Streptococcus thermophilus AK223-6 and application thereof in preparation of product for regulating intestinal tract, improving memory and improving epilepsy

Abstract

The invention discloses streptococcus thermophilus AK223-6 and application thereof in preparation of products for regulating intestinal tracts, improving memory and improving epilepsy, and belongs to the field of microorganisms and biological medicines. The invention provides streptococcus thermophilus AK223-6 which is preserved in the Guangdong Microbial Culture Collection Center on July 8, 2024, the preservation date of the streptococcus thermophilus AK223-6 is July 8, 2024, and the preservation number of the streptococcus thermophilus AK223-6 is GDMCCNO: 64835. Based on the streptococcus thermophilus, a metabiotic product is successfully researched and developed, and verification shows that the metabiotic product has the effects of regulating intestinal flora balance, promoting digestion and absorption of a human body, improving memory, improving epilepsy and reducing epilepsy times. After 100mg of a metabolite product containing 16,000,000,000 strains and metabolites is given every day, lactic acid bacteria polysaccharide, ergosterol, cephalin, vitamin A and vitamin B1 in the endophytic necessary quantity can be continuously provided in the intestinal reproduction and growth process, the memory and epilepsy can be effectively improved, the epilepsy frequency is reduced, and the practical application value is good.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms and biomedicine, and specifically relates to thermophilic streptococcus AK223-6 and its application in preparing products for regulating intestines, improving memory and improving epilepsy. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Epilepsy is one of the most common neurological disorders, affecting approximately 50 million people worldwide. Despite treatment with different classes of anti-epileptic drugs, more than 30% of people with epilepsy continue to have seizures. This is known as drug-resistant epilepsy. For these patients with drug-resistant epilepsy, a variety of complementary therapies have been proposed, including surgery, neurostimulator implantation, and ketogenic diet, but these therapies have limited effectiveness.

[0004] In addition, in traditional Chinese medicine, memory loss is called amnesia, which has a series of manifestations such as mental weakness, forgetfulness, insomnia and dreaminess. Memory loss can be caused by many factors, including normal physiological aging process, lifestyle factors, health problems and other external factors.

[0005] Studies have shown that long-term use of probiotics can effectively improve the host's memory, mainly in terms of improving sleep quality, relieving stress and anxiety, and improving health. Other studies have shown that taking probiotics has a positive effect on improving epilepsy symptoms by regulating the balance of intestinal flora. Therefore, finding a probiotic that can effectively improve memory and / or improve epilepsy can provide a new strategy for improving memory and / or improving the prevention and treatment of epilepsy. Summary of the invention

[0006] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide thermophilic Streptococcus AK223-6 and its use in preparing products for regulating the intestine, improving memory, and improving epilepsy. The thermophilic Streptococcus provided by the present invention ( Streptococcus thermophilus ) AK223-6 not only regulates the balance of intestinal flora and promotes digestion and absorption, but also has the effects of improving memory and epilepsy.

[0007] In order to achieve the above object, the technical solution of the present invention is: In a first aspect, the present invention provides a thermophilic Streptococcus ( Streptococcus thermophilus)AK223-6, deposited in Guangdong Microbiological Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, the deposit date is July 8, 2024, and the deposit number is GDMCCNO: 64835.

[0008] In a second aspect, the present invention provides a kind of above-mentioned thermophilic streptococcus ( Streptococcus thermophilus ) A derivative of AK223-6, wherein the derivative is one or more of living bacteria, dead bacteria, fermentation broth or metabolites.

[0009] In a third aspect, the present invention provides a microbial agent, comprising an active ingredient, wherein the active ingredient comprises the above-mentioned Streptococcus thermophilus ( Streptococcus thermophilus ) AK223-6 or its inactivated bacteria, fermentation broth, or metabolites.

[0010] In one or more embodiments, the microbial agent comprises inactivated Streptococcus thermophilus ( Streptococcus thermophilus )AK223-6 and its fermentation broth.

[0011] In a fourth aspect, the present invention provides a method for preparing the microbial agent, the method comprising: S1. Bacteria activation: The thermophilic Streptococcus ( Streptococcus thermophilus ) AK223-6 was inoculated into the activation medium and cultured at 35-40°C for 20-25h to obtain the expanded bacterial culture liquid; S2. Preparation of primary bacterial seed solution: inoculate the expanded bacterial seed solution prepared in step S1 into the primary seed culture solution at an inoculation amount of 0.5-5%, and culture at 35-40° C. for 10-15 hours to obtain the primary bacterial seed solution; S3, seed tank strain preparation: the primary strain liquid prepared in step S2 is inoculated into a seed tank containing a seed tank culture medium under sterile conditions, the inoculation amount is 0.5-5%, the stirring culture is 50-100r / min, the pH is natural, the temperature is 30-40°C, the tank pressure is 0.03-0.06MPa, and the culture time is 15-20h to obtain the seed tank strain; S4, thermophilic streptococcus fermentation: inoculate the seed tank strain into a fermentation tank containing sterilized fermentation culture medium, with an inoculation amount of 0.5-7%, 35-39°C, 140-170r / min, tank pressure 0.04-0.06MPa, pH 6.3-6.7, and ferment for 24-26h; cool to 19-21°C, which is the fermentation liquid; heat the fermentation liquid to 119-121°C and maintain for 4-7s for UHT inactivation treatment.

[0012] In one or more embodiments, in step S1, the composition of the activation medium is as follows, by weight: 16-23 parts of oligofructose, 4-8 parts of chitosan, 4-8 parts of beef extract powder, 0.5-1.5 parts of sodium acetate, 0.2-1 parts of Tween-80, 0.5-1.5 parts of potassium dihydrogen phosphate, 800-1200 parts of water, and the pH is adjusted to 7.0; In one or more embodiments, in step S2, the composition of the primary seed culture solution is as follows, by weight: 6-12 parts of oligofructose, 4-8 parts of peptone, 2-6 parts of beef extract powder, 1-4 parts of albumin peptone, 0.5-2 parts of sodium acetate, 0.5-2 parts of Tween-80, 800-1200 parts of tap water, and natural pH value; In one or more embodiments, in step S3, the composition of the seed tank culture medium is as follows, by weight percentage: fructooligosaccharide 2-6%, casein peptone 0.5-2%, dipotassium hydrogen phosphate 0.1-0.6%, albumin peptide 1-2%, sodium acetate 0.5-1%, Tween-80 0.05-0.3%, polyether defoamer 0.05-0.3%, tap water 90-95%, pH value natural; In one or more embodiments, in step S4, the fermentation broth comprises the following components, by weight percentage: fructooligosaccharide 2-6%, casein peptone 0.5-2%, xylo-oligosaccharide 1-3%, yeast extract 2-6%, sodium acetate 1-3%, Tween-80 0.1-0.4%, spirulina oligopeptide 1-4%, dipotassium hydrogen phosphate 0.5-2%, polyether defoamer 0.05-0.2%, tap water 80-90%; In one or more embodiments, after step S4 is completed, the obtained fermentation broth is centrifuged to collect the inactivated wet bacterial sludge metabolites, and the inactivated wet bacterial sludge metabolites are freeze-dried to obtain freeze-dried powder of the inactivated bacterial metabolites.

[0013] In a fifth aspect, the present invention provides the thermophilic Streptococcus ( Streptococcus thermophilus )AK223-6, or Streptococcus thermophilus ( Streptococcus thermophilus ) Application of derivatives of AK223-6 or microbial agents in food preparation.

[0014] In a sixth aspect, the present invention provides the thermophilic Streptococcus ( Streptococcus thermophilus )AK223-6, or Streptococcus thermophilus ( Streptococcus thermophilus ) Use of derivatives or microbial agents of AK223-6 in the preparation of any of the following products: (1) Products used to regulate intestinal flora and promote digestion; (2) Products used to improve memory; (3) Products used to improve epilepsy or reduce the frequency of epileptic seizures; (4) Products used in the production of lactic acid bacteria polysaccharides, ergosterol, cephalin, vitamin A and vitamin B1.

[0015] The product is one of a food and a medicine; The food is any one of beverages, dairy products, candy products, health foods, special dietary foods and special medical formula foods.

[0016] In a sixth aspect, the present invention provides a product comprising the above-mentioned thermophilic Streptococcus ( Streptococcus thermophilus )AK223-6, and / or the above-mentioned thermophilic Streptococcus ( Streptococcus thermophilus ) derivatives of AK223-6, and / or the above-mentioned microbial agents and acceptable adjuvants or excipients.

[0017] In some examples of this embodiment, the product is one of a food and a medicine; In some examples of this embodiment, the food is any one of a beverage, a dairy product, a candy product, a health food, a special dietary food and a special medical purpose formula food.

[0018] In some examples of this embodiment, the product comprises a postbiotic preparation.

[0019] In an eighth aspect of postbiotic products, the present invention provides a method for regulating intestinal flora, promoting digestion or improving memory or improving epilepsy, reducing the number of epileptic seizures, or for producing lactic acid bacteria polysaccharides, ergosterol, cephalin, vitamin A, and vitamin B1, using the above-mentioned Streptococcus thermophilus ( Streptococcus thermophilus )AK223-6, or Streptococcus thermophilus ( Streptococcus thermophilus ) Derivatives or microbial agents of AK223-6.

[0020] The beneficial effects of the present invention are: The above technical scheme screened and obtained a thermophilic Streptococcus ( Streptococcus thermophilus ) AK223-6, a postbiotic product was successfully developed based on the thermophilic Streptococcus. It has been proven that it can regulate the balance of intestinal flora, promote human digestion and absorption, and improve memory, improve epilepsy, and reduce the number of epileptic seizures. Daily administration of 100 mg of the postbiotic product containing 16 billion strains and metabolites of the present invention can sustainably provide the endogenous necessary amount of lactic acid polysaccharides, ergosterol, cephalin, vitamin A, and vitamin B1 during the intestinal reproduction and growth process, which can effectively improve memory and improve epilepsy, and has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0022] Figure 1 This is a result analysis diagram of the metabolites of the postbiotic product in Example 2 of the present invention; Figure 2 This is a data diagram of the NO content in the serum of mice in different groups in Example 5 of the present invention; Figure 3 The figure is a data diagram of the content of SOD in the serum of mice of different groups in Example 5 of the present invention; Figure 4 This is a data graph of the content of MDA and T-AOC in the serum of mice in different groups in Example 5 of the present invention. DETAILED DESCRIPTION

[0023] A first typical embodiment of the present invention provides a thermophilic Streptococcus ( Streptococcus thermophilus )AK223-6, deposited in Guangdong Provincial Microbiological Culture Collection Center, the preservation date is July 8, 2024, and the preservation number is GDMCCNO: 64835.

[0024] The bacterium was isolated from fresh Grifola frondosa fruiting bodies in Nanning, Guangxi Zhuang Autonomous Region, and was identified as thermophilic Streptococcus by 16srRNA method. The sequence of the bacterium is as follows: This strain has been preserved as a patented strain in the Guangdong Provincial Microbial Culture Collection Center with the preservation number GDMCCNO: 64835. This thermophilic Streptococcus strain is a probiotic strain approved for use by the National Health Commission. This strain not only has the effects of regulating the balance of intestinal flora and promoting human digestion and absorption, but also improves memory and epilepsy. 100 mg of a postbiotic product containing 16 billion strains and metabolites of the present invention can sustainably provide the endogenous necessary amount of lactic acid polysaccharides, ergosterol, cephalin, vitamin A, and vitamin B1 during the intestinal reproduction and growth process, which can improve memory and maintain a healthy nervous system.

[0025] A second typical embodiment of the present invention provides a thermophilic Streptococcus ( Streptococcus thermophilus ) A derivative of AK223-6, wherein the derivative is one or more of living bacteria, dead bacteria, fermentation broth or metabolites.

[0026] A third typical embodiment of the present invention provides a microbial agent, comprising an active ingredient, wherein the active ingredient comprises the above-mentioned Streptococcus thermophilus ( Streptococcus thermophilus ) AK223-6 or its inactivated bacteria, fermentation broth, metabolites. The metabolites include extracellular metabolites and / or intracellular metabolites.

[0027] In some examples of this embodiment, the microbial agent comprises inactivated Streptococcus thermophilus and its fermentation broth. In this case, the microbial agent is a postbiotic product.

[0028] A fourth typical embodiment of the present invention provides a method for preparing the microbial agent, the method comprising: S1. Bacteria activation: The thermophilic Streptococcus ( Streptococcus thermophilus ) AK223-6 is inoculated into the activation culture medium and cultured at 35-40°C (preferably 37°C) for 20-25h (preferably 24h) to obtain an expanded bacterial culture solution.

[0029] The activation culture medium is composed of the following parts by weight: 16-23 parts of oligofructose, 4-8 parts of chitosan, 4-8 parts of beef extract powder, 0.5-1.5 parts of sodium acetate, 0.2-1 parts of Tween-80, 0.5-1.5 parts of potassium dihydrogen phosphate, 800-1200 parts of water, and the pH is adjusted to 7.0 (alkaline solution adjustment).

[0030] More specifically, the activation culture medium is composed of the following, by weight: 20 parts of oligofructose, 6 parts of chitosan, 6 parts of beef extract powder, 0.9 parts of sodium acetate, 0.5 parts of Tween-80, 0.9 parts of potassium dihydrogen phosphate, 1000 parts of water, and the pH is adjusted to 7.0 (alkaline solution adjustment).

[0031] S2. Preparation of primary bacterial seed liquid: inoculate the expanded bacterial seed liquid prepared in step S1 into the primary seed culture liquid with an inoculation amount of 0.5-5%, and culture at 35-40° C. (preferably 37° C.) for 10-15 h (preferably 12 h) to obtain the primary bacterial seed liquid.

[0032] The first-level seed culture solution is composed of the following parts by weight: 6-12 parts of oligofructose, 4-8 parts of peptone, 2-6 parts of beef extract powder, 1-4 parts of albumin peptone, 0.5-2 parts of sodium acetate, 0.5-2 parts of Tween-80, 800-1200 parts of tap water, and a natural pH value.

[0033] More specifically, the composition of the primary seed culture solution is as follows, in parts by weight: 8 parts of oligofructose, 6 parts of peptone, 4 parts of beef extract powder, 2 parts of albumin peptone, 0.8 parts of sodium acetate, 1 part of Tween-80, 1000 parts of tap water, natural pH value.

[0034] S3. Preparation of seed tank strains: inoculate the primary strain liquid prepared in step S2 into a seed tank containing a seed tank culture medium under sterile conditions, with an inoculation amount of 0.5-5%% (preferably 5%, mass percentage), stirring culture at 50-100r / min (preferably 80r / min), natural pH, temperature 30-40°C (preferably 37°C), tank pressure 0.03-0.06MPa (preferably 0.05MPa), and culture time 15-20h (preferably 18h) to obtain seed tank strains.

[0035] The composition of the seed tank culture medium is as follows, by weight percentage: 2-6% oligofructose, 0.5-2% casein peptone, 0.1-0.6% dipotassium hydrogen phosphate, 1-2% albumin peptide, 0.5-1% sodium acetate, 0.05-0.3% Tween-80, 0.05-0.3% polyether defoamer, 90-95% tap water, and natural pH value.

[0036] More specifically, the composition of the seed tank culture medium is as follows, by weight percentage: 4% oligofructose, 0.9% casein peptone, 0.3% dipotassium hydrogen phosphate, 1.5% albumin peptide, 0.7% sodium acetate, 0.1% Tween-80, 0.1% polyether defoamer, 92.4% tap water, and natural pH.

[0037] S4, plant lactobacillus fermentation: inoculate the seed tank strain into a fermentation tank containing sterilized fermentation culture liquid, with an inoculation amount of 0.5-7% (preferably 5-7%), 35-39°C (preferably 37°C), 140-170r / min (preferably 160r / min), tank pressure 0.04-0.06MPa (preferably 0.05MPa), pH 6.3-6.7 (preferably 6.5), fermentation for 24-26h (preferably 25h); cool to 19-21°C (preferably 20°C), which is the fermentation liquid. Heat the fermentation liquid to 119-121°C (preferably 120°C) and keep it for 4-7s (preferably 6s) for UHT inactivation treatment.

[0038] The fermentation culture solution is composed of the following, by weight percentage: 2-6% fructo-oligosaccharide, 0.5-2% casein peptone, 1-3% xylo-oligosaccharide, 2-6% yeast extract, 1-3% sodium acetate, 0.1-0.4% Tween-80, 1-4% spirulina oligopeptide, 0.5-2% dipotassium hydrogen phosphate, 0.05-0.2% polyether defoamer, and 80-90% tap water.

[0039] More specifically, the composition of the fermentation culture medium is as follows, by weight percentage: 4% fructooligosaccharide, 1% casein peptone, 1.5% xylo-oligosaccharide, 4% yeast extract, 1.5% sodium acetate, 0.2% Tween-80, 2% spirulina oligopeptide, 1% dipotassium hydrogen phosphate, 0.1% polyether defoamer, and 84.7% tap water.

[0040] In some examples of this embodiment, after step S4 is completed, the obtained fermentation broth is centrifuged to collect the inactivated wet bacterial sludge metabolites, and the inactivated wet bacterial sludge metabolites are freeze-dried (the final moisture content is preferably controlled to be 4%) to obtain freeze-dried powder of inactivated bacterial metabolites.

[0041] A fifth typical embodiment of the present invention provides the thermophilic Streptococcus ( Streptococcus thermophilus )AK223-6, or Streptococcus thermophilus ( Streptococcus thermophilus ) Application of derivatives of AK223-6 or microbial agents in food preparation.

[0042] A sixth typical embodiment of the present invention provides the thermophilic Streptococcus ( Streptococcus thermophilus )AK223-6, or Streptococcus thermophilus ( Streptococcus thermophilus ) Use of derivatives or microbial agents of AK223-6 in the preparation of any of the following products: (1) Products used to regulate intestinal flora and promote digestion; (2) Products used to improve memory; (3) Products used to improve epilepsy and reduce the frequency of epileptic seizures; (4) Products used in the production of lactic acid bacteria polysaccharides, ergosterol, cephalin, vitamin A and vitamin B1.

[0043] In some examples of this embodiment, the product is one of a food and a medicine; The food is any one of beverages, dairy products, candy products, health foods, special dietary foods and special medical formula foods.

[0044] The lactic acid bacteria polysaccharide, ergosterol, cephalin, vitamin A and vitamin B1 respectively have the following effects: Lactic acid bacteria polysaccharides: can improve the gastrointestinal health function of the body, regulate the composition of intestinal flora, reshape the intestinal flora ecology, and ultimately produce anti-epileptic effects. Improve memory by preventing oxidative stress and inflammation.

[0045] Ergosterol: It plays a role in protecting the brain by promoting the growth and differentiation of neurons, anti-oxidative stress response, and anti-inflammatory response; Phospholipids: It promotes blood circulation in the brain, helps improve insufficient blood supply to the brain, and thus relieves secondary epileptic seizures caused by hypoxic-ischemic encephalopathy; it can promote the development of brain nerves and increase the activity of brain cells, thereby enhancing memory.

[0046] Vitamin A: It is involved in the formation and connection of neurons, can promote the synthesis and release of neurotransmitters, enhance the efficiency of neurotransmission, and thus improve the cognitive function of the brain.

[0047] Vitamin B1: It can assist the production of acetyl coenzyme A and acetylcholine, and has a certain auxiliary effect on the brain's memory.

[0048] A seventh typical embodiment of the present invention provides a product, comprising the thermophilic Streptococcus ( Streptococcus thermophilus )AK223-6, and / or Streptococcus thermophilus ( Streptococcus thermophilus ) derivatives of AK223-6, and / or microbial agents and acceptable adjuvants or excipients.

[0049] In some examples of this embodiment, the product is one of a food and a medicine; The food is any one of beverages, dairy products, candy products, health foods, special dietary foods and special medical formula foods.

[0050] In some examples of this embodiment, the product comprises a postbiotic product.

[0051] In some examples of this embodiment, the auxiliary agent or adjuvant can be a carrier, excipient, diluent, etc. commonly used in pharmacy. Moreover, according to the usual method, it can be made into an oral preparation in the form of powder, granule, suspension, emulsion, syrup, spray, etc.

[0052] The non-pharmaceutical active ingredients such as carriers, excipients and diluents that can be included are well known in the field, and those of ordinary skill in the art can determine that they meet clinical standards. In another specific embodiment of the present invention, the carriers, excipients and diluents include but are not limited to lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talcum powder, magnesium stearate and mineral oil.

[0053] The drug can be prepared in the form of an injection preparation or an oral preparation. The injection preparation includes liquid injection, injection powder, and injection tablet according to the physical state; and includes intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, and spinal cavity injection according to the injection part; preferably, the solvent of the injection preparation includes water for injection or physiological saline.

[0054] Formulations for oral use include tablets containing the active ingredient mixed with nontoxic pharmaceutically acceptable excipients. These excipients can be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starch including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate or sodium phosphate); granulating agents and disintegrants (e.g., cellulose derivatives including microcrystalline cellulose, starch including potato starch, cross-linked carboxymethyl cellulose sodium, alginates or alginic acid); binders (e.g., sucrose, glucose, sorbitol, gum arabic, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethyl cellulose, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinyl pyrrolidone or polyethylene glycol); and lubricants, glidants and anti-adherents (e.g., magnesium stearate, zinc stearate, stearic acid, silicon dioxide, hydrogenated vegetable oil or talc). Preparations for oral use may also be in the form of chewable tablets, or in the form of hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin), or in the form of soft gelatin capsules in which the active ingredient is mixed with water or an oil medium (e.g., peanut oil, liquid paraffin or olive oil). Powders, granules and pills can be prepared using the ingredients mentioned above under tablets or capsules in a conventional manner using, for example, a mixer, fluidized bed equipment or spray drying equipment.

[0055] Other pharmaceutically acceptable excipients for oral formulations include, but are not limited to, colorants, flavoring agents, plasticizers, humectants, and buffers. Formulations for oral use may also be in the form of chewable tablets, or in the form of hard gelatin capsules, wherein the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate, or kaolin), or in the form of soft gelatin capsules, wherein the active ingredient is mixed with water or an oil medium (e.g., peanut oil, liquid paraffin, or olive oil). Powders, granules, and pills may be prepared in a conventional manner using, for example, a mixer, a fluidized bed apparatus, or a spray drying apparatus using the ingredients mentioned above under tablets or capsules.

[0056] The eighth typical embodiment of the present invention provides a method for regulating intestinal flora, promoting digestion or improving memory or improving epilepsy, reducing the number of epileptic seizures or for producing lactic acid bacteria polysaccharides, ergosterol, cephalin, vitamin A, vitamin B1, using the above-mentioned thermophilic streptococcus ( Streptococcus thermophilus )AK223-6, or Streptococcus thermophilus ( Streptococcus thermophilus ) Derivatives or microbial agents of AK223-6.

[0057] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0058] In the examples, the "%" of the components in each culture medium (liquid) is the mass volume percentage, and the unit is g / mL. In the examples, the density of Tween-80 is 1.08 g / cm 3 .

[0059] Example 1 A method for preparing a postbiotic product comprises the following steps: Equipment used in production: strain incubator, clean workbench, constant temperature incubator, various test tubes, triangular flasks, seed tanks, fermentation tanks, vacuum freeze dryer, three-dimensional mixer, low-temperature crusher, vacuum packaging machine.

[0060] (1) Bacteria activation Bacterial strain culture: Expansion culture of bacterial strains preserved in -80℃ refrigerator.

[0061] Preparation of culture medium: 20g oligofructose, 6g chitosan, 6g beef extract powder, 0.9g sodium acetate, 0.5mL Tween-80, 0.9g potassium dihydrogen phosphate, 1000mL tap water, pH adjusted to 7.0 (alkaline solution adjustment).

[0062] Operation: Add the oligofructose and other raw materials in the above formula into 1000mL of water in the formula without any distinction, stir for 10 minutes to dissolve, and then divide into test tubes, each test tube contains 5mL, seal with kraft paper and place in a medical sterilizer for sterilization at 0.1-0.12MPa for 20 minutes. The culture medium is ready.

[0063] Inoculation: Under sterile conditions (clean bench), open the -80℃ low-temperature refrigerator to store the frozen tube of bacteria, dissolve it at room temperature, then take one sterilized test tube culture medium, add 0.5mL of the dissolved bacterial solution into the test tube, seal the tube mouth with kraft paper, shake it gently by hand, put it in a constant temperature incubator at 37℃ and culture it for 24 hours. This is called expansion culture medium.

[0064] (2) Preparation of primary bacterial culture liquid: Primary seed culture formula: 8 g fructooligosaccharide, 6 g peptone, 4 g beef extract powder, 2 g albumin peptone, 0.8 g sodium acetate, 1 mL Tween-80, 1000 mL tap water, natural pH.

[0065] Operation: Add the above raw materials into 1000mL of tap water without any distinction, start the blender, and stir for 10 minutes to dissolve them all. This is called the primary seed culture solution.

[0066] Add the stirred and dissolved first-level seed culture solution into the triangular flask according to the dosage, then wrap the bottle mouth with 6 layers of gauze and a layer of kraft paper, put it into the sterilizer for sterilization. Before sterilization, first open the exhaust valve on the sterilizer and close it when a small amount of steam is discharged. When the pressure on the sterilizer reaches 0.05MPa, open the exhaust valve on the sterilizer to exhaust for 6-8min, then close the exhaust valve and continue heating. Start timing when the steam pressure in the sterilizer reaches 0.1-0.12MPa. The sterilization time is 20min. Leave the heat source after completion and let it cool down naturally. When the pressure gauge on the sterilizer returns to zero, open the sterilizer, take out the sterilized culture medium and put it into the clean bench to cool naturally. When the temperature drops to 37°C, transfer the above cultured expanded bacterial liquid to the sterilized culture liquid in the triangular bottle. The inoculation amount is 0.5%, that is, 100mL of the first-level seed culture liquid and 0.5mL of the expanded bacterial liquid. After completion, seal the bottle mouth with the original gauze and kraft paper, shake it slightly by hand to mix the connected bacteria and culture liquid evenly, and then place it in a constant temperature box and culture it at 37°C for 12 hours. This is called the first-level bacterial liquid.

[0067] (3) Preparation of seed tank strains: Sterilization of fermentation equipment, pipelines and sterile filtration systems: First, open the valves of all inlet and outlet pipelines and sterile air pipelines, and pass 0.12-0.14MPa steam. Allow the steam to connect with the pipeline valves and discharge a small amount of steam. Ventilate for 40 minutes, then close the valves of all inlet and outlet pipelines and set aside.

[0068] Sterilization of empty seed tanks and fermentation tanks: Close all valves, open the drain valve at the bottom of the tank and the drain valve in the tank interlayer, open the direct steam valve to introduce 0.12-0.14MPa steam into the pipe, start timing when the temperature in the tank reaches 121℃, the sterilization time is 40min, then close the drain valve at the bottom of the tank and the drain valve in the interlayer, and the temperature in the tank will naturally drop to 37℃ for standby use.

[0069] Seed pot culture: Formula: 4% oligofructose, 0.9% casein peptone, 0.3% dipotassium hydrogen phosphate, 1.5% albumin peptide, 0.7% sodium acetate, 0.1% Tween-80, 0.1% polyether defoamer, 92.4% tap water, natural pH.

[0070] Specific operation: First, pour the tap water used in the formula into the sterilized seed tank, start the mixer, add the raw materials required in the formula respectively (the order of addition is not important), then stir continuously and pass steam for heating. First, use the steam in the tank interlayer to heat to 95°C, then switch to direct steam heating. When the temperature in the tank reaches 121°C, maintain it for 20 minutes to achieve the sterilization effect, then turn off the steam, and start cooling through the tap water in the tank interlayer. When the temperature in the tank reaches 37°C, it is ready for use. This is called seed culture medium. Then inoculate the above-cultured first-level bacterial culture solution into the seed tank under sterile conditions. The inoculation amount is 5% of the culture medium in the tank. 100 kg of seed tank culture medium can be inoculated with 5 kg of first-level bacterial solution. Then cover the inoculation cap on the tank and start stirring and culturing. The culture conditions are: temperature 37°C, natural pH value, stirring speed 80r / min, tank pressure 0.05MPa, and culture time 18h to meet the requirements. If the tank body is lowered, sterile air can be introduced to maintain the tank pressure. If the tank pressure is too high, the air release valve on the tank top can be used to adjust it. This is called seed tank culture.

[0071] (4) Fermentation with Streptococcus thermophilus Formula: 4% fructooligosaccharide, 1% casein peptone, 1.5% xylo-oligosaccharide, 4% yeast extract powder, 1.5% sodium acetate, 0.2% Tween-80, 2% spirulina oligopeptide, 1% dipotassium hydrogen phosphate, 0.1% polyether defoamer, and 84.7% tap water.

[0072] Specific operation: First, add the tap water used in the formula into the sterilized fermentation tank, start the mixer, add the raw materials used in the formula into the fermentation tank (the order of addition is not important), and then pass steam for heating. First, use the interlayer steam to heat to 95°C, and then switch to direct steam heating. When the temperature in the tank reaches 121°C, start timing, and keep it for 30 minutes to achieve the sterilization effect, and then cool it down with the tap water in the interlayer. When the temperature in the tank drops to 37°C, start to inoculate the strains. Before inoculation, first increase the tank pressure of the seed tank to 0.1MPa, and keep the tank pressure of the fermentation tank at 0.05MPa, then open the inoculation pipeline valve, and transport the seeds cultured in the seed tank to the fermentation tank by pressure difference, and then close the inoculation channel valve.

[0073] The inoculation amount is 5 kg of the culture medium in the seed tank for every 100 kg of the fermentation tank. The culture conditions are: culture temperature 37°C, stirring speed 160r / min, tank pressure maintained at 0.05MPa, and the pH value is maintained at 6.5 through the acid and alkali automatic control system during the fermentation process. If the tank pressure decreases, sterile air can be introduced to maintain the tank pressure until the fermentation is completed. The total fermentation is 25 hours, and then the temperature is lowered to 20°C. This is the fermentation liquid. UHT inactivation treatment is carried out at 120°C for 6 seconds.

[0074] (5) Centrifugation of fermentation broth The fermentation liquid is pumped into a storage tank for centrifugation. Operation: Start the centrifuge, adjust the speed to 10000r / min, centrifuge at a feed rate of 2kg / min, collect the inactivated wet bacterial sludge metabolites after centrifugation, and freeze-dry the collected inactivated wet bacterial sludge metabolites.

[0075] (6) Freeze-drying of bacterial sludge metabolites Preparation of bacterial suspension: Take 1000g of inactivated wet bacterial sludge metabolites, add 4000mL of sterile saline, 600g of egg white peptide, 600g of maltodextrin, 2000g of water-soluble corn starch, 50g of polyanthocyanin, and 120g of vitamin C powder.

[0076] Operation: First, take 2500mL of normal saline in the formula, add maltodextrin and stir to dissolve, then add 1000g of inactivated wet bacterial sludge metabolites, and then add the remaining normal saline and water-soluble corn starch and auxiliary materials in the formula and stir well, which is called a suspension. The suspension is freeze-dried and the moisture content is finally controlled to 4% to obtain the freeze-dried powder of the inactivated bacterial metabolites, i.e., the postbiotic product.

[0077] After testing, the freeze-dried powder obtained in Example 1 had a content of inactivated bacteria of 160 billion cells / g.

[0078] The inactivated bacteria of the freeze-dried powder will be lost or broken during the dilution process. Although the bacteria are broken, the effect of the strain will not be weakened.

[0079] The freeze-dried powder can be widely used in food, baked food, solid beverages, liquid beverages, and compressed candies.

[0080] Example 2 - Metabolomics Analysis Metabolomics analysis of mouse feces was performed using ultra-performance liquid chromatography-tandem mass spectrometry.

[0081] Experimental scheme: The mice were divided into a control group and an experimental group. The control group was gavaged with 0.5 mL of normal saline while being fed normally, and the experimental group was gavaged with 0.5 mL of postbiotic product solution (10 mg of the postbiotic product prepared in Example 1 was dissolved in 0.5 mL of sterilized water) while being fed normally, once a day. After feeding for thirty days, the fecal samples of the mice in the control group and the experimental group were placed in 400 μL of extraction solution (acetonitrile: methanol = 1: 1), vortexed for 30 s, and then subjected to low-temperature ultrasonic extraction for 30 min (5°C, 40 KHz), allowed to stand for 30 min at -20°C, placed in a 4°C centrifuge, centrifuged at 13000 r / min for 15 min, the supernatant was taken, dried with nitrogen, re-dissolved with 120 μL of reconstitution solution (acetonitrile: water = 1: 1), extracted at low temperature for 5 min (5°C, 40 KHz), placed in a 4°C centrifuge, centrifuged at 13000 r / min for 5 min, and an appropriate amount of supernatant was taken for analysis. 10 µL of sample supernatant was separated by BEHC18 column (100 mm×2.1 mm id1.8 µm) and then entered mass spectrometry detection. The separation flow rate was 0.40 mL / min and the column temperature was 40 °C. After completion, the LC-MS raw data was transferred to the computer for processing and analysis. The MS mass spectrum information was matched with the public database to obtain the main metabolite information as shown in Table 2 and Figure 1 shown.

[0082] Method for determination of lactic acid bacteria polysaccharide content: spectrophotometry; Ergosterol content detection method: high performance liquid chromatography; Method for detecting cephalin content: high performance liquid chromatography; Vitamin A content detection method: Reversed-phase high performance liquid chromatography; Vitamin B1 content detection method: high performance liquid chromatography.

[0083] Table 1 Main metabolomics analysis of fed mice

[0084] According to Table 1, daily administration of 10 mg of a postbiotic product containing 1.6 billion inactivated bacterial cells and metabolites (i.e., the freeze-dried powder prepared in Example 1) to mice can significantly increase the content of lactic acid polysaccharides, ergosterol, cephalin, vitamin A, and vitamin B1 in mouse feces, indicating that the postbiotic product provided by the present invention can sustainably provide the endogenous necessary amount of lactic acid polysaccharides, ergosterol, cephalin, vitamin A, and vitamin B1 during the intestinal reproduction and growth process. It can improve memory, improve epilepsy, and maintain a healthy nervous system.

[0085] Example 3 - Improving memory 1. Small-scale experiment to improve memory In this application trial, the subjects were people with an average age of 60 in 8 communities in Jinan City. As of December 2022, a total of 100 people were recruited to participate in the experiment. All of the above-mentioned people signed a written informed consent form. If the participant was unable to sign, the agent (usually a family member) signed it. The experimental group took 0.1 grams of probiotic product (i.e., the freeze-dried powder prepared in Example 1) once a day before going to bed. During the drinking period, the original eating habits were not changed and the diet was normal. The test results are shown in Table 2.

[0086] Table 2 Effect of improving memory application

[0087] Description of Table 2: Middle-aged: 50 people started to take the product for 3 days and the results were counted. After taking it for 3 days, 14 people felt that their slow reaction, lack of concentration, memory loss, low mood, and depression improved. After taking it for 6 days, 23 people felt that their slow reaction, lack of concentration, memory loss, low mood, and depression improved significantly. After taking it for 8 days, 34 people felt that their slow reaction, lack of concentration, memory loss, low mood, and depression improved significantly. After taking it for 10 days, 48 ​​people felt that their slow reaction, lack of concentration, memory loss, low mood, and depression improved completely. A total of 2 people did not improve after taking the Shengyuan product.

[0088] Elderly: 50 people started to take the product for 4 days and the results were counted. After 4 days, 12 people felt that their slow reaction, memory loss, depression, depression, insomnia and other conditions improved. After 8 days, 25 people felt that their slow reaction, memory loss, depression, depression, insomnia and other conditions improved significantly. After 12 days, 36 people felt that their slow reaction, memory loss, depression, depression, insomnia and other conditions improved significantly. After 15 days, 46 people felt that their slow reaction, memory loss, depression, depression, insomnia and other conditions were completely improved. A total of 4 people did not improve after taking the Shengyuan product.

[0089] 2. Mouse verification experiment to improve memory: SPF mice weighing 18-22g were selected for the experiment. The mice were divided into a control group, a model group and an experimental group. The control group and the model group were given 0.5 mL of normal saline by gavage while feeding normally, and the experimental group was given 0.5 mL of postbiotic product solution by gavage while feeding normally, once a day. Among them, the preparation method of the postbiotic product solution is: dissolving the postbiotic product prepared in Example 1 in 0.5 mL of sterilized water to obtain a postbiotic product solution. Among them, the added mass of the postbiotic product was 10 mg (experimental group 1), 15 mg (experimental group 2), and 20 mg (experimental group 3), respectively. The experiments were carried out after continuous gavage for 30 days. The model group and each dose group were intraperitoneally injected with scopolamine 5 mg / kg body weight 10 minutes before experimental training, and the injection volume was 10 mL / kg body weight. Each dose group was given maintenance feed.

[0090] (1) Platform experiment The training started the next day after the last gavage. The mice were placed in the reaction box to adapt to the environment 3 minutes before the experiment. After that, the mice were placed at the bottom of the experimental box and the circuit was turned on, so that the mice were subjected to electrical stimulation and jumped onto the insulating platform. After one training, the mice were placed on the insulating platform, and the error latency (the time of the first jump off the platform) and the number of errors in jumping off the platform within 5 minutes were recorded. The number of errors and percentages of mice in each group were calculated, and this score was used as the test period score. After 24 hours, the test was repeated, and the experimental time was changed to 3 minutes to conduct and record the retest period experiment. After 7 days of stopping training, the extinction period experiment was conducted and recorded according to the retest period experimental method.

[0091] Table 3 Effects on the error latency of the mouse jumping test (x - ±s)

[0092] The error latency and number of errors of mice in the platform jumping test can reflect the effect of the sample on improving the memory of mice. As shown in Table 3, the error latency of the two experimental groups was significantly shortened compared with the model group. As shown in Table 4, the number of errors in the experimental group was significantly reduced compared with the model control group. As shown in Tables 5 and 6, the number of animals with errors was significantly reduced in the two experimental groups compared with the model group, and the percentage of animals with errors was correspondingly reduced. This shows that in the platform jumping test, oral administration of postbiotic products can significantly improve the memory of mice.

[0093] (2) Water maze experiment The training started on the second day after the last gavage. The training was conducted twice a day. Before each training, the mice were placed in a water maze for adaptation. During the training, the mice were tested by entering the water from two quadrants every day. The timing was started. The animals found the platform within 60 seconds at most for each experiment. If the animals did not successfully land on the platform within 60 seconds, the animals were allowed to rest on the platform for 30 seconds. If the platform was not found within 60 seconds, the score was recorded as 60 seconds. The mice were then placed on the platform to rest for 30 seconds. If the animals found the platform within the specified time, the timing was started according to the time they found the platform and rested for 30 seconds. If the mice fell into the water before the rest time of 30 seconds, they swam again, the timing was stopped, and the mice were placed on the platform to rest and the timing continued for 30 seconds. The purpose was to ensure that each mouse had the same time to collect spatial information after training. Train four times in this way, and start testing from the fifth time. Each animal is measured twice in the first and fourth quadrants. Calculate the number of animals and the total number of times that each group of animals climbed onto the rescue platform, the total time to reach the rescue platform, and the total number of animals that reached the end point within 60 seconds (percentage), including the results of the test period and the retest period. After stopping training for 7 days, the extinction experiment can be carried out from the starting point at different times.

[0094] Table 7 Effects on the test period of the mouse water maze experiment

[0095] According to Tables 7, 8 and 9, compared with the model group, the number of animals that climbed onto the life-saving platform and the number of times the animals climbed onto the life-saving platform during the test period, retest period and extinction period in the experimental group were significantly increased; the time the experimental group climbed onto the life-saving platform during the test period, retest period and extinction period was significantly shortened, indicating that postbiotic products can significantly improve the memory of mice.

[0096] (3) Dark avoidance experiment The training started the next day after the last sample was given. The experimental box is divided into two parts, a spacious and bright light room and a closed dark room, with a hole in the middle connecting the two parts. The experiment starts when the mouse is placed in the light room with its face facing away from the dark room. The error latency (the time required for the first entry into the dark room to receive an electric shock) and the number of errors in receiving electric shocks within 5 minutes of each group of mice are recorded. The number of animals with errors and the percentage of animals with errors in each group of mice are calculated, and this score is used as the test period score. After 24 hours, the retest period experiment is carried out and recorded. After 7 days of stopping training, the extinction period experiment is carried out and recorded according to the retest period experiment method.

[0097] Table 10 Effects on the latency of errors in the dark avoidance test in mice (x - ±s)

[0098] The dark avoidance experiment takes advantage of the nature of mice that they like dark environments. Bright lighting environments can make mice anxious and irritable, making them eager to enter dark and enclosed spaces. When they move to a dark room, they will be subjected to electric shocks. Therefore, the error latency and number of errors of mice in the dark avoidance experiment can reflect the effect of the test substance on improving the memory of mice. As shown in Table 10, compared with the model group, the error latency of the experimental group during the retest period tended to decrease. As shown in Tables 11 and 12, compared with the model group, the number of animals with errors during the retest period in the experimental group was significantly reduced, and the percentage of animals with errors was correspondingly reduced. As shown in Table 13, compared with the model group, the number of errors in the retest period in the experimental group was significantly reduced. Therefore, the number of errors in the dark avoidance experiment of mice gavaged with postbiotic products was significantly reduced, indicating that postbiotic products can significantly improve the memory of mice.

[0099] Example 4 - Regulating intestinal flora and promoting digestion 1. Experiment on regulating the function of intestinal flora in mice: The mice were divided into a control group and an experimental group, with 10 mice in each group. The control group was gavaged with 0.5 mL of normal saline while being fed normally, and the experimental group was gavaged with 0.5 mL of postbiotic product solution (10 mg of the postbiotic product prepared in Example 1 was dissolved in 0.5 mL of sterile water) while being fed normally, once a day. After 30 days of feeding, the mouse feces were aseptically taken, weighed, diluted 10 times with a sterile diluent to make a uniform suspension, and then diluted 10 times in increments, and the appropriate dilutions were selected and inoculated on each selective culture medium. The number of bacteria per gram of wet feces was calculated, and the logarithm was taken for statistical processing. 24 hours after the last administration of the postbiotic product, rectal feces were taken, and the intestinal flora was detected as shown in Table 14.

[0100] Table 14 Suitable culture conditions and detection of intestinal flora (x-±SD)

[0101] According to Table 14, postbiotic products can increase the number of beneficial bacteria (Bifidobacterium and Lactobacillus), while the number of harmful bacteria (Enterobacter and Enterococcus) did not increase but showed a slight downward trend, indicating that postbiotic products can regulate the function of the intestinal flora of mice.

[0102] 2. Mouse gastrointestinal motility experiment The mice were divided into a control group, a model group, and an experimental group, with 10 mice in each group. The control group and the model group were given 0.5 mL of sterile water by gavage once a day while being fed normally; the experimental group was given 0.5 mL of postbiotic product solution (10 mg of the postbiotic product prepared in Example 1 dissolved in 0.5 mL of sterile water) by gavage once a day while being fed normally. After 15 days of gavage, the mice in each group were fasted for 16 hours. Afterwards, the model group and the experimental group were gavaged with compound difenocolone, and the control group was gavaged with sterile water. Thirty minutes later, all groups of mice were gavaged with gum arabic ink. Thirty minutes later, the mice were killed by cervical dissection. The abdomen was quickly incised along the midline of the abdomen to separate the mesentery. The intestinal tube was cut with the pylorus as the starting point and the ileocecal part as the end point. The small intestine was gently pulled into a straight line, and the length of the intestinal tube was measured as the "total length of the small intestine". The length from the pylorus to the front of the ink was the "ink advancement length". The ink advancement rate was calculated according to the following formula. The ink advancement rate of the small intestine is shown in Table 15.

[0103]

[0104] Table 15 Small intestinal ink propulsion rate (x-±SD)

[0105] According to Table 15, the intestinal motility inhibition model of mice was established by administering compound difenocool. Compared with the model group, the ink propulsion rate of the small intestine in the experimental group increased, indicating that the postbiotic product prepared in Example 1 can promote digestion in the small intestine.

[0106] Example 5 - Improving epilepsy and reducing the number of epileptic seizures 1. Small-scale population experiment 10 people with epilepsy aged 5-40 years old volunteered to participate in the test and were able to complete the questionnaire and record. 0.1 g of the postbiotic product (i.e., the freeze-dried powder prepared in Example 1) was taken once a day before going to bed, and the original eating habits were not changed during the drinking period, and the normal diet was maintained. The test results are shown in Table 16.

[0107] Table 16 Effect of application in reducing the number of epileptic seizures

[0108] In this application trial, the recruited subjects are people with an average age of 60 years old in 8 communities in Jinan City. As of December 2022, a total of 100 people have been recruited to participate in the experiment. All of the above persons have signed a written informed consent form. If the participant is unable to sign, an agent (usually a family member) will sign it.

[0109] Table 17 Effect of improving memory application

[0110] Description: Middle-aged: 50 people started to take the medicine for 3 days and the results were counted. After taking it for 3 days, 14 people felt that their slow reaction, lack of concentration, memory loss, low mood, and depression improved. After taking it for 6 days, 23 people felt that their slow reaction, lack of concentration, memory loss, low mood, and depression improved significantly. After taking it for 8 days, 34 people felt that their slow reaction, lack of concentration, memory loss, low mood, and depression improved significantly. After taking it for 10 days, 48 ​​people felt that their slow reaction, lack of concentration, memory loss, low mood, and depression improved completely. A total of 2 people did not improve after taking Streptococcus thermophilus.

[0111] Elderly: 50 people started taking the medicine for 4 days and the results were counted. After 4 days, 12 people felt that their slow reaction, memory loss, depression, depression, insomnia and other conditions improved. After 8 days, 25 people felt that their slow reaction, memory loss, depression, depression, insomnia and other conditions improved significantly. After 12 days, 36 people felt that their slow reaction, memory loss, depression, depression, insomnia and other conditions improved extremely significantly. After 15 days, 46 people felt that their slow reaction, memory loss, depression, depression, insomnia and other conditions were completely improved. A total of 4 people did not improve after taking Streptococcus thermophilus.

[0112] 2. Validation experiment of animal model After several rats were fed normally for 3 days, rats in other groups except the control group were intraperitoneally injected with scopolamine methyl nitrate (1 mg / kg), and procarpine (330 mg / kg) was injected intramuscularly 20 minutes later to induce status epilepticus in rats. After successful induction, diazepam (5 mg / kg) was injected intramuscularly to terminate the seizure. The number of epileptic seizures in rats was then recorded within 30 days, and the model was successfully established if the seizure occurred more than 3 times.

[0113] 50 rats with successful modeling were selected and randomly divided into a control group (10 rats), a model group (10 rats), and an experimental group (30 rats). The control group and the model group were given 0.5 mL of normal saline by gavage while feeding normally, and the experimental group was given 0.5 mL of postbiotic product solution by gavage while feeding normally, once a day. Among them, the preparation method of the postbiotic product solution is: dissolve the postbiotic product prepared in Example 1 in 0.5 mL of sterilized water to obtain a postbiotic product solution. Among them, the added mass of the postbiotic product was 10 mg (experimental group 1), 15 mg (experimental group 2), and 20 mg (experimental group 3), respectively. After continuous gavage for 30 days, the number of epileptic seizures in rats was observed. After the experiment, 90 rats were killed and the brains were taken. The brain tissue was added with 4°C normal saline at a weight volume ratio of 1:9 to prepare a 10% homogenate. The contents of NO, MDA, SOD, and T-AOC in the homogenate of rat brain tissue were detected according to the steps and requirements of the kit instructions.

[0114] Table 18 Comparison of the number of epilepsy events in each group of rats

[0115] According to Table 18, the number of epilepsy episodes in rats after oral administration of the post-biotic product was significantly reduced in a dose-dependent manner.

[0116] according to Figure 2 , Figure 3 , Figure 4 It can be seen that compared with the control group, the NO and MDA contents in the serum of the rats in the model group were significantly increased, and the SOD and T-AOC contents were decreased. After the model group was gavaged with the postbiotic products (experimental groups 1, 2, and 3), the SOD and T-AOC activities in the serum increased, and the MDA and NO contents were significantly decreased, indicating that gavage with the postbiotic products can improve the body's ability to scavenge free radicals, reduce the degree of lipid peroxidation damage, protect brain tissue, reduce the damage of free radicals to local brain tissue, and clear the free radicals produced by rats after epilepsy, thereby playing a neuroprotective role, thereby reducing the attack of free radicals on cell membranes, maintaining the stability of cell membranes, and reducing the frequency of epileptic seizures.

[0117] The above-mentioned effect verification experiments show that the postbiotic product prepared by the present invention has the functions of improving memory and improving epilepsy, reducing the number of epilepsy, regulating the function of intestinal flora and promoting digestion.

[0118] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A thermophilic Streptococcus ( Streptococcus thermophilus )AK223-6, deposited in Guangdong Microbiological Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, the deposit date is July 8, 2024, and the deposit number is GDMCCNO: 64835.

2. A thermophilic Streptococcus according to claim 1 ( Streptococcus thermophilus ) A derivative of AK223-6, characterized in that The derivative is one or more of living bacteria, dead bacteria, fermentation broth or metabolites.

3. A microbial agent, characterized in that: Comprising an active ingredient, the active ingredient comprising the thermophilic Streptococcus ( Streptococcus thermophilus ) AK223-6 or its inactivated bacteria, fermentation broth, or metabolites.

4. The microbial agent according to claim 3, characterized in that The microbial agent comprises inactivated Streptococcus thermophilus ( Streptococcus thermophilus )AK223-6 and its fermentation broth.

5. The method for preparing the microbial agent according to claim 3 or 4, characterized in that: The preparation method comprises: S1. Bacteria activation: The thermophilic Streptococcus ( Streptococcus thermophilus ) AK223-6 was inoculated into the activation medium and cultured at 35-40°C for 20-25h to obtain the expanded bacterial culture liquid; S2. Preparation of primary bacterial seed solution: inoculate the expanded bacterial seed solution prepared in step S1 into the primary seed culture solution at an inoculation amount of 0.5-5%, and culture at 35-40° C. for 10-15 hours to obtain the primary bacterial seed solution; S3, seed tank strain preparation: the primary strain liquid prepared in step S2 is inoculated into a seed tank containing a seed tank culture medium under sterile conditions, the inoculation amount is 0.5-5%, the stirring culture is 50-100r / min, the pH is natural, the temperature is 30-40°C, the tank pressure is 0.03-0.06MPa, and the culture time is 15-20h to obtain the seed tank strain; S4, thermophilic streptococcus fermentation: inoculate the seed tank strain into a fermentation tank containing sterilized fermentation culture medium, with an inoculation amount of 0.5-7%, 35-39°C, 140-170r / min, tank pressure 0.04-0.06MPa, pH 6.3-6.7, and ferment for 24-26h; cool to 19-21°C, which is the fermentation liquid; heat the fermentation liquid to 119-121°C and maintain for 4-7s for UHT inactivation treatment.

6. The preparation method according to claim 5, characterized in that: After step S4 is completed, the obtained fermentation liquid is centrifuged to collect the inactivated wet bacterial sludge metabolites, and the inactivated wet bacterial sludge metabolites are freeze-dried to obtain freeze-dried powder of the inactivated bacterial metabolites; In step S1, the activation medium is composed of the following, by weight: 16-23 parts of oligofructose, 4-8 parts of chitosan, 4-8 parts of beef extract powder, 0.5-1.5 parts of sodium acetate, 0.2-1 parts of Tween-80, 0.5-1.5 parts of potassium dihydrogen phosphate, 800-1200 parts of water, and the pH is adjusted to 7.0; In step S2, the composition of the primary seed culture solution is as follows, by weight: 6-12 parts of oligofructose, 4-8 parts of peptone, 2-6 parts of beef extract powder, 1-4 parts of albumin peptone, 0.5-2 parts of Tween-80, 0.5-2 parts of sodium acetate, 800-1200 parts of tap water, and natural pH value; In step S3, the composition of the seed tank culture medium is as follows, in weight percentage: fructooligosaccharide 2-6%, casein peptone 0.5-2%, dipotassium hydrogen phosphate 0.1-0.6%, albumin peptide 1-2%, sodium acetate 0.5-1%, Tween-80 0.05-0.3%, polyether defoamer 0.05-0.3%, tap water 90-95%, pH value natural; In step S4, the fermentation culture medium is composed of the following, in percentage by weight: 2-6% fructooligosaccharide, 0.5-2% casein peptone, 1-3% xylo-oligosaccharide, 2-6% yeast extract, 1-3% sodium acetate, 0.1-0.4% Tween-80, 1-4% spirulina oligopeptide, 0.5-2% dipotassium hydrogen phosphate, 0.05-0.2% polyether defoamer, and 80-90% tap water.

7. A thermophilic Streptococcus according to claim 1 ( Streptococcus thermophilus ) AK223-6, or the thermophilic Streptococcus according to claim 2 ( Streptococcus thermophilus ) Use of a derivative of AK223-6 or a microbial agent as described in claim 3 or 4 in preparing food.

8. A thermophilic Streptococcus according to claim 1 ( Streptococcus thermophilus ) AK223-6, or the thermophilic Streptococcus according to claim 2 ( Streptococcus thermophilus ) Use of a derivative of AK223-6 or a microbial agent according to claim 3 or 4 in the preparation of any of the following products: (1) Products used to regulate intestinal flora and promote digestion; (2) Products used to improve memory; (3) Products used to improve epilepsy or reduce the frequency of epileptic seizures; (4) Products used to produce lactic acid bacteria polysaccharides, ergosterol, cephalin, vitamin A, and vitamin B1; The product is one of a food and a medicine; The food is any one of beverages, dairy products, candy products, health foods, special dietary foods and special medical formula foods.

9. A product comprising the thermophilic Streptococcus ( Streptococcusthermophilus ) AK223-6, and / or the thermophilic Streptococcus according to claim 2 ( Streptococcusthermophilus ) A derivative of AK223-6, and / or the microbial agent according to claim 3 or 4, and an acceptable auxiliary agent or adjuvant; The product is one of a food and a medicine; The food is any one of beverages, dairy products, candy products, health foods, special dietary foods and special medical formula foods.

10. The product according to claim 9, characterized in that The products include postbiotic products.

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