CN119286730A - Streptococcus thermophilus AK223-6 and its application in preparing products for regulating intestines, improving memory and improving epilepsy - Google Patents

Abstract

The invention discloses streptococcus thermophilus AK223-6 and application thereof in preparing products for regulating intestinal tracts, improving memory and improving epilepsy, and belongs to the fields of microorganisms and biological medicines. The invention provides streptococcus thermophilus (Streptococcus thermophilus) AK223-6 which is preserved in the microorganism strain collection center of Guangdong province, the preservation date is 2024, 07 and 08, and the preservation number is GDMCCNO:64835. Based on the streptococcus thermophilus, a metaplasia product is successfully developed, and the streptococcus thermophilus has the effects of regulating the balance of intestinal flora, promoting digestion and absorption of human bodies, improving memory, improving epilepsy and reducing epileptic times. The daily administration of 100mg of metagen product containing 160 hundred million strains and metabolites of the invention can sustainably provide the necessary endogenous quantity of lactic acid bacteria polysaccharide, ergosterol, cephalin, vitamin A and vitamin B1 in the intestinal canal reproduction and growth process, can effectively improve the memory, improve the epilepsy and reduce the epilepsy frequency, and has good practical application value.

Description

Streptococcus thermophilus AK223-6 and application thereof in preparing products for regulating intestinal tract, improving memory and improving epilepsy

Technical Field

The invention belongs to the fields of microorganisms and biological medicines, and particularly relates to streptococcus thermophilus AK223-6 and application thereof in preparation of products for regulating intestinal tracts, improving memory and improving epilepsy.

Background

The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.

Epilepsy is one of the most common neurological disorders affecting around 5000 tens of thousands worldwide. Despite the different kinds of anti-epileptic medication, more than 30% of epileptic patients continue to develop seizures. This is known as drug-resistant epilepsy. For these drug resistant epileptic patients, a number of supplemental therapies have been proposed, including surgery, neurostimulator implantation, and ketogenic diet, but these therapies have limited effectiveness.

In addition, hypomnesis is called amnesia in traditional Chinese medicine, and has a series of manifestations such as mental weakness, amnesia when meeting everything, insomnia and dreaminess. Hypomnesis can be caused by a variety of factors, including normal physiological aging processes, lifestyle factors, health problems, and other some external factors.

The study shows that the long-term taking of the probiotics can effectively improve the memory of a host, mainly can improve the sleep quality, relieve the stress and anxiety, improve the health condition and the like, and the study also shows that the taking of the probiotics has positive effects on improving the epileptic symptoms by regulating the intestinal flora balance. Therefore, searching for a probiotic agent capable of effectively improving memory and/or improving epilepsy can provide a new strategy for improving memory and/or improving epileptic prevention and treatment.

Disclosure of Invention

In order to solve the defects in the prior art, the invention aims to provide streptococcus thermophilus AK223-6 and application thereof in preparing products for regulating intestinal tracts, improving memory and improving epilepsy. The streptococcus thermophilus (Streptococcus thermophilus) AK223-6 provided by the invention has the effects of regulating the balance of intestinal flora, promoting digestion and absorption of human bodies, improving memory and improving epilepsy.

In order to achieve the above purpose, the technical scheme of the invention is as follows:

In a first aspect, the invention provides streptococcus thermophilus (Streptococcus thermophilus) AK223-6 deposited at the Guangdong province microorganism strain collection center under the address of building 5, no. 59 of Dai 100 in Guangzhou martyr, with the date of deposition being 2024, 07, 08 and the deposit number being GDMCCNO:64835.

In a second aspect, the present invention provides a derivative of Streptococcus thermophilus (Streptococcus thermophilus) AK223-6 as defined above, the derivative being one or more of a live bacterium, a dead bacterium, a fermentation broth or a metabolite.

In a third aspect, the invention provides a microbial agent comprising an active ingredient, wherein the active ingredient comprises streptococcus thermophilus (Streptococcus thermophilus) AK223-6 or an inactivated thallus, fermentation liquor and metabolite thereof.

In one or more embodiments, the microbial agent comprises inactivated streptococcus thermophilus (Streptococcus thermophilus) AK223-6 and fermentation broths thereof.

In a fourth aspect, the present invention provides a method for preparing the microbial agent, which comprises:

S1, strain activation:

Inoculating the streptococcus thermophilus (Streptococcus thermophilus) AK223-6 into an activation culture medium, and culturing for 20-25h at 35-40 ℃ to obtain an expanded strain liquid;

s2, preparing a first-stage strain liquid, namely inoculating the expanded strain liquid prepared in the step S1 into a first-stage seed culture liquid, wherein the inoculum size is 0.5-5%, and culturing for 10-15h at 35-40 ℃ to obtain the first-stage strain liquid;

S3, preparing seed tank strains, namely inoculating the first-level strain liquid prepared in the step S2 into a seed tank containing a seed tank culture medium under the aseptic condition, wherein the inoculum size is 0.5-5%, the stirring culture is carried out at 50-100r/min, the pH is natural, the temperature is 30-40 ℃, the tank pressure is 0.03-0.06MPa, and the culture time is 15-20 hours, so as to obtain seed tank strains;

S4, streptococcus thermophilus fermentation, namely inoculating the seed tank strain into a fermentation tank containing sterilized fermentation culture solution, wherein the inoculation amount is 0.5-7%, the temperature of the fermentation tank is 35-39 ℃, the pressure of the fermentation tank is 140-170r/min, the pressure of the fermentation tank is 0.04-0.06MPa, the pH value of the fermentation tank is 6.3-6.7, the fermentation tank is fermented for 24-26 hours, the temperature of the fermentation tank is reduced to 19-21 ℃, the fermentation tank is heated to 119-121 ℃, and the fermentation tank is kept for 4-7S, and UHT inactivation treatment is carried out.

In one or more embodiments, in the step S1, the composition of the activation medium is as follows, in parts by weight, 16-23 parts of fructooligosaccharide, 4-8 parts of chitosan, 4-8 parts of beef extract, 0.5-1.5 parts of sodium acetate, 0.2-1 part of tween-80, 0.5-1.5 parts of potassium dihydrogen phosphate, 800-1200 parts of water, and adjusting the pH to 7.0;

In one or more embodiments, in the step S2, the primary seed culture solution comprises, by weight, 6-12 parts of fructo-oligosaccharides, 4-8 parts of peptone, 2-6 parts of beef extract, 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 the step S3, the composition of the seed tank culture medium comprises, by weight, 2-6% of fructo-oligosaccharides, 0.5-2% of casein peptone, 0.1-0.6% of dipotassium hydrogen phosphate, 1-2% of albumin peptide, 0.5-1% of sodium acetate, 0.05-0.3% of tween-80, 0.05-0.3% of polyether defoamer, 90-95% of tap water and natural pH value;

in one or more embodiments, in the step S4, the fermentation culture solution comprises, by weight, 2-6% of fructo-oligosaccharide, 0.5-2% of casein peptone, 1-3% of xylo-oligosaccharide, 2-6% of yeast extract powder, 1-3% of sodium acetate, 0.1-0.4% of tween-80, 1-4% of spirulina oligopeptide, 0.5-2% of dipotassium hydrogen phosphate, 0.05-0.2% of polyether defoamer and 80-90% of tap water;

in one or more embodiments, after step S4 is completed, the obtained fermentation broth is centrifuged, the inactivated wet bacterial sludge metabolite is collected, and the inactivated wet bacterial sludge metabolite is freeze-dried to obtain an inactivated bacterial metabolite freeze-dried powder.

In a fifth aspect, the invention provides the use of said streptococcus thermophilus (Streptococcus thermophilus) AK223-6, or a derivative or microbial agent of streptococcus thermophilus (Streptococcus thermophilus) AK223-6, in the preparation of a food product.

In a sixth aspect, the invention provides the use of said streptococcus thermophilus (Streptococcus thermophilus) AK223-6, or a derivative or microbial agent of streptococcus thermophilus (Streptococcus thermophilus) AK223-6, in the preparation of any one of the following products:

(1) A product for regulating intestinal flora and promoting digestion;

(2) A product for improving memory;

(3) Products for improving epilepsy and reducing seizure frequency;

(4) The method is used for producing lactobacillus polysaccharide, ergosterol, cephalin, vitamin A and vitamin B1.

The product is one of food and medicine;

The food is any one of beverage, dairy product, candy product, health food, special diet food and special medical purpose formula food.

In a sixth aspect, the present invention provides a product comprising the above Streptococcus thermophilus (Streptococcus thermophilus) AK223-6, and/or a derivative of the above Streptococcus thermophilus (Streptococcus thermophilus) AK223-6, and/or the above microbial agent, and an acceptable adjuvant or adjuvant.

In some examples of this embodiment, the product is one of a food product and a pharmaceutical product;

In some examples of this embodiment, the food product is any one of a beverage, a dairy product, a confectionery product, a health food product, a special meal food product, and a special medical use formula food product.

In some examples of this embodiment, the product comprises a metaproduct.

In an eighth aspect of the metazoan product, the invention provides a method for regulating intestinal flora, promoting digestion or improving memory or improving epilepsy, reducing seizure count or for producing lactobacillus polysaccharides, ergosterol, cephalin, vitamin a, vitamin B1, using the derivatives or microbial agents of streptococcus thermophilus (Streptococcus thermophilus) AK223-6 or streptococcus thermophilus (Streptococcus thermophilus) AK 223-6.

The beneficial effects of the invention are as follows:

The streptococcus thermophilus (Streptococcus thermophilus) AK223-6 is obtained by screening according to the technical scheme, and a metazoan product is successfully developed based on the streptococcus thermophilus, and the product has the effects of regulating intestinal flora balance, promoting human digestion and absorption, improving memory, improving epilepsy and reducing epileptic seizure frequency. The daily administration of 100mg of the metagen product containing 160 hundred million strains and metabolites of the invention can sustainably provide the necessary endogenous quantity of lactic acid bacteria polysaccharide, ergosterol, cephalin, vitamin A and vitamin B1 in the intestinal canal reproduction and growth process, can effectively improve the memory and improve the epilepsy, and has good practical application value.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.

FIG. 1 is a chart showing the analysis of the results of the metabolites of the metaproduct of example 2 of the present invention;

FIG. 2 is a graph showing the NO content data in the serum of different groups of mice in example 5 of the present invention;

FIG. 3 is a graph showing SOD content data in serum of different groups of mice in example 5 of the present invention;

FIG. 4 is a graph showing MDA and T-AOC content data in serum of different groups of mice in example 5 of the present invention.

Detailed Description

In a first exemplary embodiment of the present invention, there is provided Streptococcus thermophilus (Streptococcus thermophilus) AK223-6 deposited at the Cantonese microorganism strain collection at 2024, month 07 and 08 under accession number GDMCCNO:64835.

The strain is separated from fresh grifola frondosa fruit bodies in the Guangxi Zhuang nationality and Nanning market, and is identified as streptococcus thermophilus by a 16srRNA method. The sequence of the bacterium is as follows:

GGCTCCAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGATTGGCTTTAAGAGATTAGCTCGCCGTCACCGACTCGCAACTCGTTGTACCAACCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTATTACCGGCAGTCTCGCTAGAGTGCCCAACTGAATGATGGCAACTAACAATAGGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACCGATGTACCGAAGTAACTTTCTATCTCTAGAAATAGCATCGGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTTCGGCACTGAATCCCGGAAAGGATCCAACACCTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGAGAGCCGCTTTCGCCACCGGTGTTCCTCCATATATCTACGCATTTCACCGCTACACATGGAATTCCACTCTCCCCTTCTGCACTCAAGTTTGACAGTTTCCAAAGCGAACTATGGTTGAGCCACAGCCTTTAACTTCAGACTTATCAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTCGGGACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTCCCTTTCTGGTAAGCTACCGTCACAGTGTGAACTTTCCACTCTCACACCCGTTCTTGACTTACAACAGAGCTTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCGGTCAGGGTTGCCCCCATTGCCGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTATGTATCGTCGCCTAGGTGAGCCATTACCTCACCTACTAGCTAATACAACGCAGGTCCATCTTGTAGTGGAGCAATTGCCCCTTTCAAATAAATGACATGTGTCATCCATTGTTATGCGGTATTAGCTATCGTTTCCAATAGTTATCCCCCGCTACAAGGCAGGTTACCTACGCGTTACTCACCCGTTCGCAACTCATCCAAGAAGAGCAAGCTCCTCTCTTCAGCG

The strain has been subjected to patent strain preservation in the Guangdong province microorganism strain preservation center, the preservation number is GDMCCNO:64835, and the streptococcus thermophilus strain is a probiotic strain approved for use by the national Committee for Wei Jian. The strain has effects of regulating intestinal flora balance, promoting digestion and absorption, improving memory, and improving epilepsy. 100mg of metazoan product containing 160 hundred million strains and metabolites of the invention can sustainably provide the necessary amount of lactic acid bacteria polysaccharide, ergosterol, cephalin, vitamin A and vitamin B1 for endogenous growth in the intestinal tract reproduction and growth process, can improve memory and maintain a healthy nervous system.

In a second exemplary embodiment of the present invention, there is provided a derivative of Streptococcus thermophilus (Streptococcus thermophilus) AK223-6 as described above, which is one or more of a live bacterium, a dead bacterium, a fermentation broth or a metabolite.

In a third exemplary embodiment of the present invention, a microbial agent is provided, which comprises an active ingredient, wherein the active ingredient comprises streptococcus thermophilus (Streptococcus thermophilus) AK223-6 or an inactivated thallus, a fermentation broth or a metabolite thereof. The metabolites include extracellular and/or intracellular metabolites of the bacterial cell.

In some examples of this embodiment, the microbial agent comprises inactivated streptococcus thermophilus and fermentation broths thereof. At this time, the microbial agent is a metaproduct.

According to a fourth exemplary embodiment of the present invention, there is provided a method for preparing the microbial agent, the method comprising:

S1, strain activation:

Inoculating the streptococcus thermophilus (Streptococcus thermophilus) AK223-6 into an activation culture medium, and culturing at 35-40 ℃ and preferably 37 ℃ for 20-25 hours (preferably 24 hours) to obtain the expanded strain liquid.

The activating culture medium comprises, by weight, 16-23 parts of fructo-oligosaccharide, 4-8 parts of chitosan, 4-8 parts of beef extract powder, 0.5-1.5 parts of sodium acetate, 0.2-1 part of tween-80, 0.5-1.5 parts of potassium dihydrogen phosphate, 800-1200 parts of water and pH of the medium is adjusted to 7.0 (alkali liquor adjustment).

More specifically, the composition of the activation medium is as follows, in parts by weight, 20 parts of fructo-oligosaccharide, 6 parts of chitosan, 6 parts of beef extract powder, 0.9 part of sodium acetate, 0.5 part of tween-80, 0.9 part of potassium dihydrogen phosphate, 1000 parts of water, and the pH is adjusted to 7.0 (alkali liquor adjustment).

S2, preparing a primary strain liquid, namely inoculating the expanded strain liquid prepared in the step S1 into a primary seed culture liquid, wherein the inoculation amount is 0.5-5%, and culturing for 10-15 hours (preferably 12 hours) at 35-40 ℃ which is preferably 37 ℃, so as to obtain the primary strain liquid.

The primary seed culture solution comprises, by weight, 6-12 parts of fructo-oligosaccharide, 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.

More specifically, the primary seed culture solution comprises the following components in parts by weight:

8 parts of fructo-oligosaccharide, 6 parts of peptone, 4 parts of beef extract powder, 2 parts of albumin peptone, 0.8 part of sodium acetate, 1 part of tween-80, 1000 parts of tap water and natural pH value.

S3, preparing seed tank strains, namely inoculating the first-stage strain liquid prepared in the step S2 into a seed tank containing a seed tank culture medium under the aseptic condition, wherein the inoculation amount is 0.5-5% (preferably 5% by mass), the stirring culture is carried out at 50-100r/min (preferably 80 r/min), the pH is natural, the temperature is 30-40 ℃ (preferably 37 ℃), the tank pressure is 0.03-0.06MPa (preferably 0.05 MPa), and the culture time is 15-20h (preferably 18 h), so as to obtain the seed tank strains.

Wherein the seed tank culture medium comprises, by weight, 2-6% of fructo-oligosaccharide, 0.5-2% of casein peptone, 0.1-0.6% of dipotassium hydrogen phosphate, 1-2% of albumin peptide, 0.5-1% of sodium acetate, 0.05-0.3% of Tween-80, 0.05-0.3% of polyether defoamer, 90-95% of tap water and natural pH value.

More specifically, the seed tank culture medium comprises, by weight, 4% of fructo-oligosaccharide, 0.9% of casein peptone, 0.3% of dipotassium hydrogen phosphate, 1.5% of albumin peptide, 0.7% of sodium acetate, 0.1% of tween-80, 0.1% of polyether defoamer, 92.4% of tap water and natural pH value.

S4, lactobacillus plantarum fermentation, wherein the seed tank strain is inoculated into a fermentation tank containing sterilized fermentation culture solution, the inoculation amount is 0.5-7% (preferably 5-7%), 35-39 ℃ (preferably 37 ℃), 140-170r/min (preferably 160 r/min), the tank pressure is 0.04-0.06MPa (preferably 0.05 MPa), the pH is 6.3-6.7 (preferably 6.5), the fermentation time is 24-26h (preferably 25 h), and the temperature is reduced to 19-21 ℃ (preferably 20 ℃), so that the lactobacillus plantarum fermentation is the fermentation solution. The fermentation broth is heated to 119-121C (preferably 120C) for 4-7s (preferably 6 s) for UHT inactivation treatment.

Wherein the fermentation culture solution comprises, by weight, 2-6% of fructo-oligosaccharide, 0.5-2% of casein peptone, 1-3% of xylo-oligosaccharide, 2-6% of yeast extract powder, 1-3% of sodium acetate, 0.1-0.4% of tween-80, 1-4% of spirulina oligopeptide, 0.5-2% of dipotassium hydrogen phosphate, 0.05-0.2% of polyether defoamer and 80-90% of tap water.

More specifically, the fermentation culture solution comprises, by weight, 4% of fructo-oligosaccharide, 1% of casein peptone, 1.5% of xylo-oligosaccharide, 4% of yeast extract powder, 1.5% of sodium acetate, 0.2% of tween-80, 2% of spirulina oligopeptide, 1% of dipotassium hydrogen phosphate, 0.1% of polyether defoamer and 84.7% of tap water.

In some examples of this embodiment, after step S4 is completed, the resulting fermentation broth is centrifuged to collect the inactivated wet bacterial sludge metabolite, which is freeze-dried (preferably 4% of the final control moisture content) to obtain an inactivated bacterial metabolite freeze-dried powder.

In a fifth exemplary embodiment of the present invention, there is provided the use of said streptococcus thermophilus (Streptococcus thermophilus) AK223-6, or a derivative or microbial agent of streptococcus thermophilus (Streptococcus thermophilus) AK223-6, in the preparation of a food product.

In a sixth exemplary embodiment of the present invention, there is provided the use of said streptococcus thermophilus (Streptococcus thermophilus) AK223-6, or a derivative or microbial agent of streptococcus thermophilus (Streptococcus thermophilus) AK223-6, for the preparation of any one of the following products:

(1) A product for regulating intestinal flora and promoting digestion;

(2) A product for improving memory;

(3) Products for improving epilepsy and reducing seizure frequency;

(4) The method is used for producing lactobacillus polysaccharide, ergosterol, cephalin, vitamin A and vitamin B1.

In some examples of this embodiment, the product is one of a food product and a pharmaceutical product;

The food is any one of beverage, dairy product, candy product, health food, special diet food and special medical purpose formula food.

The lactobacillus polysaccharide, ergosterol, cephalin, vitamin A and vitamin B1 have the following functions respectively:

lactic acid bacteria polysaccharide can improve gastrointestinal health function of organism, regulate intestinal flora composition, remodel intestinal flora ecology, and finally produce antiepileptic effect. Memory is improved by preventing oxidative stress and inflammation.

Ergosterol plays a role in brain protection by promoting the growth and differentiation of neurons, resisting oxidative stress, resisting inflammatory reaction and the like;

The cephalin has the effects of promoting cerebral blood circulation, improving cerebral blood supply insufficiency, relieving secondary epileptic seizure caused by ischemic and anoxic encephalopathy, promoting development of cerebral nerve, and improving activation degree of brain cells, thereby playing a role in enhancing memory.

Vitamin A participates in the formation and connection of neurons, can promote the synthesis and release of neurotransmitters, and enhances the efficiency of neurotransmission, thereby improving the cognitive function of the brain.

Vitamin B1 can assist the generation of acetyl coenzyme A and acetylcholine, and has certain auxiliary effect on the memory of the brain.

In a seventh exemplary embodiment of the present invention, a product is provided comprising said streptococcus thermophilus (Streptococcus thermophilus) AK223-6, and/or a derivative of streptococcus thermophilus (Streptococcus thermophilus) AK223-6, and/or a microbial agent, and an acceptable adjuvant or adjuvant.

In some examples of this embodiment, the product is one of a food product and a pharmaceutical product;

The food is any one of beverage, dairy product, candy product, health food, special diet food and special medical purpose formula food.

In some examples of this embodiment, the product comprises a metaproduct.

In some examples of this embodiment, the auxiliary or adjuvant may be a pharmaceutically usual carrier, excipient, diluent, or the like. Further, according to a general method, it is possible to prepare an oral preparation such as powder, granule, suspension, emulsion, syrup, and spray.

The non-pharmaceutically active ingredients, such as carriers, excipients and diluents, which may be included, are well known in the art and can be determined by one of ordinary skill in the art to meet clinical criteria. In yet another embodiment of the present invention, the carriers, excipients and diluents include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil and the like.

The medicine can be prepared in the form of injection preparation or oral preparation. The injection preparation comprises liquid injection, powder for injection and tablet for injection according to physical state classification, intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection and spinal cavity injection according to injection part classification, and the solvent of the injection preparation preferably comprises water for injection or physiological saline.

Formulations for oral use include tablets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate), granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid), binders (e.g., sucrose, dextrose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, polyvinylpyrrolidone, or polyethylene glycol), and lubricants, glidants, and anti-adherents (e.g., magnesium stearate, zinc stearate, stearic acid, silicon dioxide, hydrogenated vegetable oils, or talc). Formulations for oral use may also be presented as chewable tablets, or as 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 as 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 can be prepared in a conventional manner using the ingredients mentioned above under tablets or capsules using, for example, mixers, fluidized bed equipment or spray drying equipment.

Other pharmaceutically acceptable excipients for oral formulations include, but are not limited to, coloring agents, flavoring agents, plasticizers, humectants, and buffers. Formulations for oral use may also be presented as chewable tablets, or as 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 as 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 can be prepared in a conventional manner using the ingredients mentioned above under tablets or capsules using, for example, mixers, fluidized bed equipment or spray drying equipment.

In an eighth exemplary embodiment of the present invention, the present invention provides a method for regulating intestinal flora, promoting digestion or improving memory or improving epilepsy, reducing seizure count or for producing lactobacillus polysaccharides, ergosterol, cephalin, vitamin a, vitamin B1, using the above-mentioned streptococcus thermophilus (Streptococcus thermophilus) AK223-6, or derivatives or microbial agents of streptococcus thermophilus (Streptococcus thermophilus) AK 223-6.

In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.

In the examples, "%" of the composition in each medium (liquid) is mass volume percentage, and the unit is g/mL. In the examples, tween-80 had a density of 1.08g/cm ³.

Example 1

A method for preparing a metaproduct, comprising the steps of:

The equipment used in the production comprises a strain incubator, an ultra-clean bench, a constant temperature incubator, various test tubes, triangular flasks, a seed tank, a fermentation tank, a vacuum freeze dryer, a three-dimensional stirrer, a low-temperature pulverizer and a vacuum packaging machine.

(1) Strain activation

Culturing strain, and expanding culture of preserved strain in refrigerator at-80 deg.C.

The culture medium is prepared from fructo-oligosaccharide 20g, chitosan 6g, beef extract powder 6g, sodium acetate 0.9g, tween-80 0.5mL, potassium dihydrogen phosphate 0.9g, tap water 1000mL, and pH value adjusted to 7.0 (alkali liquor adjustment).

The preparation method comprises the steps of adding the raw materials such as fructo-oligosaccharide and the like in the formula into 1000mL of water before and after the raw materials are not separated, stirring for 10min to dissolve the raw materials, then subpackaging the raw materials into test tubes, filling 5mL of each test tube with a rubber plug kraft paper, sealing, sterilizing for 20min under 0.1-0.12MPa in a medical sterilizer, and finishing the preparation of the culture medium.

Inoculating, namely opening a freezing tube for preserving bacteria in a low-temperature refrigerator at-80 ℃ under the aseptic condition (super clean workbench), dissolving at room temperature, taking 1 branch of the sterilized test tube culture medium, adding 0.5mL of the dissolved bacteria liquid into a test tube, sealing a tube orifice by using rubber plug kraft paper, shaking the tube slightly and uniformly by hands, and placing the tube in a constant-temperature incubator at 37 ℃ for standing culture for 24 hours, thus obtaining the expanded culture liquid.

(2) Preparing a first-stage strain liquid:

the primary seed culture formula comprises 8g of fructo-oligosaccharide, 6g of peptone, 4g of beef extract powder, 2g of albumin peptone, 0.8g of sodium acetate, 1000mL of tween-80, 1000mL of tap water and natural pH value.

The operation is that the raw materials are added into 1000mL tap water before and after the raw materials are not separated, a stirrer is started, and the raw materials are stirred for 10min to dissolve all the raw materials, and the raw materials are called primary seed culture solution.

Adding the stirred and melted primary seed culture solution into a triangular flask according to the amount, wrapping a bottle mouth by using 6 layers of gauze and adding a layer of kraft paper, placing the bottle mouth into a sterilizer for sterilization, opening an exhaust valve on the sterilizer and the like at first, closing the valve when a small amount of steam is discharged, opening the exhaust valve on the sterilizer for 6-8min when the pressure on the sterilizer reaches 0.05MPa, closing the exhaust valve, continuing to heat, starting timing when the steam pressure in the sterilizer reaches 0.1-0.12MPa, sterilizing for 20min, leaving a heat source after the completion, naturally cooling the bottle mouth, opening the sterilizer when a pressure meter on the sterilizer returns to zero, taking out a sterilized culture medium, placing the sterilized culture medium into an ultra-clean workbench for natural cooling, transferring the cultured expanded strain solution into the sterilized culture solution of the triangular flask when the temperature is reduced to 37 ℃, and placing the cultured expanded strain solution into the sterilized culture solution of the triangular flask, wherein the inoculation amount is 0.5%, namely 100mL of the primary seed culture solution, and placing the expanded strain solution into 0.5mL, sealing the bottle mouth by using the original gauze and kraft paper after the completion, sealing the bottle mouth when the steam pressure in the sterilizer reaches 0.1-0.12MPa, and slightly shaking the culture solution, and uniformly mixing the strain with the culture solution at the temperature for the culture solution under the condition of 37 ℃ after the first stage is called as the culture solution.

(3) Preparing seed tank strains:

Sterilization of fermentation equipment, tubing, and sterile filtration systems:

Firstly, opening valves of each inlet and outlet pipeline and a sterile air pipeline, introducing steam of 0.12-0.14MPa, enabling the steam to be communicated with the pipeline valves and a small amount of steam to be discharged, ventilating for 40min, and then closing the valves of each inlet and outlet pipeline for later use.

Empty pot sterilization of seed pot and fermentation pot:

Closing each valve, opening the drain valve at the bottom of the tank and the drain valve in the interlayer of the tank, opening the direct steam valve to introduce steam of 0.12-0.14MPa into the pipe, starting timing when the temperature in the tank reaches 121 ℃, sterilizing for 40min, and then closing the drain valve at the bottom of the tank and the drain valve in the interlayer, wherein the temperature in the tank naturally drops to 37 ℃ for standby.

Seed pot culture:

The formula comprises 4% of fructo-oligosaccharide, 0.9% of casein peptone, 0.3% of dipotassium hydrogen phosphate, 1.5% of albumin peptide, 0.7% of sodium acetate, 0.1% of tween-80, 0.1% of polyether defoamer, 92.4% of tap water and natural pH value.

The specific operation is that firstly, tap water used in the formula is put into a seed tank sterilized by an empty tank, a stirrer is started, raw materials required in the formula are respectively added (the adding sequence is not divided into the front and the rear), then, the raw materials are continuously stirred and are introduced with steam for heating, firstly, when the temperature in the tank is up to 95 ℃ by utilizing the steam in the interlayer of the tank, the direct steam is used for heating, the sterilization effect can be achieved after the temperature in the tank reaches 121 ℃ for 20min, then, the steam is closed, the tap water passing through the interlayer of the tank is cooled down, and when the temperature in the tank reaches 37 ℃ for standby, the seed culture medium is obtained. Inoculating the cultured primary strain culture solution into a seed tank under aseptic conditions, wherein the inoculum size is 5% of that of the culture medium in the tank, respectively, and 100 kg of the culture medium in the seed tank is respectively inoculated with 5 kg of the primary strain solution, then, covering an inoculating cap on the tank to start stirring culture, wherein the culture conditions are that the temperature is 37 ℃, the pH value is natural, the stirring rotation speed is 80r/min, the tank pressure is 0.05MPa, the culture time is 18h, and if the tank body is reduced, sterile air can be introduced to keep the tank pressure, and the tank pressure is too high and can be adjusted by a deflation valve at the top of the tank, thus the seed tank strain is obtained.

(4) Streptococcus thermophilus fermentation

The formula comprises 4% of fructo-oligosaccharide, 1% of casein peptone, 1.5% of xylo-oligosaccharide, 4% of yeast extract powder, 1.5% of sodium acetate, 0.2% of tween-80, 2% of spirulina oligopeptide, 1% of dipotassium hydrogen phosphate, 0.1% of polyether defoamer and 84.7% of tap water.

The specific operation is that firstly, tap water used in the formula is put into a fermentation tank sterilized by an empty tank, a stirrer is started, raw materials used in the formula are put into the fermentation tank (the adding sequence is not divided into the front and the back), and then steam is introduced for heating. Firstly, heating to 95 ℃ by using interlayer steam, then, heating by using direct steam, starting timing when the temperature in the tank reaches 121 ℃, keeping for 30min to achieve a sterilization effect, then cooling by using interlayer tap water, starting inoculating strains when the temperature in the tank is reduced to 37 ℃, firstly, increasing the tank pressure of a seed tank to 0.1MPa before inoculation, keeping the tank pressure of a fermentation tank to 0.05MPa, then opening an inoculation pipeline valve, conveying the seeds cultured in the seed tank into the fermentation tank in a pressure difference mode, and then closing an inoculation channel valve.

The inoculation amount is 5kg of strains cultured in a seed tank after inoculating each 100 kg of culture medium in the fermentation tank, wherein the culture condition is that the culture temperature is 37 ℃, the stirring speed is 160r/min, the tank pressure is kept at 0.05MPa, the pH value is kept at 6.5 through an automatic acid and alkali regulating and controlling system in the fermentation process, if the tank pressure is reduced, sterile air can be introduced to keep the tank pressure until the fermentation is completed, the total fermentation is carried out for 25 hours, and then the temperature is reduced to 20 ℃, so that the fermentation liquid is obtained. UHT inactivation treatment is carried out under 120 ℃ for 6 s.

(5) Centrifuging the fermentation broth

Pumping the fermentation liquor into a storage tank for centrifugation, starting the centrifuge, adjusting the rotation speed to 10000r/min, centrifuging at a feeding speed of 2kg/min, collecting the inactivated wet bacterial sludge metabolite after centrifugation, and freeze-drying the collected inactivated wet bacterial sludge metabolite.

(6) Freeze drying of bacterial mud metabolites

The preparation of the suspension is that 1000g of the inactivated wet bacterial sludge metabolite is taken, 4000mL of sterile physiological saline, 600g of egg white peptide, 600g of maltodextrin, 2000g of water-soluble corn starch, 50g of rhizoma polygonati peptide and 120g of vitamin C powder are added.

The preparation method comprises the steps of firstly taking 2500mL of physiological saline in a formula, adding maltodextrin, stirring and dissolving, then adding 1000g of inactivated wet bacterial sludge metabolite, then adding the rest physiological saline, water-soluble corn starch and auxiliary materials in the formula, fully and uniformly stirring to obtain suspension, freeze-drying the suspension, and finally controlling the water content to be 4%, thereby obtaining the frozen powder of the inactivated bacterial metabolite, namely a metaplasia product.

Through detection, the content of the inactivated bacteria cells in the freeze-dried powder obtained in the example 1 is 1600 hundred million/g.

The freeze-dried powder can inactivate bacteria in the dilution process, and the bacteria are broken, but the function of the strain is not weakened.

The freeze-dried powder can be widely used in foods, baked foods, solid beverages, liquid beverages and pressed candies.

EXAMPLE 2 Metabolic analysis

And performing metabonomics analysis on the mouse feces by adopting an ultra-high performance liquid chromatography-tandem mass spectrometry.

Experimental protocol mice are divided into control group and experimental group, the control group is fed with normal physiological saline in the stomach of 0.5 mL, the experimental group is fed with normal physiological saline in the stomach of 0.5 mL (10 mg of the metaproduct prepared in example 1 is dissolved in 0.5 mL of sterilized water) once daily. Thirty days after feeding, the fecal samples of the mice of the control group and the experimental group were respectively placed in 400 μl of an extraction solution (acetonitrile: methanol=1:1), after vortexing 30 s, low-temperature ultrasonic extraction 30min (5 ℃,40 KHz), -standing 30min at 20 ℃, placing in a 4 ℃ centrifuge, centrifuging 15 min at 13000 r/min, collecting supernatant, drying with nitrogen, re-dissolving 120 μl of a re-solution (acetonitrile: water=1:1), low-temperature ultrasonic extraction 5 min (5 ℃,40 KHz), placing in a 4 ℃ centrifuge, centrifuging 5 min at 13000 r/min, and collecting a proper amount of supernatant for machine analysis. 10. mu.L of sample supernatant was separated by BEHC chromatography column (100 mm X2.1 mm i.d.1.8 μm) and subjected to mass spectrometry. The separation flow rate was 0.40 mL/min and the column temperature was 40 ℃. After completion, the LC-MS raw data is transferred to a computer for processing and analysis. The MS mass spectrum information was matched via a public database to obtain the primary metabolite information as shown in table 2 and fig. 1.

The method for measuring the content of the lactobacillus polysaccharide comprises a spectrophotometry;

The ergosterol content detection method comprises high performance liquid chromatography;

the cephalin content detection method comprises high performance liquid chromatography;

the vitamin A content detection method comprises reversed phase high performance liquid chromatography;

The vitamin B1 content detection method comprises high performance liquid chromatography.

TABLE 1 Primary metabonomic analysis of fed mice

According to Table 1, it can be seen that 10mg of the metaproduct (i.e. the freeze-dried powder prepared in example 1) containing 16 hundred million inactivated bacteria cells and metabolites is administered to mice daily, so that the content of lactic acid bacteria polysaccharide, ergosterol, cephalin, vitamin A and vitamin B1 in the feces of the mice can be remarkably increased, and thus the metaproduct provided by the invention can continuously provide the required amount of lactic acid bacteria polysaccharide, ergosterol, cephalin, vitamin A and vitamin B1 for endogenous use in the intestinal reproduction and growth process. Can improve memory, improve epilepsy, and maintain healthy nervous system.

EXAMPLE 3 memory improvement

1. Small scale experiments to improve memory

In this application trial, the recruited subjects were 8 communities in Jinan city, a population of average age 60 years, and 100 total recruits were enrolled to participate in the trial by month 12 of 2022, all of whom signed written informed consent, and if the participants could not sign, by an agent (typically a family member). The experimental group took 0.1 gram of probiotic product (i.e. the lyophilized powder prepared in example 1) once a day before sleeping. The original eating habit is not changed during drinking, and the food is normally eaten. The test results are shown in Table 2.

TABLE 2 application effects of improving memory

Description of Table 2:

In middle-aged 50 people start counting after taking for 3 days, and 14 people feel insensitive, have concentration, memory decline, emotion is low, and the like after taking for 3 days. After taking for 6 days, 23 people have the conditions of insensitive feeling, inattention, memory decline, low emotion, smoldering and the like, and the situations are obviously improved. 34 people feel insensitive, concentration is not concentrated, memory is reduced, emotion is low, and the like after taking the medicine for 8 days, so that the improvement is extremely obvious. The patients with 48 persons who take the medicine for 10 days have the symptoms of slow sensory response, inattention, memory decline, low emotion, smoldering and the like, and the medicine is completely improved. The prebiotic products of 2 people in total cannot be improved after taking the medicine.

The aged 50 people begin to count results after taking for 4 days, and the conditions of slow sensory response, memory decline, deep emotion, smoldering, insomnia and the like of 12 people after taking for 4 days are improved. After 8 days of taking, the conditions of slow sensory response, memory decline, deep emotion, smoldering, insomnia and the like of 25 people are obviously improved. The improvement of 36 people with insensitive feeling, memory decline, deep emotion, smoldering and insomnia after 12 days of taking is very obvious. The feeling reaction of 46 people is slow, the memory is reduced, the emotion is low, the people feel smoldering, the insomnia and the like are completely improved after 15 days of taking the medicine. The prebiotic products of the total 4 people after taking the medicine are not improved.

2. Mouse validation experiment to improve memory:

SPF-class mice are selected, the weight of the mice is 18-22g, the mice are divided into a control group, a model group and an experimental group, the control group and the model group are fed with 0.5 mL physiological saline to irrigate the stomach while feeding normally, and the experimental group is fed with 0.5 mL metaplasia product solution to irrigate the stomach once daily while feeding normally. The preparation method of the metaproduct solution comprises the step of dissolving the metaproduct prepared in the embodiment 1 in 0.5 mL sterilized water to obtain the metaproduct solution. Wherein, the added mass of the metaproduct is respectively 10mg (experimental group 1), 15 mg (experimental group 2) and 20mg (experimental group 3). Experiments were performed after 30d of continuous gastric lavage. The model group and each dose group were intraperitoneally injected with scopolamine 5mg/kg body mass 10min before experimental training, with an injection volume of 10mL/kg body mass. Each dose group was given maintenance feed.

(1) Bench jump experiment

Training was started the next day after the last lavage. The mice were placed into the reaction box 3min before the experiment to adapt to the environment, and then placed at the bottom of the experiment box to turn on the circuit, so that the mice suffered electrical stimulation and jumped over the insulating platform. After training once, the mice are placed on an insulating platform, the error latency period (time of first jumping off the platform) and the number of errors of jumping off the platform within 5 minutes are recorded, and the number and the percentage of errors of each group of mice are calculated, so that the results are used as test period results. The test was repeated after 24 hours, and the test time was changed to 3 minutes and the retest period test was recorded. After stopping training for 7d, the remission experiments were performed and recorded as a retest experiment.

TABLE 3 influence of error latency in mouse diving experiments (x ^-.+ -. S)

The error latency and the number of errors of the mice in the diving experiment can reflect the memory improving effect of the sample on the mice. As shown in table 3, the error latency was significantly reduced in the two experimental groups compared to the model group. As shown in table 4, the number of experimental group errors was significantly reduced compared to the model control group. As shown in tables 5 and 6, the number of animals in error was significantly reduced and the percentage of animals in error was correspondingly reduced in the two experimental groups compared with the model group. In the bench jump experiment, the stomach-infused metaplasia product can obviously improve the memory of mice.

(2) Water maze test

Training was started the next day after the last lavage. The mice were trained twice daily and placed in the water maze for adaptation prior to each training. The method comprises the steps of respectively testing water from two quadrants of a mouse every day, starting timing, finding a platform for at most 60 seconds every time of experimental animals, allowing the animal to rest on the platform for 30 seconds if the animal does not successfully log on the platform within 60 seconds, recording results as 60 seconds if the animal does not find the platform within 60 seconds, then placing the mouse on the platform for 30 seconds, starting timing for 30 seconds according to the found time if the animal finds the platform within a specified time, falling into water if the rest time of the mouse is not up to 30 seconds, swimming again, stopping timing, placing the mouse on the platform for rest, continuing timing, and counting for 30 seconds. The purpose is to ensure that each mouse has the same time to collect spatial information after training. The method is trained four times, testing is started in the fifth time, the first quadrant and the fourth quadrant of each animal are measured twice, the number and total times of the animals on the life-saving table of each group are calculated, the total time for reaching the life-saving table and the total number (percentage) of the animals reaching the end point in 60 seconds are calculated, and the results of the two stages of the testing period and the retesting period are included. The regression experiment can be performed at various times from the starting point 7 days after stopping training.

TABLE 7 influence on test period of mouse water maze experiment

As can be seen from tables 7, 8 and 9, compared with the model group, the number of animals on the life-saving table and the number of animals on the life-saving table in the test period, the retest period and the regression period are obviously increased in the experimental group, the time for boarding the life-saving table in the test period, the retest period and the regression period is obviously shortened, and the postnatal product can obviously improve the memory of the mice.

(3) Dark prevention experiment

Training was started the next day after the last sample administration. The experimental box is divided into two parts, namely a spacious bright light room and a closed dark room, and a hole is formed in the middle of the experimental box to enable the two parts to be connected. The mice of each group were timed with their face facing away from the darkroom and placed in the darkroom as the start of the experiment, and the error latency (the time required to first enter the darkroom to receive an electric shock) and the number of errors that received an electric shock were recorded for each group of mice within 5 minutes. And the number of wrong animals and the percentage of wrong animals of each group of mice were calculated, and the results were used as test period results. The retest phase experiment was performed and recorded after 24 hours. After stopping training for 7d, the remission experiments were performed and recorded as a retest experiment.

Table 10 influence of error latency in the dark avoidance experiments of mice (x ^-.+ -. S)

The darkness avoidance experiment utilizes the nature that the mice like a dark environment, and the bright illumination environment can cause anxiety and dysphoria of the mice, so that the mice want to enter a dark airtight space, and the mice can suffer electric shock when the mice move to a darkroom. Therefore, the error latency and the error times of the mice in the dark-avoiding experiment can reflect the memory improving effect of the test object on the mice. As shown in table 10, the experimental group showed a tendency to decrease in retest period error latency compared to the model group. As shown in tables 11, 12, the number of erroneous animals in the retest period was significantly reduced in the experimental group compared to the model group, and the percentage of erroneous animals was reduced accordingly. As shown in table 13, the number of retest period errors was significantly reduced in the experimental group compared to the model group. Therefore, the frequency of errors in dark-avoiding experiments of mice in which the metaplasia products are used for lavage is obviously reduced, which indicates that the metaplasia products can obviously improve the memory of the mice.

Example 4-modulation of intestinal flora and digestion promotion

1. Functional experiment for regulating intestinal flora of mice:

Mice were divided into control and experimental groups of 10 mice each. The control group was fed normal with normal saline and was filled with 0.5 mL of the stomach, and the experimental group was fed normal with a solution of 0.5 mL of the metaproduct (the metaproduct 10 mg prepared in example 1 was dissolved in 0.5 mL of sterilized water) once daily. After 30 days of feeding, the mouse feces are aseptically collected, weighed, diluted 10 times by sterilized diluent to prepare uniform suspension, sequentially diluted 10 times in turn, and the proper dilutions are selected to be respectively inoculated on each selective culture medium. The bacterial count in each gram of wet feces is calculated, and the statistics treatment is carried out after taking the logarithm. 24 hours after the last administration of the metaproduct, the intestinal faeces were straightened and the intestinal flora was examined as shown in Table 14.

TABLE 14 detection of suitable culture conditions and intestinal flora (x- + -SD)

As can be seen from table 14, the metaproduct can increase the number of beneficial bacteria (bifidobacteria and lactobacilli), while the number of harmful bacteria (enterobacteria, enterococci) is not increased but shows a slight decrease trend, indicating that the metaproduct can regulate the intestinal flora function of mice.

2. Mouse gastrointestinal peristalsis experiment

Mice were divided into control, model and experimental groups of 10 mice each. The control group and the model group were administered with 0.5 mL sterilized water for gastric lavage once daily while feeding normally, and the experimental group was administered with 0.5 mL metaproduct solution (10 mg metaproduct prepared in example 1 was dissolved in 0.5 mL sterilized water) for gastric lavage once daily while feeding normally. After 15 days of gastric lavage, each group of mice fasted for 16 hours. After that, the model group and the experimental group were subjected to compound diphenox lavage, the control group was subjected to sterilization water lavage, after 30 minutes, each group of mice was subjected to acacia ink lavage, after 30 minutes, the cervical spine fracture method was lethal, the abdomen was rapidly cut along the median line of abdomen to separate the mesentery, the pylorus was used as the starting point, the ileocecum was used as the ending point, the intestinal canal was cut, the small intestine was gently pulled into a straight line, the length of the intestinal canal was measured to be the total length of the small intestine, the front edge from the pylorus to the ink was the "ink pushing length", the ink pushing rate was calculated according to the following formula, and the ink pushing rate of the small intestine was shown in table 15.

TABLE 15 small intestine ink Propulsion Rate (x- + -SD)

As can be seen from table 15, the mice produced by administering the compound diphenox had established the intestinal motility inhibition model. The increase in small intestine ink push rate compared to the model group indicates that the metaproduct prepared in example 1 can promote digestion of small intestine.

EXAMPLE 5 amelioration of epilepsy and reduction of seizure number

1. Small scale crowd experiment

10 Persons with epilepsy aged 5-40 were voluntarily tested and able to complete questionnaires and recordings. The 0.1 g metaproducts (i.e. the freeze-dried powder prepared in example 1) were taken once daily before sleeping, and the original eating habit was not changed during drinking, and the normal diet was not changed. The test results are shown in Table 16.

Table 16 effects of reducing seizure number application

In this application trial, the recruited subjects were 8 communities in Jinan city, a population of average age 60 years, and 100 total recruits were enrolled to participate in the trial by month 12 of 2022, all of whom signed written informed consent, and if the participants could not sign, by an agent (typically a family member).

Table 17 memory improving application effect

The result is counted after 50 people take the medicine for 3 days, and 14 people feel insensitive, have concentration, memory decline, emotion is low, and the like after taking the medicine for 3 days. After taking for 6 days, 23 people have the conditions of insensitive feeling, inattention, memory decline, low emotion, smoldering and the like, and the situations are obviously improved. 34 people feel insensitive, concentration is not concentrated, memory is reduced, emotion is low, and the like after taking the medicine for 8 days, so that the improvement is extremely obvious. The patients with 48 persons who take the medicine for 10 days have the symptoms of slow sensory response, inattention, memory decline, low emotion, smoldering and the like, and the medicine is completely improved. A total of 2 people failed to improve on Streptococcus thermophilus administration.

The aged 50 people begin to count results after taking for 4 days, and the conditions of slow sensory response, memory decline, deep emotion, smoldering, insomnia and the like of 12 people after taking for 4 days are improved. After 8 days of taking, the conditions of slow sensory response, memory decline, deep emotion, smoldering, insomnia and the like of 25 people are obviously improved. The improvement of 36 people with insensitive feeling, memory decline, deep emotion, smoldering and insomnia after 12 days of taking is very obvious. The feeling reaction of 46 people is slow, the memory is reduced, the emotion is low, the people feel smoldering, the insomnia and the like are completely improved after 15 days of taking the medicine. A total of 4 people failed to improve on Streptococcus thermophilus administration.

2. Verification experiment of animal model

After 3d of normal feeding of several rats, rats in other groups were treated with scopolamine methyl nitrate (1 mg/kg) by intraperitoneal injection, and after 20min, pramipexole (330 mg/kg) by intramuscular injection, status epilepticus seizure was induced in rats, and after successful induction, diazepam (5 mg/kg) was intramuscular injected to terminate the seizure. The number of seizures in the rat within 30d was then recorded, with more than 3 seizures being successful in modeling.

The 50 rats successfully molded are 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 normal saline lavage with normal feeding, and the experimental group was given 0.5 mL post-natal product solution lavage with normal feeding once daily. The preparation method of the metaproduct solution comprises the step of dissolving the metaproduct prepared in the embodiment 1 in 0.5 mL sterilized water to obtain the metaproduct solution. The added quality of the metaproduct is respectively 10mg (experimental group 1), 15 mg (experimental group 2) and 20 mg (experimental group 3). After the experiment is finished, 90 rats are sacrificed and brains are taken after the number of epileptic seizure of the rats is observed by continuously filling the stomach for 30 days, 4 ℃ physiological saline is added into brain tissues according to the weight-to-volume ratio of 1:9, 10% homogenate is prepared, and NO, MDA, SOD, T-AOC content in the rat brain tissue homogenate is detected according to the steps and requirements of a kit instruction.

Table 18 comparison of the number of epileptic seizures in rats of each group

From Table 18, it can be seen that the number of seizures in rats after drenching the raw meta-products was significantly reduced and dose dependent.

According to fig. 2,3 and 4, compared with a control group, the amounts of NO and MDA in serum of a rat in a model group are obviously increased, the amounts of SOD and T-AOC are reduced, and after the model group is drenched with the post-raw-element products (experimental groups 1,2 and 3), the activities of SOD and T-AOC in serum are increased, and the amounts of MDA and NO are obviously reduced.

The effect verification experiment shows that the metaproduct prepared by the invention has the functions of improving memory and epilepsy, reducing epileptic times, regulating intestinal flora function and promoting digestion.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A thermophilic Streptococcus thermophilus AK223-6, deposited in Guangdong Microbiological Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, with a deposit date of July 8, 2024 and a deposit number of GDMCCNO: 64835. 2. A derivative of Streptococcus thermophilus AK223-6 according to claim 1, 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 it comprises an active ingredient, wherein the active ingredient comprises the thermophilic Streptococcus thermophilus AK223-6 or its inactivated bacteria, fermentation liquid, and metabolites as described in claim 1. 4 . The microbial agent according to claim 3 , characterized in that the microbial agent comprises inactivated 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 Streptococcus thermophilus AK223-6 is inoculated into an activation culture medium, and cultured at 35-40° C. for 20-25 hours to obtain an expanded bacterial culture solution; 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 mud metabolites, and the inactivated wet bacterial mud metabolites are freeze-dried to obtain the inactivated bacterial metabolites freeze-dried powder; 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. Use of the Streptococcus thermophilus AK223-6 according to claim 1, or the derivative of the Streptococcus thermophilus AK223-6 according to claim 2, or the microbial agent according to claim 3 or 4 in preparing food. 8. Use of the Streptococcus thermophilus AK223-6 according to claim 1, or the derivative of the Streptococcus thermophilus AK223-6 according to claim 2, or the 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 Streptococcus thermophilus AK223-6 according to claim 1, and/or the derivative of Streptococcus thermophilus AK223-6 according to claim 2, and/or the microbial agent according to claim 3 or 4, and acceptable adjuvants or excipients; 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 product comprises a postbiotic product.