Lactobacillus reuteri A-1 and its application in preparing iron supplement and intestinal regulation products

Abstract

The present invention discloses Lactobacillus reuteri A-1 and its application in preparing iron supplement and intestinal regulation products, belonging to the field of microorganisms and biomedicine. The present invention provides a Lactobacillus reuteri ( Limosilactobacillus reuteri )A‑1, deposited in Guangdong Microbial Culture Collection Center, the deposit date is July 8, 2024, and the deposit number is GDMCCNO: 64837. Based on this Lactobacillus reuteri, a postbiotic product has been successfully developed. It has been verified that it has the effect of regulating the balance of intestinal flora, promoting human digestion and absorption, and effectively supplementing iron. Daily administration of 100 mg of a postbiotic product containing 16 billion strains and metabolites of the present invention can sustainably provide endogenously required amounts of glutathione, lycopene, and arginine during intestinal reproduction and growth, can effectively supplement iron, and has good practical application value.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms and biomedicine, and specifically relates to Lactobacillus reuteri A-1 and application thereof in the preparation of iron supplementation and intestinal regulation products. 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] Iron deficiency in the human body is a global problem, affecting more than 2 billion people worldwide. Iron in the body is related to oxygen transport and storage, electron transfer, redox reaction regulation, hormone synthesis, DNA replication, cell cycle recovery and control, nitrogen fixation and antioxidant effects. Good regulation of iron is the guarantee of human health, and too much or too little will have adverse effects on the human body. Disturbance of iron homeostasis is closely related to the occurrence and development of various diseases: iron deficiency can lead to host developmental delay and low immunity, while iron overload is more likely to cause inflammatory reactions and metabolic problems, and may also be closely related to the occurrence and development of cancer.

[0004] The main source of iron in the human body is dietary iron, which can be divided into heme iron and non-heme iron. Heme iron is mostly found in the blood, muscles and blood-containing organs of animals. Current studies have found that there are two main ways for heme iron to be absorbed by the intestine: one is through receptor-mediated endocytosis by intestinal epithelial cells; the other is through the heme-specific transporter protein, heme carrier protein 1 / proton-coupled folate transporter (HCP1 / PCFT). Non-heme iron is mostly in the form of inorganic Fe 3+ In plant foods, Fe 3+ Reduced to Fe by duodenal cytochrome B (DcytB) in the duodenum 2+ , absorbed into intestinal epithelial cells via the divalent metal transporter (DMT1) and subsequently enters the plasma via the ferroportin (FPN1).

[0005] Iron determines its growth, toxicity and ability to settle in the intestine. At the same time, iron also determines the proliferation ability of some intestinal microorganisms and affects the production of intestinal flora metabolites. Intestinal microorganisms can convert iron-bound ellagic acid into urea A that does not bind iron, thereby reducing the amount of iron-bound substances in the intestine. In addition, intestinal microorganisms convert iron ions into Fe 2+form. Lack of intestinal flora leads to iron deficiency in intestinal cells, reducing the absorption rate of iron in the diet. At the same time, iron also determines the proliferation capacity of some intestinal microorganisms and affects the production of intestinal flora metabolites. On the other hand, iron can react with a variety of substances in the intestine to produce certain toxicity, prompting the intestinal flora to use a variety of detoxification mechanisms to resist damage. A large number of studies have shown that different flora have various complex iron acquisition systems, which can obtain iron by secreting iron carriers to chelate iron in the surrounding environment. For example, Shigella has an iron carrier uptake system, a heme transporter uptake system, and an iron and ferrous iron transport system. Vibrio cholerae also has an iron uptake mechanism that depends on heme, catechol ferritin, etc. Under normal circumstances, the iron content in the colon is strictly regulated by the body, thereby limiting the ability of bacteria to obtain iron, but when the iron in the diet is increased or lacking, it will affect the unabsorbed iron content in the colon, thereby affecting the composition and metabolism of intestinal microorganisms in the colon. Help maintain the balance of intestinal flora.

[0006] Probiotics are a type of active microorganisms that are beneficial to human health. Long-term use of probiotics can promote iron absorption. This may be due to the anti-inflammatory effect of the colon. Long-term use of probiotics can increase the diversity and dominant bacteria of intestinal flora and reduce the number of pathogenic bacteria. Inducing intestinal immune processes and responses stimulates immune organs such as the thymus, liver, spleen, and intestines, stimulates the biological activity of macrophages, and thereby improves immunity overall by increasing the response of B and T lymphocytes to antigen cell stimulation and specific cellular immune responses. When probiotics are fermented, lactose is produced, which increases the passive absorption of metals such as iron, and produces weak organic acids, which exhibit the property of promoting mineral absorption. The production of these weak organic acids naturally lowers the pH, which helps the ferrous form of iron (Fe 3+ ) into a more bioavailable form of iron (Fe 2+ ). Long-term use of probiotics will produce more short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate. These short-chain fatty acids can induce increased proliferation of epithelial cells, ultimately increasing the absorption surface area, thereby increasing iron absorption. Therefore, providing a strain with functions such as iron supplementation is of great clinical significance. Summary of the invention

[0007] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide Lactobacillus reuteri A-1 and its application in the preparation of iron supplementation and intestinal regulation products. The Lactobacillus reuteri A-1 provided by the present invention has the effects of regulating the balance of intestinal flora and promoting human digestion and absorption, as well as supplementing iron and improving iron deficiency anemia.

[0008] In order to achieve the above object, the technical solution of the present invention is:

[0009] In a first aspect, the present invention provides a Lactobacillus reuteri ( Limosilactobacillus reuteri)A-1, 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: 64837.

[0010] In a second aspect, the present invention provides a kind of above-mentioned Lactobacillus reuteri ( Limosilactobacillus reuteri ) A derivative of A-1, wherein the derivative is one or more of living bacteria, dead bacteria, fermentation broth or metabolites.

[0011] In a third aspect, the present invention provides a microbial agent, comprising an active ingredient, wherein the active ingredient comprises the above-mentioned Lactobacillus reuteri ( Limosilactobacillus reuteri ) A-1 or its inactivated bacteria, fermentation broth, or metabolites.

[0012] In one or more embodiments, the microbial agent comprises inactivated Lactobacillus reuteri ( Limosilactobacillus reuteri )A-1 and its fermentation broth.

[0013] In a fourth aspect, the present invention provides a method for preparing the microbial agent, the method comprising:

[0014] S1. Bacteria activation:

[0015] Inoculating the Lactobacillus reuteri A-1 into an activation culture medium, and culturing at 35-40° C. for 20-25 hours to obtain an expanded bacterial culture solution;

[0016] 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 30-40 h to obtain the primary bacterial seed solution;

[0017] 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 100-150r / min, the pH is natural, the temperature is 30-40°C, the tank pressure is 0.03-0.06MPa, and the culture time is 10-15h h to obtain the seed tank strain;

[0018] S4, 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, 150-200r / min, tank pressure 0.04-0.06MPa, pH 6.0-6.5, and ferment for 24-26h; cool to 19-21°C, which is the fermentation liquid; heat the fermentation liquid to 119-121°C and keep it for 4-7s for UHT inactivation treatment.

[0019] In one or more embodiments, in step S1, the activation medium is composed of: 15-20 parts of anhydrous glucose, 1-2 parts of lactoferrin, 0.1-0.5 parts of beef extract powder, 0.5-1 parts of sodium acetate, 0.4-0.8 parts of Tween-80, 0.2-0.6 parts of potassium dihydrogen phosphate, 900-1000 parts of tap water, and the pH is adjusted to 7.0.

[0020] In one or more embodiments, in step S2, the composition of the primary seed culture solution is as follows: 2-6 parts of anhydrous glucose, 2-6 parts of bovine bone peptone, 2-6 parts of beef extract powder, 0.1-0.5 parts of D-ribose, 0.5-1 parts of sodium acetate, 0.8-1.2 parts of Tween-80, 900-1000 parts of tap water, and natural pH.

[0021] In one or more embodiments, the composition of the seed tank culture medium is as follows, in weight percentage: 2-6% anhydrous glucose, 0.1-0.5% casein peptone, 0.5-1% dipotassium hydrogen phosphate, 0.2-0.6% sodium acetate, 0.8-1.2% whey peptide, 0.05-0.15% Tween-80, 0.05-0.15% polyether defoamer, 90-95% tap water, natural pH value, totaling 100%.

[0022] In one or more embodiments, in step S4, the fermentation broth is composed of the following, in weight percentage: 2-6% anhydrous glucose, 1.2-1.6% casein peptone, 2.6-3.2% stachyose, 2-6% wheat germ powder, 2-3% yeast extract powder, 1-2% sodium acetate, 0.1-0.4% Tween-80, 0.5-1.5% sodium caseinate, 0.5-1.5% dipotassium hydrogen phosphate, 0.05-0.15% polyether defoamer, and 80-85% tap water, totaling 100%.

[0023] 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.

[0024] In a fifth aspect, the present invention provides the above-mentioned Lactobacillus reuteri ( Limosilactobacillus reuteri )A-1, or Lactobacillus reuteri ( Limosilactobacillus reuteri ) Use of derivatives of A-1 or microbial agents in the preparation of food.

[0025] In a sixth aspect, the present invention provides the above-mentioned Lactobacillus reuteri ( Limosilactobacillus reuteri )A-1, or Lactobacillus reuteri ( Limosilactobacillus reuteri )Use of a derivative or microbial agent of A-1 in the preparation of any of the following products:

[0026] (1) Products used to regulate intestinal flora and promote digestion;

[0027] (2) Products used to supplement iron and improve iron deficiency anemia;

[0028] (3) Products used in the production of glutathione, lycopene and arginine.

[0029] The product is one of a food and a medicine;

[0030] The food is any one of beverages, dairy products, candy products, health foods, special dietary foods and special medical formula foods.

[0031] In a seventh aspect, the present invention provides a product comprising the above-mentioned Lactobacillus reuteri ( Limosilactobacillus reuteri ) A-1, and / or the above-mentioned Lactobacillus reuteri ( Limosilactobacillus reuteri ) derivatives of A-1, and / or the above-mentioned microbial agents and acceptable adjuvants or excipients.

[0032] In some examples of this embodiment, the product is one of a food and a medicine;

[0033] 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.

[0034] In some examples of this embodiment, the product comprises a postbiotic preparation.

[0035] In an eighth aspect, the present invention provides a method for regulating intestinal flora, promoting digestion or supplementing iron, or for producing glutathione, lycopene, or arginine, using the above-mentioned Lactobacillus reuteri ( Limosilactobacillus reuteri )A-1, or the above-mentioned Lactobacillus reuteri ( Limosilactobacillus reuteri ) A derivative of A-1 or the above-mentioned microbial agent.

[0036] The beneficial effects of the present invention are:

[0037] This application obtains a strain of Lactobacillus reuteri ( Limosilactobacillus reuteri ) A-1, based on the Lactobacillus reuteri, a postbiotic product has been successfully developed. It has been verified that it has the effect of regulating the balance of intestinal flora, promoting human digestion and absorption, and supplementing iron and improving iron deficiency anemia. 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 glutathione, lycopene, and arginine during the intestinal reproduction and growth process, can effectively supplement iron, improve iron deficiency anemia, and maintain the health of the body, and has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] 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.

[0039] Figure 1 Analysis of metabolites after taking the postbiotic product in Example 2 of the present invention;

[0040] Figure 2 The weight change of rats before and after modeling in the construction of the animal model in Example 3 of the present invention;

[0041] Figure 3 The changes in hemoglobin content in rats before and after modeling in the animal model construction of Example 3 of the present invention;

[0042] Figure 4 The weight changes of rats in each group in the iron supplementation verification experiment using an animal model in Example 3 of the present invention;

[0043] Figure 5 The change of hemoglobin content in rats in the iron supplementation verification experiment using an animal model in Example 3 of the present invention;

[0044] Figure 6 The red blood cell count of each group of rats in the iron supplementation verification experiment using an animal model in Example 3 of the present invention;

[0045] Figure 7 The hematocrit content of each group of rats in the iron supplementation verification experiment using an animal model in Example 3 of the present invention;

[0046] Figure 8 The free protoporphyrin content of erythrocytes of rats in each group in the iron supplementation verification experiment using an animal model in Example 3 of the present invention;

[0047] Fig. 9 The serum iron content of rats in each group in the iron supplementation verification experiment using an animal model in Example 3 of the present invention;

[0048] Fig.10 The serum iron binding capacity content of rats in each group in the iron supplementation verification experiment using an animal model in Example 3 of the present invention;

[0049] Fig.11 The serum ferritin content of rats in each group in the iron supplementation verification experiment using an animal model in Example 3 of the present invention;

[0050] Fig.12 The iron saturation of each group of rats in the iron supplementation verification experiment using an animal model in Example 3 of the present invention. DETAILED DESCRIPTION

[0051] A first typical embodiment of the present invention provides a Lactobacillus reuteri ( Limosilactobacillus reuteri )A-1, deposited in Guangdong Microbiological Culture Collection Center, the preservation date is July 8, 2024, and the preservation number is GDMCCNO: 64837.

[0052] The bacterium was isolated from fresh Cordyceps sinensis meat in Nagqu City, Tibet Autonomous Region, and was identified as Lactobacillus reuteri by the 16srRNA method.

[0053] The sequence of the bacteria is as follows:

[0054]

[0055] This strain has been preserved as a patented strain in the Guangdong Provincial Microbial Culture Collection Center, with the preservation number GDMCCNO: 64837. This Lactobacillus reuteri strain is a probiotic strain approved for use by the National Health Commission. This strain has the effect of regulating the balance of intestinal flora and promoting human digestion and absorption, and also has the effect of supplementing iron. Daily administration of 100 mg of a postbiotic product containing 16 billion strains of the present invention and metabolites can sustainably provide the endogenous necessary amount of glutathione, lycopene, and arginine during the intestinal reproduction and growth process, which can effectively supplement iron and maintain the health of the body.

[0056] A second typical embodiment of the present invention provides a Lactobacillus reuteri ( Limosilactobacillus reuteri ) A derivative of A-1, wherein the derivative is one or more of living bacteria, dead bacteria, fermentation broth or metabolites.

[0057] A third typical embodiment of the present invention provides a microbial agent, comprising an active ingredient, wherein the active ingredient comprises the above-mentioned Lactobacillus reuteri ( Limosilactobacillus reuteri ) A-1 or its inactivated cells, fermentation broth, metabolites. The metabolites include extracellular metabolites and / or intracellular metabolites.

[0058] In some embodiments of this embodiment, the microbial agent comprises inactivated Lactobacillus reuteri ( Limosilactobacillus reuteri ) A-1 and its fermentation liquid. At this time, the microbial agent is a postbiotic product.

[0059] A fourth typical embodiment of the present invention provides a method for preparing the microbial agent, the method comprising:

[0060] S1. Bacteria activation:

[0061] The Lactobacillus reuteri ( Limosilactobacillus reuteri ) A-1 is inoculated into the activation medium and cultured at 35-40°C (preferably 37°C) for 20-25h (preferably 24h) to obtain an expanded bacterial culture solution.

[0062] The activation culture medium is composed of: 15-20 parts of anhydrous glucose, 1-2 parts of lactoferrin, 0.1-0.5 parts of beef extract powder, 0.5-1 parts of sodium acetate, 0.4-0.8 parts of Tween-80, 0.2-0.6 parts of potassium dihydrogen phosphate, 900-1000 parts of tap water, and the pH is adjusted to 7.0.

[0063] More specifically, the activation culture medium is composed of: 16 parts of anhydrous glucose, 1 part of lactoferrin, 0.2 parts of beef extract powder, 0.8 parts of sodium acetate, 0.5 parts of Tween-80, 0.4 parts of potassium dihydrogen phosphate, 1000 parts of tap water, and the pH is adjusted to 7.0.

[0064] S2. Preparation of primary bacterial seed liquid: inoculate the expanded bacterial seed liquid prepared in step S1 into the primary seed culture liquid, with the inoculation amount being 0.5%-5%, and culture at 35-40° C. (preferably 37° C.) for 30-40 h (preferably 36 h) to obtain the primary bacterial seed liquid.

[0065] Wherein, the composition of the primary seed culture solution is as follows:

[0066] 2-6 parts of anhydrous glucose, 2-6 parts of bovine bone peptone, 2-6 parts of beef extract powder, 0.1-0.5 parts of D-ribose, 0.5-1 parts of sodium acetate, 0.8-1.2 parts of Tween-80, 900-1000 parts of tap water, natural pH value.

[0067] More specifically, the composition of the primary seed culture solution is as follows:

[0068] 4 parts of anhydrous glucose, 4 parts of bovine bone peptone, 4 parts of beef extract powder, 0.2 parts of D-ribose, 0.6 parts of sodium acetate, 1 part of Tween-80, 1000 parts of tap water, natural pH.

[0069] 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 100-150r / min (preferably 140r / min), natural pH, temperature 30-40°C (preferably 37°C), tank pressure 0.03-0.06MPa (preferably 0.05MPa), and culture time 10-15h (preferably 12h) to obtain seed tank strains.

[0070] Wherein, the composition of the seed tank culture medium is as follows:

[0071] Anhydrous glucose 2-6%, casein peptone 0.1-0.5%, dipotassium hydrogen phosphate 0.5-1%, sodium acetate 0.2-0.6%, whey peptide 0.8-1.2%, Tween-80 0.05-0.15%, polyether defoamer 0.05-0.15%, tap water 90-95%, natural pH value, the total is 100%.

[0072] More specifically, the composition of the seed tank culture medium is as follows:

[0073] Anhydrous glucose 4%, casein peptone 0.2%, dipotassium hydrogen phosphate 0.7%, sodium acetate 0.4%, whey peptide 1%, Tween-80 0.1%, polyether defoamer 0.1%, tap water 93.5%, natural pH.

[0074] S4, 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), 150-200r / min (preferably 180r / min), tank pressure 0.04-0.06MPa (preferably 0.05MPa), pH 6.0-6.5 (preferably 6.0), fermentation for 24-26h (preferably 25h); cool to 19-21°C (preferably 20°C), this 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.

[0075] The fermentation culture medium is composed of: 2-6% anhydrous glucose, 1.2-1.6% casein peptone, 2.6-3.2% stachyose, 2-6% wheat germ powder, 2-3% yeast extract powder, 1-2% sodium acetate, 0.1-0.4% Tween-80, 0.5-1.5% sodium caseinate, 0.5-1.5% dipotassium hydrogen phosphate, 0.05-0.15% polyether defoamer, and 80-85% tap water, totaling 100%.

[0076] More specifically, the fermentation broth is composed of 4% anhydrous glucose, 1.5% casein peptone, 3% stachyose, 4% wheat germ powder, 2.5% yeast extract powder, 1.5% sodium acetate, 0.2% Tween-80, 1% sodium caseinate, 0.8% dipotassium hydrogen phosphate, 0.1% polyether defoamer, and 81.4% tap water.

[0077] 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.

[0078] Furthermore, the freeze drying of the inactivated wet bacteria mud metabolites is as follows: the inactivated wet bacteria mud metabolites are configured into a suspension and then freeze dried. The suspension comprises: 950-1050 parts of inactivated wet bacteria mud metabolites, 3900-4100 parts of sterile physiological saline, 600-800 parts of pumpkin juice powder, 1900-2100 parts of bovine colostrum, 90-110 parts of vitamin C powder, and 110-130 parts of anserine. Preferably, the inactivated wet bacteria mud metabolites are 1000 parts, 4000 parts of sterile physiological saline, 700 parts of pumpkin juice powder, 2000 parts of bovine colostrum, 100 parts of vitamin C powder, and 120 parts of anserine.

[0079] A fifth typical embodiment of the present invention provides the Lactobacillus reuteri ( Limosilactobacillus reuteri )A-1, or Lactobacillus reuteri ( Limosilactobacillus reuteri ) Use of derivatives of A-1 or microbial agents in the preparation of food.

[0080] 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 or a special medical purpose formula food.

[0081] A sixth typical embodiment of the present invention provides the above-mentioned Lactobacillus reuteri ( Limosilactobacillus reuteri )A-1, or Lactobacillus reuteri ( Limosilactobacillus reuteri )Application of derivatives or microbial agents of A-1 in the preparation of any of the following products:

[0082] (1) Products used to regulate intestinal flora and promote digestion;

[0083] (2) Products used to supplement iron and improve iron deficiency anemia;

[0084] (3) Products used in the production of glutathione, lycopene and arginine.

[0085] In some examples of this embodiment, the product is one of a food and a medicine;

[0086] The food is any one of beverages, dairy products, candy products, health foods, special dietary foods and special medical formula foods.

[0087] The glutathione, lycopene and arginine have the following effects respectively:

[0088] Glutathione: It has a reducing effect on iron and combines with iron molecules to form a soluble small molecule complex, which can promote iron absorption and prevent iron deficiency anemia.

[0089] Lycopene: On the one hand, it can increase the permeability of small intestinal mucosal epithelial cells to iron ions, thereby promoting iron absorption; on the other hand, it can also reduce the difficultly absorbed trivalent iron to easily absorbed divalent iron.

[0090] Arginine: It can chelate with iron to form small soluble monomers, prevent iron precipitation, and facilitate iron absorption.

[0091] A seventh typical embodiment of the present invention provides a product, comprising the Lactobacillus reuteri ( Limosilactobacillus reuteri )A-1, and / or Lactobacillus reuteri ( Limosilactobacillus reuteri) derivatives of A-1, and / or microbial agents and acceptable adjuvants or excipients.

[0092] In some examples of this embodiment, the product includes one of a food and a medicine.

[0093] In some embodiments of this embodiment, the product includes a postbiotic product. In some embodiments 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 formula food. In some embodiments of this embodiment, the adjuvant or excipient may be a carrier, excipient, and diluent commonly used in pharmacy. Moreover, according to the usual method, it can be made into an oral preparation such as a powder, a granule, a suspension, an emulsion, a syrup, a spray, etc.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] In an eighth typical embodiment of the present invention, the present invention provides a method for regulating intestinal flora, promoting digestion or supplementing iron, or for producing glutathione, lycopene, and arginine, using the above-mentioned Lactobacillus reuteri ( Limosilactobacillus reuteri )A-1, or Lactobacillus reuteri ( Limosilactobacillus reuteri ) A-1 derivatives or microbial agents.

[0099] 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.

[0100] 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 .

[0101] Example 1

[0102] A method for preparing a postbiotic product comprises the following steps:

[0103] 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.

[0104] (1) Bacteria activation

[0105] Bacterial strain culture: Expansion culture of bacterial strains preserved in -80℃ refrigerator.

[0106] Preparation of culture medium: 16 g anhydrous glucose, 1 g lactoferrin, 0.2 g beef extract powder, 0.8 g sodium acetate, 0.5 mL Tween-80, 0.4 g potassium dihydrogen phosphate, 1000 mL tap water, pH adjusted to 7.0.

[0107] Operation: Add the anhydrous glucose and other raw materials in the above formula into 1000mL of water in the formula without any particular order, stir for 10 minutes to dissolve, and then divide into test tubes, each test tube contains 5mL, seal with kraft paper stopper, and place in a medical sterilizer for sterilization at 0.1-0.12MPa for 20 minutes. The culture medium is ready.

[0108] 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.

[0109] (2) Preparation of primary bacterial culture liquid:

[0110] Primary seed culture formula: 4 g anhydrous glucose, 4 g bovine bone peptone, 4 g beef extract powder, 0.2 g D-ribose, 0.6 g sodium acetate, 1 mL Tween-80, 1000 mL tap water, natural pH.

[0111] Operation: Add the above raw materials into 1000mL of tap water without any distinction, start the blender, and stir for 10 minutes to completely dissolve them. This is called the first-level seed culture solution.

[0112] 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 culture 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 36 hours. This is called the first-level bacterial culture liquid.

[0113] (3) Preparation of seed tank strains:

[0114] Sterilization of fermentation equipment, pipelines and sterile filtration systems:

[0115] 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.

[0116] Sterilization of empty seed tanks and fermentation tanks:

[0117] 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.

[0118] Seed pot culture:

[0119] Formula: 4% anhydrous glucose, 0.2% casein peptone, 0.7% dipotassium hydrogen phosphate, 0.4% sodium acetate, 1% whey peptide, 0.1% Tween-80, 0.1% polyether defoamer, 93.5% tap water, natural pH value.

[0120] 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 140r / min, tank pressure 0.05MPa, and culture time 12h 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.

[0121] (4) Lactobacillus reuteri fermentation

[0122] Formula: 4% anhydrous glucose, 1.5% casein peptone, 3% stachyose, 4% wheat germ powder, 2.5% yeast extract powder, 1.5% sodium acetate, 0.2% Tween-80, 1% sodium caseinate, 0.8% dipotassium hydrogen phosphate, 0.1% polyether defoamer, and 81.4% tap water.

[0123] 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.

[0124] The inoculation amount is 6 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 180r / min, tank pressure maintained at 0.05MPa, and the pH value is maintained at 6.0 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.

[0125] (5) Centrifugation of fermentation broth

[0126] 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 8kg / min, collect the inactivated wet bacterial sludge metabolites after centrifugation, and freeze-dry the collected inactivated wet bacterial sludge metabolites.

[0127] (6) Freeze-drying of bacterial sludge metabolites

[0128] Preparation of bacterial suspension: Take 1000g of inactivated wet bacterial sludge metabolites, add 4000mL of sterile saline, 700g of pumpkin juice powder, 2000g of bovine colostrum, 100g of vitamin C powder, and 120g of anserine.

[0129] Operation: First, take 2500mL of physiological saline in the formula, add bovine colostrum to stir and dissolve, then add 1000g of inactivated wet bacterial mud metabolites, and then add the remaining physiological saline and pumpkin juice powder 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 a freeze-dried powder of inactivated bacterial metabolites, i.e., a postbiotic product. After testing, the inactivated bacterial cell content in the freeze-dried powder obtained by Example 1 is 160 billion / g.

[0130] 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.

[0131] The freeze-dried powder can be widely used in food, baked food, solid beverages, liquid beverages, and compressed candies.

[0132] Example 2 - Metabolomics Analysis

[0133] Metabolomics analysis of mouse feces was performed using ultra-performance liquid chromatography-tandem mass spectrometry.

[0134] 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, 1.8 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, and the main metabolite information was obtained as shown in Table 1.

[0135] Glutathione content determination method: ELISA method;

[0136] Lycopene content determination method: High performance liquid chromatography;

[0137] Arginine content detection method: high performance liquid chromatography.

[0138] Table 1 Main metabolomics analysis of fed mice

[0139]

[0140] It can be seen from Table 1 that daily administration of 10 mg of a postbiotic product containing 1.6 billion inactivated bacterial cells and metabolites (i.e., the lyophilized powder prepared in Example 1) to mice can significantly increase the contents of glutathione, lycopene, and arginine in the feces of mice, indicating that the postbiotic product provided by the present invention can sustainably provide the endogenous necessary amount of glutathione, lycopene, and arginine during the intestinal reproduction and growth process, improve iron elements, and maintain a healthy nervous system.

[0141] Example 3 - Iron supplementation to improve iron deficiency anemia

[0142] 1. Small-scale application experiment of iron supplementation

[0143] In this application trial, the subjects were iron-deficient infants and pregnant women aged 2-35 years old from 5 communities in Jinan City. As of June 2022, a total of 60 people were recruited to participate in the experiment. All of the above-mentioned persons 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 gram of the postbiotic product (i.e., the freeze-dried powder prepared in Example 1) once a day before going to bed. The original eating habits were not changed during the drinking period, and the diet was normal. The test results are shown in Table 2.

[0144] Table 2 Effect of iron supplementation

[0145]

[0146] Description of Table 2:

[0147] Infants and young children: 20 people started to collect the results after taking the medicine for 8 days. After taking it for 8 days, 5 people felt that their loss of appetite, dizziness, fatigue, irritability, weight loss, lack of concentration, memory loss and other conditions were improved. After taking it for 16 days, 9 people felt that their loss of appetite, dizziness, fatigue, irritability, weight loss, lack of concentration, memory loss and other conditions were significantly improved, and hemoglobin increased by 7g / L. After taking it for 24 days, 12 people felt that their loss of appetite, dizziness, fatigue, irritability, weight loss, lack of concentration, memory loss and other conditions were extremely improved. After taking it for 30 days, 18 people felt that their loss of appetite, dizziness, fatigue, irritability, weight loss, lack of concentration, memory loss and other conditions were completely improved, and the hemoglobin level increased by 20g / L. A total of 2 people did not improve after taking it.

[0148] Pregnant women: 20 people started to collect the results after taking the medicine for 8 days. After taking it for 8 days, 6 people felt that fatigue, difficulty concentrating, dizziness, paleness and other conditions improved. After taking it for 16 days, 10 people felt that fatigue, difficulty concentrating, dizziness, paleness and other conditions were significantly improved, and hemoglobin increased by 10g / L. After taking it for 24 days, 15 people had extremely obvious improvements in fatigue, difficulty concentrating, dizziness, paleness and other conditions. After taking it for 30 days, 19 people had completely improved in fatigue, difficulty concentrating, dizziness, paleness and other conditions, hemoglobin increased by 20g / L, ferritin increased by 18μg / L, and serum iron increased by 6μmol / L. A total of 1 person did not improve after taking it.

[0149] Lactating women: 20 people started taking the medicine for 8 days and the results were counted. After taking it for 8 days, 4 people felt tired, dizzy, headache, tinnitus, palpitations, shortness of breath, loss of appetite, depression, exercise intolerance, pale complexion, increased heart rate and other symptoms improved. After taking it for 16 days, 12 people felt tired, dizzy, headache, tinnitus, palpitations, shortness of breath, loss of appetite, depression, exercise intolerance, pale complexion, increased heart rate and other symptoms improved significantly, and immunity also increased. After taking it for 24 days, 14 people felt tired, dizzy, headache, tinnitus, palpitations, shortness of breath, loss of appetite, depression, exercise intolerance, pale complexion, increased heart rate and other symptoms improved significantly. After taking the medicine for 30 days, 18 people's fatigue, dizziness, headache, tinnitus, palpitations, shortness of breath, loss of appetite, depression, exercise intolerance, pale complexion, increased heart rate and other symptoms were completely improved. A total of 2 people did not improve after taking the medicine.

[0150] 2. Animal model verification experiment of iron supplementation:

[0151] (1) Construction of animal model

[0152] 60 healthy weaned male rats were selected and fed after 4 days of adaptation. The control group (10 rats) were fed with normal feed, and the model group (50 rats) were fed with low-iron feed for 28 days. Every 7 days, tail blood was collected from some rats to measure body weight and hemoglobin (Hb) to determine whether the model was successful. Results Figure 1 , Figure 2 shown.

[0153] according to Figure 1 It can be seen that before the experimental rats were fed with low-iron feed to establish the iron deficiency anemia model (i.e., 0d), the weight of rats in each group was basically the same. As the modeling time increased, the weight of rats in each group increased, but the weight of rats in the modeling group was always lower than that in the control group, which can be used as an auxiliary indicator for the success of the modeling.

[0154] according to Figure 2 It can be seen that the hemoglobin content of the experimental rats in each group was basically the same before modeling (i.e., 0 days). After the modeling group rats were fed with a low-iron diet for 7 days, the hemoglobin content was lower than that of the control group. Taking Hb < 90 g / L as the standard for determining iron deficiency anemia in rats, the hemoglobin content of the modeling group rats was 81.5 ± 3.9 g / L after being fed with a low-iron diet for 28 days, indicating that the modeling was successful.

[0155] During the modeling period, the body shape comparison results of the model group and the control group are shown in Table 3. It can be seen from Table 3 that with the increase of the amount of probiotics, the rats' appetite became better and better, their fur became smoother and smoother, their energy became more and more vigorous, their skin color, ears, claws, eyes, etc. gradually returned to normal, and the color and state of feces returned to normal.

[0156] Table 3 Body shape of rats

[0157]

[0158] (2) Experiments on iron supplementation using animal models

[0159] 40 rats with successful modeling were selected and divided into a model group and an experimental group, with 10 rats in each group. All groups continued to be fed with low-iron feed. The control group and the model group were gavaged with 0.5 mL of deionized water, and the experimental group was gavaged with a postbiotic product solution (i.e., the lyophilized powder prepared in Example 1). The preparation method of the postbiotic product solution was as follows: the postbiotic product was dissolved in 0.5 mL of sterilized water, wherein the mass percentage of the postbiotic product added was 10 mg (experimental group 1), 15 mg (experimental group 2), and 20 mg (experimental group 3). The gavage volume of rats in each group was 10 mL / kg BW. Subsequent experiments were carried out after continuous gavage for 35 days.

[0160] (1) Record the smoothness, density, luster of the fur, mental activity, diet, feces, etc. of each group of rats.

[0161] (2) Determine the body weight changes of rats in each group.

[0162] (3) After being deprived of food for 12 h, the mice were anesthetized with ether and then decapitated. Blood was collected from the neck and divided into two tubes in blood collection tubes containing sodium heparin anticoagulant. An appropriate amount of whole blood was taken from one tube and quickly shaken for the determination of hemoglobin (Hb), red blood cell count (RBC), hematocrit (Hct) and erythrocyte free protoporphyrin (FEP).

[0163] (4) Centrifuge another tube at 4°C, 3000 r / min, for 15 min, take 1 ml of each aliquot and place in micro test tubes, and store at -20°C. Use the kit to measure the contents of serum iron (SI), serum total iron binding capacity (TIBC), serum ferritin (SF), and iron saturation.

[0164] Figure 4 The figure shows the weight changes of rats in each group. Figure 4 It can be seen that the weights of rats in the model group and the experimental group are basically the same, and are significantly lower than those of rats in the control group. After intragastric administration, the final weights of rats in each experimental group were significantly higher than those in the model group and lower than those in the control group. This result shows that iron deficiency can cause a decrease in appetite, thereby slowing down growth rate, and the postbiotic product prepared in Example 1 can effectively increase rat appetite and growth rate by supplementing iron.

[0165] Figure 5 This is a graph showing changes in rat hemoglobin. Hemoglobin is a protein found in red blood cells. Its main function is to transport oxygen to the body's tissues. Iron is an important element of hemoglobin. Iron deficiency anemia may lead to a decrease in hemoglobin. The hemoglobin content reflects the body's iron deficiency. Figure 5It can be seen that the hemoglobin content of the model group showed a negative growth, while both the experimental group and the control group showed a positive growth. After 35 days of intragastric administration of the postbiotic product, the hemoglobin of the rats in the experimental group basically returned to normal, indicating that the postbiotic product prepared in Example 1 can effectively supplement iron.

[0166] Figure 6 is the number of red blood cells of rats in each group; Figure 7 is the hematocrit of rats in each group; Figure 8 is the free protoporphyrin in erythrocytes of rats in each group. Figure 6 , Figure 7 , Figure 8 It can be seen that there is no significant difference in the number of red blood cells in the experimental group 3 rats and the control group. The hematocrit of the experimental group is significantly higher than that of the model group, showing an obvious dose relationship. With the increase of the dose, it increases significantly. The free protoporphyrin in red blood cells of the control group and each experimental group is significantly lower than that of the model group.

[0167] Fig. 9 is the serum iron of rats in each group; Fig.10 is the serum iron binding capacity of rats in each group; Fig.11 is the serum ferritin of rats in each group; Fig.12 is the iron saturation of rats in each group. Fig. 9 , Fig.10 , Fig.11 , Fig.12 It can be seen that the contents of serum iron, serum ferritin and serum iron saturation of the model group animals were significantly lower than those of the control group and the experimental group, while the content of serum iron binding capacity was higher than that of the control group and the experimental group. After gavage with the postbiotic product prepared in Example 1, the contents of serum iron, serum ferritin and serum iron saturation were significantly increased compared with the model group, while the content of serum iron binding capacity was significantly reduced compared with the model group.

[0168] Example 4 - Regulating intestinal flora and promoting digestion

[0169] 1. Experiment on regulating the function of intestinal flora in mice:

[0170] 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 h after the last administration of the postbiotic product, rectal feces were taken, and the intestinal flora was detected as shown in Table 4.

[0171] Table 4 Suitable culture conditions and detection of intestinal flora (x-±SD)

[0172]

[0173] According to Table 4, 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 in mice.

[0174] 2. Mouse gastrointestinal motility experiment

[0175] 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 for gavage once a day while feeding 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) for gavage once a day while feeding normally. After 15 days of gavage, the mice in each group were fasted for 16 hours. After that, the model group and the experimental group were given compound difenoxol for gavage, and the control group was given sterile water for gavage. After 30 minutes, the mice in each group were given Arabic gum ink for gavage. After 30 minutes, the mice were killed by cervical vertebral dissection, and the abdomen was quickly cut 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 intestinal tube length was measured as the "total length of the small intestine", and 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 5.

[0176]

[0177] Table 5 Small intestinal ink propulsion rate (x-±SD)

[0178]

[0179] According to Table 5, 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.

[0180] The above effect verification experiments show that the postbiotic product prepared by the present invention has the functions of supplementing iron, improving iron deficiency anemia, regulating intestinal flora and promoting digestion.

[0181] 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 Lactobacillus reuteri ( Limosilactobacillus reuteri )A-1, 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 GDMCC NO: 64837.

2. A Lactobacillus reuteri according to claim 1 ( Limosilactobacillus reuteri ) A derivative of A-1, characterized in that The derivative is one or more of living bacteria, dead bacteria, and fermentation broth.

3. A microbial agent, characterized in that: comprising an active ingredient, wherein the active ingredient comprises the Lactobacillus reuteri ( Limosilactobacillus reuteri ) A-1 or its inactivated bacteria or fermentation broth.

4. The microbial agent according to claim 3, characterized in that The microbial agent comprises inactivated Lactobacillus reuteri ( Limosilactobacillus reuteri )A-1 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 Lactobacillus reuteri ( Limosilactobacillus reuteri ) A-1 is 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 30-40 h 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 100-150 r / min, the pH is natural, the temperature is 30-40°C, the tank pressure is 0.03-0.06MPa, and the culture time is 10-15h to obtain the seed tank strain; S4, Lactobacillus reuteri 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., 150-200 r / min, tank pressure 0.04-0.06 MPa, pH 6.0-6.5, and ferment for 24-26 hours; cool to 19-21° C., which is the fermentation liquid; heat the fermentation liquid to 119-121° C. and maintain for 4-7 seconds 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: 15-20 parts of anhydrous glucose, 1-2 parts of lactoferrin, 0.1-0.5 parts of beef extract powder, 0.5-1 parts of sodium acetate, 0.4-0.8 parts of Tween-80, 0.2-0.6 parts of potassium dihydrogen phosphate, 900-1000 parts of tap water, and the pH is adjusted to 7.0; In step S2, the composition of the primary seed culture solution is as follows: 2-6 parts of anhydrous glucose, 2-6 parts of bovine bone peptone, 2-6 parts of beef extract powder, 0.1-0.5 parts of D-ribose, 0.5-1 parts of sodium acetate, 0.8-1.2 parts of Tween-80, 900-1000 parts of tap water, and the pH value is natural; In step S3, the composition of the seed tank culture medium is as follows, in terms of weight percentage: 2-6% anhydrous glucose, 0.1-0.5% casein peptone, 0.5-1% dipotassium hydrogen phosphate, 0.2-0.6% sodium acetate, 0.8-1.2% whey peptide, 0.05-0.15% Tween-80, 0.05-0.15% polyether defoamer, 90-95% tap water, and natural pH value; In step S4, the fermentation culture medium is composed of the following, in percentage by weight: 2-6% anhydrous glucose, 1.2-1.6% casein peptone, 2.6-3.2% stachyose, 2-6% wheat germ powder, 2-3% yeast extract powder, 1-2% sodium acetate, 0.1-0.4% Tween-80, 0.5-1.5% sodium caseinate, 0.5-1.5% dipotassium hydrogen phosphate, 0.05-0.15% polyether defoamer, and 80-85% tap water.

7. A Lactobacillus reuteri according to claim 1 ( Limosilactobacillus reuteri ) A-1, or the derivative of Lactobacillus reuteri A-1 according to claim 2, or the microbial agent according to claim 3 or 4 in the preparation of food.

8. A Lactobacillus reuteri according to claim 1 ( Limosilactobacillus reuteri ) A-1, or the Lactobacillus reuteri according to claim 2 ( Limosilactobacillus reuteri ) A derivative of A-1, or the microbial agent according to claim 3 or 4, in the preparation of any of the following products: (1) It helps regulate intestinal flora and aids digestion; (2) Improve iron deficiency anemia; The product is one of a food and a medicine; The food is any one of beverages, dairy products and candy products.

9. A product comprising the Lactobacillus reuteri ( Limosilactobacillus reuteri ) A-1, and / or the Lactobacillus reuteri according to claim 2 ( Limosilactobacillus reuteri ) A-1 derivative, and / or the microbial agent according to claim 3 or 4 and acceptable auxiliary agents or excipients; The product is one of a food and a medicine; The food is any one of beverages, dairy products and candy products.

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

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