the food is solid food, liquid food or semi-solid food.

5. The use according to any one of claims 1 to 4, characterized in that: The product at least has the function of improving learning and memory, and may also have one or more of the following functions (a) to (d): (a) Neuroprotection; (b) Antioxidant; (c) fat loss; (d) Protect the intestinal barrier.

6. Use of Megasphaera elsdenii KCTC5187 or its microbial agent in the preparation of products that have the effects of improving learning and memory, neuroprotection, anti-oxidation, fat reduction, and protecting the intestinal barrier.

7. The use according to claim 6, characterized in that: The products include but are not limited to medicines, foods, food additives, feeds, feed additives, cosmetics, and health products.

8. The use according to claim 6, characterized in that: The improvement of learning and memory refers to improving the learning and memory abilities of patients with high fat content or lipid metabolism disorders; Optionally, the neuroprotection is to increase the strength of dopamine neurons, glutamate neurons, γ-aminobutyric acid neurons and / or serotonergic neurons; Optionally, the antioxidant is to reduce ROS or O 2- level; Optionally, the fat reduction is reducing lipid deposition; Optionally, the protection of the intestinal barrier is to reduce intestinal leakage and alleviate intestinal barrier damage.

9. A drug for improving learning and memory function, characterized in that: The drug contains the above-mentioned Megasphaera elsdenii KCTC5187 or its microbial agent, and pharmaceutically acceptable excipients; Optionally, the improving learning and memory function is to improve the learning and memory ability of people with high fat content or patients with lipid metabolism disorders.

10. The drug according to claim 9, characterized in that The dosage form of the drug includes any one of suspension, granules, capsules, powders, tablets, emulsions, solutions, pills, injections, suppositories, enemas, aerosols, patches or drops; the administration route of the drug includes oral administration, sublingual administration, skin mucosal administration, inhalation administration or injection.

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