Bifidobacterium longum subsp. infantis YLGB-1496 bacterial preparation, preparation method, and use thereof for intestinal health

Abstract

A Bifidobacterium longum subsp. infantis YLGB-1496 bacterial preparation, a preparation method, and a use thereof. The bacterial preparation is prepared by fermentation of a viable bacterium of Bifidobacterium longum subsp. infantis, and is prepared from the viable bacterium of Bifidobacterium longum subsp. infantis and its metabolite of specific composition; and the bacterial preparation is used for intestinal health regulation, has efficacies such as an anti-inflammatory efficacy, enhanced gut immunity, regulation of gut microbiota, and alleviation of intestinal bloating, can be used for the preparation of fermented foods, drugs, or animal feed, and has wide application prospects.

Description

Bifidobacterium longum subspecies infantis YLGB-1496 bacterial agent, preparation method and intestinal health application thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application number 2024101748526 filed with the China Patent Office on February 7, 2024, entitled “Bifidobacterium longum infantis subspecies YLGB-1496 bacterial agent, preparation method and intestinal health application thereof”, the entire contents of which are incorporated by reference into this application. Technical Field

[0003] The present disclosure relates to the technical field of probiotics, and in particular to a Bifidobacterium longum subspecies infantis YLGB-1496 bacterial agent, a preparation method thereof, and intestinal health applications thereof. Background Art

[0004] Colitis (also known as nonspecific ulcerative colitis) is an inflammatory disease of the colon caused by a variety of factors, including infections with bacteria, fungi, viruses, protozoa, parasites, genetics, immunity, antibiotic use, and radiotherapy. Currently, there are no unified diagnostic criteria for colitis. The symptoms of colitis vary depending on the specific cause of the colitis, the duration of the disease, and its severity. Common symptoms of colitis may include mild to severe abdominal pain, persistent diarrhea, bloody stools, fecal incontinence, loss of appetite, and unexplained weight loss. Severe symptoms include shortness of breath, a rapid or irregular heartbeat, and fever.

[0005] Currently, probiotics are widely used in the clinical treatment of pediatric diarrhea and other colitis. Different brands of probiotics contain different bacterial species and counts, and their clinical effects vary. Improving the prevention and treatment of enterocolitis is particularly important.

[0006] In view of this, the present disclosure is proposed. Summary of the Invention

[0007] The purpose of the present disclosure is to provide a class of steroid synthase inhibitors and their therapeutic applications to solve the above technical problems.

[0008] The present disclosure is achieved as follows:

[0009] In a first aspect, the present disclosure provides a Bifidobacterium longum subsp. infantis bacterial agent, which is prepared from live bacteria of Bifidobacterium longum subsp. infantis and metabolites thereof. The deposit number of Bifidobacterium longum subsp. infantis is CCTCC NO: M2011122, and the metabolites contain: Glu-Leu at least 0.156 mg / 100 g, tanacetin at least 0.142 mg / 100 g, agaveside A at least 0.161 mg / 100 g, transdermalin A at least 0.155 mg / 100 g, cyclopeptide A at least 0.145 mg / 100 g, and diglucoside at least 0.145 mg / 100 g.

[0010] The above-mentioned metabolites are characteristic metabolites of the Bifidobacterium longum subspecies infantis agent identified in the present disclosure. The levels of the above-mentioned metabolites are much higher than those of other metabolites. In addition, the above-mentioned metabolites in Bifidobacterium longum subspecies infantis have stable and repetitive peaks, and have clear molecular formulas and structures, which facilitate subsequent detection and quantification.

[0011] The Bifidobacterium longum subspecies infantis preparation, containing the aforementioned characteristic metabolites, exhibits at least one of the following biological functions: anti-inflammatory, effective intestinal immunity enhancement, intestinal flora regulation, and relief of intestinal flatulence or diarrhea. Furthermore, this probiotic postbiotic product exhibits excellent antioxidant properties, including DPPH and hydroxyl radical scavenging abilities, helping to enhance intestinal barrier function.

[0012] In a second aspect, the present disclosure further provides a method for preparing a Bifidobacterium longum subsp. infantis agent, which comprises inoculating Bifidobacterium longum subsp. infantis and fermenting the agent at 37±0.5° C. and a pH of 5.75±0.5.

[0013] In a third aspect, the present disclosure further provides a use of a Bifidobacterium longum subsp. infantis agent or a Bifidobacterium longum subsp. infantis agent prepared by the above-mentioned preparation method in the preparation of nutritional additives or fermented foods, wherein the fermented foods are solid foods, liquid foods, or semi-solid foods.

[0014] In a fourth aspect, the present disclosure further provides a use of a Bifidobacterium longum subsp. infantis bacterial agent or a Bifidobacterium longum subsp. infantis bacterial agent prepared by the above-mentioned preparation method in the preparation of a medicine or feed for any of the following purposes:

[0015] (1) Enhance intestinal immunity;

[0016] (2) regulating intestinal flora;

[0017] (3) Relieve intestinal flatulence;

[0018] (4) prevention and / or treatment of inflammatory diseases;

[0019] and (5) anti-aging.

[0020] Experiments have shown that the Bifidobacterium longum subsp. infantis preparation provided by the present disclosure has the efficacy of enhancing intestinal immunity, regulating intestinal flora, relieving intestinal flatulence, and preventing and / or treating inflammatory diseases. Therefore, the Bifidobacterium longum subsp. infantis preparation can be used to prepare medicines or feeds and has broad application prospects.

[0021] In a fifth aspect, the present disclosure provides a Bifidobacterium longum subsp. infantis bacterial agent or a Bifidobacterium longum subsp. infantis bacterial agent prepared by the above-mentioned preparation method, for use in any of the following purposes:

[0022] (1) Enhance intestinal immunity;

[0023] (2) regulating intestinal flora;

[0024] (3) Relieve intestinal flatulence;

[0025] (4) prevention and / or treatment of inflammatory diseases;

[0026] and (5) anti-aging.

[0027] The present disclosure has the following beneficial effects:

[0028] The live bacterial agent of Bifidobacterium longum subspecies infantis provided in the present disclosure has the effects of anti-inflammation, effectively enhancing intestinal immunity, regulating intestinal flora and relieving intestinal flatulence, can be used to prepare fermented foods, medicines or feeds, and has very broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present disclosure and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without creative work.

[0030] FIG1 is a graph showing the statistical results of the DPPH free radical scavenging ability of YLGB-1496 inactivated at different temperatures;

[0031] FIG2 is a graph showing the statistical results of the hydroxyl radical scavenging ability of YLGB-1496 inactivated at different temperatures;

[0032] FIG3 is a statistical table of characteristic metabolite compositions of YLGB-1496 at different inactivation temperatures;

[0033] Figure 4 is a diagram of an intestinal anatomical experiment;

[0034] FIG5 is a diagram showing the results of H&E pathological staining of the ileum of mice;

[0035] Figure 6 is a graph showing the statistical results of NEC scores;

[0036] FIG7 is a graph showing the statistical results of NEC survival rate;

[0037] Figure 8 is a statistical table of intestinal inflammatory factor expression levels;

[0038] Figure 9 is a PCoA diagram of the species-level composition of the intestinal flora of mice in the GB1496A group and the GB1496D group (A) and the genus-level composition of the intestinal flora (B);

[0039] Figure 10 shows the difference analysis of intestinal flora at the genus level among the GB1496A, GB1496D, and NEC groups;

[0040] Figure 11 shows the species-level composition of the intestinal flora (A) and the intestinal flora function prediction diagram (B). DETAILED DESCRIPTION

[0041] To make the purpose, technical solutions, and advantages of the embodiments of the present disclosure more clear, the technical solutions in the embodiments of the present disclosure are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer were followed. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially.

[0042] The features and performance of the present disclosure are further described in detail below with reference to the embodiments.

[0043] In a first aspect, the present disclosure provides a Bifidobacterium longum subsp. infantis bacterial agent, which is prepared from live bacteria of Bifidobacterium longum subsp. infantis and metabolites thereof. The deposit number of Bifidobacterium longum subsp. infantis is CCTCC NO: M2011122, and the metabolites contain: Glu-Leu at least 0.156 mg / 100 g, tanacetin at least 0.142 mg / 100 g, agaveside A at least 0.161 mg / 100 g, transdermalin A at least 0.155 mg / 100 g, cyclopeptide A at least 0.145 mg / 100 g, and diglucoside at least 0.145 mg / 100 g.

[0044] The above-mentioned metabolites are characteristic metabolites of the Bifidobacterium longum subspecies infantis agent identified in the present disclosure. The levels of the above-mentioned metabolites are much higher than those of other metabolites. In addition, the above-mentioned metabolites in Bifidobacterium longum subspecies infantis have stable and repetitive peaks, and have clear molecular formulas and structures, which facilitate subsequent detection and quantification.

[0045] The Bifidobacterium longum subspecies infantis preparation, containing the aforementioned characteristic metabolites, exhibits at least one of the following biological functions: anti-inflammatory, effective intestinal immunity enhancement, intestinal flora regulation, and relief of intestinal flatulence or diarrhea. Furthermore, this probiotic postbiotic product exhibits excellent antioxidant properties, including DPPH and hydroxyl radical scavenging abilities, helping to enhance intestinal barrier function.

[0046] The Bifidobacterium longum subspecies infantis with the deposit number CCTCC NO: M2011122 is the YLGB-1496 strain, and its deposit information refers to the strain information disclosed in patent CN201910604301.8.

[0047] In an optional embodiment of the present disclosure, the mass ratio of Glu-Leu, Tanacetin, Agavoside A, Permetin A, Cyclolinopeptide A and Desglucocoroloside in the metabolites is: 1.5-1.6:1.41-1.43:1.6-1.62:1.54-1.56:1.44-1.46:1.45-1.46.

[0048] Under the above ratio, the anti-inflammatory and antioxidant effects of Bifidobacterium longum subspecies infantis can be synergistically enhanced.

[0049] In optional embodiments of the present disclosure, the bacterial agent is in the form of a liquid, solid, or semisolid. Liquids include, but are not limited to, solutions, suspensions, emulsions, and semi-emulsions. Solids include, but are not limited to, granules, powders, and tablets. Semisolid dosage forms include, but are not limited to, ointments, suppositories, and pastes.

[0050] In a second aspect, the present disclosure provides a method for preparing a Bifidobacterium longum subsp. infantis agent, which comprises inoculating Bifidobacterium longum subsp. infantis and fermenting the agent at 37±0.5° C. and a pH of 5.75±0.5.

[0051] In an optional embodiment of the present disclosure, the fermentation time is 11-13 hours. Under this fermentation time, a Bifidobacterium longum subsp. infantis bacterial agent with high biological activity can be obtained.

[0052] In an optional embodiment of the present disclosure, the stirring speed of the fermentation tank is 60-80 rpm; at this stirring speed, a Bifidobacterium longum subsp. infantis agent with high biological activity can be obtained.

[0053] In an optional embodiment of the present disclosure, the fermentation is carried out under a tank pressure of 0.01 to 0.03 MPa. Under the above fermentation conditions, a Bifidobacterium longum subsp. infantis bacterial agent with high biological activity can be obtained.

[0054] In an optional embodiment of the present disclosure, 9 hours after the start of fermentation, the fermentation process begins with a natural pH drop until the fermentation ends.

[0055] In one embodiment, before inoculating the cultured seeds into the fermentation tank for fermentation, the method further includes preparing fermentation seeds, which includes: inoculating the cultured seeds into the fermentation tank and fermenting at 37° C. for 11-13 hours.

[0056] In one embodiment, during the preparation of the fermentation seeds, the pressure in the fermentation tank is 0.01-0.03 MPa, and the stirring speed of the fermentation tank is 70 rpm.

[0057] In an optional embodiment of the present disclosure, the cultured seeds are seeds that have undergone tertiary seed preparation.

[0058] In a third aspect, the present disclosure further provides a use of a Bifidobacterium longum subsp. infantis agent or a Bifidobacterium longum subsp. infantis agent prepared by the above-mentioned preparation method in the preparation of nutritional additives or fermented foods, wherein the fermented foods are solid foods, liquid foods, or semi-solid foods.

[0059] The fermented food includes but is not limited to foods for specific health purposes, health foods, and the like.

[0060] In one embodiment, the fermented food is dairy products, soy products, fruit and vegetable products;

[0061] In one embodiment, the dairy product is selected from any one of desserts, lactic acid bacteria beverages, cheese and yogurt; and the fruit and vegetable product is selected from any one of cucumber, carrot, beet, celery and cabbage products.

[0062] In one embodiment, above-mentioned food includes but is not limited to drinkable food. The form of food and drink is also not limited, and can adopt the form of all food and drink that can be circulated normally such as solid, liquid, fluid food shape, jelly shape, sheet, granule, capsule shape etc. Health food is selected from beverage, lozenge, solid beverage, chewable tablet, capsule, granule and drop. The manufacture of above-mentioned food and drink can be carried out by the conventional method of those skilled in the art. In the manufacture of above-mentioned food and drink, as long as do not hinder the growth of Bifidobacterium longum, can also add carbohydrate, protein, fat, dietary fiber, vitamins, the necessary trace metal (manganese sulfate, zinc sulfate, magnesium chloride, potassium carbonate etc.), spices or other complexes of organism.

[0063] The bacterial agent provided by the present disclosure can be prepared as general food and beverages such as dairy products and fermented milk. For example, the bacterial agent is mixed into milk or dairy products that have been heated, mixed, homogenized, sterilized, and then cooled, and then fermented and cooled to produce pure yogurt. It can also be used in combination with other bacteria and / or prebiotics.

[0064] In a fourth aspect, the present disclosure further provides a use of a Bifidobacterium longum subsp. infantis agent or a Bifidobacterium longum subsp. infantis agent prepared by the above-mentioned preparation method in the preparation of a composition for any of the following uses:

[0065] (1) Enhance intestinal immunity;

[0066] (2) regulating intestinal flora;

[0067] (3) Relieve intestinal flatulence;

[0068] (4) prevention and / or treatment of inflammatory diseases;

[0069] and (5) anti-aging.

[0070] The composition is selected from any one of health food, medicine and feed.

[0071] Experiments have shown that the Bifidobacterium longum subsp. infantis preparation provided by the present disclosure has the efficacy of enhancing intestinal immunity, regulating intestinal flora, relieving intestinal flatulence, and preventing and / or treating inflammatory diseases. Therefore, the Bifidobacterium longum subsp. infantis preparation can be used to prepare medicines or feeds and has broad application prospects.

[0072] Feed is, for example, animal feed, including but not limited to ruminant feed, poultry feed, fish feed, etc. For example, pig, sheep, cattle, horse, and fish feed.

[0073] In one embodiment, improving intestinal immunity is achieved by enhancing intestinal barrier function;

[0074] In one embodiment, enhancing the intestinal barrier function includes maintaining the integrity of the ileal villi structure and avoiding or repairing damage to the intestinal barrier.

[0075] The intestinal barrier is a crucial barrier that protects the intestine from harmful substances, such as pathogenic microorganisms and toxins, from entering other tissues, organs, and the bloodstream. The normal intestinal mucosal barrier is composed of mechanical, chemical, immune, and biological barriers. Using the Bifidobacterium longum subsp. infantis preparation provided herein to prevent or repair damage to these mechanical, chemical, immune, and / or biological barriers falls within the scope of this disclosure.

[0076] In one embodiment, the inflammatory disease is selected from chronic inflammatory diseases;

[0077] In one embodiment, the chronic inflammatory disease is selected from inflammatory bowel disease.

[0078] In one embodiment, when the chronic inflammatory disease is inflammatory bowel disease, the prevention and / or treatment of inflammatory bowel disease includes at least one of the following uses: reducing or eliminating inflammatory cell infiltration in the colon, reducing or eliminating colonic crypt abscesses or damage, and inhibiting the level of inflammation in intestinal tissue;

[0079] In one embodiment, the inflammatory bowel disease is selected from any one of acute colitis, ulcerative colitis, Crohn's disease, microscopic colitis, diversion colitis, Behçet's disease immuno-oncology colitis, chemotherapy or radiation colitis, graft-versus-host disease colitis, collagenous colitis, lymphocytic colitis, necrotizing enterocolitis and pouchitis.

[0080] In one embodiment, necrotizing enterocolitis is neonatal necrotizing enterocolitis. Probiotic intervention, in particular, effectively enhances the digestive and absorptive capacity of the intestine, promotes rapid maturation of the intestinal mucosal barrier and immune system, and thereby improves the body's immune system, which is of great significance to the healthy development of infants and young children.

[0081] In one embodiment, treating colitis comprises improving the survival rate of patients with colitis;

[0082] In one embodiment, preventing colitis comprises reducing the incidence of colitis;

[0083] In one embodiment, the expression level of at least one of the following intestinal tissue inflammatory factors is inhibited: IL-6, IL-1β, IL-10, TLR-4 and TNF-α;

[0084] In one embodiment, the abundance of Escherichia coli in the intestine is suppressed, while the abundance of Lactobacillus and Bifidobacterium is increased.

[0085] In one embodiment, the drug is in the form of a tablet, pill, powder, suspension, gel, emulsion, cream, granules, nanoparticles, capsule, suppository, injection or spray.

[0086] In one embodiment, the above-mentioned medicine is a liquid pharmaceutical preparation (such as a kind of as injection), and for example, solution, suspension and gel usually contain liquid carrier, for example water and / or pharmaceutically acceptable organic solvent.In addition, this type of liquid preparation can also include pH adjusting agent, emulsifier or dispersant, buffer, preservative, wetting agent, gelling agent (such as methylcellulose), dye and / or flavoring, for example as defined above. Medicine can be isotonic, that is, they can have the same osmotic pressure as blood. The isotonicity of medicine can be regulated by using sodium chloride and other pharmaceutically acceptable reagents, and these reagents are such as glucose, maltose, boric acid, sodium tartrate, propylene glycol and other inorganic or organic soluble substances. The viscosity of liquid composition can be regulated by pharmaceutically acceptable thickening agent such as methylcellulose. Other suitable thickening agents include such as xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer etc. The preferred concentration of thickening agent depends on selected reagent.

[0087] In an optional embodiment, the above-mentioned drug is a solid pharmaceutical preparation, such as freeze-dried bacterial powder, granular preparation, etc.

[0088] In an alternative embodiment, the medicament is formulated for oral administration, injection, or oral administration.

[0089] In a fifth aspect, the present disclosure further provides a use of a Bifidobacterium longum subsp. infantis agent or a Bifidobacterium longum subsp. infantis agent prepared by the above-mentioned preparation method in any of the following uses:

[0090] (1) Enhance intestinal immunity;

[0091] (2) regulating intestinal flora;

[0092] (3) Relieve intestinal flatulence;

[0093] (4) prevention and / or treatment of inflammatory diseases;

[0094] and (5) anti-aging.

[0095] In one embodiment, improving intestinal immunity is achieved by enhancing intestinal barrier function.

[0096] In one embodiment, enhancing the intestinal barrier function comprises:

[0097] Maintain the integrity of the ileal villus structure and avoid or repair damage to the intestinal barrier.

[0098] In one embodiment, the inflammatory disease is selected from chronic inflammatory diseases.

[0099] In one embodiment, the chronic inflammatory disease is selected from inflammatory bowel disease.

[0100] In one embodiment, when the chronic inflammatory disease is inflammatory bowel disease, the prevention and / or treatment of inflammatory bowel disease includes at least one of the following uses: reducing or eliminating inflammatory cell infiltration in the colon, reducing or eliminating colonic crypt abscesses or damage, and inhibiting the level of inflammation in intestinal tissue.

[0101] In one embodiment, the inflammatory bowel disease is selected from any one of acute colitis, ulcerative colitis, Crohn's disease, microscopic colitis, diversion colitis, Behçet's disease immuno-oncology colitis, chemotherapy or radiation colitis, graft-versus-host disease colitis, collagenous colitis, lymphocytic colitis, necrotizing enterocolitis and pouchitis.

[0102] In one embodiment, the necrotizing enterocolitis is neonatal necrotizing enterocolitis.

[0103] Example 1

[0104] This embodiment provides a Bifidobacterium longum subsp. infantis bacterial agent, the preparation method of which is as follows:

[0105] First and third level seed preparation

[0106] 1. Standard cryopreservation tubes

[0107] Prepare uniformly from the purified strains, divide into 1.5mL centrifuge tubes, no less than 50 tubes, and store in a -80℃ refrigerator with a shelf life of no more than 6 months.

[0108] 2. Activation of cryotubes

[0109] Take a portion of the bacterial strain stored at -80℃, thaw it at room temperature, aseptically take 200μL of bacterial liquid and inoculate it into 10mL of seed liquid culture medium, and culture it at 37℃ for 11-13h.

[0110] 3. Primary purification

[0111] Take the cultured bacterial liquid for dilution and coating, with dilution degrees of -4, -5, and -6. Make two MRS solid plates for each dilution, and culture them upside down at 37°C for 48h to 72h until colonies are formed on the plates. Use an inoculation loop to pick single colonies and place them in 5 tubes of 10mL MRS liquid culture medium. The colonies picked should be of uniform size and cultured at 37°C for 11 to 13h.

[0112] 4. Secondary Purification

[0113] Take the cultured purified bacterial solution and dilute it for coating. The dilution degree is -4 、 -5 、 -6For each dilution, prepare two MRS solid plates and culture them upside down at 37℃ for 48h~72h until colonies are formed on the plates. Use an inoculation loop to pick single colonies and place them in 5 tubes of 10mL MRS liquid culture medium. The colonies picked should be of uniform size and cultured at 37℃ for 11~13h.

[0114] 5. Primary Seed Preparation

[0115] Select one tube of secondary purified bacterial culture and pipette 200 μL of the bacterial culture into five tubes of 10 mL MRS liquid culture medium. Incubate at 37°C for 11-13 h.

[0116] 6. Secondary Seed Preparation

[0117] Select 4 tubes of cultured first-level seeds, aspirate 4 mL of each tube and inject into 4 bottles of 80 mL MRS liquid culture medium, and culture at 37°C for 11-13 hours.

[0118] 7. Preparation of Tertiary Seeds

[0119] The cultured secondary seeds were poured into two bottles of 1.8L MRS liquid culture medium and cultured at 37℃ for 12 to 14 hours.

[0120] 8. Temporary storage of third-level seeds

[0121] After the third-level seeds are prepared, they can be placed at 4℃ for no more than 6 hours.

[0122] 2. Fermentation Seed Preparation

[0123] 1. Vaccination

[0124] (1) Turn on the stirring paddle and temperature control program, the speed is 70 rpm, and the temperature is 37 °C.

[0125] (2) Open the nitrogen inlet valve and introduce a small flow of nitrogen into the fermenter to maintain positive pressure in the fermenter for 5 to 10 minutes.

[0126] (3) When inoculating, pour alcohol onto the inoculation loop and ignite it to form a flame circle.

[0127] (4) Unscrew the top of the inoculator and pour the cultured seeds into the sterile area above the flame, with an inoculation rate of 2.5%.

[0128] (5) After inoculation, tighten the upper cover of the inoculator, close the inoculation valve, and extinguish the alcohol flame ring.

[0129] (6) Close the nitrogen inlet valve and exhaust valve, and control the tank pressure to 0.01-0.03 MPa for pressure-maintaining fermentation.

[0130] 2. Fermentation

[0131] (1) Set the fermentation parameters to 70 rpm and 37°C.

[0132] (2) The fermentation time was 11 h to 13 h, and the fermentation status was monitored every 2 h, including pH, OD600, temperature, and rotation speed.

[0133] (3) After fermentation is complete, the fermentation tank can be inoculated or cooled to 10-20°C for storage for no more than 6 hours.

[0134] 3. Fermentation tank culture

[0135] 1. Vaccination

[0136] (1) Turn on the stirring paddle and temperature control program, the speed is 70 rpm, and the temperature is 37 °C.

[0137] (2) Steam sterilize the inoculation pipe for 30 minutes.

[0138] (3) Open the nitrogen inlet valve and fill with nitrogen for 5 to 10 minutes. Open the bottom valve of the seed tank and the inoculation valve of the fermentation tank for inoculation, with an inoculation amount of 2%.

[0139] (4) After inoculation, close the fermenter inoculation valve and clean the inoculation pipeline.

[0140] (5) Close the nitrogen inlet valve and the discharge valve, and control the tank pressure to 0.01-0.03 MPa for pressure-maintaining fermentation.

[0141] 2. Fermentation

[0142] (1) Set the fermentation parameters to 70 rpm, 37°C, and a constant pH of 5.75.

[0143] (2) The fermentation time was 11 to 13 h, and the fermentation status was monitored every 2 h, including pH, OD600, temperature, and rotation speed.

[0144] (3) After 9 hours of fermentation, the fermentation process begins with a natural pH drop. When the pH drops below 4.2, the fermentation ends and the bacteria can be inactivated. Alternatively, the temperature can be lowered to 10-20°C and stored for no more than 4 hours.

[0145] 4. Freeze-drying

[0146] The excipients are mixed with the fermentation broth and then freeze-dried.

[0147] The excipient formula is as follows:

[0148] The freeze-drying steps are as follows:

[0149] (1) Sterile workshop personnel requirements

[0150] Operators enter the production area according to the "Workshop Personnel Purification Operating Procedures".

[0151] (2) Stalls

[0152] Adjust the transfer tank pressure to 0.10-0.12 MPa. Use this pressure to pump the bacterial solution through the pipe into the freeze-drying tray. Tray weight: 1.6-1.8 kg / tray. Place the freeze-drying trays in order, insert the temperature probe, and close the door.

[0153] (3) Freeze-drying process

[0154] Turn on the freeze dryer and set the program according to the freeze drying process.

[0155] The freeze-drying process parameters are as follows:

[0156] (4) Freeze-drying is completed

[0157] After the freeze-drying program is completed, open the box air inlet valve and the trap air inlet valve. When the box returns to normal atmospheric pressure, you can open the box door to take out the material.

[0158] Example 2

[0159] This example compares the effects of different inactivation processes on the antioxidant function of the postbiotics of the strain and the hydroxyl radical scavenging ability of YLGB-1496 inactivated at different temperatures.

[0160] 1. Inactivated bacteria were prepared by the following method. The fermentation conditions were the same as those in Example 1. After the fermentation was completed, the bacteria were inactivated.

[0161] 1. Bacteria inactivation

[0162] (1) The sterilization conditions of the strain are as follows:

[0163] The sterilization conditions of Bifidobacterium longum subsp. infantis YLGB-1496 are 90°C for 15 min.

[0164] (2) After the bacteria are inactivated, they can be centrifuged or cooled to 10-20°C for storage for no more than 4 hours.

[0165] 2. Centrifugal separation

[0166] (1) Open the centrifuge operating water supply valve, the total pressure of the operating water pipeline is >3 bar, and the machine seal water pressure is maintained at 1.8~2.5 bar.

[0167] (2) Start the centrifuge and wait until the centrifuge speed reaches 11600-11800 rpm. The program will automatically perform the slag discharge operation, and the drum indicator on the main interface will be constantly on.

[0168] (3) Open the feed valve and the material enters the centrifuge drum to start centrifugation.

[0169] (4) Centrifugal process parameter settings: feed rate 600 L / h, slag discharge time 150 s.

[0170] (5) After centrifugation, add protective agent according to the weight of the bacterial sludge.

[0171] 3. Excipient Mixing

[0172] (1) Steam sterilize the excipient pipeline for 30 minutes.

[0173] (2) Add excipients at a ratio of 1:1 by weight of the bacterial sludge.

[0174] (3) After adding the excipients, stir for 5 to 10 minutes to mix thoroughly.

[0175] (4) After the excipients are mixed, the material is transferred to a transfer tank and stirred for 5 to 10 minutes before proceeding to the next step of freeze-drying and panning.

[0176] The excipient formula is as follows:

[0177] 4. Freeze drying

[0178] (1) Sterile workshop personnel requirements

[0179] Operators enter the production area according to the "Workshop Personnel Purification Operating Procedures".

[0180] (2) Stalls

[0181] Adjust the transfer tank pressure to 0.10-0.12 MPa. Use this pressure to pump the sludge through the pipe into the freeze-drying trays. Tray weight: 1.6-1.8 kg / tray. Place the freeze-drying trays in order, insert the temperature probe, and close the door.

[0182] (3) Freeze-drying process

[0183] Turn on the freeze dryer and set the program according to the freeze drying process.

[0184] The freeze-drying process parameters are as follows:

[0185] (4) Freeze-drying is completed

[0186] After the freeze-drying program is completed, open the box air inlet valve and the trap air inlet valve. When the box returns to normal atmospheric pressure, you can open the box door to take out the material.

[0187] 2. The antioxidant activity detection method is as follows:

[0188] (1) 2,2-Diphenyl-1-picrylphenylhydrazine (DPPH) free radical scavenging method

[0189] The DPPH radical scavenging assay was performed using a previously described method with slight modifications. 200 μL of 0.2 mM DPPH solution was mixed with 200 μL of bacterial suspension (109 cfu / mL) and incubated at 25°C in the dark for 30 min. An equal volume of PBS (pH 7.4) was used in the control group, and an equal volume of PBS (pH 7.4) was used in the blank group instead of the DPPH radical solution. After centrifugation at 2,000 × g for 10 min, the absorbance of the solution was measured at 517 nm. The calculation formula is as follows:

[0190] (2) Eliminate hydroxyl free radicals

[0191] A total of 1.0 mL of each sample was added to a mixture containing 2.5 mM 1,10-phenanthroline (1.0 mL), 2.5 mM FeSO₄ (1.0 mL), and PBS (1.0 mL, pH 7.4). After adding 20 mM H₂O₂ (1.0 mL), the mixture was incubated in a 37°C water bath for 90 min. The absorbance was measured at 536 nm. The hydroxyl radical scavenging activity was calculated as follows:

[0192] As shown in Figure 1 , the DPPH scavenging ability of the live YLGB-1496 bacteria was 36.96%, indicating that the live bacteria had good DPPH free radical scavenging ability.

[0193] The results are shown in FIG2 , and the hydroxyl radical scavenging ability of the YLGB-1496 live bacteria is 48.96%.

[0194] Example 3

[0195] Characteristic metabolite composition of YLGB-1496 at different inactivation temperatures. Detection of characteristic metabolites was performed using the following LC-MS parameters.

[0196] The specific method is as follows:

[0197] 1. Sample Processing

[0198] The entire sample (0.5 mL) was transferred to a 2 mL centrifuge tube, and a 6 mm diameter grinding bead was added. 400 μL of extraction solution (methanol:water = 4:1 (v:v)) containing 0.02 mg / mL of internal standard (L-2-chlorophenylalanine) was added. The sample was then ground in a cryo-tissue grinder at -10°C, 50 Hz for 6 min and subjected to low-temperature ultrasonic extraction for 30 min (5°C, 40 kHz). The sample was then allowed to stand at -20°C for 30 min, centrifuged at 13,000 g, 4°C for 15 min, and the supernatant was transferred to an inlet vial with an internal cannula for analysis. Additionally, 20 μL of the supernatant from each sample was pipetted and mixed for use as a quality control sample.

[0199] 2. LC-MS detection

[0200] The instrument platform used for LC-MS analysis was the Thermo Fisher Scientific UHPLC-Q Exactive HF-X system coupled with Fourier transform mass spectrometry.

[0201] Chromatographic conditions: The chromatographic column was an ACQUITY UPLC HSS T3 (100 mm × 2.1 mm id, 1.8 μm; Waters, Milford, USA); the mobile phase A was 95% water + 5% acetonitrile (containing 0.1% formic acid), and the mobile phase B was 47.5% acetonitrile + 47.5% isopropanol + 5% water (containing 0.1% formic acid). The injection volume was 2 μL, and the column temperature was 40°C.

[0202] The mobile phase elution gradient is as follows:

[0203] Mass spectrometry conditions: The sample was ionized by electrospray, and the mass spectrometry signals were collected in positive and negative ion scanning modes. Specific parameters are shown in the table below:

[0204] 3. Quality Control

[0205] Quality control samples (QC) are prepared by mixing equal volumes of extracts from all samples. The volume of each QC is the same as that of the sample and is processed and tested using the same method as the analytical samples. During the instrument analysis process, a QC sample is inserted into every 5-15 analytical samples to examine the stability of the entire detection process.

[0206] 4. Data Processing

[0207] Before statistical analysis, the raw data required a series of preprocessing. The raw data were imported into the metabolomics processing software Progenesis QI (Waters Corporation, Milford, USA) for baseline filtering, peak identification, integration, retention time correction, and peak alignment. This ultimately yielded a data matrix of retention time, mass-to-charge ratio, and peak intensity. The following data preprocessing was then performed:

[0208] (1) Only variables with more than 80% non-zero values ​​in any set of samples are retained;

[0209] (2) Use 1 / 2 of the minimum value in the original matrix to fill the missing values;

[0210] (3) The data were normalized using the total peak area normalization method, and then variables with a relative standard deviation (RSD) of ≥30% in QC samples were deleted;

[0211] The raw data were imported into the metabolomics processing software Progenesis QI (Waters Corporation, Milford, USA) for library search and identification analysis, and the MS and MSMS mass spectrometric information was matched with metabolic databases. The main databases used included commercial databases such as HMDB (http: / / www.hmdb.ca / ) and Metlin (https: / / metlin.scripps.edu / ), public databases, and self-built databases.

[0212] As shown in Figure 3, the results show that the live bacteria contained 1.568-1.572 g / L of γGlu-Leu, 1.423-1.425 g / L of tanacetin, 1.613-1.614 g / L of agaveside A, 1.553-1.554 g / L of transdermalin A, 1.452-1.454 g / L of cyclotide A, and 1.456-1.459 g / L of diglucoside. GF-1, GF-2, and GF-3 are live bacteria samples 1, 2, and 3.

[0213] Example 4

[0214] NEC animal experiments were conducted on inactivated bacteria of GB1496.

[0215] 1. Animal experimental methods are as follows:

[0216] The main experimental reagents are as follows:

[0217] The solution was prepared as follows:

[0218] (1) Anaerobic culture medium: Boil 1 L MRS broth and add 0.5 g L-cysteine ​​hydrochloride and mix well.

[0219] (2) 75% ethanol solution: Add 25 ml of sterile enzyme-free water to 75 ml of anhydrous ethanol.

[0220] (3) Physiological saline: Add 8.5 g of sodium chloride to 1 L of deionized water.

[0221] 1. Animal experimental methods are as follows:

[0222] A. YLGB1496 strain culture

[0223] (1) YLGB1496 was inoculated into 10 ml of MRS broth containing L-cysteine ​​hydrochloride at a 2% inoculation rate and activated for three generations in a 37°C constant temperature incubator (anaerobic tube culture).

[0224] (2) YLGB1496 live bacterial solution: 10 ml of live bacterial solution with MRS broth medium was centrifuged at 4500r for 10 min, the supernatant was discarded, and the solution was resuspended in sterile water. Repeat 2-3 times. Finally, 10 ml of sterile water was added to resuspend the solution, and 1 ml was transferred to a 15 ml centrifuge tube. Then, physiological saline was added to adjust the bacterial solution concentration to 5×107 cfu / ml and stored at 4℃ for later use.

[0225] (3) YLGB1496 sterilized solution: The previous steps are the same as above. Finally, place it in an 80℃ water bath to inactivate it for 12 minutes and store it at 4℃ for later use.

[0226] B. NEC Animal Experiment Design

[0227] (1) Experimental animal grouping:

[0228] The NEC animal experiment groups are as follows:

[0229] (2) Animal husbandry: All mice in each group were housed in a SPF environment with an indoor temperature of 22-25°C and an indoor humidity of 50%-60%. Each cage contained one mother mouse and 6-8 pups. The pups were fed the same diet as the mother mice, and the mothers themselves fed the pups. The experimental period was 4 days, starting from the time of separation from the cages. The mice were gavaged three times daily, with an interval of 8 hours between each gavage. The gavage dose was 50 μl / time on the first day and 100 μl / time on subsequent days.

[0230] (3) Mouse NEC modeling method: Only gavage treatment was performed on the first day. One hour after gavage on the second, third, and fourth days, NEC was induced. Except for the mice in the Con group, the mice in the other groups were placed in a homemade animal hypoxia experimental box after gavage, filled with 100% nitrogen to create an oxygen-deficient environment for 60 seconds. After that, ventilation was performed to restore normoxic conditions, and the mice were placed in a 4°C refrigerator for cold stimulation for 10 minutes. The model was established three times a day. The mortality rate was calculated 24 hours after the first modeling. The dead mice were immediately dissected. If it was determined that the death was not caused by gavage, it was included in the NEC death.

[0231] C. Preparation of mouse ileum NEC pathological sections

[0232] (1) Embedding and sectioning of mouse ileum tissue: After the mouse was slaughtered, 1 cm of tissue from the terminal ileum was obtained, the contents inside were rinsed with PBS, and the tissue was placed in a tissue embedding cage and placed in 4% paraformaldehyde fixative for 24 hours. After fixation, the tissue was washed 3 times with PBS to remove excess fixative and stored in 50% anhydrous ethanol. The tissue was placed in an automatic tissue dehydrator and the water was removed according to the program. Finally, the tissue was embedded in paraffin for sectioning. The embedded tissue was sliced ​​into 3.5 μm sections in a paraffin slicer and stored at room temperature.

[0233] (2) Hematoxylin eosin (H&E) staining: Place the sections in an oven at 65°C for 1 hour. Afterwards, dewax the sections by incubating in xylene I for 15 minutes, xylene II for 15 minutes, 100% ethanol I for 5 minutes, 100% ethanol II for 5 minutes, 95% ethanol for 5 minutes, 80% ethanol for 5 minutes, and 70% ethanol for 5 minutes, then placing them in distilled water. This completes the dewaxing of the samples. Afterwards, stain with hematoxylin for 5 minutes, rinse repeatedly with tap water and soak for 5 minutes. Stain with eosin for 30 seconds, rinse with tap water, and soak for 5 minutes. Dehydrate and make transparent according to the following steps: 95% ethanol I for 5 minutes, 95% ethanol II for 5 minutes, 100% ethanol I for 5 minutes, 100% ethanol II for 5 minutes, xylene I for 5 minutes, and xylene II for 5 minutes. Mount the sections with neutral gum, observe under a microscope, and take pictures.

[0234] (3) NEC tissue scoring criteria: The scores are: 0 (normal), no damage 1 (mild), slight submucosal and / or lamina propria separation 2 (moderate), moderate submucosal and / or lamina propria separation, and / or edema of the submucosal and muscle layers 3 (severe), severe submucosal and / or lamina propria separation, and / or severe edema of the submucosal and muscle layers, regional villus loss 4 (necrosis), villus loss and necrosis. If necessary, intermediate scores of 0.5, 1.5, 2.5, and 3.5 are also used to more accurately assess the level of ileal damage. To determine the incidence of NEC, animals with a histological score of less than 2 points did not develop NEC; animals with a histological score of 2 or higher developed NEC.

[0235] The NEC organization's scoring criteria are as follows:

[0236] D. Collection of blood and intestinal tissue samples and extraction of ileal RNA

[0237] (1) The entire intestinal tissue of all mice needs to be removed and straightened to photograph the intestinal tissue morphology.

[0238] (2) All mouse intestinal tissues were divided into three parts (small intestine, ileum, and colon), and the ileum was then divided into three segments of 1 cm in length; the last 1 cm segment was taken for pathological sectioning; the remaining two segments were stored at -80°C for RT-qPCR detection; all mice were decapitated and bled, and 2-3 serum samples from each group were combined into one and stored at -80°C.

[0239] (3) The intestinal contents of all mice were collected, and 2-4 samples from each group were combined into one sample, which was stored at -80°C for the measurement of intestinal flora.

[0240] E. Full-length 16S rDNA sequence amplification

[0241] ① Genomic DNA extraction: After the genomic DNA extraction is completed, the extracted genomic DNA is detected by 1% agarose gel electrophoresis.

[0242] ②PCR Amplification: Synthesize specific primers with barcodes for the designated sequencing region. To ensure the accuracy and reliability of subsequent data analysis, two conditions must be met: 1) Use the lowest possible amplification cycle number; 2) Ensure that the number of amplification cycles for each sample is consistent. Randomly select representative samples for pilot experiments to ensure that the majority of samples can amplify products of appropriate concentrations at the lowest cycle number.

[0243] ③PCR using Kapa Biosystems: KAPA HiFi DNA Polymerase; PCR instrument: ABI All samples were analyzed under formal experimental conditions, with three replicates per sample. PCR products from the same sample were mixed and analyzed by 2% agarose gel electrophoresis. PCR products were recovered by gel excision using the AxyPrep DNA Gel Recovery Kit (AXYGEN) and eluted with Tris-HCl buffer. Detection was performed by 2% agarose gel electrophoresis.

[0244] ④ Fluorescence quantification: Referring to the preliminary quantitative results of electrophoresis, the PCR products were detected and quantified using the QuantiFluorTM-ST blue fluorescence quantification system (Promega), and then mixed in corresponding proportions according to the sequencing amount requirements of each sample.

[0245] F.acBio SMRT third-generation sequencing

[0246] ①PacBio library construction: (1) End repair: First, the ends of the fragments are blunted; Attach circular linkers: Connect the two ends to the circular single strands, and the two ends of the single strands are connected to the double-stranded positive and negative strands respectively, to obtain a dumbbell-like structure ("horse ring"), called SMRT Bell; (2) Remove the sequences that are not connected to the linker; (3) Anneal the library single-stranded loop with the primer and bind it to the polymerase fixed at the bottom of the ZMW (zero-mode waveguides).

[0247] ②PacBio sequencing: Within a single-molecule real-time reaction well (SMRTCell), a reaction tube (or SMRTCell) contains numerous circular nanopores, known as ZMWs (zero-mode waveguides), with an outer diameter of just over 100 nanometers, which is smaller than the wavelength of the detection laser (hundreds of nanometers). Laser light directed from the bottom cannot penetrate the pores into the solution above, limiting the energy to a small area (20 x 10-21 L), just enough to cover the desired area. This ensures that the signal originates only from this small reaction zone, while any free nucleotide monomers outside the pore remain in darkness, minimizing background. A polymerase bound to the template DNA is immobilized at the bottom of each ZMW. When sequencing reagents are added, each base pairing produces a corresponding fluorescent signal, which is detected. A single SMRTCell contains 150,000 ZMWs, with each well housing a single DNA strand undergoing high-speed synthesis. The raw data indicates a pulse peak for each synthesized base, with a synthesis rate of >100 bases per minute. Coupled with a high-resolution optical detection system, real-time detection is possible.

[0248] G. Data Processing and Analysis

[0249] All results are expressed as mean ± standard deviation. The Student's t test was used to assess the significance of differences in microbial taxa and diversity indices. The Wilcoxon rank-sum test was used to compare key taxa between groups. Differences were considered significant when P < 0.05. SPSS 26.0 was used for statistical analysis, and all figures were created and processed using GraphPad.

[0250] The GB1496D dead bacteria are the inactivated bacteria prepared by the method of Example 2.

[0251] 2. Intestinal hypoxia and ischemia during NEC can lead to intestinal histological changes, generally including intestinal flatulence and edema, and in severe cases, intestinal hemorrhagic necrosis.

[0252] The intestinal tract of the animals was dissected, and the experimental results are shown in Figure 4. The results show that:

[0253] Compared with the NEC group, the Con group showed more flatulence in the NEC group, a pathological characteristic of NEC, indicating partial cell necrosis in the intestinal barrier. Compared with the NEC group, the live GB1496 bacteria group showed no flatulence and more normal intestinal morphology. Compared with the NEC group, the dead GB1496 bacteria group still showed a small amount of intestinal flatulence, but it had a certain alleviating effect.

[0254] 3. NEC occurs in the terminal ileum and colon. NEC can cause damage and rupture of the villi in the ileum. Therefore, the severity of NEC is often assessed based on hematoxylin and eosin (H&E) staining of the ileum. H&E pathological staining experiments were performed on the ileum of mice.

[0255] The results in Figure 5 show that the villi in the Con group were intact and the crypt structure was clear; the villi in the NEC group were severely damaged, the crypt structure was impaired, and the matrix layer was separated from the epithelial layer; both the GB1496A group and the GB1496D group were able to protect the integrity of the intestinal villi, which was closer to the Con group; comparing the GB1496A group and the GB1496D group, the integrity of the intestinal villi of the former was better than that of the latter.

[0256] The results showed that both GB1496A and GB1496D groups could effectively protect the integrity of the ileal villi structure and avoid intestinal barrier damage, and the effect of GB1496A group was better than that of GB1496D group.

[0257] 4. The incidence of NEC is primarily determined by the NEC score, which is based on the results of hematoxylin and eosin staining of terminal ileal tissue sections. A score of 2 or greater is considered a positive diagnosis of NEC. The NEC incidence results are scored as follows.

[0258] The results are shown in Figure 6.

[0259] Compared with the Con group, the incidence of NEC in the NEC group increased significantly to 100% (P<0.05), indicating that the NEC model was successfully established. The incidence of GB1496A (21.43%) and GB1496D (50%) groups was significantly lower than that in the NEC group (P<0.05).

[0260] 5.NEC survival rate experiment.

[0261] During the NEC modeling process, the body may die due to the disease. As shown in Figure 7, there was a certain mortality rate in the NEC group, GB1496A group and GB1496D group, but there was no statistically significant difference in the survival rate among the groups (P>0.05).

[0262] Overall, a trend toward protective effects was observed in the GB1496A and GB1496D groups against NEC mice, but due to limited sample size, no significant differences were found (Log-rank (Mantel-Cox) test (P=0.2350)).

[0263] 6. Intestinal inflammatory factor expression level experiment

[0264] To understand the effects of GB1496A and GB1496D on intestinal inflammatory cytokines in NEC mice, we used qPCR to detect the mRNA expression of these molecules in intestinal tissue. qPCR was used to detect the levels of IL-6, IL-1β, IL-10, TLR-4, and TNF-α molecules in intestinal blockade. The primers and methods involved in the detection are as follows:

[0265] ① RNA Extraction: RNA was extracted using the Trizol method. 50 mg of frozen ileal tissue was added to 1 mL of Trizol to lyse the cells and placed in an RNase-free homogenizer tube containing glass beads. Homogenize the sample using a low-temperature homogenizer at 4°C, 8000 RPM, and 30 seconds per cycle for a total of three cycles. After homogenization, the sample was allowed to stand at 4°C. Once foam disappeared, the supernatant was transferred to a 1.5 mL centrifuge tube and centrifuged at 12,000 g at 4°C for 5 min. The supernatant was removed. 200 μL of chloroform was added, the sample was shaken vigorously for 15 seconds, and the sample was allowed to stand at room temperature for 5 minutes. The supernatant was removed and 0.5 volumes of anhydrous ethanol were added as appropriate. The mixture was then transferred to a column-type centrifuge tube and centrifuged at 12,000 g at 4°C for 2 minutes. RNA was adsorbed to the centrifuge column. 0.5 mL of protein removal buffer was added and the sample was centrifuged at 12,000 g at 4°C for 30 seconds. Add 0.5 mL of rinse buffer, let stand for 2 minutes, centrifuge at 12,000 g at 4°C for 30 seconds, and repeat the rinse. Add 50 μL of ultrapure water to the center of the spin column, let stand for 2 minutes, and centrifuge at 12,000 g at 4°C for 2 minutes. Keep the RNA in solution on ice and measure the RNA concentration using a Nanodrop.

[0266] ② Reverse transcription: Add 4 μL of reverse transcriptase mix to a total volume of 20 μL. Dilute the RNA with enzyme-free sterile water to a total RNA concentration of 2 μg. Make up the total volume to 20 μL with enzyme-free ultrapure water. The reverse transcription cycle is: 25°C for 10 minutes; 42°C for 50 minutes; 85°C for 5 minutes; and then cool at 4°C. Store the transcribed cDNA sample at -20°C.

[0267] ③ Real-time quantitative PCR (RT-qPCR): A 20 μL reaction system includes: 10 μL TB Green Premix reagent; 0.4 μL upstream primer; 0.4 μL downstream primer; n μL cDNA template; and 9.2-n μL sterile enzyme-free water. Mix thoroughly in the dark.

[0268] ④ PCR conditions: Initial denaturation at 95°C for 600 s; three-step amplification: denaturation at 95°C for 20 s; annealing at 60–62°C for 20 s; extension at 72°C for 20 s; 40 cycles of amplification; melting at 95°C for 10 s; 65°C for 60 s; and 97°C for 1 s. Primer sequences and annealing temperatures are shown in Table 1-5. β-actin was used as an internal reference gene.

[0269] The primer sequences are as follows:

[0270] As shown in Figure 8 , compared with the Con group, the expression levels of IL-6 ( Figure 8 (A) ), IL-1β ( Figure 8 (B) ), TNF-α ( Figure 8 (D) ), and TLR-4 ( Figure 8 (C) ) in the NEC group were significantly increased, while IL-10 ( Figure 8 (E) ) was significantly decreased. Compared with the NEC group, the expression levels of IL-6 ( Figure 8 (A) ), TNF-α ( Figure 8 (D) ), and TLR-4 ( Figure 8 (C) ) in the GB1496A and GB1496D groups were significantly decreased, while the expression of IL-10 ( Figure 8 (E) ) was increased. Furthermore, the expression level of IL-1β ( Figure 8 (B) ) was significantly decreased in the GB1496D group, and also decreased in the GB1496A group.

[0271] As shown in FIG8 , from the expression levels of the above inflammatory factors, GB1496A and GB1496D have the effect of inhibiting the inflammation level.

[0272] 7. Experiment on the effects of GB1496A group and GB1496D group on the intestinal flora of mice.

[0273] ① To understand the composition of the intestinal flora of each group of mice, 16s rDNA sequencing was used to obtain the intestinal flora composition of each group. Figure 9(A) shows the top 10 bacterial species at the species level in each group: mammalian cocci, unclassified Enterobacteriaceae, Escherichia fergusonii, unclassified Lactobacillus, Lactobacillus paracasei, unclassified Pasteurella, Streptococcus danielii, Pseudomonas aeruginosa, Enterococcus faecalis, and Bifidobacterium animalis.

[0274] ② Principal coordinates analysis (PCoA) based on Bray-Curtis distance can be used to investigate similarities or differences in sample community composition. Figure 9(B) compares the β-diversity of the gut microbiota composition of the NEC, GB1496A, and GB1496D groups at the genus level. As shown in Figure 9, the gut microbiota composition of the NEC group and the other two groups differed significantly, allowing for complete separation.

[0275] ③ In order to understand the changes in the composition of the intestinal flora of NEC mice at the genus level after intervention with GB1496A and GB496D, this example compares the composition differences of the intestinal flora genus level between the GB1496A group, the GB496D group and the NEC group. Figure 10 (A) shows the differences in the intestinal flora of the GB1496A group and the NEC group at the genus level, among which the abundance of pathogenic bacteria Escherichia coli, pathogenic bacteria Streptococcus, pathogenic bacteria Pseudomonas and conditional pathogenic bacteria Acinetobacter calcoaceticus was significantly decreased in the GB1496A group compared with the model group; Lactobacillus paracasei, compared with the NEC group, its abundance was significantly increased in the 1496 live bacteria group. Figure 10 (B) shows the differences in the intestinal flora of the GB1496D group and the NEC group at the genus level, among which the abundance of pathogenic bacteria Streptococcus and pathogenic bacteria Pseudomonas was significantly decreased in the GB1496D group compared with the NEC group. The abundance of Lactobacillus paracasei and Bifidobacterium increased in the GB1496D group compared with the NEC group.

[0276] ④ To predict the functional information of the microbial communities in mouse samples, we used functional composition and abundance to further understand potential microbial functional characteristics during disease progression. PICRUST2 was used to predict clusters of orthologous groups (COGs) during disease progression. Figure 11 shows some functional information for each group, such as RNA processing and modification, chromatin structure and dynamics, energy generation and conversion, cell cycle control, cell division and chromosome division, and amino acid transport and metabolism. Industrial Applicability

[0277] The present disclosure provides a class of compounds that can be used as drugs, which have the effect of inhibiting steroid synthase with a high inhibition rate, can be used to prepare steroid synthase inhibitors, can be used as drugs for treating hormone-dependent diseases, can be used to prepare and use drugs for treating hormone-dependent diseases, and can be used to treat hormone-dependent diseases. Hormone-dependent diseases include at least one of the following diseases: congestive heart failure, hypertension, chronic kidney disease, diabetic nephropathy, hyperaldosteronism, cardiac fibrosis, renal syndrome, metabolic syndrome, Cushing's syndrome, insulin resistance, obesity, type II diabetes, breast cancer, prostate cancer, ovarian cancer, cervical cancer, diabetic foot, diabetic eye disease, diabetic ulcer, renal failure, non-alcoholic fatty liver disease, fatty liver, cirrhosis, liver fibrosis, liver cancer, pancreatic cancer, bile duct cancer, colon cancer, and rectal cancer.

Claims

1. A Bifidobacterium longum subsp. infantis bacterial agent, characterized in that The invention is prepared from live bacteria of Bifidobacterium longum subspecies infantis and metabolites thereof. The deposit number of the Bifidobacterium longum subspecies infantis is CCTCC NO: M2011122. The metabolites contain: Glu-Leu (0.156 mg / 100 g or more), tanacetin (0.142 mg / 100 g or more), agaveside A (0.161 mg / 100 g or more), transdermalin A (0.155 mg / 100 g or more), cyclopeptide A (0.145 mg / 100 g or more), and diglucoside (0.145 mg / 100 g or more).

2. The Bifidobacterium longum subspecies infantis bacterial agent according to claim 1, characterized in that The mass ratios of Glu-Leu, tanacetin, agaveside A, transdermal A, cyclopeptide A and diglucoside in the metabolites are: 1.5-1.6: 1.41-1.43: 1.6-1.62: 1.54-1.56: 1.44-1.46: 1.45-1.

46.

3. The Bifidobacterium longum subspecies infantis bacterial agent according to claim 1, characterized in that The bacterial agent is in the form of liquid, solid or semi-solid.

4. The method for preparing the Bifidobacterium longum subspecies infantis bacterial agent according to any one of claims 1 to 3, wherein The Bifidobacterium longum subspecies infantis is inoculated and fermented at 37±0.5° C. and a pH of 5.75±0.

5.

5. The use of the Bifidobacterium longum subspecies infantis bacterial agent according to any one of claims 1 to 3 or the Bifidobacterium longum subspecies infantis bacterial agent prepared by the preparation method according to claim 4 in preparing a nutritional additive or food, characterized in that, The food includes fermented food, milk powder, liquid milk, cheese, and snacks.

6. The use according to claim 5, characterized in that The fermented food is solid food, liquid food or semi-solid food.

7. The use according to claim 5 or 6, characterized in that: The fermented foods are dairy products, soy products, and fruit and vegetable products.

8. The use according to claim 7, characterized in that: The dairy product is selected from any one of desserts, lactobacillus beverages, cheese and yogurt; and the fruit and vegetable product is selected from any one of cucumber, carrot, beet, celery and cabbage products.

9. Use of the Bifidobacterium longum subspecies infantis bacterial agent according to any one of claims 1 to 3 or the Bifidobacterium longum subspecies infantis bacterial agent prepared by the preparation method according to any one of claims 4 to 5 in preparing a composition for any one of the following purposes, characterized in that: (1) Enhance intestinal immunity; (2) regulating intestinal flora; (3) Relieve intestinal flatulence; (4) prevention and / or treatment of inflammatory diseases; and (5) anti-aging.

10. The use according to claim 9, characterized in that The improvement of intestinal immunity is achieved by enhancing the intestinal barrier function.

11. The use according to claim 9 or 10, characterized in that The enhancement of intestinal barrier function includes: Maintain the integrity of the ileal villus structure and avoid or repair damage to the intestinal barrier.

12. The use according to claim 9, characterized in that The inflammatory disease is selected from chronic inflammatory diseases.

13. The use according to claim 9 or 12, characterized in that: The chronic inflammatory disease is selected from inflammatory bowel disease.

14. The use according to claim 12 or 13, characterized in that When the chronic inflammatory disease is inflammatory bowel disease, the prevention and / or treatment of inflammatory bowel disease includes at least one of the following uses: reducing or eliminating inflammatory cell infiltration in the colon, reducing or eliminating colonic crypt abscesses or damage, and inhibiting the level of intestinal tissue inflammation.

15. The use according to claim 13 or 14, characterized in that The inflammatory bowel disease is selected from any one of acute colitis, ulcerative colitis, Crohn's disease, microscopic colitis, diversion colitis, Behçet's disease immuno-oncology colitis, chemotherapy or radiation colitis, graft-versus-host disease colitis, collagenous colitis, lymphocytic colitis, necrotizing enterocolitis and pouchitis.

16. The use according to claim 15, characterized in that The necrotizing enterocolitis is neonatal necrotizing enterocolitis.

17. The use according to any one of claims 15-16, characterized in that Treating colitis involves improving the survival rate of patients with colitis.

18. The use according to any one of claims 15-16, characterized in that Prevention of colitis includes reducing the incidence of colitis.

19. The use according to any one of claims 15-16, characterized in that Inhibit the expression level of at least one of the following intestinal tissue inflammatory factors: IL-6, IL-1β, IL-10, TLR-4 and TNF-α.

20. The use according to any one of claims 15-16, characterized in that Inhibit the abundance of Escherichia coli in the intestine and increase the abundance of Lactobacillus and Bifidobacterium.

21. Use of the Bifidobacterium longum subsp. infantis agent according to any one of claims 1 to 3 or the Bifidobacterium longum subsp. infantis agent prepared by the method according to any one of claims 4 to 5 in any of the following uses, characterized in that: (1) Enhance intestinal immunity; (2) regulating intestinal flora; (3) Relieve intestinal flatulence; (4) prevention and / or treatment of inflammatory diseases; and (5) anti-aging.

22. The use according to claim 21, characterized in that The improvement of intestinal immunity is achieved by enhancing the intestinal barrier function.

23. The use according to claim 21 or 22, characterized in that The enhancement of intestinal barrier function includes: Maintain the integrity of the ileal villus structure and avoid or repair damage to the intestinal barrier.

24. The use according to claim 21, characterized in that The inflammatory disease is selected from chronic inflammatory diseases.

25. The use according to claim 24, characterized in that The chronic inflammatory disease is selected from inflammatory bowel disease.

26. The use according to claim 24 or 25, characterized in that When the chronic inflammatory disease is inflammatory bowel disease, the prevention and / or treatment of inflammatory bowel disease includes at least one of the following uses: reducing or eliminating inflammatory cell infiltration in the colon, reducing or eliminating colonic crypt abscesses or damage, and inhibiting the level of intestinal tissue inflammation.

27. The use according to claim 25 or 26, characterized in that The inflammatory bowel disease is selected from any one of acute colitis, ulcerative colitis, Crohn's disease, microscopic colitis, diversion colitis, Behçet's disease immuno-oncology colitis, chemotherapy or radiation colitis, graft-versus-host disease colitis, collagenous colitis, lymphocytic colitis, necrotizing enterocolitis and pouchitis.

28. The use according to claim 27, characterized in that The necrotizing enterocolitis is neonatal necrotizing enterocolitis.

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