Postbiotic product of lactobacillus paracasei k56, k56 bacteriocin, preparation method therefor, and use thereof in intestinal health and antioxidation

Abstract

A postbiotic product of lactobacillus paracasei K56. The postbiotic product of a probiotic comprises inactivated bacteria of the probiotic and / or metabolites thereof, and the probiotic includes lactobacillus paracasei K56, which has the preservation number of CGMCC No.15139 or DSM27447; the metabolic products comprise: 62.60 mg / 100 g or more of L-methionine and 70.1 mg / 100 g or more of short peptide; and the inactivated bacteria of the probiotic are inactivated bacteria of a probiotic after undergoing fermentation, and the inactivation conditions for the inactivated bacteria involve treating at a temperature ranging from 70°C to 121°C for 5-15 min. The bacteriocin of lactobacillus paracasei K56 comprises exocytotic materials and metabolic products of lactobacillus paracasei K56, and in the bacteriocin, the mass ratio of citric acid to L-methionine is greater than 8:1. The postbiotic product of the probiotic can be applied to intestinal health.

Description

Lactobacillus paracasei K56 postbiotic product, K56 secretin, preparation method, and intestinal health and antioxidant applications thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202410174816X, filed with the Patent Office of China on February 7, 2024, entitled “Postbiotic product of Lactobacillus paracasei K56, preparation method and application thereof for intestinal health”, and Chinese patent application No. 2024101748140, filed with the Patent Office of China on February 7, 2024, entitled “Bacteriocins of Lactobacillus paracasei K56, preparation method and antioxidant application thereof”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to the field of postbiotic technology, and in particular to a Lactobacillus paracasei K56 postbiotic product, a K56 secretin, a preparation method, and intestinal health and anti-oxidation applications thereof. Background Art

[0004] Inflammation in medicine is a complex of reactive events involving the tissues of higher animals when they come into contact with noxious agents of varying nature. The most common outcome of this process is neutralization of the noxious agent or limitation of the damage caused by it. The causes of inflammation include physical, chemical, and biological factors. The clinical manifestations of inflammation are well-known: heat (calorie), redness (rubor), tumors, pain (dolor), and functional impairment (functio laesa). These, in turn, correspond to the distinct events associated with inflammation: dilation of blood vessels with increased local circulation (congestion: redness, heat); the outflow of plasma (diapedesis), with the expulsion of blood-forming elements, such as granulocytes, lymphocytes, plasma cells, and especially erythrocytes (diapedesis), which is increased by cellular elements directly from the tissues (hystogenic reaction), leading to swelling of the inflamed area (tumor). Two main types of inflammation are distinguished based on the degree of vascular and cellular response to the inflammatory stimulus: acute inflammation and chronic inflammation.

[0005] In addition to drug therapy, CAM therapies (Complementary and Alternative Medicine) are increasingly being studied as support for the treatment of inflammation-based diseases, such as plant extracts (curcumin or Boswellia serrata), probiotics, etc.

[0006] Postbiotics are currently a hot research area attracting considerable attention. In 2021, the International Scientific Association of Probiotics and Prebiotics (ISAPP) published a consensus statement on postbiotics, which defines them as preparations of non-living microorganisms and / or their components that have beneficial effects on host health. As our understanding of intestinal health and the microbiome continues to deepen, postbiotics are being recognized as a potential functional food and health management tool, attracting significant attention from the industry.

[0007] Therefore, it is of great significance to develop a postbiotic that can fight inflammation and maintain intestinal health. Summary of the Invention

[0008] The purpose of the present disclosure is to provide a Lactobacillus paracasei K56 postbiotic product, a preparation method and its intestinal health application to solve the above technical problems.

[0009] The present disclosure is achieved as follows:

[0010] In a first aspect, the present disclosure provides a probiotic postbiotic product comprising: inactivated probiotic bacteria and / or metabolites thereof, wherein the probiotic comprises Lactobacillus paracasei K56, which has a deposit number of CGMCC No. 15139 or DSM 27447;

[0011] The metabolites include: more than 62.60 mg / 100 g of L-methionine and more than 70.1 mg / 100 g of short peptides.

[0012] The above-mentioned metabolites are the characteristic target metabolites of the K56 probiotic postbiotics identified in the present disclosure. Compared with live bacteria, the levels of the above-mentioned metabolites in the K56 probiotic postbiotics are much higher than the metabolite levels of live bacteria. In addition, the above-mentioned metabolites in the K56 probiotic postbiotics have stable and repeated peaks, and have clear molecular formulas and structures, which facilitate subsequent detection and quantification.

[0013] The probiotic postbiotic product containing the above-mentioned characteristic target metabolites has at least one of the following biological functions: enhancing intestinal barrier function, preventing and / or treating colitis. The probiotic postbiotic product also has good antioxidant effect, good DPPH free radical scavenging ability and hydroxyl free radical scavenging ability.

[0014] 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.

[0015] In a second aspect, the present disclosure provides a probiotic postbiotic product comprising: inactivated probiotic bacteria and metabolites thereof, wherein the probiotic comprises Lactobacillus paracasei K56, which has a deposit number of CGMCC No. 15139 or DSM 27447;

[0016] The inactivated probiotics are fermented probiotics; the inactivation conditions for the inactivated probiotics are 70-121° C. for 5-15 minutes.

[0017] When inactivation conditions were within the range of 70-100°C, the DPPH radical scavenging capacity of the bacteria showed a downward trend, but the overall change was minimal. Within the 70-100°C range, the hydroxyl radical scavenging capacity of the postbiotics from the K56 strain remained largely unchanged 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 postbiotics from the K56 strain.

[0018] In a third aspect, the present disclosure provides a method for preparing a probiotic postbiotic product, comprising the steps of inactivating a fermentation product of probiotics; sterilization conditions are: 70-121°C for 5-15 minutes. The probiotic postbiotic product obtained by this inactivation method has good anti-inflammatory activity.

[0019] In a fourth aspect, the present disclosure provides a composition comprising the aforementioned probiotic postbiotic product or the aforementioned probiotic postbiotic product. The composition, including but not limited to a pharmaceutical composition or a vaccine composition, has promising application prospects in preventing and / or treating inflammatory bowel disease and enhancing intestinal barrier function.

[0020] In a fifth aspect, the present disclosure provides the use of a probiotic postbiotic product, a probiotic postbiotic product or a composition in the preparation of a composition for (1) enhancing intestinal barrier function; (2) preventing and / or treating inflammatory bowel disease; and / or (3) anti-aging.

[0021] In a sixth aspect, the present disclosure provides a Lactobacillus paracasei K56 secretin, which includes exocytosis of Lactobacillus paracasei K56 and metabolites of Lactobacillus paracasei K56. The mass ratio of citric acid to L-methionine in the secretin is greater than 8:1. The preservation number of Lactobacillus paracasei K56 is CGMCC No. 15139.

[0022] In a seventh aspect, the present disclosure provides a method for preparing a secretin of Lactobacillus paracasei K56, comprising the following steps:

[0023] Bacteriocin extraction: using a solvent to extract bacterial mud separated from the fermentation broth of Lactobacillus paracasei K56 to obtain a mixed solution;

[0024] Sterilize, separate the supernatant from the mixed solution and sterilize it by heat to obtain bacteriocin.

[0025] In an eighth aspect, the present disclosure provides the use of a secretin of Lactobacillus paracasei K56 in preparing a food, wherein the food comprises at least one of a dairy product and a beverage.

[0026] In a ninth aspect, the present disclosure provides the use of a secretin of Lactobacillus paracasei K56 in the preparation of an antioxidant composition.

[0027] In a tenth aspect, the present disclosure provides the use of Lactobacillus paracasei K56 secretin in anti-oxidation.

[0028] The present disclosure has the following beneficial effects:

[0029] The probiotic postbiotic product provided by the present disclosure has anti-inflammatory and antioxidant effects, and the antioxidant function is manifested in a good scavenging ability for DPPH free radicals and hydroxyl free radicals. The probiotic postbiotic product can play a role in intestinal protection, and is expected to be used to prevent or treat proctitis and to develop anti-inflammatory drugs. The probiotic postbiotic product is prepared by heat inactivation, and the preparation method is simple and easy, which is convenient for batch production. In addition, the secretory agent of Lactobacillus paracasei K56 in the present disclosure has an antioxidant function and can be applied to food, feed and medicine. Compared with a mixture including intact dead cells + cell wall components + cell membrane components + cell-free supernatant, the secretory agent in the present disclosure is a liquid or water-soluble powder, which is more advantageous for use in some liquid beverages and medicines. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a process flow chart of K56 strain postbiotic products;

[0031] Figure 2 shows the K56 strain postbiotic powder after sieving and packaging;

[0032] FIG3 is a scanning electron micrograph of live and heat-inactivated K56;

[0033] FIG4 is a graph showing the statistical results of the DPPH free radical scavenging ability of K56 inactivated at different temperatures;

[0034] FIG5 is a graph showing the statistical results of the hydroxyl radical scavenging ability of K56 inactivated at different temperatures;

[0035] Figure 6 shows the DPPH radical scavenging ability of the cell-free supernatants of the four bacterial strains at 80°C and 121°C;

[0036] Figure 7 shows the hydroxyl radical scavenging ability of the cell-free supernatants of the four bacterial strains at 80°C and 121°C;

[0037] Figure 8 is a statistical graph showing the expression of the pro-inflammatory factor TNF-α in RAW264.7 cells after intervention with inactivated K56 bacteria;

[0038] Figure 9 is a statistical graph showing the expression of the pro-inflammatory factor TNF-α in RAW264.7 cells after intervention with K56 cell-free supernatant;

[0039] FIG10 is a graph showing the composition of non-volatile components at different thermal inactivation temperatures;

[0040] Figure 11 shows the results of TOP50 differential metabolite analysis between GB-1496 and K56 groups;

[0041] Figure 12 shows the peak area changes of six potential target substances at different heat inactivation temperatures of K56 strain;

[0042] FIG13 is a statistical table of characteristic metabolite compositions of K56 at different inactivation temperatures;

[0043] FIG14 is a liquid chromatogram of the mixed standard working solution;

[0044] 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);

[0045] Figure 16 is a flow chart of the preparation of secretory agents from Lactobacillus paracasei K56 in Example 1;

[0046] Figure 17 is a physical image of the secretory activity of Lactobacillus paracasei K56 in Example 1;

[0047] FIG18 is a liquid chromatogram of a bacteriocin sample obtained in Example 1;

[0048] FIG19 shows the hydroxyl radical scavenging ability of secretin of Lactobacillus paracasei K56 under different extraction conditions;

[0049] Figure 20 shows the DPPH radical scavenging ability of secretory agents of Lactobacillus paracasei K56 under different extraction conditions. DETAILED DESCRIPTION

[0050] In a first aspect, the present disclosure provides a probiotic postbiotic product comprising: inactivated probiotic bacteria and / or metabolites thereof, wherein the probiotic comprises Lactobacillus paracasei K56, which has a deposit number of CGMCC No. 15139 or DSM 27447;

[0051] The metabolites include: more than 62.60 mg / 100 g of L-methionine and more than 70.1 mg / 100 g of short peptides.

[0052] The preservation information of K56 strain refers to the strain information disclosed in patent CN107916236B.

[0053] The above-mentioned metabolites are the characteristic target metabolites of the K56 probiotic postbiotics identified in the present disclosure. Compared with live bacteria, the levels of the above-mentioned metabolites in the K56 probiotic postbiotics are much higher than the metabolite levels of live bacteria. In addition, the above-mentioned metabolites in the K56 probiotic postbiotics have stable and repeated peaks, and have clear molecular formulas and structures, which facilitate subsequent detection and quantification.

[0054] The probiotic postbiotic product containing the aforementioned characteristic target metabolites has at least one of the following biological functions: enhancing intestinal barrier function and preventing and / or treating colitis. The probiotic postbiotic product also exhibits excellent antioxidant effects, including excellent DPPH free radical scavenging and hydroxyl free radical scavenging capabilities.

[0055] 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.

[0056] In one embodiment of the present disclosure, the metabolites include: 62.60 to 4238.72 mg / 100 g or more of L-methionine and 70.1 to 9078.63 mg / 100 g or more of short peptides.

[0057] In one embodiment of the present disclosure, the metabolites include: 62.60 to 4046.05 mg / 100 g or more of L-methionine and 70.1 to 8615.60 mg / 100 g or more of short peptides.

[0058] In one embodiment of the present disclosure, the metabolites include: 62.60 to 3853.377 mg / 100 g or more of L-methionine and 70.1 to 8615.60 mg / 100 g or more of short peptides.

[0059] In one embodiment of the present disclosure, the short peptide is selected from at least one of GPRPK, anti-arrhythmic peptide and βAsp-Leu.

[0060] Among them, GPRPK refers to: Gly-Pro-Arg-Pro-Lys.

[0061] Antiarrhythmic peptide refers to: GP (Hyp) GAG, molecular formula C 19 H 30 N6O8.

[0062] In one embodiment of the present disclosure, the metabolites contain more than 43.79 mg / 100 g of GPRPK, more than 5.75 mg / 100 g of antiarrhythmic peptide, and more than 20.56 mg / 100 g of βAsp-Leu.

[0063] In one embodiment of the present disclosure, the metabolites contain 43.79-3840.88 mg / 100 g of GPRPK, 5.75-3840.88 mg / 100 g of antiarrhythmic peptide, and 20.56-1344.10 mg / 100 g of βAsp-Leu.

[0064] In one embodiment of the present disclosure, the metabolites contain 43.79-3666.30 mg / 100 g of GPRPK, 5.75-3666.30 mg / 100 g of antiarrhythmic peptide, and 20.56-1283.01 mg / 100 g of βAsp-Leu.

[0065] In one embodiment of the present disclosure, the metabolites contain more than 43.79-3491.71 mg / 100 g of GPRPK, more than 5.75-3491.71 mg / 100 g of antiarrhythmic peptide, and more than 20.56-1221.91 mg / 100 g of βAsp-Leu.

[0066] In one embodiment of the present disclosure, the probiotic postbiotic product further comprises 293.51 mg / 100 g or more of citric acid and 43.59 mg / 100 g or more of proline propionic acid.

[0067] In one embodiment of the present disclosure, the probiotic postbiotic product further comprises 293.51-32998.59 mg / 100 g of citric acid and 43.59-2952.02 mg / 100 g or more of proline propionic acid.

[0068] In one embodiment of the present disclosure, the probiotic postbiotic product further comprises 293.51-31498.66 mg / 100 g of citric acid and 43.59-2817.83 mg / 100 g or more of proline propionic acid.

[0069] In one embodiment of the present disclosure, the probiotic postbiotic product further comprises 293.51-29998.72 mg / 100 g of citric acid and 43.59-2683.65 mg / 100 g or more of proline propionic acid.

[0070] Proline propionic acid, CAS number 6422-36-2, molecular formula is C8H 14 N2O3, H-Pro-Ala-OH.

[0071] In one embodiment of the present disclosure, the mass ratios of proline propionic acid, L-methionine, citric acid, GPRPK, antiarrhythmic peptide and βAsp-Leu in the probiotic postbiotic product are 39.23-47.59: 56.34-68.86: 264.16-322.86: 39.41-48.17: 5.18-6.33: 18.50-22.62.

[0072] In one embodiment of the present disclosure, the mass ratios of proline propionic acid, L-methionine, citric acid, GPRPK, antiarrhythmic peptide and βAsp-Leu in the probiotic postbiotic product are 43.59:62.60:293.51:43.79:5.75:20.56.

[0073] The above substances in the probiotic postbiotic product have good anti-inflammatory and antioxidant effects at the above mass ratios.

[0074] In one embodiment of the present disclosure, the probiotic postbiotic product further comprises a highly active substance, and the highly active substance is selected from at least one of agavoside A, permetin A and isoleucine-glutamic acid.

[0075] In one embodiment of the present disclosure, the highly active substances include: 1.5 mg / kg or more of agaveside A, 1.49 mg / kg or more of transdermalin A, and 1.4 mg / kg or more of isoleucine-glutamic acid.

[0076] In one embodiment of the present disclosure, the inactivated probiotics are fermented probiotics; the inactivation conditions for the inactivated probiotics are heat inactivation, ultrasound inactivation, lysozyme inactivation, or pulsed electric field inactivation. Other inactivation methods besides heat inactivation are also within the scope of protection of the present disclosure as long as they can produce biologically active postbiotics.

[0077] The inactivation conditions for inactivated bacteria are 70-121℃, and the treatment time is 5-15min. For example, at 70℃, 71℃, 72℃, 73℃, 74℃, 75℃, 76℃, 77℃, 78℃, 79℃, 80℃, 81℃, 82℃, 83℃, 84℃, 85℃, 86℃, 87℃, 88℃, 89℃, 90℃, 91℃, 92℃, 93℃, 94℃, 95℃, 96℃, 97℃, 98℃, 99℃, 100℃, 101℃, 102℃, 103℃, 104℃, 105℃, 106℃, 107℃, 108℃, 109℃, 110℃, 111℃, 112℃, 113℃, 114℃, 115℃, 116℃, 117℃, 118℃, 119℃, 120℃, 121℃, 122℃, 123℃, 124℃, 125℃, 126℃, 127℃, 128℃, 129℃, 130℃, 131℃, 132℃, 133℃ The postbiotic product is treated at 8°C, 109°C, 110°C, 111°C, 112°C, 113°C, 114°C, 115°C, 116°C, 117°C, 118°C, 119°C, 120°C or 121°C, or any temperature point between any two of the above temperatures, for 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, or any point between 5 minutes and 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.

[0078] In one embodiment of the present disclosure, the inactivation conditions for sterilization are: 70-100° C., treatment for 10-15 minutes.

[0079] In one embodiment of the present disclosure, the inactivation conditions for sterilization are: 90-100° C., treatment for 10-15 minutes.

[0080] In a second aspect, the present disclosure further provides a probiotic postbiotic product, comprising: inactivated probiotic bacteria and metabolites thereof, wherein the probiotic bacteria comprises Lactobacillus paracasei K56, which has a deposit number of CGMCC No. 15139 or DSM 27447;

[0081] The inactivated probiotics are fermented probiotics; the inactivation conditions for the inactivated probiotics are 70-121° C. for 5-15 minutes.

[0082] 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.

[0083] The inventors found that when the inactivation conditions were within the range of 70-100°C, the DPPH radical scavenging ability of the bacteria showed a downward trend, but the overall change was not significant. Within the range of 70-100°C, as the heat inactivation temperature increased, the postbiotics of the K56 strain did not change much in their ability to scavenge hydroxyl radicals. Within the range of 70-121°C, as the heat inactivation temperature increased, the anti-inflammatory activity of the postbiotics of the K56 strain was not significantly affected. In an optional embodiment, the inactivation conditions are at 90-100°C for 10-15 minutes. At this temperature, the cell-free supernatant after K56 inactivation can significantly inhibit the level of pro-inflammatory factors, and the anti-inflammatory activity is stronger.

[0084] 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.

[0085] In one embodiment of the present disclosure, the fermentation is high-density fermentation, and the conditions for high-density fermentation are 37±0.5° C. and pH 5.75±0.5. The postbiotic product obtained under the above fermentation conditions has good biological activity.

[0086] In one embodiment of the present disclosure, the fermentation time is 11-13 hours; for example, the fermentation time is 11 hours, 12 hours or 13 hours.

[0087] In one embodiment of the present disclosure, the inactivation conditions for the inactivated bacteria are: 70-100° 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.

[0088] In one embodiment of the present disclosure, the probiotic postbiotic product is prepared from 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.

[0089] The inventors discovered that heat-killed dead bacteria, cell fragments, inactivated bacterial precipitates, and cell-free supernatants all exhibited significant inhibitory effects on proinflammatory cytokines, maintaining excellent stability in their 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 100°C for 15 minutes.

[0090] In one embodiment of the present disclosure, the probiotic postbiotic product further comprises: a carrier and / or auxiliary materials.

[0091] In one embodiment of the present disclosure, the carrier or auxiliary material is selected from:

[0092] 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.

[0093] Examples include: 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, sodium starch glycolate, sodium bicarbonate, calcium phosphate, calcium citrate; lubricants such as magnesium stearate, talc, sodium lauryl sulfate; flavorings 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.

[0094] In an optional embodiment, 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.

[0095] In one embodiment of the present disclosure, the probiotic postbiotic product is in the form of liquid, solid or semi-solid.

[0096] 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 the inactivated bacteria of Lactobacillus paracasei K56 strain. The dried product includes but is not limited to spray-dried product, freeze-dried product, vacuum-dried product, drum-dried product, etc.

[0097] In a third aspect, the present disclosure further provides a method for preparing a probiotic postbiotic product, which comprises the following steps: inactivating the fermentation product of the probiotic; the sterilization conditions are: 70-121° C., and a treatment time of 5-15 min.

[0098] 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.

[0099] 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 postbiotics of the K56 strain did not change much with increasing heat inactivation temperature. Within the 70-121°C range, increasing heat inactivation temperature had no significant effect on the anti-inflammatory activity of the postbiotics of the K56 strain.

[0100] 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.

[0101] In one embodiment of the present disclosure, the treatment is carried out at 90-100° C. for 10-15 minutes. In an alternative embodiment, the treatment is carried out at 100° C. for 15 minutes. At this temperature, the cell-free supernatant after K56 inactivation can significantly suppress the level of pro-inflammatory factors and has stronger anti-inflammatory activity.

[0102] In one embodiment of the present disclosure, the fermentation product of probiotics is a fermentation product of probiotics after high-density fermentation.

[0103] In one embodiment of the present disclosure, the high-density fermentation is performed at 37°C±0.5°C and a pH of 5.75±0.5.

[0104] In one embodiment of the present disclosure, the fermentation time is 11-13 hours.

[0105] In one embodiment of the present disclosure, the fermentation product is centrifuged, and the precipitate is mixed with a carrier and / or auxiliary material. In an alternative embodiment, 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.

[0106] In one embodiment of the present disclosure, the cell-free supernatant of the inactivated bacteria after centrifugation is used to prepare probiotic postbiotics;

[0107] In one embodiment of the present disclosure, the precipitate is mixed with a protective agent and an excipient, and then freeze-dried to produce a powder. In other embodiments, the powder can be produced by spray drying, vacuum drying, drum drying, etc. in addition to freeze drying.

[0108] In a fourth aspect, the present disclosure further provides a composition comprising the above-mentioned probiotic postbiotic product. The composition includes but is not limited to a pharmaceutical composition.

[0109] In a fifth aspect, the present disclosure further provides the use of a probiotic postbiotic product or the above composition in the preparation of a composition for (1) enhancing intestinal barrier function; (2) preventing and / or treating inflammatory bowel disease; and / or (3) anti-aging.

[0110] The composition is selected from health food, food antioxidant or medicine.

[0111] In one embodiment of the present disclosure, enhancing the intestinal barrier function includes: maintaining the integrity of the ileal villus structure and avoiding or repairing damage to the intestinal barrier.

[0112] In one embodiment of the present disclosure, the drug for preventing and / or treating inflammatory bowel disease comprises 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;

[0113] In one embodiment of the present disclosure, 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.

[0114] In one embodiment of the present disclosure, the necrotizing enterocolitis is neonatal necrotizing enterocolitis;

[0115] In one embodiment of the present disclosure, the expression level of at least one of the following intestinal tissue inflammatory factors is suppressed: IL-6, IL-1β, IL-10, TLR-4 and TNF-α.

[0116] The anti-aging functions include but are not limited to: (1) maintaining the balance of normal microbial flora on the surface of the skin, avoiding skin immune diseases and skin aging caused by imbalance of microbial flora; (2) the composition can reduce inflammation and oxidative damage induced by external stimuli, thereby leading to skin cell damage and skin aging; (3) slowing down the functional decline of keratinocytes and fibroblasts, and slowing down the degradation rate of collagen and elastin.

[0117] Anti-aging compositions include, but are not limited to, cosmetics and skincare products. For example, probiotic and postbiotic products can be used as a component of a cosmetic base, including but not limited to skin conditioners. Cosmetics include toners, lotions, serums, ointments, creams, or masks.

[0118] As an embodiment of the anti-aging cosmetic disclosed herein, the anti-aging cosmetic is in the form of liquid, emulsion, cream or solid.

[0119] When the anti-aging composition is a cosmetic, in one embodiment, it further comprises cosmetically acceptable excipients, which include at least one of an emollient, an emulsifier, a thickener, a humectant, a pH adjuster, a skin protectant, and a preservative.

[0120] In one 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.

[0121] 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 optional concentration of thickening agent depends on selected reagent.

[0122] In an optional embodiment, the above-mentioned drug is a solid pharmaceutical preparation, such as freeze-dried bacterial powder, granular preparation, etc.

[0123] In an alternative embodiment, the medicament is formulated for oral administration, injection, or oral administration.

[0124] In a sixth aspect, the present invention further provides the use of a probiotic postbiotic product or the above-mentioned composition in enhancing intestinal barrier function.

[0125] In one embodiment of the present disclosure, enhancing the intestinal barrier function includes:

[0126] Maintain the integrity of the ileal villus structure and avoid or repair damage to the intestinal barrier.

[0127] In a seventh aspect, the present invention further provides use of a probiotic postbiotic product or the above-mentioned composition in preventing and / or treating inflammatory bowel disease.

[0128] In one embodiment of the present disclosure, the prevention and / or treatment of inflammatory bowel disease comprises 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;

[0129] In one embodiment of the present disclosure, 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.

[0130] In one embodiment of the present disclosure, the necrotizing enterocolitis is neonatal necrotizing enterocolitis.

[0131] In one embodiment of the present disclosure, the expression level of at least one of the following intestinal tissue inflammatory factors is suppressed: IL-6, IL-1β, IL-10, TLR-4 and TNF-α.

[0132] In an eighth aspect, the present invention further provides the use of a probiotic postbiotic product or the above-mentioned composition in anti-aging. The anti-aging functions include, but are not limited to: (1) maintaining the normal balance of the microbiome on the skin surface, preventing skin immune diseases and skin aging caused by an imbalance of the microbiome; (2) the composition can reduce inflammation and oxidative damage induced by external stimuli, which can lead to skin cell damage and skin aging; (3) slowing the functional decline of keratinocytes and fibroblasts, and slowing the degradation rate of collagen and elastin.

[0133] In a ninth aspect, the present disclosure further provides a Lactobacillus paracasei K56 secretin, including exocytosis products of Lactobacillus paracasei K56 and metabolites of Lactobacillus paracasei K56. The mass ratio of citric acid to L-methionine in the secretin is greater than 8:1. The deposit number of Lactobacillus paracasei K56 is CGMCC No. 15139.

[0134] Exocytotic substances are a collective term for the functionally active substances released from the intracellular space into the extracellular solvent by living bacteria through exocytosis after resuspending them in pure water and allowing them to stand at a certain temperature. Exocytotic substances include, but are not limited to, phospholipid bilayers, nucleic acids, peptides, and organic acids.

[0135] The secretin of Lactobacillus paracasei K56 including the above-mentioned metabolites (citric acid and L-methionine) has antioxidant function and good DPPH free radical scavenging ability and hydroxyl free radical scavenging ability.

[0136] In one embodiment of the present disclosure, the mass ratio of citric acid to L-methionine in the secretin is 8-75:1. For example, the mass ratio of citric acid to L-methionine is 8-10:1, 10-20:1, 15-30:1, 30-40:1, 40-60:1, 50-70:1, or 50-75:1. At these ratios, the secretin prepared by fermentation of Lactobacillus paracasei K56 exhibits excellent antioxidant activity, as demonstrated by excellent DPPH free radical scavenging and hydroxyl free radical scavenging abilities.

[0137] In one embodiment of the present disclosure, the mass ratio of citric acid to L-methionine in K56 secretin is 25-40: 1. For example, the mass ratio of citric acid to L-methionine is 25:1, 30:1, 35:1 or 40:1.

[0138] In the present disclosure, bacteriocin mainly includes components such as exocytosis products 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 the bacteriocin.

[0139] The Lactobacillus paracasei K56 secretin 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 secretin disclosed in the present invention is a liquid, semi-solid or water-soluble powder, which has more advantages in use in some liquid beverages and medicines.

[0140] In one embodiment of the present disclosure, the mass ratio of citric acid to L-methionine in the bacteriocin is 8-75:1, specifically, it can be 8:1, 15:1, 25:1, 35:1, 45:1, 55:1, 65:1, 75:1 or any value between 8 and 75:1, or any value greater than 75:1.

[0141] In one embodiment of the present disclosure, the mass ratio of citric acid to L-methionine in K56 bacteriocin is 25-40:1.

[0142] In a tenth aspect, the present disclosure provides a method for preparing the aforementioned Lactobacillus paracasei K56 secretin, comprising the following steps:

[0143] Bacteriocin extraction: using a solvent to extract bacterial mud separated from the fermentation broth of Lactobacillus paracasei K56 to obtain a mixed solution;

[0144] Sterilize, separate the supernatant from the mixed solution and sterilize it by heat to obtain bacteriocin.

[0145] In one embodiment of the present disclosure, 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; in one embodiment of the present disclosure, the extraction temperature is 3-5°C.

[0146] In one embodiment of the present disclosure, the extraction time is 1-3 hours, specifically 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours or any value between 1-3 hours; in one embodiment of the present disclosure, the extraction time is 100-150 minutes.

[0147] In one embodiment of the present disclosure, the total colony count of Lactobacillus paracasei K56 in the mixed solution is 1×10 10 -5×10 11 cfu / mL, specifically 1×10 10 cfu / mL, 3×10 10 cfu / mL, 5×10 10 cfu / mL, 1×10 11 cfu / mL, 2×10 11 cfu / mL, 3×10 11 cfu / mL, 4×10 11 cfu / mL, 5×10 11 cfu / mL or 1×10 10 -5×10 11 cfu / mL; In one embodiment of the present disclosure, the total colony count of Lactobacillus paracasei K56 in the mixed solution is 7×10 10 -8×10 10 cfu / mL.

[0148] In an optional embodiment, the temperature for 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; in one embodiment of the present disclosure, the temperature for 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; in one embodiment of the present disclosure, the heat sterilization time is 10-16 min.

[0150] When the extraction concentration of Lactobacillus paracasei K56 colonies in the mixed solution was 7.5×10 10cfu / mL, the extraction temperature was 4℃, and the extraction time was 2h, the hydroxyl radical scavenging ability of mycobacteriocin was better.

[0151] In an optional embodiment, the method further comprises the following steps: culturing Lactobacillus paracasei K56 in liquid culture, and stopping the fermentation when the Lactobacillus paracasei K56 grows to the logarithmic phase;

[0152] In one embodiment of the present disclosure, the fermentation step includes: inoculating Lactobacillus paracasei K56 fermentation seed liquid into a culture medium, and fermenting for 12-14 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 Lactobacillus paracasei K56 fermentation liquid.

[0153] In one embodiment of the present disclosure, the preparation of Lactobacillus paracasei K56 fermentation seed liquid comprises: activating the Lactobacillus paracasei K56 strain, purifying, culturing in a first-level seed liquid, expanding in a second-level seed liquid, culturing in a third-level seed liquid, and preparing fermentation seeds to obtain the Lactobacillus paracasei K56 fermentation seed liquid;

[0154] In one embodiment of the present disclosure, the culture medium is MRS liquid culture medium;

[0155] In an optional embodiment, the solvent is water; in one embodiment of the present disclosure, the solvent is sterile water, specifically sterile purified water;

[0156] In one embodiment of the present disclosure, after the sterilization step, the bacteriocin is further freeze-dried to obtain freeze-dried bacteriocin.

[0157] 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 8:1, and the L-methionine concentration is greater than 0.2 mg / ml and the citric acid concentration is greater than 8 mg / ml. In some embodiments, the L-methionine concentration in the bacteriocin is 0.2-1 mg / ml and the citric acid concentration is 8-15 mg / ml.

[0158] 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 L-methionine concentration in the bacteriocin is 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1 mg / ml, or any value between 0.2-1 mg / ml, or any value greater than 1 mg / ml; the citric acid concentration is 8 mg / ml, 9 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, or any value between 8-15 mg / ml, or any value greater than 15 mg / ml. In some embodiments, the L-methionine concentration in the bacteriocin is 0.30-0.35 mg / ml and the citric acid concentration is 10-11 mg / ml.

[0159] After the sterilization step, the bacteriocin is freeze-dried to obtain the 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 8:1.

[0160] In an eleventh aspect, the present disclosure provides a use of the secretin of Lactobacillus paracasei K56 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.

[0161] The dairy product is selected from: solid dairy products or liquid dairy products; the solid dairy product is selected from infant formula milk powder, children formula milk powder or middle-aged and elderly formula milk powder, and the liquid dairy product is selected from reconstituted milk or milk beverage, etc.

[0162] The beverage is selected from the group consisting of a carbonated beverage or a non-carbonated beverage; or a beverage is selected from the group consisting of a high-calorie beverage, a medium-calorie beverage, a low-calorie beverage, or a zero-calorie beverage. In one embodiment, the beverage is selected from the group consisting of carbonated soft drinks, cola, root beer, fruit drinks, nectar, vegetable juice, sports drinks, energy drinks, enhanced water drinks, coconut water, tea-type drinks, coffee, cocoa drinks, beverages containing dairy components, beverages containing cereal extracts, and smoothies.

[0163] In a twelfth aspect, the present disclosure provides an application of the secretin of Lactobacillus paracasei K56 of the aforementioned embodiment in preparing a composition, the composition comprising an antioxidant composition; in one embodiment of the present disclosure, the composition is selected from at least one of a medicine, a health food and a feed.

[0164] Health foods are foods that have specific health benefits or are intended to supplement vitamins or minerals. They are suitable for consumption by specific groups of people, have the function of regulating body functions, are not intended to treat diseases, and do not cause any acute, subacute, or chronic harm to the human body.

[0165] Feed includes, but is not limited to, animal feed, such as poultry, fish, livestock, rodent feed, or arthropod feed.

[0166] The drug further comprises pharmaceutically acceptable excipients, which include at least one of an emollient, an emulsifier, a thickener, a moisturizer, a pH regulator, a skin protectant, and a preservative.

[0167] In a thirteenth aspect, the present disclosure provides the use of a secretin of Lactobacillus paracasei K56 in the preparation of an antioxidant composition.

[0168] In one embodiment of the present disclosure, the antioxidant composition is used as an ingredient in medicines, health foods and feeds.

[0169] In a fourteenth aspect, the present disclosure provides the use of secretin of Lactobacillus paracasei K56 in anti-oxidation.

[0170] The features and performance of the present disclosure are further described in detail below with reference to the embodiments.

[0171] Example 1

[0172] This embodiment provides a K56 strain postbiotic product. The process flow chart is shown in FIG1 . The specific preparation method is as follows:

[0173] First and third level seed preparation

[0174] 1. Standard cryopreservation tubes

[0175] 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.

[0176] 2. Activation of cryotubes

[0177] 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.

[0178] 3. Primary purification

[0179] Take the cultured bacterial solution and dilute it for coating, the dilution degree is 10 -4 , 10 -5 , 10 -6 For each dilution, prepare two MRS solid plates and culture them upside down at 37℃ for 48h~72h until colonies are formed on the plates. Use an inoculation loop to pick single colonies and place them in 5 tubes of 10mL MRS liquid culture medium. The colonies picked should be of uniform size and cultured at 37℃ for 11~13h.

[0180] 4. Secondary Purification

[0181] Take the cultured purified bacterial solution and dilute it for coating, the dilution degree is 10 -4 , 10 -5 , 10 -6 For each dilution, prepare two MRS solid plates and culture them upside down at 37℃ for 48h~72h until colonies are formed on the plates. Use an inoculation loop to pick single colonies and place them in 5 tubes of 10mL MRS liquid culture medium. The colonies picked should be of uniform size and cultured at 37℃ for 11~13h.

[0182] 5. Primary Seed Preparation

[0183] 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.

[0184] 6. Secondary Seed Preparation

[0185] 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.

[0186] 7. Preparation of Tertiary Seeds

[0187] 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.

[0188] 8. Temporary storage of third-level seeds

[0189] After the third-level seeds are prepared, they can be placed at 4℃ for no more than 6 hours.

[0190] 2. Fermentation Seed Preparation

[0191] 1. Vaccination

[0192] (1) Turn on the stirring paddle and temperature control program, the speed is 70 rpm, and the temperature is 37 °C.

[0193] (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.

[0194] (3) When inoculating, pour alcohol onto the inoculation loop and ignite it to form a flame circle.

[0195] (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%.

[0196] (5) After inoculation, tighten the upper cover of the inoculator, close the inoculation valve, and extinguish the alcohol flame ring.

[0197] (6) Close the nitrogen inlet valve and exhaust valve, and control the tank pressure to 0.01-0.03 MPa for pressure-maintaining fermentation.

[0198] 2. Fermentation

[0199] (1) Set the fermentation parameters to 70 rpm and 37°C.

[0200] (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.

[0201] (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.

[0202] 3. Fermentation tank culture and bacterial inactivation

[0203] 1. Vaccination

[0204] (1) Turn on the stirring paddle and temperature control program, the speed is 70 rpm, and the temperature is 37 °C.

[0205] (2) Steam sterilize the inoculation pipe for 30 minutes.

[0206] (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%.

[0207] (4) After inoculation, close the fermenter inoculation valve and clean the inoculation pipeline.

[0208] (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.

[0209] 2. Fermentation

[0210] (1) Set the fermentation parameters to 70 rpm, 37°C, and a constant pH of 5.75.

[0211] (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.

[0212] (3) After 9 hours of fermentation, the fermentation process begins with natural pH drop. When the pH value drops below 4.2 and the fermentation ends, the bacteria can be inactivated or the temperature can be lowered to 10-20°C and stored for no more than 4 hours.

[0213] 3. Bacteria inactivation

[0214] (1) The sterilization conditions of the strain are as follows:

[0215] The sterilization conditions of Lactobacillus paracasei K56 are 100℃, 15min.

[0216] (2) After the bacteria are inactivated, they can be centrifuged or cooled to 10-20°C for no more than 4 hours.

[0217] 4. Centrifugal separation

[0218] (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.

[0219] (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.

[0220] (3) Open the feed valve and the material enters the centrifuge drum to start centrifugation.

[0221] (4) Centrifugal process parameter settings: feed rate 600 L / h, slag discharge time 150 s.

[0222] (5) After centrifugation, add protective agent according to the weight of the bacterial sludge.

[0223] 5. Excipient Mixing

[0224] (1) Steam sterilize the excipient pipeline for 30 minutes.

[0225] (2) Add excipients at a ratio of 1:1 by weight of the bacterial sludge.

[0226] (3) After adding the excipients, stir for 5 to 10 minutes to mix thoroughly.

[0227] (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.

[0228] 6. Freeze Drying

[0229] 1. Sterile workshop personnel requirements

[0230] Operators enter the production area according to the "Workshop Personnel Purification Operating Procedures".

[0231] 2. Stall

[0232] (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.

[0233] (2) Stall weight: 1.6-1.8 kg / tray.

[0234] (3) Place the freeze-drying trays into the freeze dryer in order, insert the temperature probe, and close the door.

[0235] 3. Freeze-drying process

[0236] Turn on the freeze dryer and set the program according to the freeze drying process.

[0237] 4. End of freeze drying

[0238] 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.

[0239] 7. Freeze-dried powder off the tray

[0240] 1. Sterile workshop personnel requirements

[0241] Operators enter the production area according to the "Workshop Personnel Purification Operating Procedures".

[0242] 2. Off-plate

[0243] 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.

[0244] 8. Crushing and screening

[0245] 1. Crushing

[0246] (1) Install a crusher with a screen size of 20 mesh.

[0247] (2) Connect the compressed air and power supply and turn on the crusher.

[0248] (3) Setting parameters: speed 10 rpm, pneumatic valve feeding interval 1 s.

[0249] (4) The weight of a single crushing shall not exceed 15kg.

[0250] 2. Sieve packaging (Figure 2)

[0251] After pulverization, the freeze-dried powder was directly sieved with a sieve size of 40 mesh, and the sieved fine powder was collected separately.

[0252] Example 2

[0253] This example compares the effects of different inactivation processes on the morphological changes of postbiotics of the strain.

[0254] Heat-killed bacteria were inactivated by heating at 70°C, 80°C, 90°C, 100°C, and 121°C for 10 minutes. The morphological changes of postbiotics were observed using scanning electron microscopy (SEM).

[0255] 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).

[0256] The results, shown in Figure 3, show that as the temperature rises, the surface roughness of the bacterial cells increases, leading to the leakage of a large amount of cellular contents. As the temperature rises, bacterial cell shrinkage and damage also gradually increase (red arrows), manifesting as cell shrinkage (blue arrows). Under heat inactivation conditions at 90°C, the release of intracellular components and debris increases, accompanied by the leakage of white clumps and intracellular contents (blue circles).

[0257] Therefore, under the condition of inactivation of probiotics, postbiotics all present a combination of "dead bacterial shell + exudate contents", among which "exudate contents" are the "cell-free supernatant" often proposed by the academic community.

[0258] Example 3

[0259] 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 K56 inactivated at different temperatures.

[0260] The antioxidant activity assay for postbiotics is as follows:

[0261] (1) 2,2-Diphenyl-1-picrylphenylhydrazine (DPPH) free radical scavenging method

[0262] The DPPH free radical scavenging assay was performed using a previous method with slight modifications. 200 μL of 0.2 mM DPPH solution was mixed with 200 μL of bacterial suspension (10 9 cfu / mL) and incubated at 25°C in the dark for 30 minutes. For the control group, an equal volume of PBS (pH 7.4) was used instead of the DPPH free radical solution; for the blank group, an equal volume of PBS (pH 7.4) was used 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:

[0263] (2) Eliminate hydroxyl free radicals

[0264] A total of 1.0 mL of each sample was added to a mixture containing 2.5 mM 1,10-phenanthroline (1.0 mL), 2.5 mM FeSO₄ (1.0 mL), and PBS (1.0 mL, pH 7.4). After adding 20 mM H₂O₂ (1.0 mL), the mixture was incubated in a 37°C water bath for 90 min. The absorbance was measured at 536 nm. The hydroxyl radical scavenging activity was calculated as follows:

[0265] As shown in Figure 4, the DPPH scavenging capacity of live K56 bacteria was 35.55%. The DPPH radical scavenging rate did not change significantly with increasing temperature. However, heat treatment at 121°C significantly decreased the scavenging capacity (P < 0.0001). Therefore, the results suggest that increasing temperature may weaken the DPPH radical scavenging capacity of K56.

[0266] The results, as shown in Figure 5, show that the hydroxyl radical scavenging capacity of live K56 bacteria was 52.96%. Heat treatment at 70°C and 80°C had little effect on the hydroxyl radical scavenging capacity of K56. Heat treatment at 90°C significantly decreased the hydroxyl radical scavenging capacity, but as the temperature increased, the hydroxyl radical scavenging capacity recovered to a level similar to that of live bacteria.

[0267] Example 4

[0268] This example compares the effects of different inactivation processes on the antioxidant function of probiotic cell-free supernatant (the supernatant after centrifugation to remove bacterial residue).

[0269] In addition to K56 in Example 1, free radical scavenging experiments were also conducted on Bifidobacterium longum subsp. infantis YLGB1496 (CCTCC NO: M2011122), Lactobacillus paracasei ET-22 (CGMCC No. 15077), and Bifidobacterium animalis subsp. lactis BL99.

[0270] The cell-free supernatant was prepared as follows:

[0271] Overnight cultures of the four proprietary strains were obtained by centrifugation (4500 × g, 10 min), washed three times, and resuspended in PBS to 1 × 10 10 The concentration of CFU / mL was determined, and 1 ml of the washed bacterial suspension was aspirated for colony count. 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 remaining 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 filter for LC-MS analysis of metabolite content.

[0272] (1) DPPH free radical scavenging ability of probiotic cell-free supernatant

[0273] 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.

[0274] (2) Hydroxyl radical scavenging ability of probiotic cell-free supernatant

[0275] 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 K56 and BL-99 at 80°C and 121°C. These results suggest that high temperatures have different effects on the hydroxyl radical scavenging abilities of the cell-free supernatants of Lactobacillus paracasei and Bifidobacterium, possibly due to different substances in the cell-free supernatants of different bacterial species.

[0276] Example 5

[0277] This example compares the effects of different inactivation processes on epigenetic anti-inflammatory function.

[0278] (1) Heat-inactivated cells

[0279] 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-inactivated bacteria.

[0280] 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:

[0281] TNF-α, forward 5-CTGAACTTCGGGGTGATCGG-3 (SEQ ID NO: 1), reverse 5-GGCTTGTCACTCGAATTTTGAGA-3 (SEQ ID NO: 2);

[0282] GAPDH, forward 5-AAGCCCATCACCATCTTCCA-3 (SEQ ID NO: 3), reverse 5-CACCAGTAGACTCCACGACA-3 (SEQ ID NO: 4).

[0283] Use 2 -ΔΔCt Methods The relative expression of each target gene was determined, and all quantifications were normalized to the GADPH gene.

[0284] 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 K56 bacteria at different temperatures significantly decreased TNF-α production (P < 0.005), and temperature had little effect on the inhibitory effect of inactivated K56 bacteria. Therefore, the results indicate that inactivated K56 bacteria have a significant inhibitory effect on TNF-α.

[0285] (2) Cell-free supernatant

[0286] The preparation of the cell-free supernatant was the same as in Example 4, and the anti-inflammatory function experiment was performed on the cell-free supernatant (the supernatant after centrifugation to remove the bacterial residue) using the same method.

[0287] As shown in Figure 9, LPS stimulation significantly increased the production of the proinflammatory cytokine TNF-α in RAW264.7 cells (P < 0.0001). As temperature increased, TNF-α production significantly decreased after intervention with K56 cell-free supernatant. Therefore, the results suggest that elevated temperature may affect the inhibitory effect of K56 cell-free supernatant on TNF-α.

[0288] (3) Peptidoglycan

[0289] The extraction method of peptidoglycan is as follows:

[0290] The heat-killed bacterial pellet was resuspended in PBS buffer and sonicated at 240W for 30 minutes. The pellet was then centrifuged and boiled in 4% SDS for 30 minutes to rupture the cell membrane and denature proteins. 10% TCA was then added to remove cell wall teichoic acid, ester teichoic acid, and proteins. After a period of stagnation, a mixed solvent (sodium acetate:chloroform:methanol = 4:5:10 by volume) was added for defatting, and the pellet was centrifuged. The pellet was treated with a combination of alkaline protease and trypsin at 37°C for 12 hours to enzymatically hydrolyze endogenous proteins and nucleic acids. The pellet was then boiled in 1% SDS for 10 minutes to further remove residual proteins. The pellet was then centrifuged and washed with hot distilled water until SDS was trace. Dialysis was then performed for 2 days, with the water changed every 8 hours. The pellet was then centrifuged and freeze-dried under vacuum to obtain a white or pale yellow powder, which represents peptidoglycan.

[0291] Example 6

[0292] This example screens postbiotic detection targets (non-volatile compounds) based on LC-MS.

[0293] The specific method is as follows:

[0294] 1. Sample Processing

[0295] 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.

[0296] 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.

[0297] 2. LC-MS detection

[0298] 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.

[0299] 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.

[0300] The mobile phase elution gradient is as follows:

[0301] 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:

[0302] 3. Quality Control

[0303] 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.

[0304] 4. Data Processing

[0305] Before statistical analysis, the raw data can be preprocessed. The raw data are imported into the metabolomics processing software Progenesis QI (Waters Corporation, Milford, USA) for baseline filtering, peak identification, integration, retention time correction, and peak alignment. The resulting data matrix contains retention time, mass-to-charge ratio, and peak intensity. The following data preprocessing is then performed:

[0306] (1) Only variables with more than 80% non-zero values ​​in any set of samples are retained;

[0307] (2) Use 1 / 2 of the minimum value in the original matrix to fill the missing values;

[0308] (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;

[0309] 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.

[0310] 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 K56 ranges from 500 to 600 substances, primarily amino acids and peptides, lipids, nucleotides, organic acids, alcohols and other acids, terpenes, and sugars.

[0311] The results of TOP50 differential metabolite analysis of two bacterial strains (GB-1496 and K56) after treatment at different temperatures are shown in Figure 11.

[0312] 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.

[0313] 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.

[0314] The peak area analysis of these six potential targets at different heat inactivation temperatures was further performed:

[0315] The analysis results are shown in Figure 12. The peak areas of Proly-Alanine, L-Methionine, βAsp-Leu and βAsp-Phe in the postbiotic system of K56 strain are 10 7 The peak area of ​​βAsp-Phe can reach up to 6×10 7 The peak areas of Citric Acid and GRPPK were between 2-4×10 6. The high content of the six substances in ET-22 and K56 provides a good basis for subsequent experimental verification and the establishment of detection methods. GRPPK is expected to be a potential specific target substance of K56. Combining the test 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, Antiarrhythmic peptide), Proly-Alanine (Prolyl Alanine), GPRPK, bAsp-Leu, bAsp-Phe.

[0316] Example 7

[0317] Characteristic metabolite composition of K56 at different inactivation temperatures. The detection of characteristic metabolites refers to the LC-MS parameters described in Example 6 above.

[0318] 13 , the results showed that under 100-degree sterilization conditions, the postbiotic product contained 1.591 mg / kg of isoleucine-glutamate, 1.55 mg / kg of agaveside A, and 1.51 mg / kg of transdermalin A.

[0319] KF-1, KF-2, and KF-3 represent live bacterial samples 1, 2, and 3; K801A, K802A, and K803A represent samples 1, 2, and 3 sterilized at 80°C; K101A, K102A, and K103A represent samples 1, 2, and 3 sterilized at 100°C; and K121A, K122A, and K123A represent samples 1, 2, and 3 sterilized at 121°C. The results showed that the levels of characteristic metabolites decreased with increasing temperature.

[0320] Example 8

[0321] This example establishes a dedicated strain postbiotic detection and verification method based on LC.

[0322] 1. Materials and Reagents

[0323] Inactivated bacteria samples with different colony counts and fermentation broth samples (i.e., the product after fermentation in step 2 of the fermenter culture in step 3 of Example 1): Lactobacillus paracasei K56.

[0324] Prolylalanine (C8H14N2O3, CAS: 6422-36-2): purity ≥ 96.5%; L-methionine (methionine, C5H 11NO2S, 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, all with purity ≥98.0%; acetonitrile, methanol, and phosphoric acid (chromatographic grade, Thermo Fisher); formic acid and acetonitrile (mass spectrometry grade, Thermo Fisher); and Wahaha purified water (Wahaha Group Co., Ltd., Hangzhou) were used in the experiments.

[0325] 2. Instruments and Equipment

[0326] 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.

[0327] 3. Preparation of standard solution

[0328] Standard stock solution: Accurately weigh an appropriate amount of standard (accurate to 0.1 mg) separately, dissolve in water and prepare a standard stock solution with a concentration of ≥5 mg / mL, and store at -20°C.

[0329] 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.

[0330] 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.

[0331] 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.

[0332] 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.

[0333] 3. LC / MS analysis conditions for sample confirmation

[0334] 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.

[0335] 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.

[0336] 4. Liquid chromatography analysis conditions

[0337] 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.

[0338] Gradient elution program table

[0339] 5. Colony count detection method

[0340] Prepare inactivated bacterial samples and fermentation broth samples with different colony counts. The preparation method and colony count method refer to GB4789.2-94.

[0341] 6. Sample Pretreatment

[0342] 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.

[0343] Liquid samples: Mix the fermentation broth sample of the dedicated strain and directly aspirate 1 mL. Centrifuge at 10,000 rpm for 10 min at 4°C. Dilute the supernatant to the linear range and load onto the column for analysis.

[0344] 7. Methodological Validation

[0345] (1) Sample confirmation

[0346] 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.

[0347] (2) Standard curve, detection limit and quantification limit

[0348] Inject the above series of mixed standard working solutions into a high performance liquid chromatograph, injecting each concentration three times in parallel, 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.

[0349] 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.

[0350] (3) Accuracy and precision

[0351] 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%.

[0352] (4) Result calculation

[0353] 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.

[0354] The content of each target component in the sample is calculated according to formula (1):

[0355] Where:

[0356] 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);

[0357] ci-mass concentration of each target component in the sample solution obtained from the standard working curve, in micrograms per milliliter (μg / mL);

[0358] V-the fixed volume of the sample extraction solution, in milliliters (mL);

[0359] m-sample sampling mass or volume, in grams or milliliters (g or mL); 10-unit conversion factor;

[0360] f-dilution factor.

[0361] 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)

[0362] Where:

[0363] Y- the number of colonies in the sample, the unit is 10^9 CFU / mL;

[0364] X-the content of a target component in the sample, in mg / 100g or mg / 100mL; a, b-parameter values.

[0365] Here are the results:

[0366] A. Sample confirmation:

[0367] 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.

[0368] The UPLC-Q-TOF-MS / MS identification results are as follows:

[0369] B. Standard Curve, Limit of Detection, and Limit of Quantitation

[0370] 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.

[0371] Retention time, standard curve, correlation coefficient, linear range, LOD and LOQ statistics of 7 target components

[0372] 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.

[0373] C. Accuracy and precision

[0374] K56 fermentation broth samples with known concentrations 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 spiking. Each level 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 the table below. 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.

[0375] Statistics table of spike test results

[0376] D. Measurement of actual samples

[0377] Samples were selected for content determination. The statistical results for the target components in the inactivated K56 bacteria samples are shown in the table below. As can be seen, the K56 inactivated bacteria samples all contained multiple target components, with L-methionine, citric acid, and GPRPK peptide present at high and consistently high levels. The liquid chromatogram of the actual sample is shown in Figure 15. Note: - indicates not detected or the resolution from the sample base peak is less than 1.5.

[0378] E. Establish the correlation between target component content and colony count

[0379] The above-mentioned liquid chromatography method was used to analyze K56, a sample of inactivated bacteria with varying colony counts (1 to 300, unit: 10^9 CFU / mL). HPLC fingerprints and analysis results for the seven target components were established. The relationship between the target component content and the colony count, the primary indicator, was investigated to identify differences in the quality 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.

[0380] 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.

[0381] In summary, this paper uses high-performance liquid chromatography (HPLC) to establish a regression equation for the colony count of inactivated bacterial samples derived from a specific strain 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.

[0382] Example 9

[0383] This embodiment provides a method for preparing a secretin of Lactobacillus paracasei K56, the preparation flow chart of which is shown in FIG16 , and the preparation method comprises the following steps:

[0384] 1. Preparation of Tertiary Seeds

[0385] 1.1 Standard cryopreservation tubes

[0386] 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.

[0387] 1.2 Activation of cryotubes

[0388] Take a portion of the bacterial strain stored at -80℃, thaw it at room temperature, aseptically take 200μL of bacterial solution and inoculate it into 10mL of seed liquid culture medium, and culture it at 37℃ for 8-10h.

[0389] 1.3 Primary purification

[0390] Take the cultured bacterial liquid for dilution and coating, with dilutions 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 18-20h.

[0391] 1.4 Secondary purification

[0392] Take the cultured purified bacterial liquid for dilution and coating, with dilutions 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 are uniform in size and cultured at 37°C for 18-20h.

[0393] 1.5 Primary seed preparation

[0394] 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 8-10 h.

[0395] 1.6 Secondary seed preparation

[0396] 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 9-11 hours.

[0397] 1.7 Preparation of tertiary seeds

[0398] The cultured secondary seeds were poured into two bottles of 1.8LMRS liquid culture medium and cultured at 37°C for 11-13 hours.

[0399] 1.8 Temporary storage of third-level seeds

[0400] After the third-level seeds are prepared, they can be placed at 4℃ for no more than 6 hours.

[0401] 1.9 Process Quality Control

[0402] (1) Indicators for determining the end point of seed growth at each level: pH 4.0-4.4, OD600 ≥ 2.0.

[0403] (2) Purity test: Observe the bacterial morphology under a microscope. Under a 100x oil immersion lens, the bacterial morphology is complete, appearing as short rods, either alone or in chains.

[0404] (3) Pollutant detection: Detection of the third-level seed liquid, including Escherichia coli and non-lactic acid bacteria.

[0405] Among them, the formula of the third-level seed fermentation medium is shown in the following table.

[0406] pH value: 6.2-6.4, sterilization conditions: 121℃, 15-20min.

[0407] 2. Fermentation Seed Preparation

[0408] 2.1 Vaccination

[0409] (1) Turn on the stirring paddle and temperature control program, the speed is 70 rpm, and the temperature is 37 °C.

[0410] (2) Open the nitrogen inlet valve and introduce a small flow of nitrogen into the fermentation tank to maintain positive pressure in the fermentation tank.

[0411] (3) When inoculating, pour alcohol onto the inoculation loop and ignite it to form a flame circle.

[0412] (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%.

[0413] (5) After inoculation, tighten the upper cover of the inoculator, close the inoculation valve, and extinguish the alcohol flame ring.

[0414] (6) After inoculation, continue to introduce a small flow of nitrogen into the tank for 5-10 minutes.

[0415] (7) Close the nitrogen inlet valve and exhaust valve, and control the tank pressure to 0.01-0.03 MPa for pressure-maintaining fermentation.

[0416] 2.2 Fermentation

[0417] (1) Set the fermentation parameters to 70 rpm and 37°C.

[0418] (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.

[0419] (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.

[0420] 2.3 Process quality control

[0421] (1) Seed tank fermentation endpoint determination indicators: pH 4.0-4.4, OD600 ≥ 2.0.

[0422] (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 immersion lens, the bacterial morphology is complete, appearing as short rods, either alone or in chains.

[0423] (3) Pollutant detection: Detect the fermentation liquid at the end of the seed tank, including Escherichia coli and non-lactic acid bacteria.

[0424] See the table below for the formula of the seed tank fermentation medium.

[0425] pH value: 6.2-6.4, sterilization conditions: 121℃, 15-20min.

[0426] 3. Fermentation Broth Culture

[0427] 3.1 Vaccination

[0428] (1) Turn on the stirring paddle and temperature control program, the speed is 70 rpm, and the temperature is 37 °C.

[0429] (2) Steam sterilize the inoculation pipe for 30 minutes.

[0430] (3) Close the steam valve, open the bottom valve of the seed tank and the inoculation valve of the fermentation tank for inoculation, with an inoculation amount of 2%.

[0431] (4) After inoculation, close the fermenter inoculation valve and clean the inoculation pipeline.

[0432] 3.2 Fermentation

[0433] (1) Set the fermentation parameters to 37°C, 70 rpm, and a constant pH of 6.0.

[0434] (2) The fermentation time was 13-15 h, and the fermentation status was monitored every 2 h, including pH, OD600, temperature, and rotation speed.

[0435] (3) After fermentation, centrifugation can be performed or the temperature can be lowered to 10-20℃ and stored for no more than 4 hours.

[0436] The fermentation medium formula of the fermentation tank is shown in the table below.

[0437] pH value: 6.2-6.4, sterilization conditions: 121℃, 15-20min.

[0438] 3.3 Mushroom sludge separation

[0439] (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.

[0440] (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.

[0441] (3) Open the feed valve, and the Lactobacillus paracasei K56 fermentation liquid enters the centrifuge drum and starts centrifugation to obtain bacterial sludge.

[0442] (4) Centrifugal process parameter settings: feed rate 600 L / h, slag discharge time 200 s.

[0443] 4. Bacteriocin Extraction

[0444] 4.1 Mud transfer

[0445] The collected bacterial sludge was transferred to a sterile fermentation tank for extraction.

[0446] 4.2 Extraction conditions

[0447] The bacterial sludge was extracted and resuspended with sterile purified water to obtain a mixed solution. The total colony counts of Lactobacillus paracasei K56 in the mixed solution were 5×10 10 cfu / mL, 7.5×10 10 cfu / mL, 1.5×10 11 cfu / mL, extraction temperatures were 4°C, 25°C, and 37°C, extraction times were 1h, 2h, and 3h, and extraction speed was 70rpm.

[0448] 5. Centrifugal separation

[0449] (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.

[0450] (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.

[0451] (3) Open the feed valve and the mixed liquid enters the centrifuge drum to start centrifugation.

[0452] (4) Centrifugal process parameter settings: feed rate 600 L / h, slag discharge time 200 s.

[0453] (5) Transfer the centrifuged supernatant to a collection tank.

[0454] 6. Packaging and sterilization

[0455] The filled bottled supernatant was heat sterilized to obtain the secretory agent. The sterilization conditions of Lactobacillus paracasei K56 were 70° C. for 10 min. The actual picture of the obtained Lactobacillus paracasei K56 secretory agent is shown in FIG17 .

[0456] 7. Quantitative Detection

[0457] 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:

[0458] (1) Preparation of standard working solution

[0459] 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.

[0460] 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.

[0461] 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.

[0462] (2) Preparation of elution solution

[0463] 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.

[0464] 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.

[0465] (3) Preparation of test solution

[0466] Liquid sample: Mix the secretory sample of Lactobacillus paracasei K56 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 it onto the column for analysis.

[0467] (4) Detection and analysis

[0468] 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.

[0469] 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.

[0470] Table 1 Gradient elution program

[0471] The total number of Lactobacillus paracasei K56 colonies in the mixed solution was calculated to be 7.5×10 10 cfu / mL, the extraction temperature was 4℃, and the extraction time was 2h. The L-methionine concentration in the bacteriocin was 0.31mg / 100ml and the citric acid concentration was 10.22mg / 100ml.

[0472] (5) Calculate the number of colonies before inactivation of inactivated bacteria

[0473] Among them, citric acid and L-methionine can be used as targets to calculate the colony count in the fermentation broth before mycobacterin extraction.

[0474] When citric acid was used as the target to calculate the total number of colonies in the fermentation broth before mycobacterial extraction, the regression equation used was y = 0.465x + 1.188; where x represents the concentration of the target in mg / 100 g; y represents the number of colonies in 10 9 CFU / mL. Substitute the calculated target concentration into the regression equation to calculate the colony count before inactivation of the fermentation broth.

[0475] 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 = 14.755x + 1.2978; 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 of the fermentation broth.

[0476] Test Example 1: 2,2-Diphenyl-1-picrylphenylhydrazyl (DPPH) free radical scavenging

[0477] 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:

[0478] Test Example 2: Hydroxyl Radical Scavenging

[0479] 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:

[0480] The present disclosure detects six detection targets (Proly-Alanine, L-Methionine, Citric Acid, bAsp-Leu, bAsp-Phe, Antiarrhythmic peptide, GRPPK) in bacteriocin and finds that only citric acid and L-methionine are present in bacteriocin, while the other four detection targets are not extracted or the content is below the detection limit. Therefore, in this application, citric acid and L-methionine are used as detection targets to characterize bacteriocin.

[0481] The test results of the hydroxyl radical scavenging ability of the bacteriocin obtained in Example 9 are shown in FIG19 . As can be seen from FIG19 , when the extraction concentration is 7.5×10 10cfu / mL, the extraction temperature was 4°C, and the extraction time was 2h, 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 9 are shown in Figure 20. In Figure 20, 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. The results in Figure 20 show that the extraction conditions had relatively little effect on DPPH radicals. Industrial Applicability

[0482] The probiotic postbiotic product provided by the present disclosure has anti-inflammatory and antioxidant effects, and the antioxidant function is manifested in a good scavenging ability for DPPH free radicals and hydroxyl free radicals. The probiotic postbiotic product can play a role in intestinal protection, and is expected to be used to prevent or treat proctitis and to develop anti-inflammatory drugs. The probiotic postbiotic product is prepared by heat inactivation, and the preparation method is simple and easy, which is convenient for batch production. The Lactobacillus paracasei K56 secretin in the present disclosure has an 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 secretin in the present disclosure is a liquid or water-soluble powder, which is more advantageous for use in some liquid beverages and medicines.

Claims

1. A probiotic postbiotic product, characterized in that: It includes: Inactivated probiotic bacteria and / or metabolites thereof, wherein the probiotic bacteria include Lactobacillus paracasei K56, which has a preservation number of CGMCC No. 15139 or DSM 27447; The metabolites contain: more than 62.60 mg / 100 g of L-methionine and more than 70.1 mg / 100 g of short peptides.

2. The probiotic postbiotic product according to claim 1, characterized in that The metabolites include: L-methionine at a concentration of 62.60 to 4238.72 mg / 100 g or more, and short peptides at a concentration of 70.1 to 9078.63 mg / 100 g or more; Preferably, the metabolites contain: 62.60 to 4046.05 mg / 100 g or more of L-methionine, and 70.1 to 8615.60 mg / 100 g or more of short peptides; Preferably, the metabolites contain: 62.60 to 3853.377 mg / 100 g or more of L-methionine, and 70.1 to 8615.60 mg / 100 g or more of short peptides; Preferably, the short peptide is selected from at least one of GPRPK, antiarrhythmic peptide and βAsp-Leu; Preferably, the metabolites contain more than 43.79 mg / 100 g of GPRPK, more than 5.75 mg / 100 g of antiarrhythmic peptide, and more than 20.56 mg / 100 g of βAsp-Leu; Preferably, the metabolites contain 43.79-3840.88 mg / 100 g of GPRPK, 5.75-3840.88 mg / 100 g of antiarrhythmic peptides, and 20.56-1344.10 mg / 100 g of βAsp-Leu; Preferably, the metabolites contain 43.79-3666.30 mg / 100 g of GPRPK, 5.75-3666.30 mg / 100 g of antiarrhythmic peptides, and 20.56-1283.01 mg / 100 g of βAsp-Leu; Preferably, the metabolites contain more than 43.79-3491.71 mg / 100 g of GPRPK, more than 5.75-3491.71 mg / 100 g of antiarrhythmic peptides, and more than 20.56-1221.91 mg / 100 g of βAsp-Leu; Preferably, the probiotic postbiotic product further comprises 293.51 mg / 100 g or more of citric acid and 43.59 mg / 100 g or more of proline propionic acid; Preferably, the probiotic postbiotic product further comprises 293.51-32998.59 mg / 100 g of citric acid and 43.59-2952.02 mg / 100 g or more of proline propionic acid; Preferably, the probiotic postbiotic product further comprises 293.51-31498.66 mg / 100 g of citric acid and 43.59-2817.83 mg / 100 g or more of proline propionic acid; Preferably, the probiotic postbiotic product further comprises 293.51-29998.72 mg / 100 g of citric acid and 43.59-2683.65 mg / 100 g or more of proline propionic acid; Preferably, the mass ratio of prolylpropionic acid, L-methionine, citric acid, GPRPK, antiarrhythmic peptide and βAsp-Leu in the probiotic postbiotic product is 39.23-47.59: 56.34-68.86: 264.16-322.86: 39.41-48.17: 5.18-6.33: 18.50-22.62; Preferably, the mass ratio of proline propionic acid, L-methionine, citric acid, GPRPK, antiarrhythmic peptide and βAsp-Leu in the probiotic postbiotic product is 43.59:62.60:293.51:43.79:5.75:20.

56.

3. The probiotic postbiotic product according to claim 1 or 2, characterized in that The probiotic postbiotic product further comprises a highly active substance, wherein the highly active substance is selected from at least one of agaveside A, transdermal A and isoleucine-glutamic acid; Preferably, the highly active substances include: 1.5 mg / kg or more of agaveside A, 1.49 mg / kg or more of transdermalin A, and 1.4 mg / kg or more of isoleucine-glutamic acid; Preferably, the inactivated probiotic bacteria are fermented probiotic bacteria; the inactivation conditions of the inactivated bacteria are heat inactivation, ultrasonic inactivation, lysozyme inactivation or pulse electric field inactivation; Preferably, the inactivation conditions of the sterilization are 70-121°C and the treatment time is 5-15 minutes; Preferably, the inactivation conditions of the sterilization are: 70-100°C for 10-15 minutes; Preferably, the inactivation conditions for the sterilization are: 90-100° C., treatment for 10-15 minutes.

4. A probiotic postbiotic product, characterized in that It includes: Inactivated probiotics and their metabolites, wherein the probiotics include Lactobacillus paracasei K56, which has a deposit number of CGMCC No. 15139 or DSM 27447; The inactivated probiotics are fermented probiotics; the inactivation conditions for the inactivated probiotics are 70-121° C. for 5-15 minutes; Preferably, the inactivation conditions of the sterilization are: 70-100°C for 10-15 minutes; Preferably, the inactivation conditions of the sterilization are: 90-100°C for 10-15 minutes; Preferably, the fermentation is high-density fermentation, and the conditions of the high-density fermentation are fermentation at 37°C ± 0.5°C and a pH of 5.75 ± 0.5; Preferably, the fermentation time is 11-13 hours.

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 is prepared from 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; Preferably, the probiotic postbiotic product further comprises: a carrier and / or auxiliary materials; Preferably, 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; Preferably, the probiotic postbiotic product is in the form of liquid, solid or semi-solid.

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

7. A composition, characterized in that The invention comprises the probiotic postbiotic product according to any one of claims 1 to 3, the probiotic postbiotic product according to claim 4, or the probiotic postbiotic product according to claim 5.

8. Use of the probiotic postbiotic product according to any one of claims 1 to 3, the probiotic postbiotic product according to claim 4, the probiotic postbiotic product according to claim 5, 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; and / or (3) anti-aging; Preferably, the composition is selected from health food, food antioxidant or medicine.

9. 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.

10. The use according to claim 8, characterized in that The drug is used for preventing and / or treating inflammatory bowel disease and includes at least one of the following uses: reducing or eliminating inflammatory cell infiltration in the colon, reducing or eliminating colon crypt abscesses or damage, and inhibiting the level of inflammation in intestinal tissue; Preferably, 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; Preferably, the necrotizing enterocolitis is neonatal necrotizing enterocolitis; Preferably, 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.

11. Use of the probiotic postbiotic product according to any one of claims 1 to 3, the probiotic postbiotic product according to claim 4, the probiotic postbiotic product according to claim 5, or the composition according to claim 7 in enhancing intestinal barrier function.

12. The use according to claim 11, 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.

13. Use of the probiotic postbiotic product according to any one of claims 1 to 3, the probiotic postbiotic product according to claim 4, the probiotic postbiotic product according to claim 5, or the composition according to claim 7 in preventing and / or treating inflammatory bowel disease.

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

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 probiotic postbiotic product according to claim 5, or the composition according to claim 7 in anti-aging.

16. Lactobacillus paracasei K56 secretory factor, characterized in that The exocytosis product comprises the exocytosis product of Lactobacillus paracasei K56 and the metabolites of Lactobacillus paracasei K56. The mass ratio of citric acid to L-methionine in the exocytosis product is greater than 8:

1. The preservation number of the Lactobacillus paracasei K56 is CGMCC No.15139.

17. The Lactobacillus paracasei K56 secretin according to claim 16, characterized in that The mass ratio of citric acid to L-methionine in the bacteriocin is 8-75:1; Preferably, the mass ratio of citric acid to L-methionine in the K56 secretin is 25-40:

1.

18. The method for preparing the secretin of Lactobacillus paracasei K56 according to claim 16 or 17, characterized in that: The following steps are involved: Bacteriocin extraction: using a solvent to extract bacterial mud separated from the fermentation broth of Lactobacillus paracasei K56 to obtain a mixed solution; Sterilize, separate the supernatant from the mixed solution and sterilize it by heat to obtain the bacteriocin.

19. The method for preparing the secretin of Lactobacillus paracasei K56 according to claim 18, wherein The extraction temperature is 0-37°C; preferably, the extraction temperature is 3-5°C; And / or, the extraction time is 1-3 hours; preferably, the extraction time is 100-150 minutes.

20. The method for preparing the secretin of Lactobacillus paracasei K56 according to any one of claims 18 to 19, characterized in that: The total colony count of Lactobacillus paracasei K56 in the mixed solution was 1×10 10 -5×10 11 cfu / mL; preferably, the total colony count of Lactobacillus paracasei K56 in the mixed solution is 7×10 10 -8×10 10 cfu / mL.

21. The method for preparing the secretin of Lactobacillus paracasei K56 according to any one of claims 18 to 20, characterized in that: The temperature of the heat sterilization is 70-121°C; preferably, the temperature of the heat sterilization is 70-100°C.

22. The method for preparing the secretin of Lactobacillus paracasei K56 according to any one of claims 18 to 21, characterized in that: The heat sterilization time is 5-30 minutes; preferably, the heat sterilization time is 10-16 minutes.

23. The method for preparing the secretin of Lactobacillus paracasei K56 according to any one of claims 18 to 22, characterized in that: The method also includes a fermentation step of culturing Lactobacillus paracasei K56 in liquid culture and stopping the fermentation when the Lactobacillus paracasei K56 grows to a logarithmic phase; Preferably, the fermentation step comprises: inoculating Lactobacillus paracasei K56 fermentation seed liquid into a culture medium, and fermenting for 12-14 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 Lactobacillus paracasei K56 fermentation liquid; Preferably, the solvent is water; preferably, the solvent is sterile water; Preferably, after the sterilization step, the bacteriocin is freeze-dried to obtain freeze-dried bacteriocin.

24. Use of the secretin of Lactobacillus paracasei K56 in preparing food according to claim 16 or 17, characterized in that: The food comprises at least one of dairy products and beverages.

25. Use of the secretin of Lactobacillus paracasei K56 according to claim 16 or 17 in preparing an antioxidant composition.

26. The use according to claim 25, characterized in that The antioxidant composition is used as an ingredient in medicines, health foods and feeds.

27. Use of the secretin of Lactobacillus paracasei K56 as claimed in claim 16 or 17 in anti-oxidation.

Images