A gamma-aminobutyric acid-containing lactic acid bacterial bio-stimulant and a preparation method thereof

Abstract

This invention relates to the field of microbial technology and discloses a lactic acid bacteria postbiotic containing γ-aminobutyric acid (GABA) and its preparation method. It aims to solve the problems of GABA loss, damage to the active structure of the postbiotic cells, and insufficient synergistic utilization of GABA and GABA-containing lactic acid bacteria postbiotics during heat inactivation and drying processes. The method involves fermenting GABA-producing lactic acid bacteria, centrifuging to collect the supernatant and bacterial cells, desalting, removing impurities, and concentrating the supernatant to obtain a GABA-rich solution. The bacterial cell precipitate is washed, resuspended in a buffer solution containing calcium ions and trehalose, and allowed to stand. It is then adjusted to an acidic state to induce peptidoglycan conformation protection, followed by staged heat inactivation. After being combined with the GABA-rich solution and a composite protectant, it is dried at low temperature to obtain the GABA-containing lactic acid bacteria postbiotic. This provides a reliable technical solution for preparing highly active GABA-containing postbiotic products.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a lactic acid bacteria postbiotic containing γ-aminobutyric acid and its preparation method. Background Technology

[0002] Postbiotics are non-living microbial preparations that are beneficial to the health of the host. Compared with traditional probiotics, postbiotics have advantages such as strong stability, high safety, and the ability to exert their effects quickly without relying on intestinal colonization.

[0003] γ-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system, which has functions such as improving sleep and reducing anxiety. Lactic acid bacteria fermentation is one of the important bio-preparation routes to obtain GABA. The fermentation products usually contain GABA, lactic acid bacteria cells and metabiotic-related components such as extracellular polysaccharides, peptidoglycans, and cell wall components. Therefore, the compound preparation of GABA with lactic acid bacteria metabiotics is beneficial to the formation of functional products with both neuroregulatory activity and intestinal barrier maintenance function.

[0004] Existing processes for preparing GABA-containing lactic acid bacteria metabiotics typically involve heat inactivation and drying. However, high-temperature heat inactivation and subsequent drying have a dual impact: on the one hand, heat treatment and dehydration may cause GABA loss or lead to the co-accumulation of GABA with culture medium residues, inorganic salts, and other impurities, affecting product quality; on the other hand, heat treatment can also cause denaturation of surface proteins, rearrangement of peptidoglycan structures, degradation or conformational changes of extracellular polysaccharides, thereby reducing the ability of metabiotic bacterial components to recognize and bind to host cell receptors.

[0005] Among the various active components of lactic acid bacteria metabiotics, peptidoglycan is one of the important structural bases for maintaining functions such as immune regulation and intestinal barrier maintenance. Its biological activity is closely related to its post-translational modification state. Among them, O-acetylation modification refers to the acetylation modification of the hydroxyl group at the C6 position of N-acetylmuramic acid in the peptidoglycan backbone. This modification can affect the binding affinity of peptidoglycan fragments to host pattern recognition receptors and is one of the important indicators for evaluating the structural activity of metabiotic bacteria.

[0006] Therefore, the preparation of GABA-containing lactic acid bacteria postbiotics cannot only focus on GABA content, nor can it only focus on whether the bacterial cell inactivation is thorough. It is also necessary to achieve a balance between GABA component enrichment, bacterial cell inactivation, protection of the active structure of postbiotics, and low-heat damage during the drying process. Existing technologies have not yet proposed effective solutions for the above-mentioned multi-objective synergistic preparation process, especially lacking process design that maintains the key structural activity of postbiotics while ensuring bacterial cell inactivation and GABA retention. Summary of the Invention

[0007] The technical problem to be solved by this invention is: how to balance the complete inactivation of bacteria, the retention of GABA components, the maintenance of the active structure of the postbiotic bacteria and the synergistic utilization of both in the intestinal environment during the preparation of lactic acid bacteria postbiotics containing γ-aminobutyric acid. To this end, we propose a lactic acid bacteria postbiotic containing γ-aminobutyric acid and its preparation method.

[0008] To achieve the above objectives, this application adopts the following technical solution: a method for preparing a postbiotic of lactic acid bacteria containing γ-aminobutyric acid (GABA), comprising the following steps: S1: inoculating lactic acid bacteria into a culture medium, and obtaining a seed culture after activation; S2: inoculating the seed culture into a fermentation medium containing monosodium glutamate (MSG) for static fermentation to obtain a fermentation broth containing GABA, collecting the supernatant and bacterial precipitate containing GABA by centrifugation, desalting, removing impurities, and concentrating the supernatant to obtain a GABA-enriched solution, and washing the bacterial precipitate, resuspending it in a buffer solution, and allowing it to stand to obtain a bacterial suspension. The buffer solution contains calcium ions and trehalose; S3: Adjust the pH of the bacterial suspension to 5.0-5.5 with lactic acid solution, let it stand for 15-30 min, and then adjust the pH to 6.5-6.8 with alkaline solution; S4: Perform staged heat treatment on the bacterial suspension. In the first stage, the temperature is raised to 48-52℃ and maintained for 20-30 min. In the second stage, the temperature is raised to 65-70℃ and maintained for 15-30 min. After inactivation, the suspension is cooled; S5: Add γ-aminobutyric acid enrichment solution and composite protectant to the inactivated bacterial suspension, mix well, and then dry at low temperature to collect the finished powder.

[0009] Preferably, the lactic acid bacteria in S1 are lactic acid bacteria capable of producing γ-aminobutyric acid, and the lactic acid bacteria are selected from one of the short starter cultures: Lactobacillus, Lactobacillus plantarum, or Lactococcus lactis.

[0010] Preferably, the buffer solution in S2 further includes Tris-HCl, and the pH of the Tris-HCl is 6.8.

[0011] Preferably, the concentration of Tris-HCl in the buffer solution of S2 is 50 mmol / L, the concentration of calcium chloride is 20-40 mmol / L, and the concentration of trehalose is 100-200 mmol / L.

[0012] Preferably, the γ-aminobutyric acid enrichment solution in S2 is a liquid obtained by microfiltration, desalting, impurity removal and concentration of the supernatant, and the added volume of the γ-aminobutyric acid enrichment solution is 10-30% of the volume of the bacterial suspension.

[0013] Preferably, in the staged heat treatment in S4, the heating rate of the first stage is 2-3℃ / min, and the heating rate of the second stage is 1-2℃ / min.

[0014] Preferably, the composite protective agent in S5 is composed of reconstituted skim milk, trehalose, monosodium glutamate and sucrose.

[0015] Preferably, based on the total volume of the system after mixing the inactivated bacterial suspension, γ-aminobutyric acid enrichment solution, and composite protectant, the final concentration of the reconstituted skim milk in the composite protectant is 5-15% (v / v), the final concentration of trehalose is 3-8% (w / v), the final concentration of monosodium glutamate is 1-3% (w / v), and the final concentration of sucrose is 2-5% (w / v).

[0016] Preferably, the low-temperature drying in S5 is vacuum low-temperature spray drying, and the material being dried at a temperature not exceeding 60°C.

[0017] A lactic acid bacteria postbiotic containing γ-aminobutyric acid (GABA), wherein the lactic acid bacteria postbiotic contains GABA and the peptidoglycan O-acetylation retention rate is not less than 85%.

[0018] The application of a lactic acid bacteria postbiotic in the preparation of a liquid oral formulation, wherein the liquid oral formulation is prepared by mixing the lactic acid bacteria postbiotic with a liquid carrier, the liquid carrier comprising purified water and optionally one or more of glycerol, propylene glycol, and polyethylene glycol, and the pH of the liquid oral formulation is 4.5-7.0.

[0019] The technical effects and advantages of this invention are as follows: In this invention, a synergistic process combining GABA-containing supernatant purification, staged protection and inactivation of bacterial cell precipitation, and low-temperature drying simultaneously solves the problems of GABA enrichment and maintenance of metatrophic cell structure and activity. Specifically, after fermentation, the supernatant is first desalted, impurity removed, and concentrated to obtain a GABA-rich solution, avoiding the direct introduction of culture medium residues and inorganic salts into the final product. After the bacterial cell precipitation is treated with a buffer solution containing calcium ions and trehalose, calcium ions mediate the formation of reversible weak crosslinks between peptidoglycan chains, and trehalose reduces the water activity of the interchain microenvironment, thereby reducing the accessibility of water molecules to O-acetylated ester bonds. Subsequently, under acidic conditions, local conformational rearrangement of peptidoglycan is induced, changing the O-acetylation sites from an exposed state to a relatively shielded state, and at p After H-reversion, the protective conformation is maintained; finally, through a staged heat treatment that combines heat adaptation and inactivation, the damage to the peptidoglycan structure and other active bacterial components during the inactivation process is reduced. It should be noted that the GABA in the finished product of this invention mainly comes from the purified GABA enrichment solution and does not depend on the subsequent release of intracellular GABA from the densified cell wall. The role of bacterial structure protection is to maintain the intestinal barrier maintenance and receptor recognition activity of the metagenic bacterial components, thereby providing synergistic support for the stable utilization of GABA. Compared with conventional heat inactivation and high-temperature drying processes, this invention achieves a balance between GABA retention, protection of the activity of the metagenic bacterial structure, and low-heat-damage drying, providing an industrially feasible technical solution for the preparation of GABA-containing lactic acid bacteria metagenics. Attached Figure Description

[0020] The disclosure of this invention is illustrated with reference to the accompanying drawings. It should be understood that the drawings are for illustrative purposes only and are not intended to limit the scope of protection of this invention. In the drawings: Figure 1 This is the standard curve of γ-aminobutyric acid of the present invention; Figure 2 This is a comparison chart of the GABA content and GABA drying retention rate of the finished product of this invention; Figure 3 This is a comparison chart showing the retention rate of O-acetylation of peptidoglycan in this invention; Figure 4 This is the dose-response curve of the present invention; Figure 5 This is a graph showing the change of transmembrane resistance over time according to the present invention. Detailed Implementation

[0021] It is readily understood that, based on the technical solution of this invention, those skilled in the art can propose various interchangeable structural methods and implementations without altering the essential spirit of the invention. Therefore, the following detailed embodiments and accompanying drawings are merely illustrative examples of the technical solution of this invention and should not be considered as the entirety of the invention or as limitations or restrictions on the technical solution of this invention.

[0022] This invention provides a method for preparing a postbiotic of lactic acid bacteria containing γ-aminobutyric acid, comprising the following steps: S1: Inoculate lactic acid bacteria into a culture medium and obtain seed culture after activation culture; S2: The seed culture is inoculated into a fermentation medium containing monosodium glutamate, and fermentation is carried out statically to obtain a fermentation broth containing γ-aminobutyric acid. The supernatant containing GABA and the cell precipitate are collected by centrifugation. The supernatant containing GABA is desalted, impurities removed and concentrated to obtain a GABA-rich solution. The cell precipitate is washed and resuspended in buffer solution, and then allowed to stand to obtain a cell suspension. S3: Adjust the pH of the bacterial suspension to 5.0-5.5 with lactic acid solution, let it stand for 15-30 minutes, and then adjust the pH back to 6.5-6.8 with alkaline solution. S4: Perform staged heat treatment on the bacterial suspension. The first stage is to heat the suspension to 48-52℃ and maintain it for 20-30 minutes for heat adaptation treatment. The second stage is to continue to heat the suspension to 65-70℃ and maintain it for 15-30 minutes for inactivation treatment. After inactivation, cool the suspension. S5: Add GABA enrichment solution and composite protectant to the inactivated bacterial suspension, mix evenly, and then dry at low temperature. Collect the powder to obtain the lactic acid bacteria post-biotic product containing γ-aminobutyric acid.

[0023] The following detailed description of each step is provided in conjunction with specific implementation methods.

[0024] In S1, the lactic acid bacteria are lactic acid bacteria capable of producing γ-aminobutyric acid. These lactic acid bacteria contain a gene encoding glutamate decarboxylase, which can catalyze the conversion of monosodium glutamate into γ-aminobutyric acid during fermentation.

[0025] The lactic acid bacteria can be selected from the short-fermenting agent Lactobacillus (… Levilactobacillusbrevis Lactobacillus plantarum ( Lactiplantibacillusplantarum ), Lactococcus lactis ( Lactococcuslactis One of them.

[0026] The short-starter culture medium, Lactobacillus, is cultured using MRS medium, which includes 10g casein peptone, 10g beef extract, 5g yeast extract, 5g glucose, 5g sodium acetate, 2g diamine citrate, 1g Tween 80, 2g dipotassium hydrogen phosphate (K2HPO4), 0.2g magnesium sulfate (MgSO4·7H2O), 0.05g manganese sulfate (MnSO4·H2O), 15g agar, and 1L distilled water, with a pH of 6.5-6.8. The culture medium for *Lactobacillus plantarum* and *Lactococcus lactis* is consistent with that of the short-starter *Lactobacillus*. After inoculation, activate the culture at 30-35℃ for 18-24 hours. Pick a single activated colony and inoculate it into a container filled with culture medium. Incubate statically at 30-35℃ for 18-24 hours to obtain the seed culture. The OD of the seed culture is... 600 The value is 1.0-1.5.

[0027] In S2, the seed culture was inoculated into the fermentation medium at an inoculation rate of 2%-5%, and then statically cultured at 27±1℃ for 36-48h. During this period, samples were taken every 6-10h to determine the pH and GABA content.

[0028] Fermentation was terminated when the GABA content in the fermentation broth was ≥3.5 g / L or the pH dropped below 4.0, yielding a fermentation broth containing γ-aminobutyric acid.

[0029] The fermentation medium contains 10g beef extract, 5g yeast extract, 15g glucose, 20g monosodium glutamate, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g ammonium citrate, 0.2g magnesium sulfate, 0.05g manganese sulfate, 1mg vitamin B6, and 1mg vitamin C per liter, with a pH of 6.0-6.5.

[0030] Vitamin B6 and vitamin C were sterilized by filtration through a 0.22 μm filter membrane and then added to the fermentation medium before inoculation.

[0031] During fermentation, monosodium glutamate undergoes an α-decarboxylation reaction to generate γ-aminobutyric acid under the catalysis of intracellular glutamate decarboxylase (GAD). Vitamin B6, as a precursor of the GAD coenzyme pyridoxal-5'-phosphate, can enhance GAD enzyme activity.

[0032] The buffer solution comprises 50 mmol / L Tris-HCl, 20-40 mmol / L calcium chloride (CaCl2), and 100-200 mmol / L trehalose, wherein the pH of the Tris-HCl is 6.8.

[0033] The Tris-HCl is used to provide a buffer environment, the calcium chloride is used to provide calcium ions, and the trehalose is used as a compatible solute to regulate osmotic pressure.

[0034] The fermentation broth was centrifuged at 8000 r / min for 10 min at 4℃. The supernatant containing GABA and the cell precipitate were collected separately. The supernatant containing GABA was subjected to microfiltration to remove impurities, desalting and vacuum concentration to 10-20% of the original volume to obtain GABA-enriched solution, which was then refrigerated for later use. The bacterial cell pellet was resuspended in 0.85% NaCl saline solution, and after repeated centrifugation and washing 1-2 times, wet bacterial cells were obtained. The wet bacterial cells were then resuspended in buffer solution, and the bacterial cell concentration (OD) was adjusted. 600 To a final concentration of 10.0±1.0, the resuspended bacterial suspension was allowed to stand at 15-25℃ for 2-6 hours, with shaking every 30 minutes during this period.

[0035] During the static treatment, divalent calcium ions interact electrostatically with the negatively charged groups in peptidoglycan, forming a cross-linked bridge structure of peptidoglycan-calcium ion-peptidoglycan. This reduces the interchain spacing of peptidoglycan and creates a relatively dense microenvironment around the ester bond at the C6 position of N-acetylmuramic acid where the O-acetylation modification is located. This reduces the accessibility of water molecules to the ester bond. At the same time, trehalose accumulates in the periplasmic space of the bacterial cells, further reducing the content of free water in the microenvironment.

[0036] In S3, the pH of the bacterial suspension was adjusted to 5.0-5.5 with 1 mol / L lactic acid solution, and the pH-adjusted bacterial suspension was allowed to stand at 15-25℃ for 15-30 min.

[0037] In an acidic environment with a pH of 5.0-5.5, lactic acid bacteria enter an acid-resistant response state, and the activity of the intracellular glutamate decarboxylase system increases. At the same time, acidic treatment may change the conformation of proteins at the cell membrane-cell wall interface and the local ionic environment, promoting local conformational adjustments in the peptidoglycan layer. Local conformational adjustments change the N-acetylmuramic acid residues where O-acetylation is located from a relatively exposed state to a relatively shielded state, thereby reducing the chance of direct interaction between water molecules, hydrogen ions and other small molecules on the O-acetylated ester bond during heat treatment.

[0038] After the acid shock treatment, the pH was adjusted back to 6.5-6.8 with 1 mol / L NaOH solution. After the pH adjustment, the activity of the glutamate decarboxylase system decreased, but due to the formation of calcium ion cross-linking and the protective effect of the trehalose microenvironment, the relative shielding state of the peptidoglycan layer was maintained during the subsequent heat treatment.

[0039] In step S4, the treated bacterial suspension was transferred to a temperature-controlled water bath, and stirring was started at a speed of 50-100 r / min. The bacterial suspension was first heated to 48-52℃ at a rate of 2-3℃ / min and maintained for 20-30 min. Then, the temperature was increased to 65-70℃ at a rate of 1-2℃ / min and maintained for 15-30 min for inactivation. After inactivation, the bacterial suspension was cooled to below 25℃ to terminate the heat effect. After inactivation, samples were taken and spread on MRS solid medium. No colony growth was observed after incubation at 30-37℃ for 48 h, indicating that the viable count was below the detection limit.

[0040] In S5, the composite protectant is composed of reconstituted skim milk, trehalose, monosodium glutamate and sucrose. Based on the total volume of the system after mixing the inactivated bacterial suspension, GABA enrichment solution and composite protectant, the final concentration of each component is: 5-15% (v / v) of reconstituted skim milk, 3-8% (w / v) of trehalose, 1-3% (w / v) of monosodium glutamate and 2-5% (w / v) of sucrose. The reconstituted skim milk is an emulsion obtained by reconstituted skim milk powder at 8-12% (w / v) and sterilized.

[0041] During the spray drying process, the reconstituted skim milk forms a protective film on the surface of the bacteria, isolating the bacteria from direct contact with the high-temperature airflow. Trehalose and sucrose, through the glass transition mechanism, replace water molecules and form hydrogen bonds with the bacterial components during dehydration, maintaining the natural conformation of the bacterial components. Monosodium glutamate (MSG) also plays a role as a protective agent.

[0042] It should be noted that the total final concentration of trehalose includes the trehalose introduced by the buffer solution and the trehalose added by the compound protectant. When the trehalose introduced by the buffer solution is insufficient to reach the target final concentration, trehalose is added to reach the corresponding final concentration.

[0043] Low-temperature drying is preferably performed using vacuum low-temperature spray drying, with a drying chamber temperature of 45-60℃ and a vacuum degree of -0.06MPa to -0.09MPa, in order to reduce the heat loss of GABA during the drying stage and reduce secondary thermal damage to the active structure of the cells.

[0044] Collect the powder obtained from spray drying, sieve it through an 80-mesh standard sieve to remove large particle agglomerates, and obtain a uniformly sized powder containing γ-aminobutyric acid (GABA) lactic acid bacteria post-biotic product. Seal and store it in a desiccator at room temperature.

[0045] In one alternative application, the powdered lactic acid bacteria postbiotic product can be used directly as a raw material for powders, nutritional supplements, or functional foods, and can also be used to prepare liquid oral preparations.

[0046] The present invention also provides the application of lactic acid bacteria postbiotics containing γ-aminobutyric acid in liquid formulations, wherein the liquid formulations include oral liquids, liquid nutritional supplements, functional drinks or other liquid oral formulations.

[0047] The liquid formulation is prepared by mixing a lactic acid bacteria postbiotic product with a liquid carrier, wherein the liquid carrier includes purified water, and optionally includes one or more of glycerol, propylene glycol, and polyethylene glycol. Purified water is mainly used as a dispersion medium. Glycerin has the functions of solubilizing, thickening, preservative, and improving taste, and protects the activity of post-biotics. Propylene glycol is miscible with water or glycerin, has low toxicity, and helps dissolve fat-soluble components. It also plays a role in stabilizing the formula and promoting absorption. Polyethylene glycol can play a role in stabilizing, solubilizing, and thickening, improving taste, and is suitable for heat-sensitive post-biotics.

[0048] In a preferred embodiment, the mass-to-volume ratio of the post-biotic powder to the liquid carrier in the liquid formulation is 1:10-30. During preparation, the post-biotic powder is added to the liquid carrier and stirred and dispersed at 15-35°C for 10-60 minutes to ensure that the post-biotic powder is uniformly dispersed in the liquid system. If necessary, the liquid formulation is obtained after homogenization treatment.

[0049] When preparing liquid oral formulations, one or more of the following can be added, depending on the requirements of dispersion stability, taste, and storage stability: stabilizers, thickeners, dispersants, pH adjusters, and preservatives that are permitted for use in food or oral formulations. The stabilizer can be selected from one or more of xanthan gum, sodium carboxymethyl cellulose, and pectin; the thickener can be selected from one or more of glycerin, sorbitol, and hydroxypropyl methylcellulose; and the pH adjuster can be selected from one or more of citric acid, sodium citrate, lactic acid, and sodium lactate, and the pH of the liquid formulation is controlled within the range of 4.5-7.0.

[0050] After the postbiotic of the present invention is compounded to form a liquid formulation, its γ-aminobutyric acid still mainly comes from the aforementioned powdered lactic acid bacteria postbiotic product; at the same time, since the aforementioned preparation method protects the O-acetylated structure of peptidoglycan, the obtained liquid formulation still retains the synergistic effect of the postbiotic cell structure activity and the γ-aminobutyric acid functional component.

[0051] The present invention will be described in detail below with reference to specific embodiments. It should be noted that these embodiments are only used to explain the present invention and do not constitute any limitation on the scope of protection of the present invention. Those skilled in the art can make adaptive adjustments to the embodiments based on their understanding of the technical solutions of the present invention, and these adjustments still fall within the scope of protection of the present invention.

[0052] Example 1 This embodiment provides a method for preparing lactic acid bacteria postbiotics containing γ-aminobutyric acid, specifically including the following steps: S1: Inoculate the short-starter Lactobacillus onto the culture medium and activate it at 32℃ for 20h. Pick a single colony after activation and inoculate it into a 250mL Erlenmeyer flask containing 50mL of culture medium. Incubate at 32℃ for 20h to obtain the seed culture. Using the uninoculated liquid culture medium as a blank, the OD600 value of the seed culture was measured to be 1.3.

[0053] S2: Inoculate the seed culture into the fermentation medium at an inoculation rate of 4% (v / v), with a liquid volume of 325 mL. Incubate at 27°C. When the GABA content in the fermentation broth reaches 3.8 g / L, terminate the fermentation to obtain a fermentation broth containing γ-aminobutyric acid. The fermentation broth was centrifuged at 8000 r / min for 10 min at 4℃. The supernatant containing GABA and the cell precipitate were collected separately. The supernatant was subjected to microfiltration to remove impurities, desalting and vacuum concentration to 15% of the original volume to obtain GABA-enriched solution, which was stored at 4℃ for later use. The bacterial cell pellet was resuspended in 0.85% (w / v) NaCl saline solution, and the pellet was washed twice by centrifugation. The wet bacterial cells were collected and resuspended in buffer solution. The OD of the bacterial suspension was adjusted by changing the amount of buffer solution added. 600 The concentration was 10.4. The resuspended bacterial suspension was left to stand at 20°C for 4 hours, and shaken once every 30 minutes during the period.

[0054] The buffer solution consisted of 50 mmol / L Tris-HCl, 30 mmol / L CaCl2, and 150 mmol / L trehalose.

[0055] S3: Adjust the pH of the above bacterial suspension to 5.2 with 1 mol / L lactic acid solution, let it stand at 20℃ for 20 min, and after the treatment, adjust the pH back to 6.8 with 1 mol / L NaOH solution.

[0056] S4: Transfer the bacterial suspension to a temperature-controlled water bath, turn on the stirrer at 80 r / min, and heat the bacterial suspension to 50℃ at a heating rate of 2.5℃ / min, maintaining the temperature for 20 min for heat acclimatization treatment; then continue heating to 68℃ at a heating rate of 1.5℃ / min, maintaining the temperature for 20 min for inactivation treatment. After inactivation, cool the bacterial suspension to below 25℃ to terminate the heat effect, and verify that the viable bacterial count is below the detection limit by MRS plate culture.

[0057] S5: Add GABA enrichment solution and composite protectant to the bacterial suspension. The volume of GABA enrichment solution is 21% (v / v) of the volume of bacterial suspension. Based on the total volume of the system after mixing the inactivated bacterial suspension, GABA enrichment solution and composite protectant, the composite protectant contains 10% (v / v) reconstituted skim milk, 5% (w / v) total trehalose, 2% (w / v) monosodium glutamate and 3% (w / v) sucrose. The reconstituted skim milk was obtained by reconstituted skim milk powder at 10% (w / v) and sterilized; the total final concentration of trehalose included trehalose introduced by the buffer solution and trehalose added by the compound protectant. When the trehalose introduced by the buffer solution was insufficient to reach 5% (w / v), trehalose was added to reach the total final concentration; the GABA enrichment solution not added to the compound system was not included in the calculation of the GABA drying retention rate in this embodiment.

[0058] After stirring until all components are completely dissolved, the mixture is equilibrated at 25°C for 30 minutes and then subjected to vacuum low-temperature spray drying. The drying chamber temperature is set at 55°C, the vacuum degree at -0.08MPa, and the feed flow rate at 240mL / h. Collect the powder obtained from spray drying, sieve it through an 80-mesh standard sieve to obtain a powdered lactic acid bacteria post-biotic product containing γ-aminobutyric acid. The product is sealed and stored in a desiccator at room temperature.

[0059] Example 2 This embodiment provides a method for preparing a postbiotic of lactic acid bacteria containing γ-aminobutyric acid. The difference from Example 1 is that the concentration of CaCl2 in the buffer solution in S2 is 20 mmol / L.

[0060] Example 3 This embodiment provides a method for preparing a postbiotic of lactic acid bacteria containing γ-aminobutyric acid. The difference from Example 1 is that the concentration of CaCl2 in the buffer solution in S2 is 40 mmol / L.

[0061] Example 4 This embodiment provides a method for preparing a postbiotic of lactic acid bacteria containing γ-aminobutyric acid. The difference from Example 1 is that in S3, the pH of the bacterial suspension is adjusted to 5.0 using a lactic acid solution.

[0062] Example 5 This embodiment provides a method for preparing a postbiotic of lactic acid bacteria containing γ-aminobutyric acid. The difference from Example 1 is that in S3, the pH of the bacterial suspension is adjusted to 5.5 using a lactic acid solution.

[0063] Example 6 This embodiment provides a method for preparing a postbiotic of lactic acid bacteria containing γ-aminobutyric acid. The difference from Example 1 is that in S4, the first stage is heated to 48°C and maintained for 30 min, and the second stage is heated to 65°C and maintained for 30 min.

[0064] Example 7 This embodiment provides a method for preparing a postbiotic of lactic acid bacteria containing γ-aminobutyric acid. The difference from Example 1 is that in S4, the first stage is heated to 52°C and maintained for 20 min, and the second stage is heated to 70°C and maintained for 15 min.

[0065] Example 8 This embodiment provides a method for preparing a lactic acid bacteria postbiotic containing γ-aminobutyric acid. The difference from Example 1 is that in S1, the short fermenting agent Lactobacillus is replaced with Lactobacillus plantarum.

[0066] Example 9 This embodiment provides a method for preparing a lactic acid bacteria postbiotic containing γ-aminobutyric acid. The difference from Example 1 is that in S1, the short starter lactobacillus is replaced with lactococcus lactis.

[0067] Comparative Example 1 This comparative example provides a method for preparing a postbiotic of lactic acid bacteria containing γ-aminobutyric acid. The difference from Example 1 is that in S2, the buffer does not contain CaCl2 and trehalose, and the bacterial cells are resuspended only in 50 mmol / L Tris-HCl. The static treatment conditions are the same as in Example 1. Step S3 is omitted. Meanwhile, in S4, the bacterial suspension is directly heated to 70°C at 2.5°C / min and maintained for 30 min.

[0068] Comparative Example 2 This comparative example provides a method for preparing a postbiotic of lactic acid bacteria containing γ-aminobutyric acid. The difference from Example 1 is that step S3 is omitted, and the bacterial suspension is directly subjected to temperature-controlled inactivation.

[0069] Comparative Example 3 This comparative example provides a method for preparing a postbiotic of lactic acid bacteria containing γ-aminobutyric acid. The difference from Example 1 is that in S2, the buffer solution does not contain CaCl2, but only contains 50 mmol / L Tris-HCl and 150 mmol / L trehalose.

[0070] Comparative Example 4 This comparative example provides a method for preparing a postbiotic of lactic acid bacteria containing γ-aminobutyric acid. The difference from Example 1 is that in S2, the supernatant containing GABA is discarded after centrifugation, and the preparation of GABA-enriched solution is not performed.

[0071] To verify the technical effects of the postbiotics prepared in the above embodiments and comparative examples of the present invention, the postbiotics prepared in Examples 1-9 and Comparative Examples 1-4 were tested to evaluate the actual effects of the technical solution of the present invention in ensuring the inactivation effect of bacteria while taking into account the drying retention of GABA after entering the compound system, the protection of the O-acetylation structure of peptidoglycan, and the synergistic utilization of postbiotics and GABA.

[0072] Experimental Example 1 This experimental example aims to verify the effect of different operations on the GABA content in the finished product by measuring the γ-aminobutyric acid content in the postbiotic products obtained from each example and comparative example.

[0073] The post-genetic agents prepared in Examples 1-9 and Comparative Examples 1-4 were used as experimental subjects. At the same time, the GABA enriched solution actually added to the compound system was taken as a control sample, and its GABA content was measured as the benchmark for calculating the GABA drying retention rate. The remaining GABA enriched solution not added to the compound system was not included in the calculation of this index. Comparative Example 4 did not have the GABA enriched solution added back, so only the GABA content in the finished product was measured, and the GABA drying retention rate was not calculated.

[0074] Each experimental group weighed 0.1g of powder, placed it in a centrifuge tube, added 1.0mL of deionized water, and sonicated for 10min to fully dissolve it. The mixture was then centrifuged at 12000r / min for 5min, and the supernatant was taken for testing. The GABA enrichment solution that was actually added to the compound system was diluted under the same derivatization conditions and used as the control sample for retention rate calculation.

[0075] Take 100 μL of the supernatant and add 100 μL of 10 mg / mL dansyl chloride acetone solution and 100 μL of 0.5 mol / L sodium bicarbonate buffer solution, respectively. After vortexing and mixing, react in the dark for 30 min. After the reaction is completed, add 400 μL of ethyl acetate, vortex extract for 1 min, and after standing and separating the layers, take the upper organic phase for high performance liquid chromatography analysis.

[0076] An Agilent ZORBAX SB-C18 column was used. Mobile phase A was 10 mmol / L sodium acetate buffer, and mobile phase B was acetonitrile. The flow rate was set to 1.0 mL / min, the detection wavelength to 254 nm, the column temperature to 30 °C, and the injection volume to 10 μL. The gradient elution program is shown in Table 1 below.

[0077] Table 1 Gradient elution program Weigh out GABA standard and prepare a series of standard solutions with concentrations of 0.1 g / L, 0.2 g / L, 0.5 g / L, 1.0 g / L, 2.0 g / L, and 5.0 g / L using deionized water. Perform linear regression with peak area as the ordinate and GABA concentration as the abscissa. The results are shown in [Figure number missing]. Figure 1 As shown, the equation of the curve is: The GABA concentration in the sample was calculated from the standard curve based on the peak area, and then converted to grams of GABA per 100g of finished powder using the following formula: Where C is the measured GABA concentration (g / L), V is the extraction liquid volume (mL), m is the sample weight (g), and the GABA drying retention rate is the ratio of the total mass of GABA measured in the finished powder to the total mass of GABA actually added to the compound system in the GABA enrichment solution. The results of the determination of GABA content and GABA drying retention rate of each sample are shown in Table 2 and [Table 3]. Figure 2 As shown.

[0078] Table 2. Results of GABA content and drying retention rate of the finished product According to Table 2 and Figure 2 The results showed that the GABA content in Examples 1-9 and Comparative Examples 1-3 was in the range of 2.2-2.8 g / 100 g, and the GABA drying retention rate reached 80.6%-86.3%. However, the GABA content in Comparative Example 4 was only 0.3 g / 100 g. This indicates that the GABA in the finished product mainly comes from the GABA-enriched liquid that was actually added back to the compound system after desalting, impurity removal and concentration. The addition of the GABA-enriched liquid is the main factor in improving the GABA level of the post-genetic derivative product.

[0079] Compared with Example 1, the GABA drying retention rate of Comparative Examples 1-3 did not decrease significantly, indicating that calcium ion crosslinking, acid shock, and staged inactivation have no direct negative impact on the chemical stability of GABA itself. The GABA loss mainly occurs in the low-temperature drying stage after compounding, rather than in the pretreatment or inactivation stage of non-bacterial cells. The remaining GABA enrichment solution that was not added to the compounding system is not included in the calculation of drying loss.

[0080] Experiment Example 2 This experimental example aims to verify the inhibitory effect of the present invention on the hydrolysis of O-acetylated ester bonds of peptidoglycan during heat inactivation by measuring the retention rate of peptidoglycan O-acetylation modification in the postbiotic products obtained in each embodiment and comparative example.

[0081] The postbiotic products prepared in Examples 1-9 and Comparative Examples 1-3 were used as experimental subjects. At the same time, peptidoglycan was extracted from the bacterial suspension that had not been inactivated as a control sample.

[0082] Each experimental group weighed 300 mg of powder, added 1.5 mL of physiological saline and soaked for 10 min, then centrifuged to collect the bacterial precipitate.

[0083] The bacterial cells were suspended in an 8% trichloroacetic acid solution and treated at 95°C for 15 min to remove teichoic acid. The precipitate was collected by centrifugation and washed with ethanol, acetone and ether to remove fat. The precipitate was then dried to obtain crude cell wall.

[0084] The crude cell wall was suspended in phosphate buffer containing 1 mg / mL trypsin and enzymatically hydrolyzed at 37°C for 12 h to remove covalently bound proteins. The precipitate was collected by centrifugation, and the precipitate was desalted by dialysis and then freeze-dried to obtain pure peptidoglycan.

[0085] Take pure peptidoglycan, add mutanolysin for enzymatic hydrolysis, and react at 37℃ for 16 h to degrade peptidoglycan macromolecules into soluble cell wall peptide monomers. After centrifuging the enzymatic hydrolysate, take the supernatant and filter it through a 0.22 μm filter membrane to obtain the test solution.

[0086] A C18 reversed-phase column was used. Mobile phase A was 0.1% formic acid solution, and mobile phase B was acetonitrile containing 0.1% formic acid. The flow rate was set to 0.5 mL / min, the column temperature to 35℃, and the injection volume to 10 μL. The gradient elution program is shown in Table 3 below.

[0087] Table 3 Gradient elution program An electrospray ionization source was used in positive ion mode, with capillary voltage set at 3.5 kV, cone voltage at 40 V, ion source temperature at 120 °C, desolvation gas temperature at 350 °C, and scanning range at 400-2000 m / z.

[0088] Extracted ion chromatograms of O-acetylated MurNAc and unmodified MurNAc were obtained separately, and quantification was performed by peak area. The O-acetylation retention rate was calculated. The results of the O-acetylation retention rate determination of peptidoglycan in each sample are shown in Table 4 and 5. Figure 3 As shown.

[0089] Table 4 Results of Peptidoglycan O-acetylation Retention Rate Determination According to Table 4 and Figure 3The results showed that the O-acetylation retention rate of Comparative Example 1 was only 68.7%, while the O-acetylation retention rate of Example 1 reached 93.2%, which was 24.5% higher than that of Comparative Example 1. This indicates that the technical solution of the present invention can effectively inhibit the hydrolysis of O-acetylated ester bonds of peptidoglycan during the heat inactivation process.

[0090] The O-acetylation retention rate of Comparative Example 2 was 81.2%, which was 12.0% lower than that of Example 1. The acidic environment of pH 5.0-5.5 put the lactic acid bacteria into an acid-resistant response state and caused local conformational adjustment of the peptidoglycan layer by changing the protein conformation of the cell membrane and cell wall interface and the local ionic environment. This caused the MurNAc residues where the O-acetylation modification was located to change from an exposed orientation to a masked state and be fixed by calcium cross-linking. Comparative Example 2 relied only on calcium cross-linking and trehalose microenvironment protection, which made it difficult to form the same degree of O-acetylation site masking effect as Example 1, resulting in a decrease in retention rate.

[0091] The O-acetylation retention rate of Comparative Example 3 was 76.5%, which was 16.7% lower than that of Example 1. Divalent calcium ions interact electrostatically with the negatively charged groups in peptidoglycan to form a peptidoglycan-calcium ion-peptidoglycan cross-linked bridge structure, which reduces the inter-chain spacing of peptidoglycan and decreases the accessibility of water molecules to the O-acetylated ester bond. At the same time, it fixes the acid shock-induced masking conformation. Comparative Example 3 lacks this cross-linked network, so it cannot effectively reduce the water activity around the ester bond, nor can it fix the conformational change, resulting in a decrease in retention rate.

[0092] Experimental Example 3 This experiment aims to test the activation ability of the postbiotic products obtained from each embodiment and comparative example on the host pattern recognition receptor NOD2 signaling pathway, thereby verifying the downstream biological effects brought about by the retention of the O-acetylated structure of peptidoglycan.

[0093] The post-genetic products prepared in Examples 1-9 and Comparative Examples 1-3 were used as test samples, with muramic acyl dipeptide (MDP) as a positive control and phosphate-buffered saline (PBS) as a negative control.

[0094] HEK-Blue TM The hNOD2 reporter gene cell line was tested. This cell line was stably transfected with human NOD2 gene and NF-κB-induced secretory embryonic alkaline phosphatase reporter gene. When the NOD2 receptor is activated, the downstream NF-κB signaling pathway is initiated, driving the expression and secretion of secretory alkaline phosphatase.

[0095] Cells were cultured in DMEM high-glucose medium supplemented with 10% (v / v) heat-inactivated fetal bovine serum, 1% (v / v) penicillin-streptomycin antibiotics, and 100 μg / mL Normocin. TM They were cultured at 37°C and 5% CO2 concentration.

[0096] HEK-Blue in the logarithmic growth phase TM hNOD2 cells, at 5 × 10⁶ cells per well 4 Cells were seeded at a density of 200 μL per well in 96-well plates.

[0097] Weigh 100 mg of each post-biotic product powder, add 10 mL of sterile PBS to each, shake to dissolve, and use as a 10000 μg / mL stock solution. Take the stock solution for serial dilution, and dilute it sequentially with sterile PBS to 1000 μg / mL, 100 μg / mL, 10 μg / mL, 1 μg / mL, and 0.1 μg / mL. After sterilizing each concentration solution by filtration through a 0.22 μm filter membrane, add 20 μL to each well of the corresponding solution, and set up 3 replicates for each concentration.

[0098] Add muramyl dipeptide solution to the positive control wells to a final concentration of 0.1 μg / mL, and add an equal volume of PBS to the negative control wells. Continue culturing for 20 h after adding the samples.

[0099] After the culture is complete, take 20 μL of the supernatant from each well and transfer it to a 96-well microplate. Add 180 μL of QUANTI-Blue to each well. TM The reagents were incubated at 37°C for 2 hours, and the absorbance at 655 nm was measured using an ELISA reader.

[0100] The dose-response curve was fitted using a four-parameter Logistic model with the logarithm of sample concentration on the x-axis and absorbance on the y-axis. The results are shown in [Figure number missing]. Figure 4 As shown.

[0101] The half-maximal effective concentration (EC) was calculated using the fitted curve. 50 EC 50 The lower the value, the stronger the activation activity of the sample on the NOD2 signaling pathway, that is, the more completely the natural NOD2 recognition ability of peptidoglycan in the postbiotic product is preserved. The results of the NOD2 signaling pathway activation activity determination of each sample are shown in Table 5.

[0102] Table 5 Results of NOD2 signaling pathway activation activity assay According to the results in Table 5, the EC of Comparative Example 1 50 The value was 28.7 μg / mL, while the EC value in Example 1 was... 50 The value was 12.3 μg / mL, which was only 42.9% of that of Comparative Example 1. This means that the concentration of the post-genetic agent required to achieve the same NOD2 activation level was reduced by more than half, indicating that the technical solution of this invention enhances the activation ability of the post-genetic agent on the NOD2 signaling pathway by protecting the O-acetylated structure of peptidoglycan.

[0103] Comparative Example 2 EC 50 The value was 22.5 μg / mL, which was 82.9% higher than that in Example 1. Combined with the data from Example 2, the corresponding decrease in NOD2 activation activity was greater than the decrease in O-acetylation retention rate. This indicates that the conformational masking induced by acid shock not only improved the chemical stability of O-acetylation, but also made the spatial orientation of O-acetylation modification in peptidoglycan fragments more favorable for binding to the NOD2 receptor.

[0104] Comparative Example 3 EC 50 The value was 25.3 μg / mL, an increase of 105.7% compared to Example 1. The O-acetylation retention rate of Comparative Example 3 was 76.5%, a decrease of 16.7% compared to Example 1, EC 0.05%. 50 The increase was approximately 6.3 times the decrease in retention, indicating that the role of calcium ion crosslinking is not only to reduce water activity to protect O-acetylated ester bonds, but also to fix the masking conformation induced by acid shock. This allows the peptidoglycan fragment to maintain the spatial conformation required for high affinity binding with NOD2 during inactivation and subsequent processing. Without calcium crosslinking, even if acid shock induces conformational masking, this conformation cannot be stably maintained during subsequent processing, leading to a decrease in functional activity.

[0105] Experiment Example 4 This experiment aims to simulate intestinal barrier function using a Caco-2 cell monolayer model, to detect the effect of combined treatment with GABA-enriched solution and postbiotic bacterial components on maintaining the integrity of the intestinal epithelial barrier, and to verify the synergistic effect of postbiotics in maintaining the intestinal barrier through the NOD2 signaling pathway and thus providing intestinal microenvironmental support for the stable utilization of GABA.

[0106] The post-genetic products obtained in Example 1 and Comparative Example 1 were selected as experimental subjects. The experiment was set up in five groups, with three duplicate wells in each group.

[0107] The blank control group was supplemented with only HBSS buffer. The pure GABA group was added to an HBSS solution containing GABA, and the final concentration of GABA was 100 μmol / L. The GABA / Comparative Example 1 group was added to HBSS solutions containing GABA and the post-genetic agent of Comparative Example 1, with the final concentration of GABA being 100 μmol / L and the final concentration of the post-genetic agent of Comparative Example 1 being 100 μg / mL. GABA / Example 1 group was added to an HBSS solution containing GABA and the post-biotic of Example 1, wherein the final concentration of GABA was 100 μmol / L and the final concentration of the post-biotic of Example 1 was 100 μg / mL; In Example 1, a HBSS solution containing the post-biotic from Example 1 was added to the group alone. The final concentration of the post-biotic from Example 1 was 100 μg / mL.

[0108] Caco-2 cells were planted at 2.5 × 10⁶ cells per well. 5 Cells were seeded at a density of 1,000 cells in Transwell chambers and cultured continuously for 21 days in MEM medium at 37°C and 5% CO2 concentration, with fresh medium replaced every 3 days to form a dense monolayer.

[0109] Transmembrane resistance (TEER) was measured using an EVOM2 epithelial transmembrane resistance meter, with a TEER value ≥300 Ω·cm selected. 2 The qualified monolayers are used for subsequent experiments.

[0110] Each sample was prepared with HBSS buffer and the pH was adjusted to 7.4 and the osmotic pressure to 300±10 mOsm / kg. The original culture medium in the Transwell chamber was aspirated and the sample was gently washed twice with HBSS. 200 μL of the sample was added to the top side and 600 μL of HBSS was added to the bottom side.

[0111] TEER values ​​were measured before administration and at 2h, 4h, 6h, 12h, and 24h after administration. Each measurement was repeated three times, and the average value was taken. The TEER value at 0h was set as 100%, and the percentage of TEER at each time point relative to 0h was calculated. The decrease in TEER at the 24h endpoint was used as the primary evaluation index. The relative TEER values ​​of each experimental group at different time points are shown in Table 6. Figure 5 As shown.

[0112] Table 6. Results of relative TEER measurements for each experimental group According to Table 6 and Figure 5 The results showed that the TEER value of the pure GABA group continued to decrease after administration, with a decrease of 38.5% in 24 hours, which was higher than that of the blank control group. This indicates that GABA alone can lead to increased permeability of Caco-2 cell monolayer and decreased barrier integrity, which may be related to the increased chloride ion secretion and altered phosphorylation level of tight junction protein occludin after GABA activates GABA receptors in intestinal epithelial cells.

[0113] The decrease in GABA / Example 1 group was 12.7%, compared to a 25.8% decrease in the pure GABA group. The decrease in GABA / Comparative Example 1 group was 24.8%, compared to a 13.7% decrease in the pure GABA group. Both groups of metabiotics showed a protective effect against GABA-induced barrier damage. However, the protective effect of the metabiotic in Example 1 was better than that in Comparative Example 1, with the former showing a decrease of only 51.2% of that in Comparative Example 1. This indicates that the invention's technical solution enhances the role of metabiotics in maintaining intestinal barrier integrity by protecting the O-acetylated structure of peptidoglycan and enhancing the activation ability of the NOD2 signaling pathway.

[0114] Based on the data from Experiments 2 and 3, the O-acetylation retention rate of the post-biotic in Example 1 was 93.2%, and the NOD2 activation activity of EC was [missing information]. 50 The value was 12.3 μg / mL, while the corresponding values ​​for the postbiotic in Comparative Example 1 were 68.7% and 28.7 μg / mL, respectively. The postbiotic in Example 1 showed a 24.5% increase in O-acetylation retention rate and a 57.1% increase in NOD2 activation activity compared to Comparative Example 1, while the decrease was reduced by 48.8%. This indicates that the more complete the O-acetylation structure is retained, the stronger the NOD2 signaling pathway activation and the better the intestinal barrier protection effect. This proves that the O-acetylation structure of peptidoglycan is a key factor determining the intestinal barrier protection function of postbiotics.

[0115] The decrease in TEER in the Example 1 group was 4.5%, which was not different from the 6.2% in the blank control group. This indicates that the metabiotic itself has no adverse effect on the monolayer barrier function of Caco-2 cells at a concentration of 100 μg / mL. Combined with the result that the decrease in TEER in the GABA / Example 1 group was lower than that in the pure GABA group, it can be concluded that the metabiotic activates the downstream NF-κB and MAPK signaling cascade through the NOD2 signaling pathway, promotes the expression and membrane localization of tight junction proteins, enhances the mechanical strength of the intestinal epithelial barrier, and thus effectively antagonizes GABA-induced barrier damage.

[0116] This invention utilizes a synergistic process of GABA enrichment solution purification, bacterial cell structure protection and inactivation, and low-temperature drying to reduce GABA loss during preparation and drying, while providing a stable intestinal microenvironment for GABA absorption and utilization.

[0117] The technical scope of this invention is not limited to the content described above. Those skilled in the art can make various modifications and variations to the above embodiments without departing from the technical concept of this invention, and all such modifications and variations should fall within the protection scope of this invention.

Claims

1. A method for preparing a lactic acid bacteria postbiotic containing γ-aminobutyric acid, characterized in that, Includes the following steps: S1: Inoculate lactic acid bacteria into a culture medium and obtain seed culture after activation culture; S2: The seed culture is inoculated into a fermentation medium containing monosodium glutamate and allowed to ferment statically to obtain a fermentation broth containing γ-aminobutyric acid (GABA). The supernatant containing GABA and the cell precipitate are collected by centrifugation. The supernatant is desalted, impurities removed and concentrated to obtain a GABA-enriched solution. The cell precipitate is washed, resuspended in a buffer solution and allowed to stand to obtain a cell suspension. The buffer solution contains calcium ions and trehalose. S3: Adjust the pH of the bacterial suspension to 5.0-5.5 with lactic acid solution, let it stand for 15-30 minutes, and then adjust the pH to 6.5-6.8 with alkaline solution. S4: Perform staged heat treatment on the bacterial suspension. In the first stage, heat the temperature to 48-52℃ and maintain it for 20-30 minutes. In the second stage, continue to heat the temperature to 65-70℃ and maintain it for 15-30 minutes. After inactivation, cool the suspension. S5: Add γ-aminobutyric acid enrichment solution and composite protectant to the inactivated bacterial suspension, mix evenly, and then dry at low temperature to collect the finished powder.

2. The method for preparing a postbiotic of lactic acid bacteria containing γ-aminobutyric acid according to claim 1, characterized in that: The lactic acid bacteria mentioned in S1 are lactic acid bacteria capable of producing γ-aminobutyric acid, and the lactic acid bacteria are selected from one of the short-starter cultures Lactobacillus, Lactobacillus plantarum, or Lactococcus lactis.

3. The method for preparing a postbiotic of lactic acid bacteria containing γ-aminobutyric acid according to claim 1, characterized in that: The buffer solution in S2 further includes Tris-HCl, and the pH of the Tris-HCl is 6.

8.

4. The method for preparing a postbiotic of lactic acid bacteria containing γ-aminobutyric acid according to claim 3, characterized in that: The buffer solution described in S2 contains Tris-HCl at a concentration of 50 mmol / L, calcium chloride at a concentration of 20-40 mmol / L, and trehalose at a concentration of 100-200 mmol / L.

5. The method for preparing a postbiotic containing γ-aminobutyric acid (GABA) from lactic acid bacteria according to claim 1, characterized in that: The γ-aminobutyric acid enrichment solution mentioned in S2 is a liquid obtained by microfiltration, desalting, impurity removal and concentration of the supernatant. The volume of the γ-aminobutyric acid enrichment solution added is 10-30% of the volume of the bacterial suspension.

6. The method for preparing a postbiotic of lactic acid bacteria containing γ-aminobutyric acid according to claim 1, characterized in that: In the staged heat treatment described in S4, the heating rate of the first stage is 2-3℃ / min, and the heating rate of the second stage is 1-2℃ / min.

7. The method for preparing a postbiotic of lactic acid bacteria containing γ-aminobutyric acid according to claim 1, characterized in that: The composite protective agent described in S5 consists of reconstituted skim milk, trehalose, monosodium glutamate, and sucrose.

8. The method for preparing a postbiotic of lactic acid bacteria containing γ-aminobutyric acid according to claim 7, characterized in that: Based on the total volume of the system after mixing the inactivated bacterial suspension, γ-aminobutyric acid enrichment solution, and composite protectant, the final concentration of the reconstituted skim milk in the composite protectant is 5-15% (v / v), the final concentration of trehalose is 3-8% (w / v), the final concentration of monosodium glutamate is 1-3% (w / v), and the final concentration of sucrose is 2-5% (w / v).

9. The method for preparing a postbiotic of lactic acid bacteria containing γ-aminobutyric acid according to claim 1, characterized in that: The low-temperature drying described in S5 is vacuum low-temperature spray drying, and the material being dried at a temperature not exceeding 60°C.

10. A lactic acid bacteria postbiotic prepared by the method for preparing a lactic acid bacteria postbiotic containing γ-aminobutyric acid according to any one of claims 1-9, characterized in that, The lactic acid bacteria postbiotic contains γ-aminobutyric acid, and the O-acetylation retention rate of peptidoglycan is not less than 85%.

11. The application of the lactic acid bacteria postbiotic of claim 10 in the preparation of a liquid oral formulation, characterized in that, The liquid oral formulation is prepared by mixing lactic acid bacteria postbiotics with a liquid carrier, wherein the liquid carrier includes purified water and optionally one or more of glycerol, propylene glycol, and polyethylene glycol, and the pH of the liquid oral formulation is 4.5-7.0.

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