Bifidobacterium longum subsp. infantis YLGB-1496 postbiotic product, bacteriocin, preparation method, and use

Abstract

A Bifidobacterium longum subsp. infantis YLGB-1496 postbiotic product, a bacteriocin, a preparation method, and a use, specifically relating to the technical field of probiotic postbiotics. The provided probiotic postbiotic product has the following biological functions: enhancing the intestinal barrier function, preventing and / or treating inflammatory bowel diseases, regulating gut flora, relieving intestinal flatulence and resisting oxidation, and has application prospects in preparing health-care foods or drugs for regulating gut flora and resisting oxidation.

Description

Bifidobacterium longum subspecies infantis YLGB-1496 postbiotic product, secretory agent, preparation method and application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This disclosure claims priority to Chinese patent application number 202410174810.2 filed with the Chinese Patent Office on February 7, 2024, entitled “Bifidobacterium longum subsp. infantis YLGB-1496 secretin, preparation method and antioxidant application thereof”;

[0003] And the priority of the Chinese patent application with application number 2024101748530 filed with the China Patent Office on February 7, 2024, entitled "Postbiotic preparation of Bifidobacterium longum subsp. infantis YLGB-1496, preparation method and application thereof for intestinal health", all of which are incorporated by reference into this disclosure. Technical Field

[0004] The present disclosure relates to the technical field of probiotic postbiotics, and in particular to a postbiotic product, secretin, preparation method and application of Bifidobacterium longum subsp. infantis YLGB-1496. Background Art

[0005] At present, postbiotics are attracting much attention as a hot research field. In 2021, the International Scientific Association of Probiotics and Prebiotics (ISAPP) published a consensus statement on postbiotics. Postbiotics refer to preparations of inanimate microorganisms and / or their components that are beneficial to host health. As derivatives of prebiotics and probiotics, postbiotics have multiple potential benefits. They can regulate intestinal flora, enhance intestinal barrier function, regulate intestinal inflammatory responses, etc., thereby having a positive impact on human intestinal health. In addition, the biological activity of postbiotics is not limited to the intestine. It also has biological activities such as inhibiting oral pathogens and regulating lung inflammatory responses. As people's understanding of intestinal health and microbiome continues to deepen, postbiotics are considered to be a potential functional food and health management method, and have received special attention from the industry.

[0006] Currently, reports on the bioactivity of postbiotics mainly refer to the bioactivity of probiotics or bacteria. Bifidobacterium longum subsp. infantis YLGB-1496 is a typical postbiotic strain. Previous studies have found that Bifidobacterium longum subsp. infantis YLGB-1496 has potential in regulating sleep.

[0007] However, the processing methods of postbiotics are still in the early stages of research. The specific form and preparation method of the product will affect its biological activity. There are no research reports on the effects of different inactivation methods and inactivation conditions on the biological activity of postbiotics.

[0008] In view of this, the present disclosure is proposed. Summary of the Invention

[0009] The purpose of the present disclosure is to provide a postbiotic product of Bifidobacterium longum subspecies infantis YLGB-1496, a bacteriocin, a preparation method and an application thereof, so as to obtain a product having antioxidant properties and capable of improving intestinal health.

[0010] The Bifidobacterium longum subsp. infantis YLGB-1496 secretin disclosed in the present invention refers to a product obtained by extracting water-soluble metabolites of Bifidobacterium longum subsp. infantis YLGB-1496 in the bacterial sludge separated from the fermentation broth of Bifidobacterium longum subsp. infantis YLGB-1496 and the exocytosis of Bifidobacterium longum subsp. infantis YLGB-1496.

[0011] The present disclosure is achieved as follows:

[0012] The purpose of the present disclosure is to provide a postbiotic product, bacteriocin, preparation method and application of Bifidobacterium longum subspecies infantis YLGB-1496 to solve the above technical problems.

[0013] The present disclosure is achieved as follows:

[0014] In a first aspect, the present disclosure provides a probiotic postbiotic product comprising: inactivated probiotic bacteria and / or metabolites thereof, wherein the probiotic comprises Bifidobacterium longum subsp. infantis YLGB-1496 strain, which has a deposit number of CCTCC NO: M2011122;

[0015] The metabolites include: more than 128.81 mg / 100 g of L-methionine and more than 764 mg / 100 g of short peptides.

[0016] In a second aspect, the present disclosure further provides a probiotic postbiotic product, comprising: inactivated probiotic bacteria and metabolites thereof, wherein the probiotic comprises Bifidobacterium longum subsp. infantis YLGB-1496 strain, which has a deposit number of CCTCC NO: M2011122;

[0017] The inactivated probiotics are inactivated probiotics after high-density fermentation; the inactivation conditions of the inactivated probiotics are 70-121° C. and the treatment time is 5-15 minutes.

[0018] In a third aspect, the present disclosure further provides a method for preparing a probiotic postbiotic product, which comprises the following steps: inactivating a fermentation product of the probiotic.

[0019] In a fourth aspect, the present disclosure further provides a secretory agent of Bifidobacterium longum subsp. infantis YLGB-1496, comprising exocytosis products of Bifidobacterium longum subsp. infantis YLGB-1496 and metabolites of Bifidobacterium longum subsp. infantis YLGB-1496, wherein the mass ratio of citric acid to L-methionine in the secretory agent is greater than 25:1, and the deposit number of Bifidobacterium longum subsp. infantis YLGB-1496 is CCTCC No. M2011122.

[0020] In a fifth aspect, the present disclosure also provides a composition comprising a probiotic postbiotic product.

[0021] In a sixth aspect, the present disclosure further provides a method for preparing the aforementioned Bifidobacterium longum subsp. infantis YLGB-1496 secretin, comprising the following steps:

[0022] Bacteriocin extraction: using a solvent to extract the bacterial sludge separated from the fermentation liquid to obtain a mixed liquid;

[0023] Sterilize, separate the supernatant from the mixed solution and sterilize it by heat to obtain the bacteriocin.

[0024] In a seventh aspect, the present disclosure further provides a composition comprising at least one of the aforementioned probiotic postbiotic product, the aforementioned probiotic postbiotic product, and the aforementioned Bifidobacterium longum subsp. infantis YLGB-1496 secretin.

[0025] In an eighth aspect, the present disclosure further provides the use of the aforementioned probiotic postbiotic product, the aforementioned probiotic postbiotic product, the aforementioned Bifidobacterium longum subsp. infantis YLGB-1496 secretin, or the aforementioned composition in the preparation of a composition for (1) enhancing intestinal barrier function; (2) preventing and / or treating inflammatory bowel disease; (3) regulating intestinal flora; (4) relieving intestinal flatulence; (5) anti-aging; and / or (6) anti-oxidation.

[0026] In a ninth aspect, the present disclosure further provides the use of the aforementioned probiotic postbiotic product, the aforementioned probiotic postbiotic product, the aforementioned Bifidobacterium longum subsp. infantis YLGB-1496 secretin, or the aforementioned composition in (1) enhancing intestinal barrier function; (2) preventing and / or treating inflammatory bowel disease; (3) regulating intestinal flora; (4) relieving intestinal flatulence; (5) anti-aging; and / or (6) anti-oxidation.

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

[0028] The probiotic postbiotic product provided by the present disclosure has the following biological functions: enhancing intestinal barrier function, preventing and / or treating inflammatory bowel disease, regulating intestinal flora, anti-oxidation and alleviating intestinal flatulence.

[0029] Through testing, the inventors found that the Bifidobacterium longum subsp. infantis YLGB-1496 strain effectively converts culture medium substrates into L-methionine and short peptides, which can effectively enhance intestinal barrier function, prevent and / or treat inflammatory bowel disease, regulate intestinal flora, and relieve intestinal flatulence. In addition, the probiotic postbiotic product provided by this disclosure has excellent antioxidant effects, with good DPPH free radical scavenging ability and hydroxyl free radical scavenging ability. It can be used as an antioxidant in food or applied to the skin to improve the aging condition of the skin.

[0030] The metabolites contain L-methionine and short peptides, which can not only significantly inhibit pathogenic bacteria, but also these antibacterial substances can maintain the stability of colon cells at a certain level.

[0031] The bacteriocin of Bifidobacterium longum subsp. infantis YLGB-1496 disclosed in the present invention has antioxidant function and can be used in food and medicine. Compared with a mixture including intact dead cells + cell wall components + cell membrane components + cell-free supernatant, the bacteriocin disclosed in the present invention is a liquid or water-soluble powder, which has more advantages for use in some liquid beverages and medicines. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] 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.

[0033] Figure 1 is a process flow chart of the YLGB-1496 strain postbiotic product;

[0034] Figure 2 shows the YLGB-1496 strain postbiotic powder after sieving and packaging;

[0035] FIG3 is a scanning electron micrograph of live and heat-killed Bifidobacterium longum subsp. infantis YLGB1496;

[0036] FIG4 is a graph showing the statistical results of the DPPH free radical scavenging ability of YLGB-1496 inactivated at different temperatures;

[0037] FIG5 is a graph showing the statistical results of the hydroxyl radical scavenging ability of YLGB-1496 inactivated at different temperatures;

[0038] Figure 6 shows the DPPH radical scavenging ability of the cell-free supernatants of the four bacterial strains at 80°C and 121°C;

[0039] Figure 7 shows the hydroxyl radical scavenging ability of the cell-free supernatants of the four bacterial strains at 80°C and 121°C;

[0040] Figure 8 is a statistical graph showing the expression of the pro-inflammatory factor TNF-α in RAW264.7 cells after intervention with inactivated YLGB-1496 bacteria;

[0041] Figure 9 is a statistical graph showing the expression of the pro-inflammatory factor TNF-α in RAW264.7 cells after intervention with YLGB-1496 cell-free supernatant;

[0042] FIG10 is a graph showing the composition of non-volatile components at different thermal inactivation temperatures;

[0043] Figure 11 shows the results of TOP50 differential metabolite analysis between YLGB-1496 and K56 groups;

[0044] Figure 12. Changes in peak areas of six potential target substances at different heat inactivation temperatures of YLGB-1496 strain;

[0045] FIG13 is a statistical table of characteristic metabolite compositions of YLGB-1496 at different inactivation temperatures;

[0046] FIG14 is a liquid chromatogram of the mixed standard working solution;

[0047] Figure 15 is a liquid chromatogram of inactivated bacteria (1-prolylalanine; 2-L-methionine; 3-citric acid; 4-GPRPK; 5-GP(Hyp)GAG; 6-bAsp-Leu; 7-bAsp-Phe);

[0048] Figure 16 is a flow chart of the preparation of secretin from Bifidobacterium longum subsp. infantis YLGB-1496 in Example 1;

[0049] Figure 17 is a physical image of the secretin of Bifidobacterium longum subsp. infantis YLGB-1496 in Example 1;

[0050] FIG18 is a liquid chromatogram of a bacteriocin sample obtained in Example 1;

[0051] FIG19 shows the hydroxyl radical scavenging ability of secretins of Bifidobacterium longum subsp. infantis YLGB-1496 under different extraction conditions;

[0052] FIG20 shows the DPPH radical scavenging ability of secretin from Bifidobacterium longum subsp. infantis YLGB-1496 under different extraction conditions;

[0053] Figure 21 is a diagram of an intestinal anatomical experiment;

[0054] FIG22 is a diagram showing the results of H&E pathological staining of the ileum of mice;

[0055] Figure 23 is a graph showing the statistical results of NEC scores;

[0056] FIG24 is a graph showing the statistical results of NEC survival rate;

[0057] Figure 25 is a statistical table of intestinal inflammatory factor expression levels;

[0058] Figure 26 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);

[0059] Figure 27 shows the difference analysis of intestinal flora at the genus level among the GB1496A, GB1496D, and NEC groups;

[0060] Figure 28 shows the species-level composition of the intestinal flora (A) and the intestinal flora function prediction diagram (B). DETAILED DESCRIPTION

[0061] 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.

[0062] The features and performance of the present disclosure are further described in detail below with reference to the embodiments.

[0063] Reference will now be made in detail to embodiments of the present disclosure, one or more examples of which are described below. Each example is provided to illustrate, not to limit, the present disclosure. Indeed, it will be apparent to those skilled in the art that various modifications and variations may be made to the present disclosure without departing from the scope or spirit of the present disclosure. For example, features illustrated or described as part of one embodiment may be used in another embodiment to produce further embodiments.

[0064] Practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. The technique is fully explained in the literature, for example, in Molecular Cloning: A Laboratory Manual, 2nd ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); and PCR: The Polymerase Chain Reaction. Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0065] 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.

[0066] The present disclosure provides a probiotic postbiotic product comprising: inactivated probiotic bacteria and / or metabolites thereof, wherein the probiotic comprises Bifidobacterium longum subsp. infantis YLGB-1496 strain, which has a deposit number of CCTCC NO: M2011122;

[0067] The metabolites include: more than 128.81 mg / 100 g of L-methionine and more than 764 mg / 100 g of short peptides.

[0068] The preservation information of the YLGB-1496 strain refers to the strain information disclosed in patent CN201910604301.8.

[0069] The above-mentioned metabolites are the characteristic target metabolites of the YLGB-1496 probiotic postbiotic identified in the present disclosure. Compared with live bacteria, the levels of the above-mentioned metabolites in the YLGB-1496 probiotic postbiotic are much higher than the metabolite levels of live bacteria. In addition, the above-mentioned metabolites in the YLGB-1496 probiotic postbiotic have stable peaks and appear repeatedly, with clear molecular formulas and structures, which facilitate subsequent detection and quantification.

[0070] Probiotic postbiotic products containing these characteristic target metabolites have at least one of the following biological functions: enhancing intestinal barrier function, preventing and / or treating inflammatory bowel disease, regulating intestinal flora, and alleviating intestinal flatulence. Furthermore, these probiotic postbiotic products exhibit excellent antioxidant properties, including DPPH and hydroxyl radical scavenging abilities. They can be used as antioxidants in foods or applied to the skin to improve aging.

[0071] The short peptides in the present disclosure refer to protein fragments composed of a small number of amino acids, which have a small molecular weight and are easily absorbed by the human body. They usually refer to short-chain peptides composed of less than 10 amino acid residues, sometimes also called oligopeptides.

[0072] In a preferred embodiment of the present disclosure, the short peptide is selected from at least one of proline propionic acid, GPRPK, antiarrhythmic peptide, βAsp-Leu and βAsp-Phe.

[0073] Proline propionic acid, CAS number 6422-36-2, molecular formula is C8H 14 N2O3, H-Pro-Ala-OH.

[0074] GPRPK refers to: Gly-Pro-Arg-Pro-Lys.

[0075] Antiarrhythmic peptide refers to: GP (Hyp) GAG, molecular formula C 19 H 30N6O8.

[0076] In a preferred embodiment of the present disclosure, the metabolites contain more than 673.72 mg / 100 g of proline propionic acid, more than 44.55 mg / 100 g of GPRPK, more than 9.79 mg / 100 g of antiarrhythmic peptide, more than 20.1 mg / 100 g of dipeptide Asp-Leu, and more than 15.84 mg / 100 g of dipeptide Asp-Phe.

[0077] The mass ratios of prolylpropionic acid, L-methionine, GPRPK, antiarrhythmic peptide, dipeptide Asp-Leu and dipeptide Asp-Phe in probiotic postbiotic products are 620-680:120-130:40-50:8-12:18-22:13-16.

[0078] The mass ratios of proline propionic acid, L-methionine, GPRPK, antiarrhythmic peptide, dipeptide Asp-Leu and dipeptide Asp-Phe in probiotic postbiotic products were 673.72:128.81:44.55:9.79:20.1:15.84.

[0079] In a preferred embodiment of the present disclosure, the probiotic postbiotic product further contains a highly active substance, and the highly active substance is selected from at least one of γGlu-Leu, Tanacetin, Agavoside A, Permetin A, Cyclolinopeptide A and Desglucocoroloside.

[0080] In an optional embodiment, the highly active substances include: Glu-Leu above 1.6 mg / kg, tanacetin above 1.46 mg / kg, agaveside A above 1.64 mg / kg, transdermalin A above 1.6 mg / kg, cyclopeptide A above 1.5 mg / kg and diglucoside above 1.5 mg / kg.

[0081] The highly active substances are derived from inactivated probiotic bacteria and / or their metabolites.

[0082] The present disclosure also provides a probiotic postbiotic product, which includes: inactivated probiotic bacteria and metabolites thereof, wherein the probiotic includes Bifidobacterium longum subsp. infantis YLGB-1496 strain, which has a deposit number of CCTCC NO: M2011122;

[0083] The inactivated probiotics are inactivated probiotics after high-density fermentation; the inactivation conditions of the inactivated probiotics are 70-121° C. and the treatment time is 5-15 minutes.

[0084] In other embodiments, the inactivated bacteria may also be inactivated by ultrasonic inactivation, lysozyme inactivation, pulsed electric field inactivation, and other inactivation methods. As long as biologically active postbiotics can be obtained, inactivation methods other than heat inactivation are also within the scope of protection of the present disclosure.

[0085] The inventors found that when the inactivation temperature ranged from 70°C to 100°C, the DPPH radical scavenging ability of the bacteria showed a downward trend, but the overall change was small. Within the 70-100°C range, the hydroxyl radical scavenging ability of the YLGB-1496 postbiotics did not change much with increasing heat inactivation temperatures. Within the 70-121°C range, increasing heat inactivation temperatures had no significant effect on the anti-inflammatory activity of the YLGB-1496 postbiotics.

[0086] The inactivation conditions can be 70-115° C., 70-110° C., 70-105° C., 70-100° C., 75-121° C., 75-115° C., 75-110° C., 75-100° C., 75-90° C., 80-90° C., 85-90° C., or any point in the above ranges. The treatment time can be 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, or any point in the range from 5 to 15 min.

[0087] In an optional embodiment, the high-density fermentation is carried out at 37° C.±0.5° C. and a pH of 5.75±0.5. The postbiotic product obtained under the above fermentation conditions has good biological activity.

[0088] In an optional embodiment, the fermentation time is 11-13 hours.

[0089] Before high-density fermentation, the YLGB-1496 strain was inoculated into MRS broth containing L-cysteine ​​hydrochloride and activated and cultured for three generations in a constant temperature incubator at 37°C ± 0.5°C; the inoculation was performed at an inoculum size of 1-2%.

[0090] In an optional embodiment, the inactivation conditions for the inactivated bacteria are: 90-100° C. for 10-15 minutes. Under the above inactivation conditions, the prepared postbiotic product has high hydroxyl radical scavenging ability, DPPH free radical scavenging ability and anti-inflammatory activity.

[0091] In a preferred embodiment of the present disclosure, the probiotic postbiotic product includes at least one of dead bacteria of inactivated bacteria, cell disrupted products of inactivated bacteria, precipitates of inactivated bacteria, and cell-free supernatants of inactivated bacteria.

[0092] The inventors discovered that heat-killed dead bacteria, cell fragments, inactivated bacterial precipitates, and cell-free supernatants all exhibited a uniform and significant inhibitory effect on proinflammatory factors, maintaining excellent stability in anti-inflammatory activity. Furthermore, heating had no significant effect on the anti-inflammatory activity of these prebiotic products. The optimal process is heat inactivation at 90°C for 10 minutes.

[0093] In an optional embodiment, the probiotic postbiotic product further comprises: a carrier and / or auxiliary materials.

[0094] In an optional embodiment, the carrier or auxiliary material is selected from:

[0095] At least one of a protective agent, an excipient, a binder, a disintegrant, a lubricant, a flavor, a preservative, a stabilizer, a suspending agent, a dispersant, and a diluent.

[0096] Examples include: protective agents, such as; excipients, such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate; binders, such as cellulose, methylcellulose, hydroxypropyl cellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, starch; disintegrants, such as starch, hydrolyzed starch, carboxymethyl cellulose, carboxymethyl cellulose calcium salt, hydroxypropyl starch, ethylene glycol starch sodium, sodium bicarbonate, calcium phosphate, calcium citrate; lubricants, such as magnesium stearate, talc, sodium lauryl sulfate; flavors, such as citric acid, menthol , glycine, orange powder; preservatives such as sodium benzoate, sodium bisulfite, parabens (such as methyl paraben, ethyl paraben, propyl paraben, butyl paraben); stabilizers such as citric acid, sodium citrate, acetic acid and polycarboxylic acids from the titriplex series, such as diethylenetriaminepentaacetic acid (DTPA); suspending agents such as methylcellulose, polyvinylpyrrolidone, aluminum stearate; dispersants; diluents such as water, organic solvents; waxes, fats and oils such as beeswax, cocoa butter; polyethylene glycol; white petrolatum, etc.

[0097] Alternatively, every 100 kg of excipients contains 10 kg of skim milk powder, 5 kg of trehalose, 1 kg of glycerol, 0.5 kg of sodium erythorbate and 83.5 kg of water.

[0098] In an optional embodiment, the probiotic postbiotic product is in the form of liquid, solid or semi-solid.

[0099] In an optional embodiment, the probiotic postbiotic product is at least one of a concentrate, gelatinized product, dried product, liquid product, diluted product, and crushed product of inactivated bacteria of Bifidobacterium longum subsp. infantis YLGB-1496. The dried product includes but is not limited to spray-dried, freeze-dried, vacuum-dried, drum-dried, and the like.

[0100] The disclosed embodiments also provide a method for preparing a probiotic postbiotic product, which comprises the following steps: inactivating a fermentation product of the probiotic;

[0101] In an optional embodiment, the sterilization conditions are: 70-121° C., and treatment for 5-15 minutes.

[0102] In other embodiments, the inactivated bacteria may also be inactivated by ultrasonic inactivation, lysozyme inactivation, pulsed electric field inactivation, and other inactivation methods. As long as biologically active postbiotics can be obtained, inactivation methods other than heat inactivation are also within the scope of protection of the present disclosure.

[0103] The inventors found that when the inactivation temperature ranged from 70°C to 100°C, the DPPH radical scavenging ability of the bacteria showed a downward trend, but the overall change was small. Within the 70-100°C range, the hydroxyl radical scavenging ability of the YLGB-1496 postbiotics did not change much with increasing heat inactivation temperatures. Within the 70-121°C range, increasing heat inactivation temperatures had no significant effect on the anti-inflammatory activity of the YLGB-1496 postbiotics.

[0104] The inactivation conditions can be 70-115° C., 70-110° C., 70-105° C., 70-100° C., 75-121° C., 75-115° C., 75-110° C., 75-100° C., 75-90° C., 80-90° C., 85-90° C., or any point in the above ranges. The treatment time can be 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, or any point in the range from 5 to 15 min.

[0105] In an optional embodiment, the treatment is carried out at 90-100°C for 10-15 min; in a more preferred embodiment, the treatment is carried out at 90°C for 10 min.

[0106] In an optional embodiment, the fermentation product of the probiotics is a fermentation product of the probiotics after high-density fermentation.

[0107] In an optional embodiment, the high-density fermentation conditions are fermentation at 37°C ± 0.5°C and a pH of 5.75 ± 0.5;

[0108] In an optional embodiment, the fermentation time is 11-13h;

[0109] In an optional embodiment, the fermentation product is centrifuged, and the precipitate is mixed with a carrier and / or auxiliary material. Alternatively, the precipitate is first mixed with a protective agent to obtain a bacterial sludge, which is then mixed with an excipient, with the mixing mass ratio of the excipient to the bacterial sludge being 1:1.

[0110] Optionally, before high-density fermentation, the YLGB-1496 strain is inoculated into MRS broth containing L-cysteine ​​hydrochloride and activated and cultured for three generations in a constant temperature incubator at 37°C ± 0.5°C;

[0111] Alternatively, the inoculation is performed at an inoculum size of 1-2%.

[0112] In an optional embodiment, the cell-free supernatant of the inactivated bacteria after centrifugation is used to prepare probiotic postbiotics.

[0113] In an optional embodiment, the precipitate is mixed with a protective agent and an excipient; and then freeze-dried to produce a powder.

[0114] In other embodiments, in addition to freeze-drying, powdering can also be spray drying, vacuum drying, drum drying, etc.

[0115] The present disclosure also provides a composition comprising a probiotic postbiotic product, including but not limited to a pharmaceutical composition.

[0116] The term "adjuvant" refers to a substance that can modify or enhance the immune response to an antigen. In other words, the immune response to an antigen can be higher or different in the presence of an adjuvant than in the absence of the adjuvant (including when the response is modified, for example, the subset of T cells activated in the presence of an adjuvant is different from the subset activated in the absence of the adjuvant). The adjuvant is, for example, selected from lipid adjuvants.

[0117] The present disclosure also provides a use of a probiotic postbiotic product or composition in preparing a composition for (1) enhancing intestinal barrier function; (2) preventing and / or treating inflammatory bowel disease; (3) regulating intestinal flora; (4) relieving intestinal flatulence; and / or (5) anti-aging.

[0118] Optionally, the composition is selected from health food, food antioxidant or medicine.

[0119] Experiments have shown that the probiotic postbiotic products provided by the present disclosure have the efficacy of regulating intestinal flora, enhancing intestinal barrier function, preventing and / or treating inflammatory bowel disease, and alleviating intestinal flatulence. Therefore, on the one hand, probiotic postbiotic products can be used to prepare health foods that regulate intestinal flora, and on the other hand, probiotic postbiotic products can be used to prepare pharmaceuticals.

[0120] As health food, it may be food for specific health use or the like.

[0121] Food includes but is not limited to drinkable food. The form of food and drink is also not restricted, and can adopt the form of all food and drink that can circulate usually 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 ordinary 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 lactic acid bacteria, 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.

[0122] The postbiotic products provided by the present disclosure can be prepared as general food and beverages of dairy products and fermented milk. For example, the postbiotic products are mixed into milk or dairy products that have been heated, mixed, homogenized, sterilized, and cooled, and then fermented and cooled to make pure yogurt. They can also be used in combination with other bacteria and / or prebiotics.

[0123] In a preferred embodiment of the present disclosure, enhancing the intestinal barrier function includes:

[0124] Maintain the integrity of the ileal villus structure and avoid or repair damage to the intestinal barrier.

[0125] In a preferred embodiment of the present disclosure, the drug for preventing and / or treating 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.

[0126] In an alternative 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.

[0127] In an alternative embodiment, the necrotizing enterocolitis is neonatal necrotizing enterocolitis.

[0128] In an alternative embodiment, treating colitis comprises improving the survival rate of a patient suffering from colitis.

[0129] In an alternative embodiment, preventing colitis comprises reducing the incidence of colitis.

[0130] In an optional 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;

[0131] In an optional embodiment, the abundance of Escherichia coli in the intestine is suppressed, and the abundance of Lactobacillus and Bifidobacterium is increased.

[0132] In a preferred embodiment of the present disclosure, the dosage form of the drug is tablets, pills, powders, suspensions, gels, emulsions, creams, granules, nanoparticles, capsules, suppositories, injections or sprays.

[0133] In an optional 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, emulsifying agent 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.

[0134] In an optional embodiment, the above-mentioned drug is a solid pharmaceutical preparation, such as freeze-dried bacterial powder, granular preparation, etc.

[0135] In an alternative embodiment, the medicament is formulated for oral administration, injection, or oral administration.

[0136] The present disclosure also provides a secretory agent of Bifidobacterium longum subsp. infantis YLGB-1496, comprising exocytosis products of Bifidobacterium longum subsp. infantis YLGB-1496 and metabolites of Bifidobacterium longum subsp. infantis YLGB-1496. The mass ratio of citric acid to L-methionine in the secretory agent is greater than 25:1. The deposit number of Bifidobacterium longum subsp. infantis YLGB-1496 is CCTCC No. M2011122.

[0137] In the present disclosure, bacteriocin mainly includes components such as exocytosis and bacterial metabolites. As the extraction of bacteriocin proceeds, the concentrations of citric acid and L-methionine in the bacteriocin gradually increase. Therefore, in the present disclosure, the concentrations of L-methionine and citric acid are used to characterize bacteriocin.

[0138] The bacteriocin of Bifidobacterium longum subsp. infantis YLGB-1496 disclosed in the present invention has antioxidant function and can be used in food and medicine. Compared with a mixture including intact dead cells + cell wall components + cell membrane components + cell-free supernatant, the bacteriocin disclosed in the present invention is a liquid or water-soluble powder, which has more advantages for use in some liquid beverages and medicines.

[0139] In an optional embodiment, the mass ratio of citric acid to L-methionine in the bacteriocin is 25-140:1, specifically 25:1, 40:1, 60:1, 80:1, 100:1, 120:1, 140:1 or any value between 25 and 140:1, or any value greater than 140:1.

[0140] Optionally, the mass ratio of citric acid to L-methionine in the bacteriocin is 90-105:1.

[0141] The present disclosure also provides a method for preparing the aforementioned Bifidobacterium longum subsp. infantis YLGB-1496 secretin, comprising the following steps:

[0142] Bacteriocin extraction: using a solvent to extract the bacterial sludge separated from the fermentation liquid to obtain a mixed liquid;

[0143] Sterilize, separate the supernatant from the mixed solution and sterilize it by heat to obtain the bacteriocin.

[0144] In an optional embodiment, the solvent is water; optionally, the solvent is sterile water, specifically sterile purified water.

[0145] In an optional embodiment, the extraction temperature is 0-37°C, specifically 0°C, 2°C, 4°C, 6°C, 8°C, 10°C, 15°C, 20°C, 25°C, 30°C, 37°C or any value between 0-37°C; optionally, the extraction temperature is 3-5°C.

[0146] In an optional embodiment, the extraction time is 0.5-3 h, specifically 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h or any value between 1-3 h; optionally, the extraction time is 30-90 min.

[0147] In an optional embodiment, the total colony count of Bifidobacterium longum subspecies infantis YLGB-1496 in the mixed solution is 0.5×10 10-3×10 11 cfu / mL, specifically 0.5×10 10 cfu / mL, 1×10 10 cfu / mL, 0.5×10 11 cfu / mL, 0.7×10 11 cfu / mL, 0.9×10 11 cfu / mL, 1×10 11 cfu / mL, 2×10 11 cfu / mL, 3×10 11 cfu / mL or 0.5×10 11 -3×10 11 cfu / mL; Optionally, the total colony count of Bifidobacterium longum subspecies infantis YLGB-1496 in the mixed solution is 1×10 11 -2×10 11 cfu / mL.

[0148] In an optional embodiment, the temperature of heat sterilization is 70-121°C, specifically 70°C, 80°C, 90°C, 100°C, 110°C, 121°C or any value between 70-121°C; optionally, the temperature of heat sterilization is 70-100°C.

[0149] In an optional embodiment, the heat sterilization time is 5-30 min, specifically 5 min, 10 min, 15 min, 20 min, 25 min, 30 min or any value between 5-30 min; optionally, the heat sterilization time is 10-16 min.

[0150] In an optional embodiment, the present invention further comprises a fermentation step of culturing Bifidobacterium longum subsp. infantis YLGB-1496 in liquid culture and stopping the fermentation when the Bifidobacterium longum subsp. infantis YLGB-1496 grows to the logarithmic phase;

[0151] Optionally, the fermentation step comprises: inoculating a fermentation seed solution of Bifidobacterium longum subsp. infantis YLGB-1496 into a culture medium, and first fermenting for 14-16 hours at a temperature of 30-40° C., a rotation speed of 60-80 rpm, and a pH of 5.8-6.2 to obtain a fermentation solution of Bifidobacterium longum subsp. infantis YLGB-1496;

[0152] Optionally, the preparation of the fermentation seed liquid of Bifidobacterium longum subsp. infantis YLGB-1496 comprises: activating, purifying, culturing the Bifidobacterium longum subsp. infantis YLGB-1496 strain with a first-level seed liquid, expanding with a second-level seed liquid, culturing with a third-level seed liquid, and preparing fermentation seeds to obtain the fermentation seed liquid of Bifidobacterium longum subsp. infantis YLGB-1496;

[0153] Optionally, the culture medium is MRS liquid culture medium;

[0154] Optionally, the solvent is water; Optionally, the solvent is sterile water;

[0155] Optionally, after the sterilization step, the bacteriocin is freeze-dried to obtain freeze-dried bacteriocin.

[0156] In the present disclosure, the bacteriocin obtained after sterilization is a liquid, wherein the mass ratio of citric acid to L-methionine is greater than 25:1, and the L-methionine concentration is greater than 0.05 mg / ml and the citric acid concentration is greater than 5 mg / ml. In some embodiments, the bacteriocin has an L-methionine concentration of 0.05-0.2 mg / ml and a citric acid concentration of 5-7 mg / ml.

[0157] Specifically, the separation of the bacterial sludge and the supernatant in the present disclosure can adopt existing separation methods, such as selecting a centrifugal method to achieve solid-liquid separation; in some embodiments, the concentration of L-methionine in the bacteriocin is 0.05 mg / ml, 0.07 mg / ml, 0.09 mg / ml, 0.11 mg / ml, 0.13 mg / ml, 0.15 mg / ml, 0.17 mg / ml, 0.19 mg / ml, 0.2 mg / ml or any value between 0.05-0.2 mg / ml, or any value greater than 0.2 mg / ml; the concentration of citric acid is 5 mg / ml, 5.5 mg / ml, 6 mg / ml, 6.5 mg / ml, 7 mg / ml or any value between 5-7 mg / ml, or any value greater than 7 mg / ml. In some embodiments, the concentration of L-methionine in the bacteriocin is 0.05-0.0.1 mg / ml and the concentration of citric acid is 5.5-6 mg / ml.

[0158] After the sterilization step, the bacteriocin is freeze-dried to obtain a freeze-dried bacteriocin, in which the solvent is removed, and the mass ratio of citric acid to L-methionine in the freeze-dried bacteriocin is greater than 25:1.

[0159] Specifically, in some embodiments, the secretin of Bifidobacterium longum subsp. infantis YLGB-1496 is prepared by the following steps:

[0160] 1. Preparation of Tertiary Seeds

[0161] 1.1 Standard cryopreservation tubes

[0162] 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.

[0163] 1.2 Activation of cryotubes

[0164] 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.

[0165] 1.3 Primary purification

[0166] 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-72h until obvious 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 20-24h.

[0167] 1.4 Secondary purification

[0168] Take the cultured purified bacterial liquid for dilution and coating, with dilution degrees of -4, -5, and -6. Make two MRS solid plates for each dilution. Invert and culture at 37°C for 48h-72h until obvious 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 are uniform in size and cultured at 37°C for 20-24h.

[0169] 1.5 Primary seed preparation

[0170] 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 hours.

[0171] 1.6 Secondary seed preparation

[0172] Select 4 tubes of cultured first-level seeds, aspirate 4 mL of each and inject into 4 bottles of 80 mL MRS liquid culture medium, and culture at 37°C for 11-13 hours.

[0173] 1.7 Preparation of tertiary seeds

[0174] The cultured secondary seeds were poured into two bottles of 1.8LMRS liquid culture medium and cultured at 37°C for 12-14 hours.

[0175] 1.8 Temporary storage of third-level seeds

[0176] After the third-level seeds are prepared, they can be placed at 4℃ for no more than 6 hours.

[0177] 1.9 Process Quality Control

[0178] (1) Indicators for determining the end point of seed growth at each level: pH 4.4-4.8, OD600 ≥ 1.8.

[0179] (2) Purity test: Observe the bacterial morphology under a microscope. Under a 100x oil immersion lens, the bacterial morphology is complete, appearing as a long rod, slightly curved, and arc-shaped. After a long period of decolorization, the bacterial body becomes transparent and relatively blurry.

[0180] (3) Pollutant detection: Detection of the third-level seed liquid, including Escherichia coli and non-lactic acid bacteria.

[0181] 2. Fermentation Seed Preparation

[0182] 2.1 Vaccination

[0183] (1) Turn on the stirring paddle and temperature control program, the speed is 70 rpm, and the temperature is 37 °C.

[0184] (2) Open the nitrogen inlet valve and introduce a small flow of nitrogen into the fermenter to maintain positive pressure in the fermenter for 5-10 minutes.

[0185] (3) When inoculating, pour alcohol onto the inoculation loop and ignite it to form a flame circle.

[0186] (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%.

[0187] (5) After inoculation, tighten the upper cover of the inoculator, close the inoculation valve, and extinguish the alcohol flame ring.

[0188] (6) Close the nitrogen inlet valve and exhaust valve, and control the tank pressure to 0.01-0.03 MPa for pressure-maintaining fermentation.

[0189] 2.2 Fermentation

[0190] (1) Set the fermentation parameters to 70 rpm and 37°C.

[0191] (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.

[0192] (3) After fermentation, the fermentation broth can be cultured or cooled to 10-20℃ and stored for no more than 6 hours.

[0193] 2.8 Process Quality Control

[0194] (1) Seed tank fermentation endpoint determination indicators: pH 4.4-4.8, OD600 ≥ 1.8.

[0195] (2) Purity test: Observe the bacterial morphology of the fermentation liquid at the end of the seed tank under a microscope. Under a 100x oil lens, the bacterial morphology is complete, long rods, slightly curved, and arc-shaped.

[0196] (3) Pollutant detection: Detect the fermentation liquid at the end of the seed tank, including Escherichia coli and non-lactic acid bacteria.

[0197] 3. Fermentation Broth Culture

[0198] 3.1 Vaccination

[0199] (1) Turn on the stirring paddle and temperature control program, the speed is 70 rpm, and the temperature is 37 °C.

[0200] (2) Steam sterilize the inoculation pipe for 30 minutes.

[0201] (3) Open the nitrogen inlet valve and fill with nitrogen for 5-10 minutes. Open the bottom valve of the seed tank and the inoculation valve of the fermentation tank for inoculation. The inoculation amount is 5%.

[0202] (4) After inoculation, close the fermenter inoculation valve and clean the inoculation pipeline.

[0203] (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.

[0204] 3.2 Fermentation

[0205] (1) Set the fermentation parameters to 30-40°C, 60-80 rpm, and a constant pH of 5.8-6.2.

[0206] (2) The fermentation time was 14 to 16 h, and the fermentation status was monitored every 2 h, including pH, OD600, temperature, and rotation speed.

[0207] (3) After fermentation, centrifuge or cool to 10-20°C for no more than 4 hours. 3.3 Mud separation.

[0208] (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.

[0209] (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.

[0210] (3) Open the feed valve, and the fermentation liquid of Bifidobacterium longum subsp. infantis YLGB-1496 enters the centrifuge drum and starts centrifugation to obtain bacterial sludge.

[0211] (4) Centrifugal process parameter settings: feed rate 600 L / h, slag discharge time 180 s.

[0212] 4. Bacteriocin Extraction

[0213] 4.1 Mud transfer

[0214] The collected bacterial sludge was transferred to a sterile fermentation tank for extraction.

[0215] 4.2 Extraction conditions

[0216] The bacterial sludge was extracted and sterile purified water was used to mix the bacterial sludge to obtain a mixed solution. The total colony counts of Bifidobacterium longum subspecies infantis YLGB-1496 in the mixed solution were 0.5×10 11 -3×10 11 cfu / mL, extraction temperature was 0-37°C, extraction time was 0.5-3h, and extraction speed was 50-100rpm.

[0217] It should be noted that in the actual production process, the OD600 value of the fermentation broth of Bifidobacterium longum subspecies infantis YLGB-1496 in step 3.2 can be adjusted, and the amount of sterile purified water added in the extraction step can be controlled so that the volume of the mixed solution is 1% of the volume of the fermentation broth of Bifidobacterium longum subspecies infantis YLGB-1496, so as to more conveniently adjust the total colony count of Bifidobacterium longum subspecies infantis YLGB-1496 in the mixed solution.

[0218] 5. Centrifugal separation

[0219] (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.

[0220] (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.

[0221] (3) Open the feed valve and the mixed liquid enters the centrifuge drum to start centrifugation.

[0222] (4) Centrifugal process parameter settings: feed rate 600 L / h, slag discharge time 180 s.

[0223] (5) Transfer the centrifuged supernatant to a collection tank.

[0224] 6. Packaging and sterilization

[0225] The filled bottled supernatant is heat sterilized to obtain the bacteriocin. The sterilization conditions of Bifidobacterium longum subsp. infantis YLGB-1496 are 70-121° C. for 5-30 min.

[0226] The present disclosure also provides a use of the secretin of Bifidobacterium longum subsp. infantis YLGB-1496 according to any one of the aforementioned embodiments in preparing food, wherein the food comprises at least one of a dairy product and a beverage.

[0227] The present disclosure also provides a use of the secretin of Bifidobacterium longum subsp. infantis YLGB-1496 according to any one of the aforementioned embodiments in the preparation of a composition, wherein the composition comprises an antioxidant composition; preferably, the composition is selected from at least one of a medicine, a health food, and a feed.

[0228] The present disclosure also provides a composition comprising at least one of the aforementioned probiotic postbiotic product, the aforementioned probiotic postbiotic product, and the aforementioned Bifidobacterium longum subsp. infantis YLGB-1496 secretin.

[0229] The present disclosure also provides the use of the aforementioned probiotic postbiotic product, the aforementioned probiotic postbiotic product, the aforementioned Bifidobacterium longum subsp. infantis YLGB-1496 secretin, or the aforementioned composition in the preparation of a composition for (1) enhancing intestinal barrier function; (2) preventing and / or treating inflammatory bowel disease; (3) regulating intestinal flora; (4) relieving intestinal flatulence; (5) anti-aging; and / or (6) anti-oxidation.

[0230] The present disclosure also provides the use of the aforementioned probiotic postbiotic product, the aforementioned probiotic postbiotic product, the aforementioned Bifidobacterium longum subsp. infantis YLGB-1496 secretin, or the aforementioned composition in (1) enhancing intestinal barrier function; (2) preventing and / or treating inflammatory bowel disease; (3) regulating intestinal flora; (4) relieving intestinal flatulence; (5) anti-aging; and / or (6) anti-oxidation.

[0231] The features and performance of the present disclosure are further described in detail below with reference to the embodiments.

[0232] Example 1

[0233] This embodiment provides a YLGB-1496 strain postbiotic product. The process flow chart is shown in FIG1 . The specific preparation method is as follows:

[0234] First and third level seed preparation

[0235] 1. Standard cryopreservation tubes

[0236] 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.

[0237] 2. Activation of cryotubes

[0238] 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.

[0239] 3. Primary purification

[0240] 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 obvious 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.

[0241] 4. Secondary Purification

[0242] Take the cultured purified bacterial solution and dilute it for coating. The dilution degree is -4 、 -5 、 -6 For each dilution, prepare two MRS solid plates and culture them upside down at 37℃ for 48h~72h until obvious 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.

[0243] 5. Primary Seed Preparation

[0244] The formula of the seed tank fermentation medium is as follows:

[0245] pH value: 6.2~6.4, sterilization conditions: 121℃, 15~20min.

[0246] 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.

[0247] 6. Secondary Seed Preparation

[0248] 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.

[0249] 7. Preparation of Tertiary Seeds

[0250] 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.

[0251] 8. Temporary storage of third-level seeds

[0252] After the third-level seeds are prepared, they can be placed at 4℃ for no more than 6 hours.

[0253] 2. Fermentation Seed Preparation

[0254] 1. Vaccination

[0255] (1) Turn on the stirring paddle and temperature control program, the speed is 70 rpm, and the temperature is 37 °C.

[0256] (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.

[0257] (3) When inoculating, pour alcohol onto the inoculation loop and ignite it to form a flame circle.

[0258] (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%.

[0259] (5) After inoculation, tighten the upper cover of the inoculator, close the inoculation valve, and extinguish the alcohol flame ring.

[0260] (6) Close the nitrogen inlet valve and exhaust valve, and control the tank pressure to 0.01-0.03 MPa for pressure-maintaining fermentation.

[0261] 2. Fermentation

[0262] (1) Set the fermentation parameters to 70 rpm and 37°C.

[0263] (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.

[0264] (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.

[0265] 3. Fermentation tank culture and bacterial inactivation

[0266] 1. Vaccination

[0267] (1) Turn on the stirring paddle and temperature control program, the speed is 70 rpm, and the temperature is 37 °C.

[0268] (2) Steam sterilize the inoculation pipe for 30 minutes.

[0269] (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%.

[0270] (4) After inoculation, close the fermenter inoculation valve and clean the inoculation pipeline.

[0271] (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.

[0272] 2. Fermentation

[0273] (1) Set the fermentation parameters to 70 rpm, 37°C, and a constant pH of 5.75.

[0274] (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.

[0275] (3) After 9 hours of fermentation, the fermentation process begins with a natural pH drop. When the pH value 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.

[0276] In this step, the fermentation tank culture medium formula is as follows:

[0277] pH value: 6.2~6.4.

[0278] 3. Bacteria inactivation

[0279] (1) The sterilization conditions of the strain are as follows:

[0280] The sterilization conditions of Bifidobacterium longum subsp. infantis YLGB-1496 are 90°C for 15 min.

[0281] (2) After the bacteria are inactivated, they can be centrifuged or cooled to 10-20°C for storage for no more than 4 hours.

[0282] 4. Centrifugal separation

[0283] (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.

[0284] (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.

[0285] (3) Open the feed valve and the material enters the centrifuge drum to start centrifugation.

[0286] (4) Centrifugal process parameter settings: feed rate 600 L / h, slag discharge time 150 s.

[0287] (5) After centrifugation, add protective agent according to the weight of the bacterial sludge.

[0288] 5. Excipient Mixing

[0289] (1) Steam sterilize the excipient pipeline for 30 minutes.

[0290] (2) Add excipients at a ratio of 1:1 by weight of the bacterial sludge.

[0291] (3) After adding the excipients, stir for 5 to 10 minutes to mix thoroughly.

[0292] (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.

[0293] The excipient formula is as follows:

[0294] 6. Freeze Drying

[0295] 1. Sterile workshop personnel requirements

[0296] Operators enter the production area according to the "Workshop Personnel Purification Operating Procedures".

[0297] 2. Stall

[0298] (1) Adjust the transfer tank pressure to 0.10-0.12 MPa, and use the pressure to pump the bacterial sludge into the freeze-drying tray through the pipeline.

[0299] (2) Stall weight: 1.6-1.8 kg / tray.

[0300] (3) Place the freeze-drying trays into the freeze dryer in order, insert the temperature probe, and close the door.

[0301] 3. Freeze-drying process

[0302] Turn on the freeze dryer and set the program according to the freeze drying process.

[0303] The freeze-drying process parameters are as follows:

[0304] 4. End of freeze drying

[0305] 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.

[0306] 7. Freeze-dried powder off the tray

[0307] 1. Sterile workshop personnel requirements

[0308] Operators enter the production area according to the "Workshop Personnel Purification Operating Procedures".

[0309] 2. Off-plate

[0310] Take out the freeze-dried plates from the freeze dryer one by one, and use the discharge table to transfer the materials into the collection bucket to prepare for the next step of crushing.

[0311] 8. Crushing and screening

[0312] 1. Crushing

[0313] (1) Install a crusher with a screen size of 20 mesh.

[0314] (2) Connect the compressed air and power supply and turn on the crusher.

[0315] (3) Setting parameters: speed 10 rpm, pneumatic valve feeding interval 1 s.

[0316] (4) The weight of a single crushing shall not exceed 15kg.

[0317] 2. Screening and packaging

[0318] After pulverization, the freeze-dried powder was directly sieved with a 40-mesh sieve, and the sieved fine powder was collected separately (Figure 2).

[0319] Example 2

[0320] This example compares the effects of different inactivation processes on the morphological changes of postbiotics of the strain.

[0321] Heat-killed bacteria were inactivated by heating at 70°C, 80°C, 90°C, 100°C, and 121°C for 10 min. The morphological changes of postbiotics were observed using scanning electron microscopy (SEM).

[0322] Morphological changes in live and heat-killed bacteria were observed using a scanning electron microscope. Live or inactivated cells were fixed in 2.5% glutaraldehyde at 4°C and then dehydrated in varying ethanol gradients (50%, 60%, 70%, 80%, 90%, and 100%). After critical point drying, slides were mounted on pillars and sputtered with gold. Morphological changes were then examined using a SU8020 SEM (HITACHI, Japan).

[0323] The results are shown in Figure 3. As for Bifidobacterium longum, we can see that it has a long bacterial morphology. The live ET-22 cells appear smooth and regular, with intact cell membranes. As the temperature rises, the bacterial cells shrink and break more (red arrows), manifesting as cell shrinkage (blue arrows). Under heat inactivation conditions at 90°C, the release of intracellular components and fragments increases, accompanied by white clumps and leakage of intracellular contents (blue circles), indicating that it has low tolerance to high temperatures.

[0324] At heat inactivation temperatures above 70°C for 10 minutes, significant morphological changes occur, with increased surface roughness and exudation of contents. As the temperature continues to rise, the bacteria rupture more and more, and the amount of exuded contents gradually increases. Therefore, under probiotic inactivation conditions, postbiotics exhibit a combination of "dead bacterial shell + exuded contents," where "exuded contents" is often referred to as "cell-free supernatant" in academia.

[0325] Example 3

[0326] 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.

[0327] The antioxidant activity assay for postbiotics is as follows:

[0328] (1) 2,2-Diphenyl-1-picrylphenylhydrazine (DPPH) free radical scavenging method

[0329] 200 μL of 0.2 mM DPPH solution was mixed with 200 μL of mixed solution (10 9 cfu / mL) as raw material for the bacteriocin or inactivated bacteria and incubate at 25°C in the dark for 30 minutes. The control group was replaced with an equal volume of PBS (pH 7.4), while the blank group was replaced with an equal volume of PBS (pH 7.4) instead of the DPPH free radical solution. After centrifugation at 2,000 × g for 10 minutes, the absorbance of the solution was measured at 517 nm. The calculation formula is as follows:

[0330] (2) Eliminate hydroxyl free radicals

[0331] A total of 1.0 mL of the mixture (10 9 cfu / mL) as the starting material for the bacteriocin preparation or inactivated bacteria. 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:

[0332] As shown in Figure 4, the DPPH scavenging capacity of live YLGB-1496 bacteria was 36.96%. Heat treatment of YLGB-1496 at 70-100°C did not significantly reduce its scavenging capacity compared to live bacteria. However, heat treatment at 121°C significantly reduced its scavenging capacity (P < 0.05). Therefore, these results suggest that high temperatures may weaken the DPPH free radical scavenging capacity of YLGB-1496.

[0333] As shown in Figure 5, the hydroxyl radical scavenging capacity of live YLGB-1496 bacteria was 48.96%. While heat treatment at 70-90°C did not significantly change the scavenging capacity compared to live bacteria, heat treatment at 100°C significantly increased the scavenging capacity (P < 0.05). Heating at 121°C significantly weakened the hydroxyl radical scavenging capacity of YLGB-1496 (P < 0.0001). Therefore, these results suggest that high temperatures may weaken the hydroxyl radical scavenging capacity of YLGB-1496.

[0334] In summary, the inactivation temperature has a significant effect on the antioxidant capacity of postbiotics. In the heating range of 70-100℃, the antioxidant capacity of the bacterial components showed a downward trend, but the overall change was not significant. However, when the inactivation temperature exceeded 100℃, the antioxidant capacity of YLGB-1496 showed a significant decline, and the antioxidant activity of postbiotics was more sensitive to temperature.

[0335] Example 4

[0336] This example compares the effects of different inactivation processes on the antioxidant function of probiotic cell-free supernatants.

[0337] In addition to Bifidobacterium longum subsp. infantis YLGB1496 of Example 1, free radical scavenging experiments were also conducted on Lactobacillus paracasei ET-22, Lactobacillus paracasei K56, and Bifidobacterium animalis subsp. lactis BL99.

[0338] The cell-free supernatant was prepared as follows:

[0339] Overnight cultures of four proprietary strains were obtained by centrifugation (4500 × g, 10 minutes), washed three times, and resuspended in PBS to a concentration of 1 × 10 10 CFU / mL. 1 ml of the washed bacterial solution was aspirated for colony counts. For the preparation of postbiotics, heat-killed bacteria were inactivated by heating at 70°C, 80°C, 90°C, 100°C, and 121°C for 10 minutes, and the number of residual viable cells was counted by MRS plate counting. After centrifugation at 10,000 × g for 10 minutes at 4°C, the cell-free supernatant was collected and filtered through a 0.22 μm sterile water membrane for LC-MS analysis of metabolite content.

[0340] (1) DPPH free radical scavenging ability of probiotic cell-free supernatant

[0341] The experimental method was similar to that in Example 3. As shown in Figure 6 , the DPPH scavenging ability of the cell-free supernatant of YLGB-1496 was stronger than that of the other three strains. There was no significant difference in the DPPH free radical scavenging ability of the cell-free supernatants of the four strains at 80°C and 121°C. This result suggests that temperature has little effect on the DPPH free radical scavenging ability of the cell-free supernatants.

[0342] (2) Hydroxyl radical scavenging ability of probiotic cell-free supernatant

[0343] The experimental method was similar to that in Example 3. As shown in Figure 7, the hydroxyl radical scavenging ability of the cell-free supernatant of ET-22 at 121°C was stronger than that at 80°C, while the hydroxyl radical scavenging ability of the cell-free supernatant of YLGB-1496 at 80°C was stronger than that at 121°C. There was no significant difference in the hydroxyl radical scavenging ability of the cell-free supernatants of ET-22 and BL-99 at 80°C and 121°C. This suggests that high temperature has different effects on the hydroxyl radical scavenging ability of the cell-free supernatants of Lactobacillus paracasei and Bifidobacterium, possibly due to different substances in the cell-free supernatants of different bacterial species.

[0344] Example 5

[0345] This example compares the effects of different inactivation processes on epigenetic anti-inflammatory function.

[0346] (1) Heat-inactivated cells

[0347] The anti-inflammatory cell test method is as follows: RAW 264.7 macrophages were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. In order to determine its anti-inflammatory activity, RAW 264.7 macrophages (1×10 6 cells / mL) were seeded in 12-well plates and then stimulated with LPS (1 μg / mL, Sigma-Aldrich) for 24 hours. Subsequently, different postbiotics were incubated with cells for 24 hours. The viable cell count before inactivation (5×10 8 cfu / mL) to calculate the intervention concentration of postbiotics, including heat-killed cells and cell-free supernatant.

[0348] To investigate the effects of different postbiotics on inflammatory cytokines, total RNA was extracted using TRIzol reagent (Invitrogen, Waltham, MA, USA), and cDNA was obtained by reverse transcription. Real-time quantitative PCR was performed using SYBR Green PCR Master Mix (TaKaRa, Shiga, Japan). Specific primer sequences are as follows:

[0349] TNF-α, forward 5-CTGAACTTCGGGGTGATCGG-3 (SEQ ID NO: 11), reverse 5-GGCTTGTCACTCGAATTTTGAGA-3 (SEQ ID NO: 12);

[0350] GAPDH, forward 5-AAGCCCATCACCATCTTCCA-3 (SEQ ID NO: 13), reverse 5-CACCAGTAGACTCCACGACA-3 (SEQ ID NO: 14).

[0351] Use 2 -ΔΔCtMethods The relative expression of each target gene was determined, and all quantifications were normalized to the GADPH gene.

[0352] As shown in Figure 8, LPS stimulation significantly increased the production of the proinflammatory cytokine TNF-α in RAW264.7 cells (P < 0.0001). Treatment with inactivated YLGB-1496 at different temperatures significantly decreased TNF-α production (P < 0.005), and temperature had little effect on the inhibitory effect of inactivated YLGB-1496 on TNF-α. Therefore, the results indicate that inactivated YLGB-1496 has a significant inhibitory effect on TNF-α.

[0353] (2) Cell-free supernatant

[0354] The preparation of the cell-free supernatant was the same as in Example 4, and the anti-inflammatory function test of the cell-free supernatant was performed using the same method.

[0355] The results are shown in Figure 9. The production of the proinflammatory factor TNF-α in RAW264.7 cells under LPS stimulation was significantly increased (P<0.0001). As the temperature increased, the inhibitory effect of YLGB-1496 on TNF-α weakened. The inhibitory effects on the expression of TNF-α after heat treatment at 100°C and 121°C were similar (P<0.01). Therefore, the results show that YLGB-1496 cell-free supernatant has a significant inhibitory effect on TNF-α, and temperature may affect the inhibitory effect.

[0356] In summary, excessive heating significantly inhibited the antioxidant activity of the four postbiotics derived from the exclusive strains, particularly at temperatures exceeding 100°C, which significantly decreased their DPPH and hydroxyl radical scavenging abilities. However, heating had no significant effect on the anti-inflammatory activity of the four postbiotics derived from the exclusive strains, maintaining excellent stability in both the cell-free supernatant, peptidoglycan, and S-layer proteins. Taking into account the antioxidant and anti-inflammatory activity results, and ensuring complete bacterial inactivation during production, we optimized the optimal process to be 90°C for 15 minutes of heat inactivation.

[0357] Example 6

[0358] This example screens postbiotic detection targets (non-volatile compounds) based on LC-MS.

[0359] The specific method is as follows:

[0360] 1. Sample Processing

[0361] 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 frozen 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 placed at -20 °C for 30 min and centrifuged for 15 min.

[0362] The supernatant was transferred to an inlet vial with an insert for analysis. Additionally, 20 μL of the supernatant from each sample was pipetted and mixed for use as a quality control sample.

[0363] 2. LC-MS detection

[0364] 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.

[0365] 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.

[0366] The mobile phase elution gradient is as follows:

[0367] 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:

[0368] 3. Quality Control

[0369] 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.

[0370] 4. Data Processing

[0371] 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:

[0372] (1) Only variables with more than 80% non-zero values ​​in any set of samples are retained;

[0373] (2) Use 1 / 2 of the minimum value in the original matrix to fill the missing values;

[0374] (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;

[0375] 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.

[0376] The main species composition of bacterial non-volatile substances is shown in Figure 10. The pre-processed data were further sorted and the peak areas greater than 10 4 The species composition of YLGB-1496 ranges from 500 to 600 substances, primarily amino acids and peptides, lipids, nucleotides, organic acids, alcohols and other acids, terpenes, and sugars.

[0377] The results of TOP50 differential metabolite analysis of two bacterial strains (YLGB-1496 and K56) after treatment at different temperatures are shown in Figure 11.

[0378] The presence of live bacterial components or their content in the postbiotic components is much higher than that in the live bacterial components, and they are stable in different heat inactivation processes (content variation range ≤ 20%). Substances that meet both of these requirements can be considered potential detection targets for postbiotic components.

[0379] By analyzing the TOP50 differential metabolites of the two strains, we identified postbiotic detection targets for the two strains (Figure 11). Comparing the raw data with the TOP50 differential metabolites, we identified six substances that could serve as potential detection targets for subsequent validation. These six substances are: Proly-Alanine, L-Methionine, Citric Acid, bAsp-Leu, bAsp-Phe, Antiarrhythmic Peptide, and GRPPK.

[0380] The peak area analysis of these six potential targets at different heat inactivation temperatures was further performed:

[0381] The analysis results are shown in Figure 12. The peak areas of the four substances L-Methionine, bAsp-Leu, bAsp-Phe and Antiarrhythmic peptide in the YLGB-1496 strain postbiotic system are 10 7 The peak area of ​​bAsp-Phe can reach up to 1×10 8 The peak area of ​​Citric Acid is 2-3×10 6 The YLGB-1496 live bacteria fraction also contains 6×10 5 Proly-Alanine may be removed in the future. The high content of five substances, L-Methionine, bAsp-Leu, bAsp-Phe, Citric Acid and Antiarrhythmic peptide, in YLGB-1496 provides a good basis for subsequent experimental verification and the establishment of detection methods. Antiarrhythmic peptide is expected to serve as a potential specific target substance of YLGB-1496. Combining the detection results of the above four strains, we screened a total of 7 characteristic substances that meet the screening requirements of postbiotic detection targets, namely: L-Methionine (L-methionine), Citric Acid (citric acid) and GP (Hyp) GAG (Antiarrhythmic peptide, Proly-Alanine (Prolyl Alanine), GPRPK, bAsp-Leu, bAsp-Phe.

[0382] Example 7

[0383] Characteristic metabolite composition of YLGB-1496 at different inactivation temperatures.

[0384] The detection of characteristic metabolites was carried out according to the LC-MS parameter conditions described in Example 6 above.

[0385] 13 , the results showed that the postbiotic preparation contained 1.6 mg / kg of γGlu-Leu, 1.46 mg / kg of tanacetin, 1.64 mg / kg of agaveside A, 1.6 mg / kg of transdermalin A, 1.5 mg / kg of cyclotide A, and 1.5 mg / kg of diglucoside.

[0386] GF-1, GF-2, and GF-3 represent live bacteria samples 1, 2, and 3; G801A, G802A, and G803A represent samples 1, 2, and 3 sterilized at 80°C; G101A, G102A, and G103A represent samples 1, 2, and 3 sterilized at 100°C; and G121A, G122A, and G123A represent samples 1, 2, and 3 sterilized at 121°C. The results showed that the levels of characteristic metabolites increased with increasing temperature.

[0387] Example 8

[0388] This example establishes a dedicated strain postbiotic detection and verification method based on LC.

[0389] 1. Materials and Reagents

[0390] Inactivated bacteria samples with different colony counts: Bifidobacterium longum subsp. infantis YLGB-1490.

[0391] Prolylalanine (C8H14N2O3, CAS: 6422-36-2): purity ≥ 96.5%; L-methionine (methionine, C5H 11 NO2S, CAS: 63-68-3): purity ≥ 98.5%; citric acid (C6H8O7, CAS: 77-92-9): purity ≥ 99.5%; βAsp-Leu peptide (aspartic acid-leucine), βAsp-Phe peptide (aspartic acid-phenylalanine), GPRPK peptide (glycine-proline-arginine-proline-lysine), and GP(Hyp)GAG peptide (glycine-proline (hydroxyproline)-glycine-alanine-glycine) standards, purity ≥ 98.0%; acetonitrile, methanol, and phosphoric acid (chromatographic grade, Thermo Fisher); formic acid and acetonitrile (mass spectrometry grade, Thermo Fisher); and pure water were used in the experiments.

[0392] 2. Instruments and Equipment

[0393] High-performance liquid chromatography with diode array detectors: Shimadzu LC-20A, SPD-M20A; high-resolution quadrupole time-of-flight liquid chromatography-mass spectrometry: Agilent 6546LC / Q-TOF; water-bath nitrogen purging apparatus; vortex mixer; high-speed centrifuge; ultrasonic cleaner; analytical balance.

[0394] 3. Preparation of standard solution

[0395] Standard stock solution: Accurately weigh appropriate amounts of standard substances (i.e., L-methionine and citric acid, etc., accurate to 0.1 mg), dissolve in water and prepare standard stock solutions with a concentration of ≥5 mg / mL, and store at -20°C.

[0396] Mixed standard intermediate solution: Accurately pipette appropriate volumes of standard stock solutions, dilute to volume with water, prepare a mixed standard intermediate solution with a concentration of 500 μg / mL, and store at 4°C.

[0397] Mixed standard working solution: dilute the mixed standard intermediate solution step by step with water as needed to prepare mixed standard working solutions with concentrations of 1μg / mL, 5μg / mL, 10μg / mL, 20μg / mL, 50μg / mL, 100μg / mL, and 200μg / mL, respectively. Prepare them before use.

[0398] 0.1% phosphoric acid aqueous solution: Take 1 mL of phosphoric acid, dilute it with water and make up to 1000 mL, mix thoroughly, and use immediately. 0.1% phosphoric acid acetonitrile solution: Take 1 mL of phosphoric acid, dilute it with acetonitrile and make up to 1000 mL, mix thoroughly, and use immediately.

[0399] 0.1% Formic Acid Aqueous Solution: Take 0.5 mL of formic acid, dilute it with water and make up to 500 mL, mix thoroughly, and use immediately. 0.1% Formic Acid Acetonitrile Solution: Take 0.5 mL of formic acid, dilute it with acetonitrile and make up to 500 mL, mix thoroughly, and use immediately.

[0400] 3. LC / MS analysis conditions for sample confirmation

[0401] An Agilent 1290 liquid chromatograph was used. The chromatographic column was a Poroshell 120Aq-C18 column (4.6 mm × 150 mm, 2.7 μm); mobile phase A consisted of 0.1% formic acid in water; mobile phase B consisted of 0.1% formic acid in acetonitrile, with a gradient elution (0 min, 0% B, 10 min, 0% B, 11 min, 8% B, 25 min, 8% B); the flow rate was 0.6 mL / min; the column temperature was 30°C; and the injection volume was 2 μL.

[0402] An Agilent 6546Q-TOF mass spectrometer was used. The ion source was an electrospray ionization (ESI) source, operating in positive and negative ion scan modes. The sheath gas temperature was 380°C, with a sheath gas flow rate of 11 L / min. The capillary voltages were 4000 V (positive) and 3500 V (negative). The nozzle voltage was 1000 V. The nebulizer pressure was 40 psi. The drying gas temperature was 340°C, with a drying gas flow rate of 10 L / min. The collision energy (CE) was 15–30 V. The TOF-MS scan range was m / z 100–1700, and the product ion scan range was 50–700 m / z.

[0403] 4. Liquid chromatography analysis conditions

[0404] An LC-20A analytical system was used. The chromatographic column was a Poroshell 120Aq-C18 column (4.6 mm × 150 mm, 2.7 μm); mobile phase A consisted of 0.1% phosphoric acid in water; mobile phase B consisted of 0.1% phosphoric acid in acetonitrile; the gradient elution program was as shown in the table below; the flow rate was 0.7 mL / min; the detection wavelength was 210 nm; the column temperature was 30°C; and the injection volume was 5 μL.

[0405] Gradient elution program table

[0406] 5. Colony count detection method

[0407] Prepare inactivated bacterial samples of different strains with different colony counts. The preparation method and colony count method refer to GB4789.2-94.

[0408] 6. Sample Pretreatment

[0409] Solid sample: Accurately weigh 0.3 g of inactivated bacteria supernatant into a centrifuge tube, add 6 mL of methanol-acetonitrile-water solution (4:4:2), oscillate and mix, extract at low temperature ultrasonically for 30 min (40 kHz), let stand at -20 ° C for 30 min, centrifuge at 10000 rpm / min at 4 ° C for 10 min, collect the supernatant and slowly blow dry with nitrogen at 40 ° C, make up to volume with 0.2 mL of pure water, vortex mix for 1 min, ultrasonicate for 2 min, centrifuge at 10000 rpm / min for 10 min, aspirate the supernatant and dilute it to the linear range, and analyze on the column.

[0410] 7. Methodological Validation

[0411] (1) Sample confirmation

[0412] To confirm that the sample contains the desired target component, qualitative analysis is performed using the information on the molecular mass and structure of the chromatographic peak provided by the mass spectrometer, thereby obtaining more and more reliable information than qualitative analysis based solely on retention time or increased spectral similarity. The mass spectrometry analysis results must simultaneously meet the following two points: (1) The difference between the retention time of the sample to be tested and the retention time of the standard is within ±2.5%; 2) The European Commission has published mass spectrometry method indicators, which recommend that when confirming the sample to be tested, a minimum confirmation score of 4 points is required, with one parent ion being 1 point and one daughter ion being 1.5 points.

[0413] (2) Standard curve, detection limit and quantification limit

[0414] Inject the mixed standard working solution into the HPLC instrument three times for each concentration and measure the corresponding chromatogram peak area. Plot a standard curve with the concentration of the standard working solution as the abscissa and the chromatogram peak area as the ordinate.

[0415] The limit of detection (LOD) and limit of quantitation (LOQ) are used to evaluate the sensitivity of a method. The limit of detection is the lowest concentration at which the analyte can be detected, and the limit of quantitation is the lowest concentration at which the analyte can be quantitatively detected. By continuously injecting a mixed standard at decreasing concentrations, the LOD is the target component concentration at which the signal-to-noise ratio (S / N) is ≥3, while the LOQ is the target component concentration at which the S / N is ≥10.

[0416] (3) Accuracy and precision

[0417] Trueness is evaluated by recovery testing, and precision is assessed by the coefficient of variation (RSD). This test measures recovery after adding three different concentrations of standard solutions to the test sample. This is the ratio of the standard addition indicated by the test to the actual standard addition, expressed as a percentage. Three replicates were performed at each concentration level, and the recovery and coefficient of variation were calculated. According to the requirements of "GB / T 27417: Guidelines for Validation and Verification of Chemical Analysis Methods for Conformity Assessment," the spike recovery should be between 80% and 110%, and the precision should be less than 20%.

[0418] (4) Result calculation

[0419] Inject the prepared sample solution into a high-performance liquid chromatograph, record the retention time and peak area of ​​the chromatographic peak, and quantify using the external standard method. The response value of the target compound in the sample solution should be within the linear range of the instrument's quantitative determination. If it exceeds the linear range, dilute the sample appropriately based on the concentration before analysis. The result is expressed as the arithmetic mean of three independent determinations obtained under repeatability conditions, rounded to three significant figures.

[0420] The content of each target component in the sample is calculated according to formula (1):

[0421] Where:

[0422] X—the content of a target component in the sample, in milligrams per 100 grams or milligrams per 100 milliliters (mg / 100g or mg / 100mL);

[0423] ci—mass concentration of each target component in the sample solution obtained from the standard working curve, in micrograms per milliliter (μg / mL);

[0424] V—the fixed volume of the sample extraction solution, in milliliters (mL);

[0425] m—sample mass or volume, in grams or milliliters (g or mL); 10—unit conversion factor;

[0426] f—dilution factor.

[0427] The feasibility of predicting the colony count of the sample based on the content of the target component was explored, and a linear regression equation was established to show the correlation between the colony count and the target component: Y = a + bX………………(2)

[0428] Where:

[0429] Y—the number of colonies in the sample, in units of 10^9 CFU / mL;

[0430] X—the content of a target component in the sample, in mg / 100g or mg / 100mL; a, b—parameter values.

[0431] Here are the results:

[0432] A. Sample confirmation:

[0433] UPLC-Q-TOF-MS / MS qualitative analysis was performed on a mixture of seven standard substances, a sample solution of an inactivated bacterial strain, and a fermentation broth sample. By comparing the mass spectra, characteristic fragment ions, and chromatographic retention times of each reference standard with the information on the characteristic components detected in these eight samples using the MassHunter METLIN Metabolite PCLD database, it was found that the retention times of the seven target components were within ±2.5% of those of the reference standard. Since each target component had two product ions and one parent ion, the confirmation score was 4, meeting the confirmation requirements stipulated by the European Union. The mass spectrometric data for the seven target components in ESI+ / - mode are shown in the table below.

[0434] The UPLC-Q-TOF-MS / MS identification results are as follows:

[0435] B. Standard Curve, Limit of Detection, and Limit of Quantitation

[0436] Under the above-mentioned LC conditions, a series of mixed standard working solutions were analyzed on the column. The peak area (Y) was recorded as the ordinate, and the concentration (X (μg / mL)) was recorded as the abscissa for linear regression. The limit of detection (LOD) and limit of quantification (LOQ) of the analytical method were determined at concentrations where the signal-to-noise ratio was ≥3 and 10, respectively.

[0437] Retention time, standard curve, correlation coefficient, linear range, LOD and LOQ statistics of 7 target components

[0438] The results showed that the seven target components exhibited good linear relationships within their respective linear ranges, with correlation coefficients ≥0.999 and LOQs ranging from 0.15 to 5.00 μg / mL, demonstrating that this method can be used for accurate quantitative analysis of the components. The chromatogram is shown in Figure 14: 1-prolylalanine; 2-L-methionine; 3-citric acid; 4-GPRPK; 5-GP(Hyp)GAG; 6-bAsp-Leu; and 7-bAsp-Phe.

[0439] C. Accuracy and precision

[0440] K56 fermentation broth samples were used as spiked samples. For the components L-methionine and citric acid, three spiked concentrations (low (approximately 50%), medium (approximately 100%), and high (approximately 150%)) were selected for the experiments. Each concentration was replicated three times. The spiked samples were also pretreated and quantitatively analyzed. Chromatograms were examined and peak areas were recorded. Accuracy was expressed as recovery: (measured value - background value) / actual spiked concentration × 100%. The results are shown in Table 3.4-3. The average recoveries of L-methionine and citric acid ranged from 80.0% to 102.4%, with relative standard deviations (RSDs) within 9.2%, meeting the requirements of GB / T 27417. This method demonstrates good accuracy and precision.

[0441] Statistics table of spike test results

[0442] D. Measurement of actual samples

[0443] Samples were selected for content determination. The statistical results for the target components in the inactivated bacteria YLGB-1496 are shown in the table below. As can be seen from the table, all four inactivated bacteria samples contained multiple target components, with L-methionine, citric acid, and GPRPK peptide present at high and consistently high levels. The liquid chromatograms of the actual samples are shown in Figure 15. Note: - indicates not detected or the resolution from the sample base peak is less than 1.5.

[0444] E. Establish the correlation between target component content and colony count

[0445] The above-mentioned liquid chromatography method was used to analyze inactivated YLGB-1496 bacteria samples with varying colony counts (1 to 300, unit: 10^9 CFU / mL) and their fermentation broth samples. HPLC fingerprints and analysis results for the seven target components were established. The relationship between the target component content and the colony count of the main indicator was studied to identify the quality differences of postbiotic products. The overall analysis results are shown in the table below. The correlation coefficient of the regression equation was greater than 0.9, indicating a significant correlation.

[0446] Correlation equation between the contents of seven target components and the number of colonies in the inactivated bacteria sample of the exclusive strain Note: — indicates that the separation degree from the sample base peak is lower than 1.5, so this component is not selected for quantification.

[0447] Correlation equation between the content of L-methionine and citric acid in the fermentation broth sample of the exclusive strain and the number of colonies

[0448] In summary, this paper uses high-performance liquid chromatography (HPLC) to establish regression equations for the colony counts of inactivated bacteria samples and fermentation broth samples, based on the contents of seven target components. This method is simple to operate, with good accuracy and precision, and can provide a reference for future analytical testing and quality evaluation of postbiotic products.

[0449] Example 9

[0450] This embodiment provides a secretin of Bifidobacterium longum subsp. infantis YLGB-1496, as shown in FIG16 , and its preparation method comprises the following steps:

[0451] 1. Preparation of Tertiary Seeds

[0452] 1.1 Standard cryopreservation tubes

[0453] 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.

[0454] 1.2 Activation of cryotubes

[0455] 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.

[0456] 1.3 Primary purification

[0457] 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-72h until obvious 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 20-24h.

[0458] 1.4 Secondary purification

[0459] Take the cultured purified bacterial liquid for dilution and coating, with dilution degrees of -4, -5, and -6. Make two MRS solid plates for each dilution. Invert and culture at 37°C for 48h-72h until obvious 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 are uniform in size and cultured at 37°C for 20-24h.

[0460] 1.5 Primary seed preparation

[0461] 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 hours.

[0462] 1.6 Secondary seed preparation

[0463] Select 4 tubes of cultured first-level seeds, aspirate 4 mL of each and inject into 4 bottles of 80 mL MRS liquid culture medium, and culture at 37°C for 11-13 hours.

[0464] 1.7 Preparation of tertiary seeds

[0465] The cultured secondary seeds were poured into two bottles of 1.8LMRS liquid culture medium and cultured at 37°C for 12-14 hours.

[0466] 1.8 Temporary storage of third-level seeds

[0467] After the third-level seeds are prepared, they can be placed at 4℃ for no more than 6 hours.

[0468] 1.9 Process Quality Control

[0469] (1) Indicators for determining the end point of seed growth at each level: pH 4.4-4.8, OD600 ≥ 1.8.

[0470] (2) Purity test: Observe the bacterial morphology under a microscope. Under a 100x oil immersion lens, the bacterial morphology is complete, appearing as a long rod, slightly curved, and arc-shaped. After a long period of decolorization, the bacterial body becomes transparent and relatively blurry.

[0471] (3) Pollutant detection: Detection of the third-level seed liquid, including Escherichia coli and non-lactic acid bacteria.

[0472] Formula of three-stage seed fermentation medium

[0473] pH value: 6.2~6.4, sterilization conditions: 121℃, 15~20min.

[0474] 2. Fermentation Seed Preparation

[0475] 2.1 Vaccination

[0476] (1) Turn on the stirring paddle and temperature control program, the speed is 70 rpm, and the temperature is 37 °C.

[0477] (2) Open the nitrogen inlet valve and introduce a small flow of nitrogen into the fermenter to maintain positive pressure in the fermenter for 5-10 minutes.

[0478] (3) When inoculating, pour alcohol onto the inoculation loop and ignite it to form a flame circle.

[0479] (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%.

[0480] (5) After inoculation, tighten the upper cover of the inoculator, close the inoculation valve, and extinguish the alcohol flame ring.

[0481] (6) Close the nitrogen inlet valve and exhaust valve, and control the tank pressure to 0.01-0.03 MPa for pressure-maintaining fermentation.

[0482] 2.2 Fermentation

[0483] (1) Set the fermentation parameters to 70 rpm and 37°C.

[0484] (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.

[0485] (3) After fermentation, the fermentation broth can be cultured or cooled to 10-20℃ and stored for no more than 6 hours.

[0486] 2.8 Process Quality Control

[0487] (1) Seed tank fermentation endpoint determination indicators: pH 4.4-4.8, OD600 ≥ 1.8.

[0488] (2) Purity test: Observe the bacterial morphology of the fermentation liquid at the end of the seed tank under a microscope. Under a 100x oil lens, the bacterial morphology is complete, long rods, slightly curved, and arc-shaped.

[0489] (3) Pollutant detection: Detect the fermentation liquid at the end of the seed tank, including Escherichia coli and non-lactic acid bacteria.

[0490] The formula of seed tank fermentation medium is as follows.

[0491] pH value: 6.2~6.4, sterilization conditions: 121℃, 15~20min.

[0492] 3. Fermentation Broth Culture

[0493] 3.1 Vaccination

[0494] (1) Turn on the stirring paddle and temperature control program, the speed is 70 rpm, and the temperature is 37 °C.

[0495] (2) Steam sterilize the inoculation pipe for 30 minutes.

[0496] (3) Open the nitrogen inlet valve and fill with nitrogen for 5-10 minutes. Open the bottom valve of the seed tank and the inoculation valve of the fermentation tank for inoculation. The inoculation amount is 5%.

[0497] (4) After inoculation, close the fermenter inoculation valve and clean the inoculation pipeline.

[0498] (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.

[0499] 3.2 Fermentation

[0500] (1) Set the fermentation parameters to 37°C, 70 rpm, and a constant pH of 6.

[0501] (2) The fermentation time was 14 to 16 h, and the fermentation status was monitored every 2 h, including pH, OD600, temperature, and rotation speed.

[0502] (3) After fermentation, centrifugation can be performed or the temperature can be lowered to 10-20°C and stored for no more than 4 hours.

[0503] The fermentation medium formula of the fermentation tank is shown in the following table.

[0504] pH value: 6.2~6.4, sterilization conditions: 121℃, 15~20min.

[0505] 3.3 Mushroom sludge separation

[0506] (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.

[0507] (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.

[0508] (3) Open the feed valve, and the fermentation liquid of Bifidobacterium longum subsp. infantis YLGB-1496 enters the centrifuge drum and starts centrifugation to obtain bacterial sludge.

[0509] (4) Centrifugal process parameter settings: feed rate 600 L / h, slag discharge time 180 s.

[0510] 4. Bacteriocin Extraction

[0511] 4.1 Mud transfer

[0512] The collected bacterial sludge was transferred to a sterile fermentation tank for extraction.

[0513] 4.2 Extraction conditions

[0514] The bacterial sludge was extracted and sterile purified water was used to mix the bacterial sludge to obtain a mixed solution. The total colony counts of Bifidobacterium longum subspecies infantis YLGB-1496 in the mixed solution were 0.5×10 10 cfu / mL, 5×10 10 cfu / mL, 7.5×10 10 cfu / mL, 1.5×10 11 cfu / mL, the extraction temperatures were 4°C, 25°C, and 37°C, the extraction times were 1h, 2h, and 3h, and the extraction speed was 70rpm.

[0515] 5. Centrifugal separation

[0516] (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.

[0517] (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.

[0518] (3) Open the feed valve and the mixed liquid enters the centrifuge drum to start centrifugation.

[0519] (4) Centrifugal process parameter settings: feed rate 600 L / h, slag discharge time 180 s.

[0520] (5) Transfer the centrifuged supernatant to a collection tank.

[0521] 6. Packaging and sterilization

[0522] The filled bottled supernatant was heat sterilized to obtain bacteriocin. The sterilization conditions of Bifidobacterium longum subsp. infantis YLGB-1496 were 75° C. for 10 min. The obtained bacteriocin of Bifidobacterium longum subsp. infantis YLGB-1496 was shown in FIG17 .

[0523] 7. Quantitative Detection

[0524] The concentrations of L-methionine and citric acid in the product obtained in Example 1 were detected, and the detection method comprised the following steps:

[0525] (1) Preparation of standard working solution

[0526] Standard stock solution: Accurately weigh appropriate amounts of standard substances (i.e., L-methionine and citric acid, accurate to 0.1 mg), dissolve in water and prepare standard stock solutions with a concentration of 5 mg / mL, and store at -20°C.

[0527] Mixed standard intermediate solution: Accurately pipette appropriate volumes of standard stock solutions, dilute to volume with water, prepare a mixed standard intermediate solution with a concentration of 500 μg / mL, and store at 4°C.

[0528] Mixed standard working solution: dilute the mixed standard intermediate solution step by step with water as needed to prepare mixed standard working solutions with concentrations of 1μg / mL, 5μg / mL, 10μg / mL, 20μg / mL, 50μg / mL, 100μg / mL, and 200μg / mL, respectively. Prepare them before use.

[0529] (2) Preparation of elution solution

[0530] 0.1% phosphoric acid aqueous solution: Take 1 mL of phosphoric acid, dilute it with water and make up to 1000 mL, mix well, and use it immediately.

[0531] 0.1% phosphoric acid acetonitrile solution: Take 1 mL of phosphoric acid, dilute with acetonitrile and make up to 1000 mL, mix well, and use immediately.

[0532] (3) Preparation of test solution

[0533] Liquid sample: Mix the secretory sample of Bifidobacterium longum subsp. infantis YLGB-1496 and directly pipette 1 mL of it. Centrifuge at 10,000 rpm for 10 min at 4°C. Dilute the supernatant to within the linear range and load onto the column for analysis.

[0534] (4) Detection and analysis

[0535] The sample solution and the standard working solution were tested by high performance liquid chromatography, and the corresponding chromatogram peak areas were measured. A standard curve was plotted with the concentration of the standard working solution as the abscissa and the chromatogram peak area as the ordinate. Based on the test results of the sample solution in step (3) (as shown in FIG. 18 , where 1 is L-methionine and 2 is citric acid), the concentrations of L-methionine and citric acid in the bacteriocin extraction step were calculated in combination with the standard curve.

[0536] Chromatographic analysis conditions: An LC-20A analytical system was used, with a Poroshell 120Aq-C18 column (4.6 mm × 150 mm, 2.7 μm); mobile phase A was 0.1% phosphoric acid in water; mobile phase B was 0.1% phosphoric acid in acetonitrile; the gradient elution program is shown in Table 1; the flow rate was 0.7 mL / min; the detection wavelength was 210 nm; the column temperature was 30°C; and the injection volume was 5 μL.

[0537] Table 1 Gradient elution program

[0538] The total number of colonies of Bifidobacterium longum subspecies infantis YLGB-1496 in the mixed solution was calculated to be 1.5×10 11 cfu / mL, the extraction temperature was 4℃, and the extraction time was 1h. The L-methionine concentration in the bacteriocin was 0.06mg / 100ml and the citric acid concentration was 5.83mg / 100ml.

[0539] (5) Calculate the number of colonies before inactivation of inactivated bacteria

[0540] Among them, citric acid and L-methionine can be used as targets to calculate the colony count in the fermentation broth before mycobacterin extraction.

[0541] When citric acid was used as the target to calculate the corresponding colony count in the fermentation broth before mycobacterial extraction, the regression equation used was y = 0.899x-0.065; where x represents the concentration of the target in mg / 100 g; y represents the colony count in 10 9 CFU / mL. Substitute the calculated target concentration into the regression equation to calculate the colony count before inactivation.

[0542] When L-methionine was used as the target to calculate the corresponding colony count in the fermentation broth before mycobacterial extraction, the regression equation used was y = 99.140x + 2.441; where x represents the concentration of the target in mg / 100 g; y represents the colony count in 10 9 CFU / mL. Substitute the calculated target concentration into the regression equation to calculate the colony count before inactivation.

[0543] In the present disclosure, LC-MS was used to detect the components of the inactivated bacteria obtained in Example 1, and the peak areas greater than 10 4The components of the bacteriocin were screened to obtain targets that characterize the inactivated bacteria components. The target screening criteria include: ① it does not exist before heat sterilization, or the content of the component before heat sterilization is much lower than the content of the component after heat sterilization; ② it can stably exist under different heat sterilization conditions within the experimental range (content change range ≤ 20%). It was found that 6 substances can be used as potential detection targets, namely: Proly-Alanine, L-Methionine, Citric Acid, bAsp-Leu, bAsp-Phe, Antiarrhythmic peptide, GRPPK. At the same time, the 6 detection targets in the bacteriocin were detected, and it was found that only citric acid and L-methionine were present in the bacteriocin. The other four detection targets were not extracted or the content was below the detection line. Therefore, in the present disclosure, citric acid and L-methionine are used as detection targets to characterize the bacteriocin.

[0544] Example 9

[0545] This example compares the scavenging abilities of the samples prepared in Example 8 for DPPH radicals and hydroxyl radicals. The detection method is as described in Example 3.

[0546] The test results of the hydroxyl radical scavenging ability of the bacteriocin obtained in Example 1 are shown in FIG19 . As can be seen from FIG19 , when the extraction concentration is 1.5×10 11 cfu / mL, the extraction temperature was 4°C, and the extraction time was 1h, the hydroxyl radical scavenging ability of the bacteriocin was better; the test results of the DPPH radical scavenging ability of the bacteriocin obtained in Example 1 are shown in FIG20 , in which the extraction temperatures and times of K1-K9 were 4°C, 1h, 4°C, 2h, 4°C, 3h, 25°C, 1h, 25°C, 2h, 25°C, 3h, 37°C, 1h, 37°C, 2h, and 37°C, 3h, respectively. The extraction temperatures and times for K10-K18 were 4°C, 1h, 4°C, 2h, 4°C, 3h, 25°C, 1h, 25°C, 2h, 25°C, 3h, 37°C, 1h, 37°C, 2h, and 37°C, 3h, respectively; and the extraction temperatures and times for K19-K27 were 4°C, 1h, 4°C, 2h, 4°C, 3h, 25°C, 1h, 25°C, 2h, 25°C, 3h, 37°C, 1h, 37°C, 2h, and 37°C, 3h, respectively. As shown in Figure 20, the effects of the extraction conditions on DPPH radicals were relatively small.

[0547] Example 10

[0548] NEC animal experiments were conducted on inactivated bacteria of GB1496.

[0549] The main experimental reagents are as follows:

[0550] The solution was prepared as follows:

[0551] (1) Anaerobic culture medium: Boil 1 L MRS broth and add 0.5 g L-cysteine ​​hydrochloride and mix well.

[0552] (2) 75% ethanol solution: Add 25 ml of sterile enzyme-free water to 75 ml of anhydrous ethanol.

[0553] (3) Physiological saline: Add 8.5 g of sodium chloride to 1 L of deionized water.

[0554] 1. Animal experimental methods are as follows:

[0555] A. YLGB1496 strain culture

[0556] (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).

[0557] (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.

[0558] (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.

[0559] B. NEC Animal Experiment Design

[0560] (1) Experimental animal grouping:

[0561] The NEC animal experiment groups are as follows:

[0562] (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.

[0563] (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.

[0564] C. Preparation of mouse ileum NEC pathological sections

[0565] (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.

[0566] (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.

[0567] (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.

[0568] The NEC organization's scoring criteria are as follows:

[0569] D. Collection of blood and intestinal tissue samples and extraction of ileal RNA

[0570] (1) The entire intestinal tissue of all mice needs to be removed and straightened to photograph the intestinal tissue morphology.

[0571] (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.

[0572] (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.

[0573] E. Full-length 16S rDNA sequence amplification

[0574] ① Genomic DNA extraction: After the genomic DNA extraction is completed, the extracted genomic DNA is detected by 1% agarose gel electrophoresis.

[0575] 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.

[0576] 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.

[0577] ④ Fluorescence quantification: refer to the preliminary quantitative results of electrophoresis, and PCR products were quantified by QuantiFluor TM -ST blue fluorescence quantitative system (Promega) was used for detection and quantification, and then the samples were mixed in corresponding proportions according to the sequencing amount requirements of each sample.

[0578] F.acBio SMRT third-generation sequencing

[0579] 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).

[0580] 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.

[0581] G. Data Processing and Analysis

[0582] 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.

[0583] 2. Perform intestinal dissection on the animal. During NEC, intestinal hypoxia and ischemia can lead to intestinal histological changes, generally including intestinal flatulence and edema, and in severe cases, intestinal hemorrhagic necrosis.

[0584] The experimental results are shown in Figure 21, which show that:

[0585] Compared with the NEC group, the Con group showed more pronounced flatulence in the NEC group, a pathological hallmark of NEC, indicating partial cell necrosis in the intestinal barrier. Compared with the NEC group, the live GB1496 group showed no significant flatulence, with more normal intestinal morphology. Compared with the NEC group, the dead GB1496 group still showed a small amount of intestinal flatulence, but it had a moderately relieving effect.

[0586] 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.

[0587] The results in Figure 22 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.

[0588] 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.

[0589] 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.

[0590] The scoring criteria are as follows:

[0591] As shown in Figure 23, the incidence of NEC in the NEC group was significantly increased to 100% compared with the Con group (P < 0.05), indicating that the NEC model was successfully established. The incidence of GB1496A (21.43%) and GB1496D (50%) groups was significantly reduced compared with the NEC group (P < 0.05).

[0592] 5.NEC survival rate experiment.

[0593] During the NEC modeling process, the body may die due to the disease. As shown in Figure 24, the NEC group, GB1496A group and GB1496D group all had a certain mortality rate, but there was no statistically significant difference in the survival rate among the groups (P>0.05).

[0594] 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)).

[0595] 6. Intestinal inflammatory factor expression level experiment

[0596] 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:

[0597] 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.

[0598] 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.

[0599] 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.

[0600] 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.

[0601] The primer sequences are as follows:

[0602] As shown in Figure 25 , compared with the Con group, the expression levels of IL-6 ( Figure 25 (A)), IL-1β ( Figure 25 (B)), TNF-α ( Figure 25 (D)), and TLR-4 ( Figure 25 (C)) in the NEC group were significantly increased, while IL-10 ( Figure 25 (E)) was significantly decreased. Compared with the NEC group, the expression levels of IL-6 ( Figure 25 (A)), TNF-α ( Figure 25 (D)), and TLR-4 ( Figure 25 (C)) in the GB1496A and GB1496D groups were significantly decreased, while the expression of IL-10 ( Figure 25 (E)) was increased. In addition, the expression level of IL-1β ( Figure 25 (B)) was significantly decreased in the GB1496D group, and the GB1496A group also showed a downward trend.

[0603] In summary, judging from the expression levels of the above inflammatory factors, GB1496A and GB1496D have the effect of inhibiting the inflammatory level.

[0604] 7. Experiment on the effects of GB1496A group and GB1496D group on the intestinal flora of mice.

[0605] 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 26(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.

[0606] ② Principal coordinates analysis (PCoA) based on Bray-Curtis distance can be used to study the similarities or differences in the composition of sample communities. Figure 26 (B) compares the β-diversity of the intestinal flora composition of the NEC group, GB1496A group, and GB1496D group at the genus level. As shown in Figure 26, the community composition of the intestinal flora of the NEC group and the other two groups is significantly different and can be completely separated.

[0607] 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 GB1496A group, GB496D group and NEC group at the genus level of intestinal flora. Figure 27 (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 27 (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.

[0608] 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 28 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.

[0609] The above description is merely a preferred embodiment of the present disclosure and is not intended to limit the present disclosure. Those skilled in the art will readily appreciate that various modifications and variations of the present disclosure are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present disclosure shall be included within the scope of protection of the present disclosure. Industrial Applicability

[0610] The probiotic postbiotic product provided by the present disclosure has the following biological functions: enhancing intestinal barrier function, preventing and / or treating inflammatory bowel disease, regulating intestinal flora, anti-oxidation and alleviating intestinal flatulence.

Claims

1. A probiotic postbiotic product, characterized in that: It includes: Inactivated probiotic bacteria and / or metabolites thereof, wherein the probiotic bacteria include Bifidobacterium longum subsp. infantis YLGB-1496 strain, whose deposit number is CCTCC NO: M2011122; The metabolites contain: more than 128.81 mg / 100 g of L-methionine and more than 764 mg / 100 g of short peptides.

2. The probiotic postbiotic product according to claim 1, characterized in that The short peptide is selected from at least one of prolyl propionic acid, GPRPK, antiarrhythmic peptide, βAsp-Leu and βAsp-Phe; Optionally, the metabolites contain more than 673.72 mg / 100 g of proline propionic acid, more than 44.55 mg / 100 g of GPRPK, more than 9.79 mg / 100 g of antiarrhythmic peptide, more than 20.1 mg / 100 g of dipeptide Asp-Leu, and more than 15.84 mg / 100 g of dipeptide Asp-Phe; Optionally, the mass ratio of prolylpropionic acid, L-methionine, GPRPK, antiarrhythmic peptide, dipeptide Asp-Leu and dipeptide Asp-Phe in the probiotic postbiotic product is 620-680:120-130:40-50:8-12:18-22:13-16; Optionally, the mass ratio of proline propionic acid, L-methionine, GPRPK, antiarrhythmic peptide, dipeptide Asp-Leu and dipeptide Asp-Phe in the probiotic postbiotic product is 673.72:128.81:44.55:9.79:20.1:15.

84.

3. The probiotic postbiotic product according to claim 1, characterized in that The probiotic postbiotic product further contains a highly active substance, wherein the highly active substance is selected from at least one of γGlu-Leu, tanacetin, agaveside A, transdermalin A, cyclopeptide A and diglucoside; Optionally, the highly active substances include: Glu-Leu at a concentration of more than 1.6 mg / kg, Tanacetin at a concentration of more than 1.46 mg / kg, Agave A at a concentration of more than 1.64 mg / kg, Transdermal A at a concentration of more than 1.6 mg / kg, Cyclic Peptide A at a concentration of more than 1.5 mg / kg, and Diglucoside at a concentration of more than 1.5 mg / kg; The highly active substance is derived from inactivated probiotic bacteria and / or metabolites thereof.

4. A probiotic postbiotic product, characterized in that It includes: Inactivated probiotics and their metabolites, wherein the probiotics include Bifidobacterium longum subsp. infantis YLGB-1496 strain, whose deposit number is CCTCC NO: M2011122; The inactivated probiotics are inactivated probiotics obtained through high-density fermentation; the inactivation conditions for the inactivated probiotics are 70-121° C. for 5-15 minutes; Optionally, the high-density fermentation is carried out at 37°C ± 0.5°C and a pH of 5.75 ± 0.5; Optionally, the fermentation time is 11-13 hours; Optionally, the inactivation sterilization conditions are: 90-100° C., treatment for 10-15 minutes.

5. The probiotic postbiotic product according to any one of claims 1 to 3 or the probiotic postbiotic product according to claim 4, characterized in that: The probiotic postbiotic product comprises at least one of the dead bacteria of the inactivated bacteria, the cell disrupted product of the inactivated bacteria, the precipitate of the inactivated bacteria, and the cell-free supernatant of the inactivated bacteria; Optionally, the probiotic postbiotic product further comprises: a carrier and / or auxiliary materials; Optionally, the carrier or auxiliary material is selected from: at least one of a protective agent, an excipient, a binder, a disintegrant, a lubricant, a flavor, a preservative, a stabilizer, a suspending agent, a dispersant, and a diluent; Optionally, the probiotic postbiotic product is in the form of liquid, solid or semi-solid.

6. The method for preparing the probiotic postbiotic product according to any one of claims 1 to 3 or the probiotic postbiotic product according to claim 4, characterized in that: The method comprises the following steps: inactivating the fermentation product of the probiotics; the sterilization conditions are: 70-121° C., 5-15 min; Optionally, treating at 90-100°C for 10-15 minutes; Optionally, the fermentation product of the probiotics is a fermentation product of the probiotics after high-density fermentation; Optionally, the high-density fermentation is carried out at 37°C ± 0.5°C and a pH of 5.75 ± 0.5; Optionally, the fermentation time is 11-13 hours; Optionally, the fermentation product is centrifuged, and the precipitate is mixed with a carrier and / or auxiliary materials; Optionally, the cell-free supernatant of the inactivated bacteria after centrifugation is used to prepare probiotic postbiotics; Optionally, the precipitate is mixed with a protective agent and an excipient; and then freeze-dried to prepare a powder.

7. A secretin of Bifidobacterium longum subsp. infantis YLGB-1496, characterized in that The invention comprises the exocytosis product of Bifidobacterium longum subspecies infantis YLGB-1496 and the metabolites of Bifidobacterium longum subspecies infantis YLGB-1496. The mass ratio of citric acid to L-methionine in the secretory agent is greater than 25:

1. The preservation number of Bifidobacterium longum subspecies infantis YLGB-1496 is CCTCC No. M2011122.

8. The secretin of Bifidobacterium longum subsp. infantis YLGB-1496 according to claim 1, characterized in that The mass ratio of citric acid to L-methionine in the bacteriocin is 25-140:1; Optionally, the mass ratio of citric acid to L-methionine in the bacteriocin is 90-105:

1.

9. A method for preparing the secretin of Bifidobacterium longum subsp. infantis YLGB-1496 according to claim 1 or 2, characterized in that: The following steps are involved: Bacteriocin extraction: using a solvent to extract the bacterial sludge separated from the fermentation liquid to obtain a mixed liquid; Sterilize, separate the supernatant from the mixed solution and sterilize it by heat to obtain the bacteriocin.

10. The method for preparing the secretin of Bifidobacterium longum subsp. infantis YLGB-1496 according to claim 3, characterized in that: The extraction temperature is 0-37°C; optionally, the extraction temperature is 3-5°C; And / or, the extraction time is 0.5-3h; optionally, the extraction time is 30-90min.

11. The method for preparing the secretin of Bifidobacterium longum subsp. infantis YLGB-1496 according to claim 3, characterized in that: The total colony count of Bifidobacterium longum subspecies infantis YLGB-1496 in the mixed solution was 0.5×10 10 -3×10 11 cfu / mL; Optionally, the total colony count of Bifidobacterium longum subspecies infantis YLGB-1496 in the mixed solution is 1×10 11 -2×10 11 cfu / mL.

12. The method for preparing the secretin of Bifidobacterium longum subsp. infantis YLGB-1496 according to claim 3, characterized in that: The temperature of heat sterilization is 70-121°C; optionally, the temperature of heat sterilization is 70-100°C; And / or, the heat sterilization time is 5-30 minutes; optionally, the heat sterilization time is 10-16 minutes.

13. The method for preparing the secretin of Bifidobacterium longum subsp. infantis YLGB-1496 according to claim 3, characterized in that: The invention also includes a fermentation step of culturing Bifidobacterium longum subspecies infantis YLGB-1496 in liquid culture and stopping the fermentation when the Bifidobacterium longum subspecies infantis YLGB-1496 grows to a logarithmic phase; Optionally, the fermentation step comprises: inoculating a fermentation seed solution of Bifidobacterium longum subsp. infantis YLGB-1496 into a culture medium, and first fermenting for 14-16 hours at a temperature of 30-40° C., a rotation speed of 60-80 rpm, and a pH of 5.8-6.2 to obtain a fermentation solution of Bifidobacterium longum subsp. infantis YLGB-1496; Optionally, the preparation of the fermentation seed liquid of Bifidobacterium longum subsp. infantis YLGB-1496 comprises: activating, purifying, culturing the Bifidobacterium longum subsp. infantis YLGB-1496 strain with a first-level seed liquid, expanding with a second-level seed liquid, culturing with a third-level seed liquid, and preparing fermentation seeds to obtain the fermentation seed liquid of Bifidobacterium longum subsp. infantis YLGB-1496; Optionally, the culture medium is MRS liquid culture medium; Optionally, the solvent is water; Optionally, the solvent is sterile water; Optionally, after the sterilization step, the bacteriocin is freeze-dried to obtain freeze-dried bacteriocin.

14. A composition, characterized in that The invention comprises at least one of the probiotic postbiotic product according to any one of claims 1 to 3, the probiotic postbiotic product according to claim 4, and the secretin of Bifidobacterium longum subsp. infantis YLGB-1496 according to claim 7 or 8.

15. Use of the probiotic postbiotic product according to any one of claims 1 to 3, the probiotic postbiotic product according to claim 4, the secretin of Bifidobacterium longum subsp. infantis YLGB-1496 according to claim 7 or 8, or the composition according to claim 7 in the preparation of a composition for (1) enhancing intestinal barrier function; (2) preventing and / or treating inflammatory bowel disease; (3) regulating intestinal flora; (4) relieving intestinal flatulence; (5) anti-aging; and / or (6) anti-oxidation; Optionally, the composition is selected from health food, food antioxidant, feed or medicine.

16. The use according to claim 8, 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.

17. The use according to claim 8, characterized in that The drug for preventing and / or treating 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; Optionally, 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; Optionally, the necrotizing enterocolitis is neonatal necrotizing enterocolitis; Optionally, said treating colitis comprises improving the survival rate of patients with colitis; Optionally, the prevention of colitis comprises reducing the incidence of colitis; Optionally, 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; Optionally, the abundance of Escherichia coli in the intestine is suppressed, while the abundance of Lactobacillus and Bifidobacterium is increased.

18. Use of the probiotic postbiotic product according to any one of claims 1 to 3, the probiotic postbiotic product according to claim 4, the secretin of Bifidobacterium longum subsp. infantis YLGB-1496 according to claims 1 to 8, or the composition according to claim 7 in (1) enhancing intestinal barrier function; (2) preventing and / or treating inflammatory bowel disease; (3) regulating intestinal flora; (4) relieving intestinal flatulence; (5) anti-aging; and / or (6) anti-oxidation.

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