Postbiotic composition microcapsules, methods of making the same, and use in hangover liver protection products

Abstract

This invention proposes a postbiotic composition microcapsule and its preparation method, as well as its application in hangover relief and liver protection products, relating to the field of postbiotic preparation technology. The postbiotic composition is obtained by fermenting and then inactivating *Lactobacillus paracasei* Postbio-Q7, *Lactobacillus paracasei* Postbio-P6, and *Lactobacillus paracasei* Postbio-P8. The preparation of the postbiotic composition microcapsule includes: 1. Adding sodium alginate, shellac, guarana extract, and protamine sulfate to sterile deionized water to prepare microcapsule shells; 2. Mixing the microcapsule shells with the above-mentioned postbiotic composition to prepare postbiotic composition microcapsules. The postbiotic composition of this invention has excellent hangover relief and liver protection effects. The prepared microcapsules can better protect the postbiotic composition, facilitating its storage and transportation, protecting internal components, reducing oxidation value, and thus improving the antibacterial and antimicrobial effects of the postbiotic.

Description

Technical Field

[0001] This invention relates to the field of post-biotic preparation technology, and in particular to a post-biotic composition microcapsule and its preparation method, and its application in hangover relief and liver protection products. Background Technology

[0002] In May 2021, the International Society for the Study of Probiotics and Prebiotics (ISAPP) released a consensus statement on postbiotics, officially establishing postbiotics as a highly regarded category in the global gut microbiota field. The statement defines postbiotics as: "preparations of non-living microorganisms and / or components thereof that are beneficial to host health." Research has shown that postbiotics play important roles in maintaining the intestinal barrier, regulating immune function, anti-oxidation, and antihypertensive effects. Furthermore, due to their "non-living" nature, although research on postbiotics lags far behind that of probiotics, it has not hindered the fascination of academia and industry. Industry considers postbiotics a novel microecological product following probiotics, prebiotics, and synbiotics, possessing broad application prospects.

[0003] my country has a long and rich drinking culture that continues to this day. Alcohol has become an important part of our culture, daily life, and work. However, excessive drinking is common in daily life for various reasons, causing serious damage to the liver. When alcohol enters the body, alcohol dehydrogenase in the liver breaks down ethanol into acetaldehyde, and acetaldehyde dehydrogenase breaks down acetaldehyde into acetic acid. Subsequently, the tricarboxylic acid cycle breaks down acetaldehyde into non-toxic water and carbon dioxide, which are then excreted from the body. Long-term excessive drinking increases the burden on the liver, reducing its ability to break down ethanol and acetaldehyde. Excessive acetaldehyde production inhibits the tricarboxylic acid cycle, causing lipid metabolism disorders and damaging liver tissue. Therefore, long-term excessive drinking easily leads to alcoholic liver disease, cirrhosis, liver fibrosis, and even liver cancer.

[0004] Some probiotics have been shown to have beneficial effects on alcoholic fatty liver disease. Currently, the most effective probiotics studied are lactic acid bacteria and bifidobacteria, including *Lactobacillus rhamnosus* and *Bifidobacterium animalis* subsp. *rhamnosus*. Clinical studies have found that *Lactobacillus rhamnosus* can enhance the phagocytic function of neutrophils in patients with alcoholic cirrhosis. Animal studies have also confirmed that *Lactobacillus rhamnosus* can improve alcoholic liver damage by reducing liver inflammation and maintaining intestinal mucosal integrity. Chinese patent publication CN115895950A discloses the use of *Lactobacillus paracasei* SMNLBK for hangover relief or addition to alcoholic fermented beverages. In this prior art, the probiotics used are live bacteria, not postbiotics prepared from fermentation bacteria. Chinese patent CN117487690A discloses a method for preparing a probiotic starter using at least one of the following strains: *Lactobacillus rhamnosus* MC-1, *Lactobacillus rhamnosus* SSN-13, and *Limosilactobacillus fermentum* G-1-46. This starter is used to ferment a traditional Chinese medicine composition to prepare a beverage with hangover-relieving and liver-protecting effects. In this prior art, on the one hand, live probiotics are used; on the other hand, these live probiotics are used for fermentation, rather than the probiotic post-biotics being directly used for hangover relief and liver protection.

[0005] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention proposes a postbiotic composition microcapsule and its preparation method, and its application in hangover relief and liver protection products. The postbiotic composition with liver protection and hangover relief functions is prepared by staged fermentation of three probiotics: Lactobacillus mucosa Postbio-Q7, Lactobacillus paracasei Postbio-P6, and Lactobacillus paracasei Postbio-P8.

[0007] The technical solution of the present invention is implemented as follows: The present invention provides a postbiotic composition, which is obtained by fermentation and inactivation of *Lactobacillus mucilaginosus* Postbio-Q7, *Lactobacillus paracasei* Postbio-P6, and *Lactobacillus paracasei* Postbio-P8.

[0008] This invention also provides a microcapsule of an epigenetic composition, comprising a microcapsule shell and a microcapsule core, wherein the microcapsule core is the epigenetic composition of claim 1. It is understood that the microcapsule shell can be a commonly used raw material for preparing pharmaceutical microcapsules. Encapsulating the epigenetic composition in microcapsules can protect the components within the epigenetic composition and improve the stability of the epigenetic composition in the microcapsule core.

[0009] The present invention also provides a method for preparing the above-mentioned post-biotic composition microcapsules, comprising the following steps: S10. Add sodium alginate, shellac, guarana extract and protamine to sterile deionized water and stir continuously at 50~60℃ for 30~60 min to obtain a solution. Of which, based on a total mass of 100%, sodium alginate 1%~4%, shellac 0.5%~2%, guarana extract 0.1%~1%, protamine 0.1%~1%, and the remainder is sterile deionized water; S20. Stir the solution at 10000~15000rpm for 10-15 minutes to obtain a homogeneous solution. Filter the solution through a 0.1µm~0.4µm filter to obtain the microcapsule solution. S30. Mix the microcapsule shell solution with the post-genetic composition, homogenize, and dry; the mass ratio of the microcapsule solution to the post-genetic composition is (1~4):(2~6), the homogenization pressure is 20-40 MPa, and the drying temperature is 160-190℃ to obtain the post-genetic composition microcapsules.

[0010] This preparation method uses sodium alginate, shellac, guarana extract, and protamine to prepare the shell of microcapsules, thereby improving the stability and antioxidant capacity of the post-biotic composition. Sodium alginate, a natural polysaccharide with good water solubility, mainly plays a role in stabilizing the microcapsule shell structure and emulsifying. Shellac primarily functions as a film-forming material in the microcapsule shell, providing good film-forming properties and stability, extending the shelf life of the raw materials, protecting the internal components (post-biotic composition), reducing oxidation value, and enhancing the moisture resistance of the post-biotic composition microcapsules. Protamine mainly improves the antibacterial properties and stability of the post-biotic composition microcapsules, extending their shelf life. The film-forming material shellac forms a cross-linked aggregated layer with sodium alginate and protamine, and the microcapsule shell structure is further strengthened through the interaction of the biochemical substances of guarana extract with the polymers constituting the aggregated layer. The four components mentioned above work together synergistically to form post-biotic composition microcapsules with higher stability. Compared with post-biotic composition microcapsules prepared using some of the four components or post-biotic compositions without microcapsules, the solubility of post-biotic composition microcapsules can be significantly increased, their oxidation value reduced, and the flowability and moisture resistance of post-biotic composition microcapsules increased.

[0011] Based on the above technical solutions, preferably, in step S10, based on a total mass of 100%, sodium alginate is 2%, shellac is 0.5%, guarana extract is 0.5%, protamine sulfate is 0.2%, and the remainder is sterile deionized water. Preferably, in step S20, a 0.2µm filter is used for filtration.

[0012] This invention also provides the application of the aforementioned epigenetic composition microcapsules in the preparation of a hangover relief and liver protection product. For example, the epigenetic composition microcapsules are combined with pharmaceutically available excipients to prepare a drug for hangover relief and liver protection.

[0013] The present invention also provides a method for preparing the above-mentioned postbiotic composition, comprising the following steps: S1. Activate *Lactobacillus fermentum* Postbio-Q7, *Lactobacillus paracasei* Postbio-P6, and *Lactobacillus paracasei* Postbio-P8 to obtain activated bacterial solutions of *Lactobacillus fermentum* Postbio-Q7, with a viable cell count of (5~10)×10⁻⁶. 6 cfu / mL; Postbio-P6 activated bacterial culture of Lactobacillus paracasei, viable count of (5~10)×10⁻⁶. 6 cfu / mL; and Postbio-P8 activated bacterial culture of Lactobacillus paracasei, with a viable count of (5~10)×10⁻⁶. 6 cfu / mL; S2. Inoculate the activated Postbio-Q7 bacterial solution of Lactobacillus fermentum into the fermentation medium at an inoculation rate of 2%~4% v / v to carry out the first fermentation and obtain the first fermentation broth. S3. Inoculate both the Postbio-P6 and Postbio-P8 activated bacterial cultures of Lactobacillus paracasei at an inoculation rate of 2%~4% v / v into the first fermentation broth and carry out a second fermentation to obtain the second fermentation broth. S4. The second fermentation broth is inactivated to obtain the post-biotic composition.

[0014] After a first fermentation with *Lactobacillus mucinus* Postbio-Q7, a second fermentation is performed by adding *Lactobacillus paracasei* Postbio-P6 and Postbio-P8. This allows for adjustments to the conditions of the first and second fermentations, thereby enhancing the antibacterial effect of the prepared postbiotic composition.

[0015] It should be noted that the inactivation treatment in step S4 is as follows: the second fermentation broth can be pasteurized at 95℃ for 40-100 min to obtain the epibiotic fermentation broth. The epibiotic fermentation broth is then clarified, impurity removed, concentrated, and desalted to obtain the epibiotic composition. Furthermore, since the obtained epibiotic composition is in a liquid state, it can be freeze-dried for easy storage and transportation to obtain epibiotic freeze-dried powder.

[0016] Based on the above technical solutions, preferably, step S3 is as follows: adding fed growth factor to the first fermentation broth; inoculating both the activated bacterial solution of Lactobacillus paracasei Postbio-P6 and the activated bacterial solution of Lactobacillus paracasei Postbio-P8 into the first fermentation broth with added growth factor at an inoculation rate of 2%~4% v / v, and carrying out a second fermentation to obtain the second fermentation broth; The volume of the supplemental growth factor is 0.04% to 0.06% of the volume of the first fermentation broth. Preferably, the volume of the supplemental growth factor is 0.05% of the volume of the first fermentation broth.

[0017] Thus, after the first fermentation, before adding probiotics (Lactobacillus paracasei Postbio-P6 activated bacterial solution and Lactobacillus paracasei Postbio-P8) for the second time, the supplementary addition of growth factor can improve the efficiency of the second fermentation culture, thereby improving the hangover relief and liver protection effect of the prepared postbiotic composition.

[0018] Based on the above technical solution, preferably, in step S2, the fermentation conditions for the first fermentation are: fermentation at 37℃ for 20~30h, rotation speed of 120~180rpm / min, and fermentation pH of 4.0~4.5. In step S3, the fermentation conditions for the second fermentation are: fermentation at 37°C for 10-20 hours, rotation speed of 120-180 rpm / min, and fermentation pH of 3.2-3.8.

[0019] Based on the above technical solutions, preferably, in step S3, the supplementary growth factors include, by mass parts, 0.5 parts of Tween 80, 1 part of disodium hydrogen phosphate, 0.01 parts of manganese sulfate, 0.1 parts of magnesium sulfate, 10 parts of yeast powder, 0.02 parts of folic acid, 0.01 parts of biotin, and 0.01 parts of vitamin K.

[0020] This invention also provides an application of the above-mentioned post-biotic composition in the preparation of a hangover relief and liver protection product. For example, the post-biotic composition is combined with pharmaceutically available excipients to prepare a product for hangover relief and liver protection.

[0021] The postbiotic compositions and postbiotic composition microcapsules disclosed herein have the following advantages over the prior art: 1. The postbiotic composition provided in this disclosure uses Postbio-Q7 for the first stage of fermentation, and then adds Postbio-P6 and Postbio-P8 for the second stage of fermentation. The fermentation conditions of each fermentation stage can be adjusted separately, thereby making the obtained postbiotic composition have excellent hangover relief and liver protection effects.

[0022] 2. Microencapsulation technology can be used to prepare microencapsulated metabiotic compositions. By optimizing the combination and synergistic effects of the raw materials used to prepare the microencapsulation shell, the stability, antioxidant properties, moisture resistance, and flowability of the microencapsulation shell structure can be improved. A stable microencapsulation shell can provide better protection for the metabiotic composition, facilitating its storage and transportation, protecting the internal components (metabiotic composition), reducing oxidation value, and thus enhancing the antibacterial and antimicrobial effects of the metabiotic. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a schematic diagram comparing the survival rates of liver cancer cells in different groups in one embodiment of this disclosure.

[0025] Figure 2 This is a schematic diagram of the half-maximal inhibition rate curve of human liver cancer cells in one embodiment of this disclosure.

[0026] Figure 3 This is a schematic diagram of the FDA fluorescence signal intensity of human liver cancer cells in one embodiment of the present disclosure.

[0027] Figure 4 This is a schematic diagram illustrating the behavior of zebrafish after different treatments in one embodiment of the present disclosure of a zebrafish intoxication model.

[0028] Figure 5 This is a schematic diagram showing the comparison of zebrafish movement distances after different treatments in one embodiment of the present disclosure.

[0029] Figure 6 This is a schematic diagram showing the intensity of zebrafish liver fat staining after processing in one embodiment of this disclosure.

[0030] Figure 7 This is a schematic diagram showing the comparison of the staining intensity (pixels) of fat in different groups of zebrafish in one embodiment of this disclosure.

[0031] Figure 8 This is a schematic diagram showing the comparison of alcohol dehydrogenase content in serum samples from different groups in one embodiment of this disclosure.

[0032] Figure 9 This is a schematic diagram showing the comparison of acetaldehyde dehydrogenase content in serum samples from different groups in one embodiment of this disclosure.

[0033] Figure 10 This is a schematic diagram of the principal component analysis results of transcription sequencing of each group of samples in one embodiment of this disclosure.

[0034] Figure 11 In one embodiment of this disclosure, a volcano diagram is used to screen differentially expressed genes between the model control group and the post-genetic group.

[0035] Figure 12 In one embodiment of this disclosure, a volcano diagram is used to screen differentially expressed genes between the model control group and the positive control group.

[0036] Figure 13 This is a schematic diagram illustrating the enrichment results of the upregulated differentially regulated KEGG pathway in the model group and the post-genetic group in one embodiment of this disclosure.

[0037] Figure 14 This is a schematic diagram illustrating the enrichment results of the differentially regulated KEGG pathway in the model group and the post-genetic group in one embodiment of this disclosure.

[0038] Figure 15 This image shows a comparison of HE staining results of mouse liver tissue sections in one embodiment of the present disclosure, where metabiotics are used to protect against acute liver injury (×400).

[0039] Figure 16 In one embodiment of this disclosure, metabiotics are used to repair alcoholic liver injury. The results of HE staining of mouse liver tissue sections are shown in the image (×400). Detailed Implementation

[0040] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0041] The *Limosilactobacillus fermentum* Postbio-Q7, *Lactobacillus paracasei* Postbio-P6, and *Lactobacillus paracasei* Postbio-P8 used in this invention are all deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Their accession numbers are CGMCC No. 26238, CGMCC No. 26237, and CGMCC No. 31112, respectively, and their accession dates are December 25, 2022, December 25, 2022, and June 27, 2024, respectively.

[0042] The following specific embodiments further illustrate the above-mentioned epigenetic composition and epigenetic composition microcapsules.

[0043] The basic formulation of the fermentation medium used in the following examples is as follows: 20g glucose, 15g whey protein, 20g soybean flour, 10g skim milk powder, 10g yeast powder, 1L distilled water, initial pH=7.0. Before inoculation and fermentation, the fermentation medium needs to be sterilized at 105℃ for 30 minutes.

[0044] The supplemental growth factor formula is as follows: Tween 80 0.5g, disodium hydrogen phosphate 1g, manganese sulfate 0.01g, magnesium sulfate 0.1g, yeast powder 10g, folic acid 0.02g, biotin 0.01g, and vitamin K 0.01g.

[0045] All materials used in this invention were purchased from the market, and the guarana extract was purchased from Shandong Tianxingjian Biochemical Co., Ltd.

[0046] Example 1 S1. Inoculate *Lactobacillus fermentum* Postbio-Q7, *Lactobacillus paracasei* Postbio-P6, and *Lactobacillus paracasei* Postbio-P8 into MRS liquid medium and incubate at 37°C for 18 hours. Subculture twice to achieve a viable count of (5~10) × 10⁻⁶ for each strain after activation. 6 cfu / mL.

[0047] S2. The activated bacterial solution of *Lactobacillus mucinus* Postbio-Q7 was inoculated into the fermentation medium at an inoculation rate of 2% v / v. The culture was fermented at 37°C for 25 h at a rotation speed of 150 rpm / min and a fermentation pH of 4.0-4.5 to obtain the first fermentation broth.

[0048] S3. Add 0.05% v / v of fed growth factor to the first fermentation broth. Inoculate both the activated Lactobacillus paracasei Postbio-P6 and Postbio-P8 bacterial cultures at an inoculation rate of 2% v / v into the first fermentation broth with added growth factor, and carry out a second fermentation to obtain the second fermentation broth.

[0049] S4. After the second fermentation broth is completed, it is pasteurized at 95℃ for 40-100 minutes to obtain the epigenetic fermentation broth. The epigenetic fermentation broth is then clarified, purified, concentrated, and desalted to obtain an epigenetic composition solution, which is labeled as epigenetic composition 1.

[0050] Example 2 The difference between this embodiment and Example 1 is that no supplemental growth factor was added in step S3, while the remaining steps were the same as in Example 1, and the obtained post-genetic composition solution was labeled as post-genetic composition 2.

[0051] Example 3 The difference between this embodiment and Example 1 is that *Lactobacillus flavus* Postbio-Q7, *Lactobacillus paracasei* Postbio-P6, and *Lactobacillus paracasei* Postbio-P8 were simultaneously inoculated into the fermentation medium at an inoculation rate of 2% v / v. Fermentation was carried out at 37°C for 25 hours at a rotation speed of 150 rpm / min, with a fermentation pH of 4.0-4.5. After one fermentation, the sterilization, clarification, and concentration / desalination processes in step S4 were directly performed. In other words, steps S2 and S3 were combined, and only one fermentation was conducted. The remaining steps were the same as in Example 1, and the resulting metabiotic composition solution was labeled as metabiotic composition 3.

[0052] The moisture content, solubility, oxidative stability, flowability, and moisture resistance of the post-biotic compositions prepared in Examples 1 to 3 were tested.

[0053] Solubility measurement method: Accurately weigh 1 g of the post-genetic composition sample prepared in the above example, dissolve it in 25 mL of deionized water, transfer the entire sample solution into a centrifuge tube and centrifuge at 3000×g for 10 min, discard the supernatant, add a small amount of deionized water, centrifuge again to separate the supernatant, transfer the precipitate into a petri dish that has been dried to constant weight, and dry it in an oven at 105℃ to constant weight. The initial mass of the sample minus the mass of the precipitate is the solubility of the sample.

[0054] Moisture resistance test method: Weigh 5 g of the post-genetic composition sample prepared in the above example, place it at 20°C and 75% humidity for 12 hours, measure its weight change, calculate the weight gain rate, and calculate the moisture resistance of the corresponding sample based on the change in weight gain rate.

[0055] In this embodiment, the moisture content of the postbiotic composition samples was determined using the oven drying method according to GB / T5528-2008. The oxidative stability of the postbiotic composition samples was tested using the colorimetric method in GB / T 5009.37-2003. The flowability of the postbiotic composition samples was determined by reacting the concentration (CI) and hauschnaby (HR) values. The results are shown in Table 1.

[0056] Regarding flowability, generally speaking, CI < 10 and HR between 1.00 and 1.11 indicate excellent powder flowability; CI < 15 and HR between 1.12 and 1.18 indicate good powder flowability; CI > 16 and HR > 1.2 indicate average or poor powder flowability. Oxidative stability is expressed in meq / kg, which stands for milligram equivalents per kilogram. meq is an abbreviation for milligram equivalents, a unit of measurement representing the chemical activity or reactivity of a substance.

[0057] Table 1. Performance comparison of post-biotic compositions 1-3

[0058] As shown in Table 1, compared with postbiotic composition 2 and postbiotic composition 3, the postbiotic composition obtained by culturing Lactobacillus mucilaginosus Postbio-Q7 with Lactobacillus paracasei Postbio-P6 and Lactobacillus paracasei Postbio-P8 in stages has lower water content, higher solubility, stronger oxidative stability, and improved moisture resistance.

[0059] The following uses the post-biotic composition prepared in Example 1 to verify the hangover relief and liver protection effects of the post-biotic composition.

[0060] I. Cytotoxicity Detection of Metabiotic Compositions The metabiotic composition obtained in Example 1 was centrifuged at 4000 rpm / min for 10 min, and the supernatant was collected. The supernatant was then filtered through a 0.22 μm filter membrane to obtain the metabiotic. The metabiotic was prepared into solutions with concentrations of 3.125%, 6.25%, 12.5%, 25%, 50%, and 100%. For example, the 50% metabiotic solution was obtained by diluting the metabiotic by one-fold with culture medium; the 100% metabiotic solution was obtained by directly filtering the metabiotic through a 0.22 μm filter membrane.

[0061] Human liver cancer cells were treated at 5×10 4 Cells were seeded into 96-well plates at a seeding density of 1 cell per well and incubated overnight in an incubator (37 °C, 5% CO2) until the cell deposition rate in the 96-well plates reached 40%~60%. Then, post-biotics were added for the experiment.

[0062] Experimental group: Divided into 6 groups according to different concentrations of post-genetic solutions added, with each group having 3.125%, 6.25%, 12.5%, 25%, 50%, and 100% post-genetic solutions added respectively; 200 μL of the corresponding concentration of post-genetic solution was added to each well.

[0063] Blank control group: Add 200 μL of culture medium to each well.

[0064] Positive control group: Add 200 μL of culture medium containing 10% DMSO to each well.

[0065] After drug administration, the 96-well plates were placed in an incubator (37 ℃, 5% CO2) and cultured for 24 h. After 24 h of cell incubation, the supernatant was discarded, and MTT working solution (0.5 mg / mL) was added. The plates were incubated at 37 ℃ in the dark for 4 h. After incubation, the supernatant was discarded, and 150 µL of DMSO was added to each well and shaken for 15 min. The OD value was read at 490 nm.

[0066] like Figure 1 As shown, when metabiotics were added to human hepatocellular carcinoma cells for co-culture, the cell survival rate was over 90% when the metabiotic concentration was within the range of 6.25%, showing no significant cytotoxicity to hepatocellular carcinoma cells compared to the blank control group. However, when the metabiotic concentration was 12.5% ​​or higher, the cell survival rate showed a significant difference compared to the blank control group, and the cell survival rate decreased in a concentration-dependent manner. Even with 100% metabiotic concentration, the cell survival rate was still higher than the positive control group. This experiment indicates that metabiotics did not cause significant damage to cells within the 6.25% concentration range, but when the concentration increased to 12.5%, metabiotics began to exhibit cytotoxicity against hepatocellular carcinoma cells. Figure 2 As shown, based on the half-maximal inhibition rate curve, when 50% of human liver cancer cells undergo apoptosis, the metagenerogen concentration is 33.6%.

[0067] II. Effects of the post-biotic composition on cell membrane permeability Fluorescein diacetate (FDA) staining is used to assess cell viability and survival status. The basic principle is that FDA is a lipid-soluble substance that is non-fluorescent and can freely cross the cell membrane into living cells. It is broken down by intracellular esterases to produce fluorescein, a green fluorescent substance, which then accumulates within the cell.

[0068] The metabiotic composition obtained in Example 1 was centrifuged at 4000 rpm / min for 10 min, and the supernatant was collected. The supernatant was filtered through a 0.22 μm filter membrane to obtain the metabiotic. The metabiotic was prepared into metabiotic solutions with contents of 3.125%, 6.25%, 12.5%, 25%, 50%, and 100%, respectively.

[0069] Human liver cancer cells were treated at 5×10 4Cells were seeded into 96-well plates at a seeding density of 1 cell per well and incubated overnight in an incubator (37 °C, 5% CO2) until the cell deposition rate in the 96-well plates reached 40%~60%. Then, post-biotics were added for the experiment.

[0070] Experimental group: Divided into 6 groups according to different concentrations of post-genetic solutions added, with each group having 3.125%, 6.25%, 12.5%, 25%, 50%, and 100% post-genetic solutions added respectively; 200 μL of the corresponding concentration of post-genetic solution was added to each well.

[0071] Control group (0% Postbiotic): 200 μL of culture medium was added to each well.

[0072] After drug administration, the 96-well plates were incubated in an incubator (37 ℃, 5% CO2) for 24 h. The cells were then removed and washed twice with PBS to remove interfering components from the culture medium and metabiotics. FDA solution was added to each cell, and the human liver cancer cells were incubated with FDA at 37 ℃ for 15 min, ensuring the FDA solution covered the cell surface. After 15 min, the FDA solution was removed, and the cells were washed twice with PBS to remove any FDA solution that had not entered the cells. The cells were observed under a fluorescence microscope, and the fluorescence signal was recorded. Live cells should show a green fluorescent signal, while dead cells should not show fluorescence.

[0073] See results Figure 3 After adding a 12.5% ​​postbiotic solution (12.5% ​​Postbiotic in the figure), the cell viability rate was over 90% compared to the normal group without postbiotic (0% Postbiotic), indicating that the cells did not exhibit significant cytotoxicity at a postbiotic solution concentration of 12.5%. As the concentration of the postbiotic solution increased, the green fluorescence signal significantly decreased at 25% (25% Postbiotic in the figure) and 50% (50% Postbiotic in the figure), indicating a significant increase in the cytotoxicity of the postbiotic solution. This suggests that the postbiotic solution can act on human liver cancer cells, causing inhibition of their activity.

[0074] III. The hangover-relieving effect of post-alcoholic combination The metabiotic composition obtained in Example 1 was centrifuged at 4000 rpm / min for 10 min, and the supernatant was collected. The supernatant was filtered through a 0.22 μm filter membrane to obtain the metabiotic. The metabiotic was prepared into metabiotic solutions with concentrations of 0.78 µl / ml, 1.56 µl / ml, and 3.12 µl / ml using water.

[0075] Establishing an animal model of alcohol intoxication: Zebrafish of the Albino strain, a gene mutant, were randomly selected 5 days after fertilization (5 dpf) and treated in 6-well plates, with 30 zebrafish in each well. After induction with anhydrous ethanol for 1 hour, the ethanol was removed to establish the zebrafish alcohol intoxication model.

[0076] Experimental group: includes three groups ( Figure 4 The following were shown: postbiocare 0.78µl / ml, postbiocare 1.56µl / ml, postbiocare 3.12µl / ml. 200µl of postbiocare solution with concentrations of 0.78µl / ml, 1.56µl / ml, and 3.12µl / ml were added to each group, and the zebrafish intoxication model was treated at 28℃ for 1 h.

[0077] Model control group: 200 µl of water was added to the well containing the zebrafish intoxication model and treated at 28 °C for 1 h.

[0078] Positive control group (RU21 100 µg / mL): 200 µl of RU21 (RU-21 Antipro natural plant extract) solution with a concentration of 100 µg / mL was added to the wells with zebrafish intoxication model and treated at 28℃ for 1 h.

[0079] Normal control group: 200 µl of water was added to the holes of normal zebrafish and treated at 28 °C for 1 h.

[0080] Ten zebrafish were randomly selected from each group and placed in a 96-well plate, one zebrafish per well, with a volume of 200 μL per well. The total movement distance of the zebrafish was measured using a behavior analyzer. The statistical analysis results of this index were used to evaluate the hangover relief efficacy of the samples. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software, and p < 0.05 was considered statistically significant.

[0081] The results are as follows Figure 4 As shown, the black line represents the distance of slow movement, the green line represents the distance of medium movement, and the red line represents the distance of fast movement. When alcohol was added, the zebrafish in the model group exhibited predominantly low-speed behavior, accompanied by a small amount of medium-speed behavior, while the blank control group and positive control group (…) Figure 4 The RU21 100 µg / mL group (illustrated) exhibited predominantly low- and medium-speed behaviors, accompanied by a small amount of high-speed behavior. When the postbiotic dose reached 3.12 µl / ml, the zebrafish's locomotor behavior showed no significant difference compared to the blank control group and the positive control group. See also Figure 5When the metabiotic concentration reached 3.12 µl / ml, the total movement distance of the intoxicated zebrafish was basically the same as that of the blank control group and the positive control group, but significantly different from that of the model group. This indicates that the metabiotic at a dose of 3.12 µl / ml has a significant effect in relieving alcohol intoxication. Thus, it is shown that adding a high concentration of metabiotic solution can have a sobering effect, making the behavior of zebrafish more normal.

[0082] IV. The Liver-Protecting Effect of Post-Alcoholic Combinations The metabiotic composition obtained in Example 1 was centrifuged at 4000 rpm / min for 10 min, and the supernatant was collected. The supernatant was filtered through a 0.22 μm filter membrane to obtain the metabiotic. The metabiotic was prepared into metabiotic solutions with concentrations of 0.78 µl / ml, 1.56 µl / ml, and 3.12 µl / ml using water.

[0083] Zebrafish of the Albino strain with a gene mutation 5 days after fertilization (5 dpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well.

[0084] Experimental group: consisting of three groups, each of which was given 200 µl of post-genetic agent solution at concentrations of 0.78 µl / ml, 1.56 µl / ml, and 3.12 µl / ml, respectively, followed by anhydrous ethanol, and treated at 28 °C for 1 day (1 d).

[0085] Model control group: Anhydrous ethanol was added to the corresponding wells and treated at 28°C for 1 day (1d).

[0086] Positive control group: RU21 at a concentration of 100 µg / mL was added to the corresponding solution, followed by anhydrous ethanol, and the solution was treated at 28°C for 1 day (1d).

[0087] Blank control group: only aqueous solution was added to the corresponding well.

[0088] After the above treatment, serum samples from zebrafish in each group were collected for the detection of alcohol dehydrogenase and aldehyde dehydrogenase levels. The fish were fixed and stained with Oil Red O (a fat-soluble dye). After staining, 10 zebrafish from each experimental group were randomly selected and photographed under a dissecting microscope. Data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software. The intensity of liver fat staining was analyzed, and the statistical analysis results of this index were used to evaluate the protective efficacy of the samples against alcoholic fatty liver. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software; p < 0.05 indicated statistical significance.

[0089] See results Figures 6-9 ,according to Figure 6The experimental results showed that after alcohol treatment, the liver fat content was reduced in both the positive control group and the experimental group with an epibiotic concentration of 1.56 µl / ml compared to the model control group. This indicates that the alcohol-induced fatty liver in zebrafish was milder in the positive control group and the experimental group with an epibiotic concentration of 1.56 µl / ml. In other words, the addition of RU21 and epibiotic solution can reduce the induction of fatty liver by alcohol, and the epibiotic solution at a concentration of 1.56 µl / ml has a protective effect on the liver. Figure 7 As shown, except for the blank control group, the experimental group with an epigenetic concentration of 1.56 µl / ml showed a significant difference compared with the model control group, indicating that the epigenetic at 1.56 µl / ml had a better liver-protective effect. Figure 8 and Figure 9 The experimental results showed that the levels of alcohol dehydrogenase and aldehyde dehydrogenase in zebrafish were significantly increased after the addition of postbiotics compared with the model group. The experimental group with a postbiotic concentration of 1.56 µl / ml showed the most significant changes in the levels of alcohol dehydrogenase and aldehyde dehydrogenase, proving that the liver-protective effect was high when the postbiotic solution concentration was 1.56 µl / ml, and that the postbiotic solution has the optimal concentration range for liver protection.

[0090] V. Effects of the metabiotic composition on gene transcription and expression in zebrafish The metabiotic composition obtained in Example 1 was centrifuged at 4000 rpm / min for 10 min, and the supernatant was collected. The supernatant was filtered through a 0.22 μm filter membrane to obtain the metabiotic. The metabiotic was prepared into metabiotic solutions with concentrations of 0.78 µl / ml, 1.56 µl / ml, and 3.12 µl / ml using water.

[0091] Zebrafish of the Albino strain with a gene mutation 5 days after fertilization (5 dpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well.

[0092] Experimental group (post-genetic group): 200 µl of post-genetic solution with a concentration of 1.56 µl / ml was added, followed by anhydrous ethanol, and the mixture was treated at 28 °C for 1 day (1 d).

[0093] Model control group: Anhydrous ethanol was added to the corresponding wells and treated at 28°C for 1 day (1d).

[0094] Positive control group: RU21 at a concentration of 100 µg / mL was added to the corresponding solution, followed by anhydrous ethanol, and the solution was treated at 28°C for 1 day (1d).

[0095] Blank control group: only aqueous solution was added to the corresponding well.

[0096] Four zebrafish from each group were randomly selected after the above treatment and stored in frozen RNA buffer for subsequent RNA extraction and transcription sequencing. Sequencing was performed using the Lumina HiSeq platform, reference sequence alignment analysis was performed using Bowtie 2 software, and gene expression levels and differential expression were calculated using HTSeq and DESeq 2 software. Results are as follows: Figure 10 As shown, the post-genetic group exhibits low sample variability and clusters together, indicating the reliability of the experimental data. Compared to the model control group, the post-genetic group shows greater data separation, demonstrating a significant difference between the post-genetic treatment and the model control group.

[0097] Differentially expressed genes were screened using a Fold Change ≥ 1.5 and a P-value < 0.05 as the screening criteria. Figure 11 As shown, compared with the model control group, the post-genetic group had a total of 479 differentially expressed genes (DEGs), of which 325 DEGs were upregulated and 154 DEGs were downregulated. Figure 12 As shown, compared with the model control group, the positive control group had a total of 3365 DEGs, of which 156 DEGs were upregulated and 209 DEGs were downregulated.

[0098] KEGG enrichment analysis was performed on significantly upregulated and downregulated differentially regulated genes in the postgenetic group. Significantly upregulated differentially regulated genes were enriched in 88 metabolic pathways, while significantly downregulated differentially regulated genes were enriched in 61 metabolic pathways. Figure 13 As shown, the significantly upregulated gene enrichment was mainly concentrated in pathways such as Primary bile acid biosynthesis, Cytokine-cytokinereceptor interaction, Cell adhesion molecules, Glycosaminoglycan biosynthesis-chondroitin sulfate / dermatan sulfate, Glycosaminoglycan biosynthesis-keratan sulfate, and One-carbon pool by folate. The upregulation of these pathways plays an important role in fatty acid metabolism, intracellular DNA synthesis, repair and methylation, signal transduction, extracellular matrix structure and functional stability, cellular antioxidant capacity, and tissue repair. Figure 14As shown, the significantly downregulated genes were significantly enriched in pathways such as Endocytosis, Tightjunction, MAPK signaling pathway, and Calcium signaling pathway. These pathways have important effects on intracellular signal transduction, calcium ion homeostasis, and responses to various external stimuli.

[0099] VI. Effects of the post-biotic composition on acute alcoholic liver injury in mice The experimental animals were SPF-grade KM mice. After one week of acclimatization feeding, they were randomly divided into groups of 8 mice each and administered the drug by gavage at a dose of 10 mL / kg / mouse.

[0100] Control group: Distilled water was administered by gavage. After 7 days of gavage, distilled water was administered by gavage at a single dose of 14 mL / kg / mouse. Mice were fasted for 12 hours and then sacrificed.

[0101] Model group: Distilled water was administered by gavage. After 7 days of gavage, mice were given a single gavage of 14 mL / kg / mouse of 50% ethanol solution (analytical grade). They were then fasted for 12 hours and sacrificed.

[0102] Positive group: Biphenyl diester drops were administered by gavage. After 7 days of gavage, mice were given a single gavage of 14 mL / kg / mouse of 50% ethanol solution (analytical grade). They were then fasted for 12 hours and euthanized.

[0103] Low-dose post-biotic group: 10.835 ml / 10g post-biotic solution was administered by gavage. After 7 days of gavage, 14 mL / kg / mouse was administered by gavage with a 50% ethanol solution (analytical grade). The mice were fasted for 12 hours and then sacrificed.

[0104] Mid-dose post-biotic group: 21.67 ml / 10 g of post-biotic solution was administered by gavage. After 7 days of gavage, 14 mL / kg / mouse was administered by gavage with a 50% ethanol solution (analytical grade). The mice were fasted for 12 hours and then sacrificed.

[0105] High-dose post-biotic group: 43.34 ml / 10g post-biotic solution was administered by gavage. After 7 days of gavage, 14 mL / kg / mouse was administered by gavage with a 50% ethanol solution (analytical grade). The mice were then fasted for 12 hours and euthanized.

[0106] After the mice in each group were sacrificed, their livers were collected and fixed in paraformaldehyde for 4 days. The fixed livers were then removed, and a section approximately 0.5 cm thick with intact structure was selected and rinsed under running tap water for about 24 hours. The livers were then dehydrated using a gradient of alcohols at 45°C. After completion, the sections were cleared with xylene and prepared into paraffin sections. These sections were then stained with hematoxylin and eosin (HE) and observed.

[0107] The results are as follows Figure 15 As shown, in the control group, the liver cells of mice were arranged regularly and uniformly, with abundant cytoplasm and no symptoms. In the model group, the liver cells were irregularly shaped, with numerous hepatocytes showing vacuolar degeneration, swelling, and loose, pale cytoplasm around the central vein and portal areas. The surrounding sinusoids were compressed, and significant vascular congestion was observed, indicating obvious pathological changes. In the high-dose metastatic agent group, the hepatocytes in the central vein and portal areas were arranged regularly and uniformly, with occasional mild vacuolation within the hepatocytes. The low-dose group showed no obvious vascular congestion, and the high-dose group also showed a significant reduction in congestion. Compared with the model group, the symptoms were significantly alleviated. This indicates that metastatic agents have a significant protective effect against acute alcoholic liver injury.

[0108] VII. The repairing effect of the post-biotic composition on alcoholic liver injury in mice. The experimental animals were SPF-grade KM mice, which were randomly divided into groups of 8 mice after one week of acclimatization feeding.

[0109] Control group: Mice were administered distilled water at a dose of 6 g / kg / mouse by gavage every 12 hours for a total of 3 times. Then, they were administered distilled water at a dose of 10 mL / kg / mouse by gavage for 7 days. After 7 days of gavage, the mice were fasted for 12 hours and then sacrificed.

[0110] Model group: Mice were administered 40% ethanol (analytical grade) by gavage at a dose of 6 g / kg / mouse every 12 hours for a total of 3 times. Then, they were administered distilled water by gavage at a dose of 10 mL / kg / mouse for 7 days. After gavage, the mice were fasted for 12 hours and then sacrificed.

[0111] Positive control group: Mice were administered 40% ethanol (analytical grade) by gavage at a dose of 6 g / kg / mouse every 12 hours for a total of 3 times. Then, they were administered biphenyl diester drops by gavage at a dose of 10 mL / kg / mouse for 7 days. After gavage, the mice were fasted for 12 hours and then sacrificed.

[0112] Low-dose metabiotic group: 40% ethanol (analytical grade) was administered by gavage at a dose of 6 g / kg / mouse every 12 hours for a total of 3 times. Then, 10.835 mL / 10 g of metabiotic solution was administered by gavage at a dose of 10 mL / kg / mouse for 7 days. After gavage, the mice were fasted for 12 hours and then sacrificed.

[0113] Medium-dose post-biotic group: 6 g / kg / mouse was administered by gavage with 40% ethanol (analytical grade) every 12 hours for a total of 3 times. Then, 21.67 mL / 10 g of post-biotic solution was administered by gavage at a dose of 10 mL / kg / mouse. After 7 days of gavage, the mice were fasted for 12 hours and then sacrificed.

[0114] High-dose metabiotic group: 6 g / kg / mouse was administered by gavage with 40% ethanol (analytical grade) every 12 hours for a total of 3 times. Then, 43.34 mL / 10 g of metabiotic solution was administered by gavage at a dose of 10 mL / kg / mouse for 7 days. Mice were then fasted for 12 hours and euthanized.

[0115] After the mice in each group were sacrificed, their livers were collected and fixed in paraformaldehyde for 4 days. The fixed livers were then removed, and a section approximately 0.5 cm thick with intact structure was selected and rinsed under running tap water for about 24 hours. The livers were then dehydrated using a gradient of alcohols at 45°C. After completion, the sections were cleared with xylene and prepared into paraffin sections. These sections were then stained with hematoxylin and eosin (HE) and observed.

[0116] The results are as follows Figure 16 As shown, in the control group, the liver cells of mice were neatly arranged, with uniform intercellular spaces, abundant cytoplasm, and distinct nuclei, and no obvious symptoms were observed. In the model group, the liver cells were disordered, with uneven intercellular spaces, irregular cell edges, numerous intracellular vacuoles, and occasional mild cell swelling and cytoplasmic extravasation. In the high-dose post-biotic group, the liver cells were relatively neatly arranged, with uniform intercellular spaces, regular and distinct cell edges, and few mild intracellular vacuoles. Compared with the model group, the symptoms were significantly reduced. This indicates that post-biotics have a repairing effect on acute alcoholic liver injury.

[0117] To further improve the stability of metabiotics, the metabiotic composition prepared in Example 1 can be encapsulated using microencapsulation technology to obtain a stable metabiotic composition microcapsule with good antibacterial properties.

[0118] Example 4 S10. Add 2g sodium alginate, 0.5g shellac, 0.5g guarana extract and 0.2g protamine to 96.8g sterile deionized water and stir continuously at 60℃ for 40min to obtain a solution.

[0119] S20. The solution is stirred and homogenized at 12000 rpm for 15 minutes to obtain a homogeneous solution. After filtration through a 0.2µm filter, the microcapsule solution is obtained.

[0120] S30. Mix the microcapsule shell solution with the post-genetic composition 1, homogenize, and dry; homogenization pressure 30 MPa, spray drying temperature 180°C, to obtain post-genetic composition microcapsules labeled as post-genetic composition microcapsules 1.

[0121] Example 5 The difference between this embodiment and Embodiment 4 is as follows: In step S10, 2g of sodium alginate, 0.5g of shellac, and 0.5g of guarana extract were added to 97g of sterile deionized water and stirred continuously at 60°C for 40 minutes to obtain a solution. The other steps are the same as in Embodiment 4, and the resulting post-biotic composition microcapsules are labeled as post-biotic composition microcapsules 2.

[0122] Example 6 The difference between this embodiment and Embodiment 4 is as follows: In step S10, 2g of sodium alginate, 0.5g of shellac, and 0.2g of protamine sulfate are added to 97.3g of sterile deionized water and stirred continuously at 60°C for 40 minutes to obtain a solution. The other steps are the same as in Embodiment 4, and the resulting post-biotic composition microcapsules are labeled as post-biotic composition microcapsules 3.

[0123] Example 7 The difference between this embodiment and Embodiment 4 is as follows: In step S10, 2g of sodium alginate and 0.5g of shellac are added to 97.5g of sterile deionized water and stirred continuously at 60°C for 40 minutes to obtain a solution. The other steps are the same as in Embodiment 4, and the resulting post-biotic composition microcapsules are labeled as post-biotic composition microcapsules 4.

[0124] Example 8 The difference between this embodiment and embodiment 4 is that the post-genetic composition 1 is freeze-dried to obtain post-genetic composition freeze-dried powder, and the performance of the post-genetic composition freeze-dried powder is compared with that of the post-genetic composition microcapsules 1-4 prepared in the above embodiment.

[0125] The moisture content, solubility, oxidative stability, flowability, and moisture resistance of the postbiotic composition microcapsules 1-4 and the postbiotic composition lyophilized powder were tested respectively, and the results are shown in Table 2.

[0126] Table 2. Performance comparison of post-biotic composition microcapsules 1-4 and post-biotic composition lyophilized powder

[0127] Table 2 shows that, compared with the freeze-dried powder of the post-biotic composition, the microcapsules (post-biotic composition microcapsules 1) formed by encapsulating the post-biotic composition in microcapsules prepared with sodium alginate, shellac, guarana extract, and protamine sulfate exhibit significantly improved properties such as antioxidant activity, moisture resistance, and solubility. This indicates that the microcapsule shells formed using this formula can protect the post-biotic composition, thereby enhancing its hangover-relieving and liver-protecting effects.

[0128] Compared with postbiotic composition microcapsules 2, 3, and 4, postbiotic composition microcapsules 1 exhibits superior performance. This is because the microcapsule shell of postbiotic composition microcapsule 2 does not contain protamine, which reduces the stability and antibacterial properties of the microcapsule. Postbiotic composition microcapsules 3 and 4 also lack certain components in their microcapsule shells, which similarly affects the cross-linking strength and stability of the microcapsule shell, thereby reducing the protective effect on the postbiotic composition.

[0129] In summary, the microcapsules of the epigenetic composition prepared by synergistically combining sodium alginate, shellac, guarana extract, and protamine can better protect the epigenetic composition by providing antioxidant and moisture-proof effects, thereby reducing the difficulty of storage and transportation and providing greater convenience for its application.

[0130] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing microcapsules of an epigenetic composition, characterized in that, The epigenetic composition microcapsules include a microcapsule shell and a microcapsule core; the microcapsule core is an epigenetic composition. Specifically, the following steps are included: S10. Add sodium alginate, shellac, guarana extract and protamine to sterile deionized water and stir continuously at 50~60℃ for 30~60 min to obtain a solution. The composition, based on a total mass of 100%, includes 1%–4% sodium alginate, 0.5%–2% shellac, 0.1%–1% guarana extract, 0.1%–1% protamine sulfate, and the remainder is sterile deionized water. S20. Stir the solution at 10000~15000rpm for 10-15 minutes to obtain a homogeneous solution. Filter the solution through a 0.1μm~0.4μm filter to obtain the microcapsule solution. S30. The microcapsule shell solution and the post-genetic composition are mixed, homogenized, and dried. The mass ratio of the microcapsule shell solution to the post-genetic composition is (1~4):(2~6). The homogenization pressure is 20-40 MPa, and the drying temperature is 160-190℃ to obtain the post-genetic composition microcapsules. The post-biotic composition includes the following steps: S1. Activate *Lactobacillus fermentum* Postbio-Q7, *Lactobacillus paracasei* Postbio-P6, and *Lactobacillus paracasei* Postbio-P8 to obtain activated bacterial solutions of *Lactobacillus fermentum* Postbio-Q7, with a viable cell count of (5~10)×10⁻⁶. 6 cfu / mL; Lactobacillus paracasei Postbio-P6 activated bacterial culture, viable count (5~10)×10⁻⁶ 6 cfu / mL; and Postbio-P8 activated bacterial culture of Lactobacillus paracasei, with a viable count of (5~10)×10⁻⁶. 6 cfu / mL; S2. Inoculate the activated Postbio-Q7 bacterial solution of Lactobacillus mucinus into the fermentation medium at an inoculation rate of 2%~4% v / v to carry out the first fermentation and obtain the first fermentation broth. S3. Inoculate both the activated bacterial cultures of Lactobacillus paracasei Postbio-P6 and Lactobacillus paracasei Postbio-P8 into the first fermentation broth at an inoculation rate of 2%~4% v / v, and carry out a second fermentation to obtain the second fermentation broth. S4. The second fermentation broth is inactivated to obtain the post-biotic composition; Step S3 involves: adding a supplemental growth factor to the first fermentation broth; inoculating both *Lactobacillus paracasei* Postbio-P6 and Postbio-P8 activated bacterial solutions into the first fermentation broth with the added growth factor at an inoculation rate of 2% to 4% v / v, and performing a second fermentation to obtain a second fermentation broth; wherein the volume of the supplemental growth factor is 0.04 to 0.06% of the volume of the first fermentation broth.

2. The method for preparing the post-biotic composition microcapsules according to claim 1, characterized in that, In step S10, based on a total mass of 100%, sodium alginate is 2%, shellac is 0.5%, guarana extract is 0.5%, protamine is 0.2%, and the remainder is sterile deionized water.

3. The preparation method according to claim 1, characterized in that: In step S2, the fermentation conditions for the first fermentation are: fermentation at 37°C for 20-30 hours, rotation speed of 120-180 rpm / min, and fermentation pH of 4.0-4.

5. In step S3, the fermentation conditions for the second fermentation are: fermentation at 37°C for 10-20 hours, rotation speed of 120-180 rpm / min, and fermentation pH of 3.2-3.

8.

4. The preparation method according to claim 1, characterized in that: In step S3, the supplemental growth factor, by mass fraction, includes 0.5 parts of Tween 80, 1 part of disodium hydrogen phosphate, 0.01 parts of manganese sulfate, 0.1 parts of magnesium sulfate, 10 parts of yeast powder, 0.02 parts of folic acid, 0.01 parts of biotin, and 0.01 parts of vitamin K.

5. A microcapsule of postbiotic composition prepared by the preparation method according to any one of claims 1 to 4.

6. The use of the post-biotic composition microcapsules according to claim 5 in the preparation of hangover relief and liver protection products.

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