Bifidobacterium animalis subspecies lactis 9360-2 and its application in preventing and / or treating cardiovascular and cerebrovascular diseases

Abstract

The present invention discloses an animal Bifidobacterium animal milk subspecies 9360-2 and its application in preventing and / or treating cardiovascular and cerebrovascular diseases, belonging to the technical field of microorganisms and biomedicine. Bifidobacterium animalis subsp.lactis ) 9360‑2, and successfully developed a postbiotic product based on this strain. It has been proven that it has the effect of regulating the balance of intestinal flora, promoting human digestion and absorption, and preventing and / or treating cardiovascular and cerebrovascular diseases. Daily administration of 200 mg of a postbiotic product containing 30 billion strains of the present invention and its metabolites can sustainably provide the endogenous necessary amount of caffeic acid, β‑sitosterol and linoleic acid during the intestinal reproduction and growth process, which can effectively prevent and / or treat cardiovascular and cerebrovascular diseases and maintain the health of the body.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and biomedicine, and specifically relates to Bifidobacterium animalis subspecies lactis 9360-2 and application thereof in preventing and / or treating cardiovascular and cerebrovascular diseases. 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] Cardiovascular and cerebrovascular diseases are characterized by ischemic or hemorrhagic lesions in the brain, heart and systemic tissues, and are one of the diseases that seriously threaten human life and health. Hypertension, diabetes, dyslipidemia, dietary habits, etc. are preventable factors that affect cardiovascular and cerebrovascular diseases.

[0004] Recent studies have shown that long-term supplementation of probiotics can effectively prevent and alleviate cardiovascular and cerebrovascular diseases. This is mainly manifested in the following aspects: Probiotics can lower cholesterol and reduce high blood pressure. Probiotics can prevent or delay the onset of diabetes.

[0005] Patent KR20160100649A discloses a bifidobacterium CBG-C12 that can produce conjugated linoleic acid (CLA). The strain has excellent acid resistance, bile resistance and antibiotic resistance, and can be used to prevent or treat cancer, atherosclerosis, diabetes and obesity. The strain can only produce conjugated linoleic acid, and cannot produce other ingredients that are beneficial to health. Therefore, strains with multiple functions and the ability to produce multiple substances are of great clinical significance. Summary of the invention

[0006] In order to overcome the deficiencies of the prior art, the object of the present invention is to provide an animal Bifidobacterium animal milk subspecies 9360-2 and its use in preventing and / or treating cardiovascular and cerebrovascular diseases. The animal Bifidobacterium animal milk subspecies provided by the present invention has the effect of regulating the balance of intestinal flora and promoting human digestion and absorption, as well as preventing and / or treating cardiovascular and cerebrovascular diseases.

[0007] In order to achieve the above object, the technical solution of the present invention is:

[0008] The first aspect of the present invention provides a Bifidobacterium animalis subsp. Bifidobacterium animalis subsp.lactis )9360-2, deposited in Guangdong Microbiological Culture Collection Center, the deposit date is July 8, 2024, and the deposit number is GDMCC No: 64834.

[0009] The second aspect of the present invention provides a kind of Bifidobacterium animalis subspecies animalis ( Bifidobacterium animalis subsp.lactis ) A derivative of 9360-2, wherein the derivative is one or more of living bacteria, dead bacteria, fermentation broth or metabolites.

[0010] The third aspect of the present invention provides a microbial agent, comprising an active ingredient, wherein the active ingredient comprises the Bifidobacterium animalis subsp. Bifidobacterium animalis subsp.lactis ) 9360-2 or its inactivated bacteria, fermentation broth, or metabolites.

[0011] A fourth aspect of the present invention provides a method for preparing the microbial agent, the method comprising:

[0012] S1, the animal Bifidobacterium animal milk subspecies ( Bifidobacterium animalis subsp.lactis ) 9360-2 was inoculated into the activation medium and cultured at 35-40°C for 15-20 h to obtain the expanded bacterial culture liquid;

[0013] S2, inoculating the expanded bacterial culture liquid obtained in step S1 into the sterile primary seed culture liquid, with an inoculation amount of 0.5-5%, and culturing at 35-40° C. for 30-40 h to obtain the primary bacterial culture liquid;

[0014] S3, inoculating the primary bacterial 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%, stirring culture at 50-100 r / min, natural pH, temperature 30-40°C, tank pressure 0.03-0.06 MPa, and culture time 11-13 h to obtain seed tank bacterial strains;

[0015] S4. 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, 100-150 r / min, tank pressure of 0.04-0.06 MPa, pH of 6.3-6.7, and ferment for 25-35 h; cool to 19-21°C, increase the temperature to 119-121°C and maintain for 4-6 s for inactivation treatment.

[0016] A fifth aspect of the present invention provides a kind of Bifidobacterium animalis subspecies lactis ( Bifidobacterium animalis subsp.lactis ) 9360-2, or the animal Bifidobacterium animal milk subspecies ( Bifidobacterium animalis subsp.lactis ) Use of the derivatives of 9360-2 or the microbial agents in the preparation of products for regulating intestinal flora and promoting digestion;

[0017] The product is a kind of health care product and medicine.

[0018] In a sixth aspect of the present invention, there is provided a kind of Bifidobacterium animalis subspecies lactis ( Bifidobacterium animalis subsp.lactis ) 9360-2, or the animal Bifidobacterium animal milk subspecies ( Bifidobacterium animalis subsp.lactis ) Use of a derivative of 9360-2 or the microbial agent in the preparation of any of the following products:

[0019] (1) Products used to prevent and / or treat cardiovascular and cerebrovascular diseases;

[0020] (2) Products used to continuously provide endogenous caffeic acid, β-sitosterol, and linoleic acid during intestinal reproduction and growth;

[0021] The method for preventing and / or treating cardiovascular and cerebrovascular diseases includes reducing the duration of cerebral ischemia and hypoxia, reducing thrombosis, reducing the rate of ventricular fibrillation, inhibiting platelet aggregation, reducing blood sugar, reducing blood lipids, and inhibiting the expression of TNF-α and / or TNF-1β;

[0022] The product described is a pharmaceutical product.

[0023] A seventh aspect of the present invention provides a product, comprising the Bifidobacterium animalis subsp. Bifidobacterium animalis subsp.lactis ) 9360-2, and / or the Bifidobacterium animalis subsp. Bifidobacterium animalis subsp.lactis ) derivatives of 9360-2, and / or the microbial agents and acceptable adjuvants or excipients;

[0024] The product is one of a health product, a postbiotic product and a medicine.

[0025] The beneficial effects of the present invention are:

[0026] The above technical solution screened and obtained a strain of animal Bifidobacterium animal milk subspecies 9360-2, and a postbiotic product was successfully developed based on the animal Bifidobacterium animal milk subspecies. It has been verified that it has the effect of regulating the balance of intestinal flora and promoting human digestion and absorption, while providing endogenously required caffeic acid, β-sitosterol and linoleic acid during intestinal reproduction and growth, and preventing and / or treating cardiovascular and cerebrovascular diseases. Daily administration of 200 mg of a postbiotic product containing 30 billion strains and metabolites of the present invention can sustainably provide endogenously required amounts of caffeic acid, β-sitosterol and linoleic acid during intestinal reproduction and growth, which can effectively prevent and / or treat cardiovascular and cerebrovascular diseases and maintain the health of the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] 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.

[0028] Figure 1 The metabolomics analysis results of mouse feces in Example 1 of the present invention;

[0029] Figure 2 This is a statistical chart of the number of gasps and gasp duration of each group of mice after cerebral ischemia and hypoxia in Example 1 of the present invention;

[0030] Figure 3 The platelet aggregation rate and inhibition rate of each group of mice in Example 1 of the present invention are statistical graphs;

[0031] Figure 4 This is a statistical graph of blood glucose levels in each group of mice in Example 1 of the present invention;

[0032] Figure 5 This is a statistical graph of blood lipid concentrations of each group of mice in Example 1 of the present invention;

[0033] Figure 6 This is a statistical graph of TNF-α expression levels of each group of cells in Example 1 of the present invention;

[0034] Figure 7 This is a statistical chart of the IL-1β expression levels of each group of cells in Example 1 of the present invention. DETAILED DESCRIPTION

[0035] A first typical embodiment of the present invention provides a Bifidobacterium animalis subsp. Bifidobacterium animalis subsp.lactis )9360-2, deposited in Guangdong Microbial Culture Collection Center, the deposit address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, the deposit date is July 8, 2024, and the deposit number is GDMCC No: 64834.

[0036] The bacteria was isolated from fresh milk (squeezed less than 30 min) and identified as Bifidobacterium animalis subsp. animalis by 16srRNA method. The sequence of the bacteria is as follows:

[0037]

[0038] A second typical embodiment of the present invention provides the above-mentioned Bifidobacterium animalis subspecies animalis ( Bifidobacterium animalis subsp.lactis ) A derivative of 9360-2, wherein the derivative is one or more of living bacteria, dead bacteria, fermentation broth or metabolites.

[0039] A third typical embodiment of the present invention provides a microbial agent, comprising an active ingredient, wherein the active ingredient comprises the above-mentioned Bifidobacterium animalis subspecies animalis ( Bifidobacterium animalis subsp.lactis ) 9360-2 or its inactivated bacteria, fermentation broth, or metabolites.

[0040] In some examples of this embodiment, the microbial agent comprises inactivated Bifidobacterium animalis subspecies animalis and its fermentation broth. The fermentation broth contains extracellular polysaccharides, polypeptides and amino acids. The microbial agent is a postbiotic product.

[0041] In some examples of this embodiment, the microbial agent further comprises a dressing, and the auxiliary material comprises one or more of lactulose, maltodextrin, anhydrous glucose, trehalose, broccoli powder, and L-carnitine.

[0042] A fourth typical embodiment of the present invention provides a method for preparing the microbial agent, the method comprising:

[0043] S1, bacterial activation: the animal Bifidobacterium animalis subsp. Bifidobacterium animalis subsp.lactis ) 9360-2 is inoculated into the activation medium and cultured at 35-40°C (preferably 37°C) for 15-20 h (preferably 16 h) to obtain an expanded bacterial culture solution.

[0044] The activation culture medium is composed of: 10-20 parts of stachyose, 1-5 parts of maltitol, 1-5 parts of beef extract powder, 1-5 parts of sodium acetate, 0.5-0.6 parts of Tween-80, 0.5-1 parts of potassium dihydrogen phosphate, 1000 parts of water, and the pH is adjusted to 7.0.

[0045] More specifically, the activation medium is composed of: 15 parts of stachyose, 3 parts of maltitol, 4 parts of beef extract powder, 1.5 parts of sodium acetate, 0.54 parts of Tween-80, 0.8 parts of potassium dihydrogen phosphate, 1000 parts of water, and the pH is adjusted to 7.0.

[0046] S2. Preparation of primary bacterial seed liquid: inoculate the expanded bacterial seed liquid prepared in step S1 into a sterile primary seed culture liquid with an inoculation amount of 0.5-5% (preferably 0.5%), and culture at 35-40° C. (preferably 37° C.) for 30-40 h (preferably 36 h) to obtain a primary bacterial seed liquid.

[0047] The composition of the primary seed culture solution is as follows: 0.5-1.5 parts of isomaltulose, 1-3 parts of polydextrose, 3-7 parts of peptone, 1-3 parts of dextran, 1-5 parts of beef extract powder, 1-3 parts of arginine, 0.5-1.5 parts of sodium acetate, 0.5-1.5 parts of vitamin A, 1-1.1 parts of Tween-80, 1000 parts of water, and the pH value is natural.

[0048] More specifically, the composition of the primary seed culture solution is as follows: 1 part of isomaltulose, 2 parts of polydextrose, 5 parts of peptone, 2 parts of dextran, 4 parts of beef extract powder, 2 parts of arginine, 1 part of sodium acetate, 1 part of vitamin A, 1.08 parts of Tween-80, 1000 parts of water, and the pH value is natural.

[0049] 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%), stirring culture at 50-100 r / min (preferably 80 r / min), natural pH, temperature 30-40°C (preferably 37°C), tank pressure 0.03-0.06 MPa (preferably 0.05 MPa), and culture time 11-13 h (preferably 12 h) to obtain seed tank strains.

[0050] The composition of the seed tank culture medium is as follows: 1-5% isomaltulose, 0.5-1.4% casein peptone, 0.2-0.6% oligofructose, 0.2-0.6% dipotassium hydrogen phosphate, 0.5-1.0% sodium acetate, 0.05-0.15% arginine, 0.05-0.15% Tween-80, 0.05-0.15% polyether defoamer, 94-96% water, and natural pH value.

[0051] More specifically, the composition of the seed tank culture medium is as follows: 3% isomaltulose, 0.9% casein peptone, 0.4% oligofructose, 0.4% dipotassium hydrogen phosphate, 0.8% sodium acetate, 0.1% arginine, 0.1% Tween-80, 0.1% polyether defoamer, 94.2% water, and natural pH.

[0052] S4. Fermentation of Bifidobacterium animalis subsp. animalis: inoculate the seed tank strain into a fermentation tank containing sterilized fermentation culture medium, with an inoculation amount of 0.5-7% (preferably 6%), 35-39°C (preferably 37°C), 100-150 r / min (preferably 140 r / min), tank pressure of 0.04-0.06 MPa (preferably 0.05 MPa), pH of 6.3-6.7 (preferably 6.5), and fermentation for 25-35 h (preferably 30 h); cool to 19-21°C (preferably 20°C), increase the temperature to 119-121°C (preferably 120°C) and maintain for 4-6 s (preferably 6 s) for UHT inactivation treatment.

[0053] The fermentation culture solution is composed of: 1-5% anhydrous glucose, 0.5-1.0% casein peptone, 0.5-1.5% cane molasses, 1-2% isomaltooligosaccharide, 1-2% stachyose, 0.5-1.5% yeast extract powder, 0.1-0.5% sodium acetate, 0.1-0.3% Tween-80, 0.5-1.5% whey protein powder, 0.5-1.0% dipotassium hydrogen phosphate, 0.05-0.15% polyether defoamer, and 87-90% water.

[0054] More specifically, the fermentation broth is composed of: 3% anhydrous glucose, 0.7% casein peptone, 1% cane molasses, 1.5% isomaltooligosaccharide, 1.8% stachyose, 1% yeast extract, 0.3% sodium acetate, 0.2% Tween-80, 1% whey protein powder, 0.9% dipotassium hydrogen phosphate, 0.1% polyether defoamer, and 88.5% water.

[0055] 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.

[0056] A fifth typical embodiment of the present invention provides a Bifidobacterium animalis subspecies lactis animalis ( Bifidobacterium animalis subsp.lactis ) 9360-2, or the animal Bifidobacterium animal milk subspecies ( Bifidobacterium animalis subsp.lactis ) Use of the derivatives of 9360-2 or the microbial agents in the preparation of products for regulating intestinal flora and promoting digestion;

[0057] The product is a kind of health care product and medicine.

[0058] A sixth typical embodiment of the present invention provides a Bifidobacterium animalis subspecies lactis ( Bifidobacterium animalis subsp.lactis ) 9360-2, or the animal Bifidobacterium animal milk subspecies ( Bifidobacterium animalis subsp.lactis) Use of a derivative of 9360-2 or the microbial agent in the preparation of any of the following products:

[0059] (1) Products used to prevent and / or treat cardiovascular and cerebrovascular diseases;

[0060] (2) Products used to continuously provide endogenous caffeic acid, β-sitosterol and linoleic acid during intestinal reproduction and growth.

[0061] In some examples of this embodiment, the method for preventing and / or treating cardiovascular and cerebrovascular diseases includes reducing the time of cerebral ischemia and hypoxia, reducing thrombosis, reducing ventricular fibrillation rate, inhibiting platelet aggregation, lowering blood sugar, lowering blood lipids, and inhibiting the expression of TNF-α and / or TNF-1β.

[0062] In some examples of this embodiment, the product is a pharmaceutical product.

[0063] The caffeic acid, β-sitosterol and linoleic acid have the following effects respectively:

[0064] Caffeic acid exhibits circoprotective effects by inhibiting oxidative stress, leukocyte infiltration, platelet aggregation, platelet-leukocyte interaction, vascular remodeling and apoptosis, as well as regulating glucose and lipid metabolism and vasodilation in the cardiovascular system.

[0065] β-Sitosterol can improve blood lipids by reducing the levels of triglycerides (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) while increasing the level of high-density lipoprotein cholesterol (HDL-C), thereby having an anti-atherosclerotic effect.

[0066] Linoleic acid is an essential fatty acid for the human body. In the human body, linoleic acid can be metabolized into arachidonic acid, which can lower blood cholesterol and reduce the occurrence of coronary heart disease. Linoleic acid can be further metabolized into carboxylic acid, which has the effects of lowering blood lipids, preventing coronary atherosclerosis and reducing cardiac tissue ischemia, and can ensure the heat and nutritional needs of brain cells.

[0067] A seventh typical embodiment of the present invention provides a product, comprising the Bifidobacterium animalis subsp. Bifidobacterium animalis subsp.lactis ) 9360-2, and / or the Bifidobacterium animalis subsp. Bifidobacterium animalis subsp.lactis ) derivatives of 9360-2, and / or the microbial agent and acceptable adjuvants or excipients.

[0068] In some examples of this embodiment, the product is one of a health product, a postbiotic product, and a medicine.

[0069] In some examples of this embodiment, the auxiliary agent or adjuvant can be a carrier, excipient, diluent, etc. commonly used in pharmacy. Moreover, according to the usual method, it can be made into an oral preparation in the form of powder, granule, suspension, emulsion, syrup, spray, etc.

[0070] The non-drug active ingredients such as carriers, excipients and diluents that may be included are well known in the art, and those skilled in the art can determine whether they meet clinical standards.

[0071] In another specific embodiment of the present invention, the carrier, excipient and diluent include but are not limited to lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0072] 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.

[0073] 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 .

[0074] Example 1

[0075] A method for preparing a postbiotic product for preventing and / or treating cardiovascular and cerebrovascular diseases comprises the following steps:

[0076] 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.

[0077] (1) Bacteria activation:

[0078] Bacterial strain culture: Expansion culture of bacterial strains preserved in -80℃ refrigerator.

[0079] Preparation of culture medium: 15.0 g stachyose, 3.0 g maltitol, 4.0 g beef extract powder, 1.5 g sodium acetate, 0.5 mL Tween-80, 0.8 g potassium dihydrogen phosphate, 1000 mL tap water, pH adjusted to 7.0 (alkaline solution adjustment).

[0080] Operation: Add the stachyose and other raw materials in the above formula into 1000 mL of water in the formula without any particular order, stir for 10 min to dissolve, and then divide into test tubes, each test tube contains 5 mL, seal with kraft paper and place in a medical sterilizer for sterilization at 0.1-0.12 MPa for 20 min. The culture medium is ready.

[0081] Inoculation: Under sterile conditions (clean bench), open the -80℃ low-temperature freezer to store the frozen tube of bacteria, and dissolve it at room temperature. Then take one sterilized test tube culture medium, add 0.5 mL of the dissolved bacterial solution into the test tube, seal the tube mouth with a rubber stopper and kraft paper, shake it gently by hand, and place it in a constant temperature incubator at 37℃ for 16 hours. This is called expansion culture medium.

[0082] (2) Preparation of primary bacterial culture solution:

[0083] Primary seed culture formula: 1 g isomaltulose, 2 g polydextrose, 5 g peptone, 2 g dextran, 4 g beef extract powder, 2 g arginine, 1 g sodium acetate, 1 g vitamin A, 1 mL Tween-80, 1000 mL tap water, natural pH.

[0084] Operation: Add the above raw materials into 1000 mL of tap water without any distinction, start the blender, and stir for 10 minutes to dissolve them all. This is called the primary seed culture medium.

[0085] Add the stirred and dissolved first-level seed culture solution into the conical flask according to the dosage, then wrap the bottle mouth with 6 layers of gauze and one layer of kraft paper, and 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 0.05 MPa, open the exhaust valve on the sterilizer to exhaust for 6-8 minutes, then close the exhaust valve and continue heating. When the steam pressure in the sterilizer reaches 0.1-0.12 MPa, start timing. The sterilization time is 20 minutes. After completion, leave the heat source 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 for natural cooling. When the temperature drops to 37°C, transfer the above cultured expansion culture solution to the conical flask containing the sterilized culture solution. The inoculation amount is 0.5%, that is, 100 mL of first-level seed culture solution and 0.5 mL of expansion culture solution. mL. After completion, seal the bottle mouth with the original gauze and kraft paper, shake it slightly by hand to mix the inoculated bacteria and culture solution evenly, and then place it in a constant temperature box and culture it at 37°C for 36 hours. This is called the primary culture solution.

[0086] (3) Preparation of seed tank strains:

[0087] Sterilization of fermentation equipment, pipelines and sterile filtration systems:

[0088] First, open the valves of all inlet and outlet pipelines and sterile air pipelines, and pass 0.12-0.14 MPa 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.

[0089] Sterilization of empty seed tanks and fermentation tanks:

[0090] 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 0.12-0.14 MPa steam into the tank, start timing when the temperature in the tank reaches 121°C, the sterilization time is 40 min, 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°C for standby use.

[0091] Formula: 3% isomaltulose, 0.9% casein peptone, 0.4% fructooligosaccharide, 0.4% dipotassium hydrogen phosphate, 0.8% sodium acetate, 0.1% arginine, 0.1% Tween-80, 0.1% polyether defoamer, 94.2% tap water, natural pH value.

[0092] 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 80 r / min, tank pressure 0.05 MPa, and culture time 12 h 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.

[0093] (4) Fermentation of Bifidobacterium animalis subsp. animalis:

[0094] Formula: 3% anhydrous glucose, 0.7% casein peptone, 1% cane molasses, 1.5% isomaltooligosaccharide, 1.8% stachyose, 1% yeast extract powder, 0.3% sodium acetate, 0.2% Tween-80, 1% whey protein powder, 0.9% dipotassium hydrogen phosphate, 0.1% polyether defoamer, and 88.5% tap water.

[0095] 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 strain. Before inoculation, first increase the tank pressure of the seed tank to 0.1 MPa, 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.

[0096] 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 140 r / min, tank pressure maintained at 0.05 MPa, and the pH value is maintained at 6.5 through the acid and alkali automatic control system during the fermentation process. If the tank pressure decreases, sterile air can be introduced to maintain the tank pressure until the fermentation is completed. The total fermentation is 30 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 s.

[0097] (5) Centrifugation of fermentation broth:

[0098] The fermentation liquid is pumped into a storage tank for centrifugation. Operation: Start the centrifuge, adjust the speed to 10000 r / min, centrifuge at a feed rate of 2kg / min, collect the inactivated wet bacterial sludge metabolites after centrifugation, and freeze-dry the collected inactivated wet bacterial sludge metabolites.

[0099] (6) Freeze-drying of bacterial sludge metabolites:

[0100] Preparation of bacterial suspension: Take 1000 g of inactivated wet bacterial sludge metabolites, add 4000 mL of sterile saline, 800 g of trehalose, 2000 g of oat bran powder, 20 g of manganese sulfate, and 80 g of zinc sulfate.

[0101] Operation: First, take 2500 mL of normal saline in the formula, add trehalose and stir to dissolve, then add 1000 g of inactivated wet bacterial sludge metabolites, and then add the remaining normal saline and oat bran powder and auxiliary materials in the formula and stir thoroughly to obtain a suspension. The suspension is freeze-dried and the moisture content is finally controlled to 4% to obtain the freeze-dried powder of inactivated bacterial metabolites, i.e., the postbiotic product.

[0102] After testing, the freeze-dried powder obtained by Example 1 had an extracellular polysaccharide content of 29%, a polypeptide content of 6%, an amino acid content of 28%, and a bacterial cell content of 150 billion / g.

[0103] 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.

[0104] The freeze-dried powder prepared by the above method was subjected to the following effect verification.

[0105] 1. Metabolomics analysis:

[0106] Metabolomics analysis of mouse feces was performed using ultra-performance liquid chromatography-tandem mass spectrometry.

[0107] 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 (20 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, fecal samples of 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, extracted with low-temperature ultrasound for 30 min (5°C, 40 KHz), left to stand at -20°C for 30 min, 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 with low-temperature ultrasound 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 mmi.d. 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 to obtain the main metabolite information as shown in Table 1 and Figure 1 shown.

[0108] Caffeic acid content determination method: HPLC;

[0109] β-Sitosterol content detection method: liquid chromatography tandem mass spectrometry;

[0110] Linoleic acid content detection method: high performance liquid chromatography.

[0111] Table 1 Main metabolomics analysis of mice

[0112]

[0113] As can be seen from Table 1, daily administration of 20 mg of a postbiotic product containing approximately 3 billion inactivated bacterial cells and metabolites to mice can significantly increase the content of caffeic acid, β-sitosterol and linoleic acid in the feces of the mice, indicating that the postbiotic product provided by the present invention can sustainably provide the endogenously required amount of caffeic acid, β-sitosterol and linoleic acid during the intestinal reproduction and growth process.

[0114] 2. Regulate intestinal flora and promote digestion

[0115] (1) Experiment on regulating the function of mouse intestinal flora:

[0116] 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 (20 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 2.

[0117] Table 2 Suitable culture conditions and detection of intestinal flora (x-±SD)

[0118]

[0119] According to Table 2, 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 have the function of regulating the intestinal flora of mice.

[0120] (2) Mouse gastrointestinal motility experiment

[0121] The mice were divided into a control group, a model group and an experimental group, with 10 mice in each group. The control group and the model group were given 0.5 mL of sterile water by gavage once a day while feeding normally; the experimental group was given 0.5 mL of postbiotic product solution (20 mg of the postbiotic product prepared in Example 1 dissolved in 0.5 mL of sterile water) by 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 by gavage, and the control group was given sterile water by gavage. After 30 minutes, the mice in each group were given Arabic gum ink by 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 3.

[0122]

[0123] Table 3 Small intestinal ink propulsion rate (x-±SD)

[0124]

[0125] As shown in Table 3, the intestinal motility inhibition model of mice was established by administering compound difenocool. Compared with the model group, the small intestinal ink propulsion rate increased in the experimental group, indicating that postbiotic products can promote small intestinal motility and digestion.

[0126] 3. Prevention and / or treatment of cardiovascular and cerebrovascular diseases

[0127] (1) Effects on cerebral ischemia and hypoxia in mice

[0128] The mice were divided into a control group, a model group, an experimental group 1, an experimental group 2, and an experimental group 3. The control group and the model group were gavaged with 0.5 mL of normal saline while being fed normally, and the experimental group was gavaged with 0.5 mL of the postbiotic product solution while being fed normally, once a day. The preparation method of the postbiotic product solution is as follows: the postbiotic product prepared in Example 1 is dissolved in 0.5 mL of sterilized water to obtain a postbiotic product solution. The added mass of the postbiotic product is 15 mg (experimental group 1), 20 mg (experimental group 2), and 25 mg (experimental group 3), respectively.

[0129] After 7 days of feeding, 30 minutes after the last oral administration, the mice were quickly decapitated behind the ears with scissors, and the duration of open-mouthed panting of the mice in each group was recorded as the duration of cerebral ischemia and hypoxia.

[0130] Results Figure 2After gavage with postbiotic products, the number and duration of gasping in mice increased, indicating that postbiotic products can reduce the duration of cerebral ischemia and hypoxia and reduce the harm caused by cerebral ischemia and hypoxia.

[0131] (2) Effects on thrombosis in mice

[0132] The mice were divided into a control group, a model group, an experimental group 1, an experimental group 2, and an experimental group 3. The control group and the model group were gavaged with 0.5 mL of normal saline while being fed normally, and the experimental group was gavaged with 0.5 mL of the postbiotic product solution while being fed normally, once a day. The preparation method of the postbiotic product solution is as follows: the postbiotic product prepared in Example 1 is dissolved in 0.5 mL of sterilized water to obtain a postbiotic product solution. The added mass of the postbiotic product is 15 mg (experimental group 1), 20 mg (experimental group 2), and 25 mg (experimental group 3), respectively.

[0133] After 7 days of feeding, 30 min after the last oral administration, the mice in the model group, experimental group 1, experimental group 2, and experimental group 3 were intravenously injected with an inducer made of collagen (430 μg / kg)-epinephrine (20 μg / kg), and the death of animals in each group within 15 min was observed and recorded.

[0134] Table 4 Effects on thrombosis in mice

[0135]

[0136] According to Table 4, the number of deaths of mice gavaged with postbiotic products was significantly lower than that of the model group, while the number of survivals increased, and the survival rate increased accordingly, indicating that postbiotic products can significantly counteract collagen-epinephrine-induced thrombosis in mice and significantly reduce the mortality rate of animals.

[0137] (3) Effects on chloroform-induced cardiac fibrillation in mice

[0138] The mice were divided into a control group, a model group, an experimental group 1, an experimental group 2, and an experimental group 3. The control group and the model group were gavaged with 0.5 mL of normal saline while being fed normally, and the experimental group was gavaged with 0.5 mL of the postbiotic product solution while being fed normally, once a day. The preparation method of the postbiotic product solution is as follows: the postbiotic product prepared in Example 1 is dissolved in 0.5 mL of sterilized water to obtain a postbiotic product solution. The added mass of the postbiotic product is 15 mg (experimental group 1), 20 mg (experimental group 2), and 25 mg (experimental group 3), respectively.

[0139] After 7 days of feeding, 30 min after the last gavage, the mice in the model group, experimental group 1, experimental group 2, and experimental group 3 were placed one by one in an inverted glass beaker containing chloroform cotton balls. After the animals stopped breathing, the chest was immediately opened, and the incidence of ventricular fibrillation of mice in each group was observed and recorded with the naked eye.

[0140] Table 5 Effects on cardiac arrhythmia in mice

[0141]

[0142] According to Table 5, the number of positive mice gavaged with postbiotic products was significantly lower than that of the model group, while the number of negative mice increased. Correspondingly, the ventricular fibrillation rate was significantly reduced, indicating that the postbiotic products have an antagonistic effect on chloroform-induced atrial fibrillation and have the effect of antiarrhythmic drugs.

[0143] (4) Effects on platelet aggregation in mice

[0144] The mice were divided into a control group, a model group, an experimental group 1, an experimental group 2, and an experimental group 3. The control group and the model group were gavaged with 0.5 mL of normal saline while being fed normally, and the experimental group was gavaged with 0.5 mL of the postbiotic product solution while being fed normally, once a day for 7 days. The preparation method of the postbiotic product solution is as follows: the postbiotic product prepared in Example 1 is dissolved in 0.5 mL of sterilized water to obtain a postbiotic product solution. The added mass of the postbiotic product is 15 mg (experimental group 1), 20 mg (experimental group 2), and 25 mg (experimental group 3), respectively.

[0145] After the experiment, the mice were anesthetized with 3.5% chloral hydrate, and blood was collected from the abdominal aorta and placed in a centrifuge tube containing sodium citrate solution (3.5wt%) (1:9 with blood volume). The anticoagulated blood sample was centrifuged at 600 r / min for 15 min, and the upper layer was taken to obtain platelet-rich plasma. The remaining blood was centrifuged at 3000 r / min for 10 min, and the upper layer was taken to obtain platelet-poor plasma, which was stored at room temperature and completed within 2 h. 500 μL of platelet-rich plasma and platelet-poor plasma from each mouse in each group were accurately drawn into the test cup, incubated in a preheated bath at 37°C for 5 min, and 5 μL of the inducer ADP solution was added. The maximum platelet aggregation rate within 5 min was measured on a platelet aggregation coagulation factor analyzer according to the instrument manual.

[0146] according to Figure 3 It can be seen that compared with the control group, postbiotic products can significantly inhibit platelet aggregation in mice in a dose-dependent manner, indicating that postbiotic products can inhibit platelet aggregation and lead to thrombosis, thereby causing cardiovascular diseases.

[0147] (5) Blood glucose testing

[0148] The mice were divided into a control group, a model group, an experimental group 1, an experimental group 2, and an experimental group 3. After fasting for 24 h, the mice in the model group and the experimental group were intraperitoneally injected with 60 mg / kg of streptozotocin (STZ), and the control group was intraperitoneally injected with an equal volume of 0.1 mM citric acid solution. After 7 days of feeding, gavage began. The control group and the model group were gavaged with 0.5 mL of normal saline while feeding normally, and the experimental group was gavaged with 0.5 mL of postbiotic product solution while feeding normally, once a day, for 30 days. Among them, the preparation method of the postbiotic product solution is: dissolving the postbiotic product prepared in Example 1 in 0.5 mL of sterilized water to obtain a postbiotic product solution. Among them, the added mass of the postbiotic product was 15 mg (experimental group 1), 20 mg (experimental group 2), and 25 mg (experimental group 3), respectively. After fasting for 16 hours, the mice were anesthetized to take blood from the tail tip, and blood glucose was tested with a blood glucose meter.

[0149] according to Figure 4 It can be seen that the blood glucose content of the model group is much higher than that of the control group, indicating that the hyperglycemia model has been successfully constructed. The blood glucose concentration of the experimental group is significantly lower than that of the model group, and is dose-dependent, indicating that postbiotic products can control the blood glucose of mice and prevent and treat diseases such as hyperglycemia.

[0150] (6) Blood lipid test

[0151] The mice were divided into control group, model group, experimental group 1, experimental group 2, and experimental group 3. The mice in the control group were fed with ordinary feed, and the mice in the model group and experimental group were fed with high-fat feed for 30 days. Blood was drawn once a week, and food but water was not allowed for 12 hours before blood collection. The blood samples were centrifuged at 4℃ for 10 min, 3000 r / min, and the upper serum was taken for TC, TG, HDL-C, and LDL-C determination. Statistical analysis was performed using statistical software. The control group was compared with the model group and the experimental group, and P<0.05, indicating successful modeling.

[0152] High-fat feed configuration: 78.8% basic feed, 10% yellow powder, 10% pig egg oil, 1% edible grade cholesterol, 0.2% pig bile salt (cholate). Basic feed can be obtained from the market or made by yourself. The composition ratio is: 25% corn flour, 25% oatmeal, 25% flour, 10% soybean meal, 7% fish meal, 4% bone meal, 2% yeast powder, 1% salt, 1% vitamin. First, heat the lard slightly to make it liquid, add egg yolk powder, cholesterol, pig bile salt and stir evenly, then add it to the good basic feed and stir evenly. During this period, add a small amount of water to make the feed shape.

[0153] After the hyperlipidemia model was successfully constructed, gavage was started. The control group and the model group were gavaged with 0.5 mL of normal saline while feeding normally, and the experimental group was gavaged with 0.5 mL of postbiotic product solution while feeding normally, once a day, for 30 days. Among them, the preparation method of the postbiotic product solution is: dissolve the postbiotic product prepared in Example 1 in 0.5 mL of sterilized water to obtain a postbiotic product solution. Among them, the added mass of the postbiotic product was 15 mg (experimental group 1), 20 mg (experimental group 2), and 25 mg (experimental group 3), respectively. Take the mouse heart blood, water bath at 37°C for 5 min, centrifuge at 4000 rpm for 5 min, take the supernatant, and use an automatic biochemical analyzer to detect the levels of TC, TG, LDL-C and HDL-C in serum.

[0154] according to Figure 5 It can be seen that the blood lipids (TC, TG, LDC-C) of mice gavaged with postbiotic products were significantly lower than those of the model group mice, and HDL-C was significantly higher than that of the model group mice, indicating that postbiotic products can improve blood lipids.

[0155] (7) Effects on pro-inflammatory mediators in mouse macrophages

[0156] RAW264.7 cells with good growth status were digested and counted, with a concentration of 2×10 5 / mL of cell density was inoculated in a 24-well plate, 1 mL per well. It was divided into a control group, a model group, an experimental group 1, an experimental group 2, and an experimental group 3. After culture in an incubator, 0.5mL of 100 ng / mL LPS was added to the model group and the experimental group after adherence, and at the same time, 0.5 mL of postbiotic product solution was added to the experimental group. Among them, the preparation method of the postbiotic product solution is: dissolve the postbiotic product prepared in Example 1 in 0.5 mL of sterilized water to obtain a postbiotic product solution. Among them, the added mass of the postbiotic product is 15 mg (experimental group 1), 20 mg (experimental group 2), and 25 mg (experimental group 3), respectively.

[0157] according to Figure 6 , Figure 7 It can be seen that compared with the model group, the levels of TNF-α and IL-1β in the experimental group were significantly reduced, indicating that postbiotic products can inhibit the expression of TNF-α and IL-1β and have a certain anti-inflammatory effect.

[0158] Through the above experiments, it can be known that the postbiotic product provided in Example 1 of the present invention can reduce the time of cerebral ischemia and hypoxia, fight thrombosis, fight atrial fibrillation, inhibit platelet aggregation, lower blood sugar and blood lipids, and has anti-inflammatory effects. Therefore, it can be used to prevent and / or treat cardiovascular and cerebrovascular diseases.

[0159] (8) Small-scale clinical trials

[0160] The subjects were people with cardiovascular and cerebrovascular diseases in five communities in Jinan City with an average age of 48 years old. As of June 2009, a total of 120 people were recruited and participated in the experiment after stopping the medication for 3 months. All the above-mentioned people signed a written informed consent form. If the participant was unable to sign, the agent (usually a family member) signed it. Take 0.2 grams of the postbiotic product prepared in Example 1 once a day before going to bed.

[0161] Table 6 Effect of application in the treatment of cardiovascular and cerebrovascular diseases

[0162]

[0163] Note: The placebo in Table 6 is maltodextrin.

[0164] Note: Cardiovascular disease: 30 people started to collect the results after taking it for 8 days. After taking it for 8 days, 8 people felt that palpitations, shortness of breath, chest tightness, dyspnea, edema, cyanosis, syncope, coughing and coughing up blood, weakness, belching, upper abdominal pain, nausea, vomiting, left back pain, left arm pain, etc. were improved. After taking it for 16 days, 15 people felt palpitations, shortness of breath, chest tightness, dyspnea, edema, cyanosis, syncope, coughing and coughing up blood, weakness, belching, upper abdominal pain, nausea, vomiting, left back pain, left arm pain, etc. were significantly improved. After taking it for 24 days, 22 people felt that palpitations, shortness of breath, chest tightness, dyspnea, edema, cyanosis, syncope, coughing and coughing up blood, weakness, belching, upper abdominal pain, nausea, vomiting, left back pain, left arm pain, etc. were extremely improved. After taking the medicine for 30 days, 28 people had completely improved their palpitations, shortness of breath, chest discomfort, dyspnea, edema, cyanosis, syncope, coughing and hemoptysis, weakness, belching, upper abdominal pain, nausea, vomiting, left back pain, left arm pain, etc. A total of 2 people had no improvement in cardiovascular disease after taking the medicine.

[0165] Cerebrovascular disease: 30 people started to collect the results after taking the medicine for 8 days. After taking it for 8 days, 6 people had improvement in hemianopsia, aphasia, nystagmus, dysphagia, ataxia, dizziness, limb weakness, numbness, language disorder, memory loss, urinary incontinence, imbalance, impaired consciousness, headache or nausea and vomiting, dizziness, tinnitus, etc. After taking it for 16 days, 12 people had significant improvement in hemianopsia, aphasia, nystagmus, dysphagia, ataxia, dizziness, limb weakness, numbness, language disorder, memory loss, urinary incontinence, imbalance, impaired consciousness, headache or nausea and vomiting, dizziness, tinnitus, etc. After taking it for 24 days, 20 people had extremely significant improvement in hemianopsia, aphasia, nystagmus, dysphagia, ataxia, dizziness, limb weakness, numbness, language disorder, memory loss, urinary incontinence, imbalance, impaired consciousness, headache or nausea and vomiting, dizziness, tinnitus, etc. After taking the medicine for 30 days, 27 people's sensory hemianopsia, aphasia, nystagmus, dysphagia, ataxia, vertigo, limb weakness, numbness, language disorder, memory loss, urinary incontinence, imbalance, consciousness disorder, headache or nausea and vomiting, dizziness, tinnitus, etc. were completely improved. A total of 3 people's cerebrovascular diseases did not improve after taking the medicine.

[0166] 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 Bifidobacterium animalis subsp. animalis ( Bifidobacterium animalis subsp.lactis )9360-2, deposited in Guangdong Microbiological Culture Collection Center, the deposit date is July 8, 2024, and the deposit number is GDMCC No: 64834.

2. A Bifidobacterium animalis subsp. animalis described in claim 1 ( Bifidobacterium animalis subsp.lactis ) A derivative of 9360-2, characterized in that The derivative is living bacteria or its fermentation broth.

3. A microbial agent, characterized in that: The active ingredient comprises the Bifidobacterium animalis subspecies lactis of claim 1. Bifidobacterium animalis subsp.lactis )9360-2 or its fermentation broth.

4. The microbial agent according to claim 3, characterized in that The microbial agent comprises inactivated Bifidobacterium animalis subspecies lactis ( Bifidobacterium animalis subsp.lactis )9360-2 and its fermentation broth.

5. A method for preparing the microbial agent according to claim 3 or 4, characterized in that: The preparation method comprises: S1, the animal Bifidobacterium animal milk subspecies ( Bifidobacterium animalis subsp.lactis ) 9360-2 was inoculated into the activation medium and cultured at 35-40°C for 15-20 h to obtain the expanded bacterial culture liquid; S2, inoculating the expanded bacterial culture liquid obtained in step S1 into the sterile primary seed culture liquid, with an inoculation amount of 0.5-5%, and culturing at 35-40° C. for 30-40 h to obtain the primary bacterial culture liquid; S3, inoculating the primary bacterial 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%, stirring culture at 50-100 r / min, natural pH, temperature 30-40°C, tank pressure 0.03-0.06 MPa, and culture time 11-13 h to obtain seed tank bacterial strains; S4. 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, 100-150 r / min, tank pressure of 0.04-0.06 MPa, pH of 6.3-6.7, and ferment for 25-35 h; cool to 19-21°C, increase the temperature to 119-121°C and maintain for 4-6 s for 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: 10-20 parts of stachyose, 1-5 parts of maltitol, 1-5 parts of beef extract powder, 1-5 parts of sodium acetate, 0.5-0.6 parts of Tween-80, 0.5-1 parts of potassium dihydrogen phosphate, 1000 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: 0.5-1.5 parts of isomaltulose, 1-3 parts of polydextrose, 3-7 parts of peptone, 1-3 parts of dextran, 1-5 parts of beef extract powder, 1-3 parts of arginine, 0.5-1.5 parts of sodium acetate, 0.5-1.5 parts of vitamin A, 1-1.1 parts of Tween-80, 1000 parts of water, and the pH value is natural; In step S3, the composition of the seed tank culture medium is as follows: 1-5% isomaltulose, 0.5-1.4% casein peptone, 0.2-0.6% fructooligosaccharide, 0.2-0.6% dipotassium hydrogen phosphate, 0.5-1.0% sodium acetate, 0.05-0.15% arginine, 0.05-0.15% Tween-80, 0.05-0.15% polyether defoamer, 94-96% water, and natural pH value; In step S4, the fermentation culture medium is composed of: 1-5% anhydrous glucose, 0.5-1.0% casein peptone, 0.5-1.5% cane molasses, 1-2% isomaltooligosaccharide, 1-2% stachyose, 0.5-1.5% yeast extract powder, 0.1-0.5% sodium acetate, 0.1-0.3% Tween-80, 0.5-1.5% whey protein powder, 0.5-1.0% dipotassium hydrogen phosphate, 0.05-0.15% polyether defoamer, and 87-90% water.

7. The preparation method according to claim 6, characterized in that: In step S1, the composition of the activation medium is as follows: 15 parts of stachyose, 3 parts of maltitol, 4 parts of beef extract powder, 1.5 parts of sodium acetate, 0.54 parts of Tween-80, 0.8 parts of potassium dihydrogen phosphate, 1000 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: 1 part of isomaltulose, 2 parts of polydextrose, 5 parts of peptone, 2 parts of dextran, 4 parts of beef extract powder, 2 parts of arginine, 1 part of sodium acetate, 1 part of vitamin A, 1.08 parts of Tween-80, 1000 parts of water, and the pH value is natural; In step S3, the composition of the seed tank culture medium is as follows: 3% isomaltulose, 0.9% casein peptone, 0.4% oligofructose, 0.4% dipotassium hydrogen phosphate, 0.8% sodium acetate, 0.1% arginine, 0.1% Tween-80, 0.1% polyether defoamer, 94.2% water, and natural pH value; In step S4, the fermentation culture medium is composed of: 3% anhydrous glucose, 0.7% casein peptone, 1% cane molasses, 1.5% isomaltooligosaccharide, 1.8% stachyose, 1% yeast extract, 0.3% sodium acetate, 0.2% Tween-80, 1% whey protein powder, 0.9% dipotassium hydrogen phosphate, 0.1% polyether defoamer, and 88.5% water.

8. A Bifidobacterium animalis subsp. lactis of claim 1 ( Bifidobacterium animalis subsp.lactis ) 9360-2, or the animal Bifidobacterium animal milk subspecies described in claim 2 ( Bifidobacterium animalis subsp. lactis ) Use of a derivative of 9360-2 or a microbial agent as claimed in claim 3 or 4 in the preparation of a product for regulating intestinal flora and promoting digestion; The product is a kind of health care product and medicine.

9. A Bifidobacterium animalis subsp. animalis lactis according to claim 1 ( Bifidobacterium animalis subsp.lactis ) 9360-2, or the animal Bifidobacterium animal milk subspecies described in claim 2 ( Bifidobacterium animalis subsp. lactis ) A derivative of 9360-2, or a microbial agent according to claim 3 or 4, in the preparation of any of the following products: (1) Products used to prevent and / or treat cardiovascular and cerebrovascular diseases; (2) Products used to continuously provide endogenous caffeic acid, β-sitosterol, and linoleic acid during intestinal reproduction and growth; The method for preventing and / or treating cardiovascular and cerebrovascular diseases includes reducing the duration of cerebral ischemia and hypoxia, reducing thrombosis, reducing the rate of ventricular fibrillation, inhibiting platelet aggregation, reducing blood sugar, reducing blood lipids, and inhibiting the expression of TNF-α and / or TNF-1β; The product described is a pharmaceutical product.

10. A product, characterized in that The Bifidobacterium animalis subspecies lactis of claim 1 Bifidobacterium animalis subsp.lactis ) 9360-2, and / or the animal Bifidobacterium animalis subsp. lactis described in claim 2 ( Bifidobacterium animalis subsp.lactis ) derivatives of 9360-2, and / or the microbial agent according to claim 3 or 4 and acceptable adjuvants or excipients; The product is one of a health product, a postbiotic product and a medicine.

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