Preparation method and application of metachion for relieving colitis, diarrhea and constipation

Abstract

This invention provides a method for preparing and applying postbiotics that can alleviate colitis, diarrhea, and constipation, belonging to the field of microbial fermentation technology. The technical problem to be solved is to provide a method for preparing postbiotics that can alleviate ulcerative colitis, chemotherapy-induced diarrhea, and constipation. The key technical solution is a method for preparing postbiotics, comprising: S1, inoculating *Lactobacillus acidophilus*, *Bifidobacterium animalis* subsp. *lactobacter*, and *Lactobacillus rhamnosus* into a fermentation medium according to an inoculation ratio for fermentation; S2, after fermentation, heat inactivation and drying to obtain a compound postbiotic containing *Lactobacillus acidophilus*, *Bifidobacterium animalis* subsp. *lactobacter*, and *Lactobacillus rhamnosus*. The main uses of the postbiotics prepared by this invention include the preparation of products for alleviating colitis, diarrhea, and constipation.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology and relates to the preparation of postbiotics, specifically to the preparation method and application of postbiotics that can relieve colitis, diarrhea and constipation. Background Technology

[0002] Gut microbiota dysbiosis is one of the core contributing factors to the development of ulcerative colitis, chemotherapy-induced diarrhea, and constipation. In recent years, the application of microbial therapy in the treatment of intestinal diseases has received widespread attention. Among them, probiotics are widely used in the field of intestinal health due to their beneficial functions such as regulating gut microbiota, strengthening the intestinal barrier, and inhibiting intestinal inflammation. However, the application of probiotics has obvious limitations: probiotics are live bacterial preparations, and their activity is easily affected by external environmental factors such as temperature, humidity, gastric acid, and bile, making storage and transportation difficult; the colonization capacity of live bacteria in the intestine is limited, and there may be a potential risk of causing bacteremia. For special populations such as immunocompromised cancer patients and patients with inflammatory bowel disease, the safety needs to be further improved; at the same time, the function of a single probiotic strain is relatively singular, making it difficult to simultaneously alleviate the complex symptoms of multiple intestinal diseases.

[0003] To overcome the limitations of live probiotic preparations, postbiotics have gradually become a research hotspot in the field of gut health. Compared with probiotics, postbiotics have advantages such as high stability, no need for colonization, and convenient storage and transportation. They also avoid the risk of spreading virulence factors and drug resistance genes that live bacteria may bring, resulting in higher safety. Their probiotic functions are similar to or even better than those of probiotics. They can play a protective role in gut health through multiple mechanisms, such as regulating the balance of gut microbiota, enhancing the function of the intestinal epithelial barrier, regulating the immune system, and inhibiting intestinal inflammatory responses.

[0004] In the existing technology, some studies have reported that single strains can alleviate intestinal inflammation and improve diarrhea or constipation.

[0005] Relevant patent documents retrieved: The country of origin is China, publication number CN120699852A, publication date 2025.09.26, this document discloses a strain of Lactobacillus acidophilus ( Lactobacillus acidophilus Lactobacillus acidophilus LIHUO 1978, with accession number GDMCC No:65514, possesses good safety, in vitro probiotic properties, and in vitro immunomodulatory properties. It can alleviate chemotherapy-induced diarrhea, improve constipation, and regulate intestinal flora, showing great promise for application in the preparation of drugs to alleviate chemotherapy-induced diarrhea and / or products to improve constipation and regulate intestinal flora.

[0006] However, the existing strains have relatively limited functions, and most can only specifically alleviate one type of intestinal disease. They cannot effectively improve three different types of conditions at the same time: ulcerative colitis, chemotherapy-induced diarrhea, and constipation. Furthermore, probiotics are live bacteria preparations, and their activity is easily affected by the environment.

[0007] Therefore, developing a simple, stable, and safe method for preparing compound postbiotics that can simultaneously and effectively alleviate three intestinal conditions—ulcerative colitis, chemotherapy-induced diarrhea, and constipation—would solve the problems of existing postbiotics having single functions, failing to meet the needs of synergistic improvement of multiple diseases, and having limited probiotic effects. This has significant clinical significance and application value. Summary of the Invention

[0008] The purpose of this invention is to provide: A method for preparing postbiotics that can relieve colitis, diarrhea and constipation, and its application, and related technologies, to solve technical problems such as a method for preparing postbiotics that can relieve ulcerative colitis, chemotherapy-induced diarrhea and constipation, or a combination thereof.

[0009] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0010] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0011] Definitions of standard chemical terms can be found in the references *Molecular Cloning: A Laboratory Manual*, Cold Spring Harbor Laboratory Science Press, 4th edition, 2017; and *Microbiology Experiments*, Higher Education Press, 4th edition, 2016.

[0012] Unless otherwise stated, conventional methods within the scope of the art, such as strain culture, strain fermentation, spray drying, etc., shall be used.

[0013] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0014] The term “CFU (colony forming unit)” used in this article refers to colony forming unit.

[0015] The term "culture medium" used in this article refers to the nutrient substrate for the growth and reproduction of microorganisms. Based on the different contents of the solidifying agent, it can be divided into three types: solid culture medium, semi-solid culture medium (semi-liquid culture medium), and liquid culture medium.

[0016] In a first aspect, the present invention provides: a method for preparing an epigenetic agent, comprising the following steps: S1. Inoculate Lactobacillus acidophilus, Bifidobacterium animalis subsp. lactis and Lactobacillus rhamnosus into the fermentation medium according to the inoculation ratio and carry out fermentation. S2. After fermentation, heat inactivation and drying are performed to obtain a compound post-biotic containing Lactobacillus acidophilus, Bifidobacterium animalis subsp. lactis, and Lactobacillus rhamnosus. Lactobacillus acidophilus includes Lactobacillus acidophilus LIHUO 1978; Bifidobacterium animalis subsp. lactis includes Bifidobacterium animalis subsp. lactis JZ-FJ01; Lactobacillus rhamnosus is Lactobacillus rhamnosus JZ-FJ02; The accession number for Lactobacillus acidophilus LIHUO 1978 is GDMCC No:65514; the accession number for Bifidobacterium animalis subsp. lactis JZ-FJ01 is CGMCC No.37148; and the accession number for Lactobacillus rhamnosus JZ-FJ02 is CGMCC No.37149.

[0017] The technical features include: Lactobacillus acidophilus, Bifidobacterium animalis subsp. lactis, Lactobacillus rhamnosus, inoculation ratio, inoculation amount, fermentation medium, fermentation conditions, heat inactivation, drying, etc.

[0018] The technical characteristic inoculation ratio is as follows: the inoculation ratio of Lactobacillus acidophilus, Bifidobacterium animalis subsp. lactis and Lactobacillus rhamnosus is 10:(2-20):(2-10).

[0019] Preferably, the inoculation ratio of Lactobacillus acidophilus, Bifidobacterium animalis subsp. lactis, and Lactobacillus rhamnosus is 10:15:5.

[0020] Among them, the technical characteristic inoculation amount is: the inoculation amount of compound Lactobacillus acidophilus, Bifidobacterium animalis subsp. lactis and Lactobacillus rhamnosus powder is 0.025‰-0.75‰; Furthermore, the inoculation amount of the compound Lactobacillus acidophilus, Bifidobacterium animalis subsp. lactis and Lactobacillus rhamnosus powder is 0.025‰, 0.05‰, 0.1‰, 0.15‰, 0.2‰, 0.25‰, 0.3‰, 0.35‰, 0.4‰, 0.45‰, 0.5‰, 0.55‰, 0.6‰, 0.65‰, 0.7‰, 0.75‰, and any intermediate value and any range between two points can be selected.

[0021] Furthermore, the inoculation amounts of the compound Lactobacillus acidophilus, Bifidobacterium animalis subsp. lactis, and Lactobacillus rhamnosus were 0.1‰, 0.25‰, 0.5‰, and 0.75‰, respectively.

[0022] Preferably, the inoculation amount of the compound Lactobacillus acidophilus, Bifidobacterium animalis subsp. lactis and Lactobacillus rhamnosus is 0.1‰.

[0023] Furthermore, the viable count of the Lactobacillus acidophilus is (100-10000)×10⁻¹⁰. 8 CFU / g; the viable count of the *Bifidobacterium lactis* subsp. described is (100-20000) × 10⁻¹⁰. 8 CFU / g; the viable count of the *Lactobacillus rhamnosus* is (100-10000) × 10⁻¹⁰. 8 CFU / g.

[0024] According to some embodiments of the present invention, the inoculation amounts of Lactobacillus acidophilus, Bifidobacterium animalis subsp. lactis, and Lactobacillus rhamnosus are 0.25 × 10⁻⁶. 8 CFU / g, 0.375×10 8 CFU / g and 0.125×10 8 CFU / g.

[0025] Among them, the technical feature is the fermentation medium: the fermentation medium includes one or more of skim milk powder, soy powder, and soy protein isolate; Furthermore, the fermentation medium includes skim milk powder and soybean flour; or; Including skim milk powder and soy protein isolate; or; Including skim milk powder.

[0026] Furthermore, when the fermentation medium includes skim milk powder, the content of skim milk powder in the medium is 8-30% (w / w); preferably, the content of skim milk powder in the medium is 20% (w / w).

[0027] Furthermore, when the fermentation medium includes skim milk powder and soybean flour, the content of skim milk powder in the medium is 8-30% (w / w), and the content of soybean flour is 1-5% (w / w). Preferably, the content of skim milk powder in the culture medium is 15% (w / w) and the content of soybean flour is 3% (w / w), or the content of skim milk powder in the culture medium is 20% (w / w) and the content of soybean flour is 3% (w / w).

[0028] Furthermore, when the fermentation medium includes skim milk powder and soy protein isolate, the content of skim milk powder in the medium is 8-30% (w / w), and the content of soy protein isolate is 1-5% (w / w). Preferably, the content of skim milk powder in the culture medium is 20% (w / w), and the content of soy protein isolate powder is 3% (w / w).

[0029] The fermentation medium is prepared by adding the corresponding components according to the medium formula, adding water to make up the volume, and then adding enzymes for enzymatic hydrolysis.

[0030] Furthermore, the enzyme is selected from one or more of neutral protease, acidic protease, papain, bromelain, and flavor protease.

[0031] Furthermore, the amount of enzyme added is 1 / 10,000 (w / w) to 10 / 10,000 (w / w). The amount of enzyme added can be selected from 0.01% (w / w), 0.02% (w / w), 0.03% (w / w), 0.04% (w / w), 0.05% (w / w), 0.06% (w / w), 0.07% (w / w), 0.08% (w / w), 0.09% (w / w), 0.01% (w / w), and any intermediate value or a range between any two values.

[0032] Preferably, the amount of enzyme added is 0.05% (w / w).

[0033] Furthermore, the enzymatic hydrolysis temperature is 37-60℃, and the enzymatic hydrolysis time is 1-6 hours; Furthermore, the enzymatic hydrolysis temperature was 50℃, and the enzymatic hydrolysis time was 2 hours.

[0034] Furthermore, the enzymatic hydrolysis process also includes a sterilization and enzyme inactivation step. Furthermore, the sterilization and enzyme inactivation conditions are: sterilization and enzyme inactivation at 90℃ for 30 minutes.

[0035] Among them, the technical characteristics of fermentation conditions are: anaerobic fermentation at 120-250 rpm and 35-40℃ for 15-72 hours; Preferably, the fermentation conditions are anaerobic, at 180 rpm and 37°C for 48 hours.

[0036] Among them, the technical feature is thermal inactivation: the thermal inactivation conditions are 75-105℃, and the inactivation time is 5-40 minutes; According to some embodiments of the present invention, the heat inactivation temperature can be selected from 75°C, 80°C, 85°C, 90°C, 95°C, 100°C, 105°C, and any intermediate value or a range between any two values.

[0037] According to some embodiments of the present invention, the heat inactivation time can be selected as 5 min, 10 min, 20 min, 25 min, 30 min, 35 min, 40 min, or any intermediate value or a range between any two values.

[0038] Preferably, the heat inactivation conditions are 90°C for 30 minutes.

[0039] Among them, the technical feature of drying is selected from any one of spray drying, hot air drying, vacuum drying, freeze drying, microwave drying, and infrared drying; Preferably, the drying is spray drying.

[0040] Furthermore, the spray drying conditions are: inlet air temperature 165-175℃, outlet air temperature 75-85℃, feed rate 10-15 rpm, and atomizer speed 15000-20000 rpm. Preferably, the spray drying conditions are: inlet air temperature 170°C, outlet air temperature 80°C, feed rate 12 rpm, and atomizer speed 18000 rpm.

[0041] Secondly, the present invention provides a postgenetic agent prepared by the above-described preparation method.

[0042] Among them, the technical features include postgenetics.

[0043] Furthermore, the post-biotic contains a high content of small molecule amino acids and metabolites; Furthermore, the small molecule amino acids mentioned include, but are not limited to, L-asparagine, acetylspermine, and N-succinyl-L-glutamic acid.

[0044] Furthermore, the various metabolites mentioned include, but are not limited to, succinic acid, D-piperidine acid, 14-hydroxymyristic acid, indolelactic acid, and 3-indolecarboxaldehyde.

[0045] Thirdly, the present invention provides the application of the above-mentioned post-biotic in the preparation of a medicament for treating colitis.

[0046] The colitis mentioned includes, but is not limited to, ulcerative colitis, Crohn's disease, bacterial colitis, viral colitis, antibiotic-associated colitis, ischemic colitis, radiation colitis, and allergic colitis.

[0047] Furthermore, the colitis mentioned is ulcerative colitis.

[0048] Specifically, the drug also includes a pharmaceutically acceptable carrier.

[0049] Furthermore, the pharmaceutically acceptable carrier is selected from any one or more of excipients, buffers, emulsifiers, stabilizers, diluents, binders, preservatives, lubricants, pH adjusters, cryoprotectants, flavoring agents, and fillers.

[0050] Specifically, the dosage form of the drug is drops, tincture, powder, tablet, capsule, granule, ointment, powder, emulsion, pill, lyophilized powder for injection, gel, suppository or aerosol.

[0051] Specifically, the drug has at least one of the following effects: (1) Relieves weight loss; (2) Relieves diarrhea and bloody stools; (3) Relieves the shortening of colon length; (4) Relieves splenomegaly; (5) Relieves colonic injury; (6) Reduce the levels of inflammatory factors IL-6 and TNF-α; (7) Increase the level of anti-inflammatory factor IL-10.

[0052] Fourthly, the present invention provides the application of the above-mentioned post-biotic in the preparation of products that promote bowel movement and relieve constipation.

[0053] The products mentioned include pharmaceuticals and health foods.

[0054] Furthermore, when the product is a medicine, the medicine can lubricate the intestines and relieve constipation.

[0055] Specifically, the drug also includes a pharmaceutically acceptable carrier.

[0056] Furthermore, the pharmaceutically acceptable carrier is selected from any one or more of excipients, buffers, emulsifiers, stabilizers, diluents, binders, preservatives, lubricants, pH adjusters, cryoprotectants, flavoring agents, and fillers.

[0057] Specifically, the dosage form of the drug is drops, tincture, powder, tablet, capsule, granule, ointment, powder, emulsion, pill, lyophilized powder for injection, gel, suppository or aerosol.

[0058] Furthermore, the health food also includes conventional excipients for health food.

[0059] Specifically, the product has at least one of the following functions: (1) Increase the water content of feces; (2) Increase bowel movement frequency; (3) Improve intestinal propulsion rate; (4) Increase serum levels of Gas, MTL, and VIP; (5) Reduce SS levels.

[0060] Fifthly, the present invention provides the use of the above-mentioned metagenin in the preparation of a medicament for treating chemotherapy-induced diarrhea.

[0061] Specifically, the drug also includes a pharmaceutically acceptable carrier.

[0062] Furthermore, the pharmaceutically acceptable carrier is selected from any one or more of excipients, buffers, emulsifiers, stabilizers, diluents, binders, preservatives, lubricants, pH adjusters, cryoprotectants, flavoring agents, and fillers.

[0063] Specifically, the dosage form of the drug is drops, tincture, powder, tablet, capsule, granule, ointment, powder, emulsion, pill, lyophilized powder for injection, gel, suppository or aerosol.

[0064] Specifically, the drug has at least one of the following effects: (1) Relieves diarrhea; (2) Increase spleen index; (3) Reduce the levels of pro-inflammatory factors IL-1β and IFN-γ; (4) Increase the level of the anti-inflammatory factor IL-10; (5) Increase the length of the small intestine.

[0065] The present invention has at least the following beneficial effects: This invention provides a method for preparing metabiotics. By fermenting a combination of *Lactobacillus acidophilus*, *Bifidobacterium animalis* subsp. *lactobacter*, and *Lactobacillus rhamnosus*, and adjusting the preparation parameters, the method overcomes the problem of competitive inhibition in multi-strain fermentation, achieving synergistic and complementary fermentation. The fermentation acidity, viable cell count, various small-molecule amino acid components, and various metabolites are all significantly improved compared to single-strain fermentation. The prepared metabiotics show good effects in relieving ulcerative colitis, chemotherapy-induced diarrhea, and constipation.

[0066] Considering the possibility of this invention entering other countries, this invention also provides the following technical solutions: A method for treating colitis includes administering a therapeutically effective amount of the aforementioned postbiotic to a subject.

[0067] A method for relieving constipation includes administering an effective amount of the aforementioned post-biotic to a subject.

[0068] A method for treating chemotherapy-induced diarrhea includes administering a therapeutically effective amount of the aforementioned postbiotic to a subject.

[0069] Subjects include living organisms (e.g., mammals) that can elicit an immune response. Examples of subjects include humans, primates, cattle, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and their transgenic species.

[0070] Therapeutic effective dose refers to the pharmaceutically considered effective dosage, that is, the amount of active drug sufficient to significantly improve the condition without causing serious side effects. The dosage depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight, personality and individual response of the patient or animal, the route of administration, the frequency of administration, and the purpose of treatment. Therefore, the dosage of the present invention can vary widely.

[0071] Preservation Instructions Preserved strain: Bifidobacterium animalis subsp. lactis JZ-FJ01; Classification and nomenclature: Bifidobacterium animalis subsp. lactis Bifidobacterium animalis subsp. lactis; Accession number: CGMCC No. 37148; Preservation period: December 23, 2025; Preservation Institution: China General Microbiological Culture Collection Center (CGMCC); Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0072] Preservation Instructions Preserved strain: Lactobacillus rhamnosus JZ-FJ02; Classification and nomenclature: Lactobacillus rhamnosus Lactobacillus rhamnosus ; Accession number: CGMCC No. 37149; Preservation period: December 23, 2025; Preservation Institution: China General Microbiological Culture Collection Center (CGMCC); Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0073] Figure 1The graph shows the changes in acidity, pH value, and viable cell count at different fermentation times.

[0074] Figure 2 AcidGut for the relief of DSS-induced ulcerative colitis; A: Body weight; B: Disease activity index (DAI); C: Colon length; D: HE staining of colon tissue; In the figure, ** p <0.01.

[0075] Figure 3 Figure 1 shows the spleen index (A), serum IL-6 level (B), serum IL-10 level (C), and serum TNF-α level (D) of mice with DSS-induced ulcerative colitis induced by AcidGut. In the figure, ns indicates no significant difference, * p <0.05,** p <0.01.

[0076] Figure 4 AcidGut was used to alleviate loperamide-induced constipation in SD rats; A: intestinal propulsion rate; B: number of fecal pellets excreted at 6 hours; C: fecal water content; D: time to first black feces excretion. In the figure, compared to the model group, * p <0.05,** p <0.01.

[0077] Figure 5 The effect of AcidGut on serum gastrointestinal factors in loperamide-induced constipation rats; A: serum Gas level; B: serum MTL level; C: serum VIP level; D: serum SS level. In the figure, compared with the model group, * p <0.05,** p <0.01.

[0078] Figure 6 Evaluating the efficacy of AcidGut in alleviating 5-fluorouracil (5-FU)-induced chemotherapy-induced colitis in mice; A: Diarrhea score; B: Spleen index; C: Small intestine length; D: IL-1β level in jejunal tissue; E: IFN-γ level in jejunal tissue; F: IL-10 level in jejunal tissue. In the figure, ns indicates no significant difference. * p <0.05,** p <0.01. Detailed Implementation

[0079] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0080] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0081] Data analysis and statistical analysis were performed using professional data processing software. One-way ANOVA was used for significance analysis, and P<0.05 was considered to indicate a significant difference.

[0082] Lactobacillus acidophilus LIHUO 1978 has been disclosed in patent CN120699852A.

[0083] Example 1: Effect of different inoculation ratios on fermentation (1) Preparation of fermentation medium: Weigh skim milk powder and soybean powder at a ratio of 20% and 3% respectively, add warm water to dissolve, stir thoroughly and then add the remaining water, add 0.05% (w / w) of neutral protease (100,000 U / g), and enzymatically hydrolyze at 50℃ for 2 hours. After the enzymatic hydrolysis is completed, sterilize and inactivate the enzyme at 90℃ for 30 minutes.

[0084] (2) Inoculation and fermentation: The viable count of Lactobacillus acidophilus LIHUO 1978 bacterial powder was 1400 × 10⁻⁶. 8 CFU / g, the viable count of Bifidobacterium animalis subsp. lactis JZ-FJ01 was 12000 × 10⁻⁶ CFU / g. 8 CFU / g, the viable count of Lactobacillus rhamnosus JZ-FJ02 was 2200 × 10⁻⁶ CFU / g. 8 CFU / g. Inoculate the fermentation medium according to the inoculation ratio and amount shown in Table 1, mix well, and place in an anaerobic culture bag for fermentation at 180 rpm and 37℃ for 48 h.

[0085] (3) Results: As can be seen from the results in Table 1, the 15 inoculation ratios set have certain differences in terms of fermentation acid production and viable cell count. Among them, the 10:15:5 ratio group has higher acidity and viable cell count.

[0086] Table 1. Effects of different inoculation ratios on fermentation acidity and viable cell count.

[0087] Example 2: Effect of different inoculum sizes on fermentation The fermentation medium was prepared according to the instructions in Example 1. *Lactobacillus acidophilus* LIHUO 1978 (viable count 1400 × 10⁻⁶) was used. 8CFU / g), Bifidobacterium animalis subsp. lactis JZ-FJ01 (live count 12000×10⁻⁶) 8 CFU / g) and Lactobacillus rhamnosus JZ-FJ02 bacterial powder (live count 2200×10⁻⁶) 8 The bacterial powder (CFU / g) was mixed to form a compound bacterial powder of 10:15:5 based on the viable cell count. After thorough mixing, it was inoculated into the fermentation medium at different inoculation amounts (0.75‰, 0.5‰, 0.25‰, 0.1‰, 0.075‰, 0.05‰, and 0.025‰), and fermentation was carried out according to the instructions in Example 1. The results in Table 2 show that inoculation in the range of 0.1‰-0.75‰ had little impact on acid production during fermentation. Considering the cost of the bacterial powder, the fermentation effect was best at an inoculation amount of 0.1‰.

[0088] Table 2. Effects of different inoculum sizes on acidity and viable cell count.

[0089] Example 3: Effects of different fermentation media on fermentation (1) Culture medium preparation: The formulations of fermentation media 1-7 are shown in Table 3 below. Weigh the skim milk powder and soybean powder, add them to warm water to dissolve, stir thoroughly, and then add the remaining water to make up the volume. Then carry out enzymatic hydrolysis. The amount of neutral protease added is 0.05%, and the other proteases are added according to the same enzyme activity units. The enzymatic hydrolysis conditions are the same as in Example 1. Fermentation media 8 is different from fermentation media 1 in that the main formulation is 20% skim milk powder and 3% soybean protein isolate, and the other conditions and methods are the same.

[0090] (2) Inoculation and fermentation: Lactobacillus acidophilus LIHUO 1978 (live count of 1400 × 10⁻⁶) 8 CFU / g), Bifidobacterium animalis subsp. lactis JZ-FJ01 (live count 12000×10⁻⁶) 8 CFU / g) and Lactobacillus rhamnosus JZ-FJ02 bacterial powder (live count 2200×10⁻⁶) 8 The bacterial powder (CFU / g) was mixed into a compound bacterial powder of 10:15:5 according to the number of live bacteria. The compound bacterial powder was inoculated at an inoculation rate of 0.1‰ and fermented for 48 hours under anaerobic conditions at 180 rpm and 37℃.

[0091] (3) Results: The results are shown in Table 3. Different fermentation media had a certain impact on fermentation acid production and viable cell count, but all of them could reach a relatively high level. For example, fermentation media 7 and fermentation media 5 achieved fermentation acidity of 327.3%. and 324.9 The viable bacterial count reached 48.5 × 10⁻⁶. 8 CFU / g and 43.5×10 8 CFU / g.

[0092] Table 3. Effects of different fermentation medium formulations on fermentation

[0093] Example 4: Effect of different fermentation times on fermentation The fermentation medium was prepared according to the proportions and methods of fermentation medium 1 in Example 3, containing Lactobacillus acidophilus LIHUO 1978 (viable count of 1400 × 10⁻⁶). 8 CFU / g), Bifidobacterium animalis subsp. lactis JZ-FJ01 (live count 12000×10⁻⁶) 8 CFU / g) and Lactobacillus rhamnosus JZ-FJ02 bacterial powder (live count 2200×10⁻⁶) 8 The bacterial powder (CFU / g) was mixed into a compound bacterial powder of 10:15:5 according to the number of live bacteria. The compound bacterial powder was inoculated at an inoculation rate of 0.1‰ and fermented under anaerobic conditions at 180 rpm and 37℃. Samples were taken and tested at different time points.

[0094] from Figure 1 It can be seen that acidity increases most rapidly in the first 24 hours of fermentation, and then continues to increase at a certain rate until 52 hours. Acidity changes more slowly in the later stages. The number of Bifidobacteria and Lactobacilli shows exponential growth in the first 12 hours, then enters a stable period. After 24 hours, the number of Bifidobacteria gradually decreases, while Lactobacilli experience a second growth, reaching a maximum total viable count of 68 × 10⁻⁶ at 60 hours. 8 CFU / g, then showed a downward trend.

[0095] Comparative Example 1: Effects of different combinations of Lactobacillus acidophilus LIHUO 1978, Bifidobacterium animalis subsp. lactis JZ-FJ01, and Lactobacillus rhamnosus JZ-FJ02 on fermentation The fermentation medium was prepared according to the proportions and methods of fermentation medium 1 in Example 3. *Lactobacillus acidophilus* LIHUO1978, *Bifidobacterium animalis* subsp. *lactobacter* JZ-FJ01, and *Lactobacillus rhamnosus* JZ-FJ02 were inoculated sequentially according to the inoculation amounts in Table 4, ensuring consistent inoculation amounts for each strain in different fermentation groups. Fermentation was carried out under anaerobic conditions at 180 rpm and 37°C for 48 hours. The results showed that the acidity and viable cell count of the three tested strains were higher after fermentation with two strains compared to fermentation with a single strain. The acidity and viable cell count were further significantly increased after fermentation with the three strains combined.

[0096] Table 4. Effects of different combinations on fermentation

[0097] Comparative Example 2: Comparison of fermentation of Lactobacillus acidophilus LIHUO 1978 with different strains The fermentation medium was prepared according to the proportions and methods of fermentation medium 1 in Example 3, and the compound fermentation was carried out according to the methods in Table 5. The strains with CICC numbers in Table 5 were obtained from the China Industrial Microbial Culture Collection Center. The strains were activated twice with MRS broth medium (with added L-cysteine ​​hydrochloride), and the OD600 value was adjusted to 2.10 before inoculation at a rate of 2.5% (v / v). *Lactobacillus reuteri* G2, *Lactobacillus bulgaricus* G3, and *Lactobacillus paracasei* G7 bacterial powders were obtained from Shaanxi Yijun Biotechnology Co., Ltd., and *Streptococcus thermophilus* TS01 bacterial powder was obtained from Xi'an Yatu Biotechnology Co., Ltd., all at a rate of 0.125 × 10⁻⁶. 8 Inoculate with an amount of CFU / g. After mixing thoroughly, ferment under anaerobic conditions at 180 rpm and 37°C for 48 hours.

[0098] As shown in Table 5, there were significant differences in acidity and viable cell count after fermentation with different strains of *Lactobacillus acidophilus* LIHUO 1978. Furthermore, the acidity and viable cell count after fermentation with most strains were lower than those after fermentation with a single strain, indicating competition and fermentation inhibition among the strains. For example, the lowest acidity (173.0%) was achieved after fermentation of *Lactobacillus acidophilus* LIHUO 1978 with *Bifidobacterium longum* subsp. *infantica* CICC6069 and *Streptococcus thermophilus*. .

[0099] Table 5 Comparison of Lactobacillus acidophilus LIHUO 1978 with different strains in fermentation

[0100] Comparative Example 3: Comparison of fermentation processes using different Lactobacillus acidophilus and Bifidobacterium animalis subsp. lactis. The fermentation medium was prepared according to the proportions and methods of fermentation medium 1 in Example 3, and inoculated according to the scheme in Table 6. The *Lactobacillus acidophilus* NCFM powder was obtained from Danisco, and the inoculation amount was consistent with *Lactobacillus acidophilus* LIHUO 1978 at 0.25 × 10⁻⁶. 8 CFU / g. The Bifidobacterium animalis subsp. lactis Bb-12 bacterial powder was obtained from Chr. Hansen, with the inoculum amount consistent with that of Bifidobacterium animalis subsp. lactis JZ-FJ01 at 0.375 × 10⁻⁶ CFU / g. 8 CFU / g.

[0101] The results show that different combinations of Lactobacillus acidophilus, Bifidobacterium animalis subsp. lactis, and Lactobacillus rhamnosus can all promote acid production and increase the number of viable bacteria, and different strains can ferment synergistically.

[0102] Table 6 Comparison of fermentation processes using different Lactobacillus acidophilus and Bifidobacterium animalis subsp. lactis.

[0103] Example 5: Analysis of the synergistic fermentation mechanism based on metabolomics differences between single-strain fermentation and compound fermentation The fermentation medium was prepared according to the proportions and methods of fermentation medium 1 in Example 3. Single and triple inoculations of *Lactobacillus acidophilus* LIHUO1978, *Bifidobacterium animalis* subsp. *lactobacter* JZ-FJ01, and *Lactobacillus rhamnosus* JZ-FJ02 bacterial powders were respectively inoculated into the fermentation medium at inoculation amounts of 0.25 × 10⁻⁶. 8 CFU / g, 0.375×10 8 CFU / g and 0.125×10 8 CFU / g. After thorough mixing, fermentation was carried out under anaerobic conditions at 180 rpm and 37℃ for 48 h. After fermentation, heat inactivation was performed at 90℃ for 30 min. The material was then spray-dried with an inlet air temperature of 170℃, an outlet air temperature of 80℃, a feed rate of 12 rpm, and an atomizer speed of 18000 rpm, to obtain AcidGut postbiotic prepared by combining Lactobacillus acidophilus LIHUO 1978 postbiotic, Bifidobacterium animalis subsp. lactis JZ-FJ01 postbiotic, Lactobacillus rhamnosus JZ-FJ02 postbiotic, and the three strains.

[0104] The metabolomics analysis of the above-mentioned postbiotic samples was performed at Shanghai Ouyi Biomedical Technology Co., Ltd. The results (Table 7) show that *Lactobacillus acidophilus* LIHUO 1978, *Bifidobacterium animalis* subsp. *lactotrichum* JZ-FJ01, and *Lactobacillus rhamnosus* JZ-FJ02 exhibit complementary and synergistic effects in the utilization of nutrients such as amino acids and vitamins. For example, *Bifidobacterium animalis* subsp. *lactotrichum* JZ-FJ01 produced relatively abundant ornithine, L-asparagine, N-acetyl-L-methionine, and 3,4-dihydroxy-DL-phenylalanine after fermentation, while the content of these amino acids decreased after fermentation of the other two strains. Literature indicates that ornithine and phenylalanine can significantly promote the growth of *Lactobacillus acidophilus*. *Lactobacillus acidophilus* LIHUO 1978 produced abundant L-histidine, L-lysine, L-arginine, L-proline, and malic acid after fermentation. Lysine and arginine can promote the growth and acid production of *Lactobacillus rhamnosus*, while malic acid can indirectly provide energy to other bacteria. Lactobacillus rhamnosus can accumulate a large amount of L-glutamic acid, N-acetyleucine and vitamin B2 after fermentation, which are essential nutrients for the growth of the other two strains.

[0105] Table 7. Differences in fermentation components between single and combined strains based on metabolomics analysis.

[0106] Note: In the table, the pattern pos is [M+H]. + Neg stands for [MH] -Level refers to the hierarchical score. Level 1 is defined as retention time ±0.3 min (18 s) and fragmentation score ≥ 45 points. Level 2 is defined as retention time ±0.3 min (18 s) and fragmentation score ≤ 0 points < 45 points. The values ​​in the table are peak areas converted using log2. Missing values ​​(0 values) within a group were replaced with half of the minimum ion intensity of all samples before conversion. The converted value is 11.979.

[0107] Example 6: Effect of compound fermentation on the accumulation of active ingredients based on metabolomics analysis The fermentation, preparation, and detection methods for the post-biotic samples were the same as in Example 5. The experimental results are shown in Table 8. As can be seen from the results in Table 8, the content of various organic acids, amino acids, polyunsaturated fatty acids, soybean saponins, tryptophan metabolites, and lysine metabolites was significantly increased after compound fermentation. For example, the content of polyunsaturated fatty acids such as α-linolenic acid, acetylsemidine, and 17-octadecenoic acid was significantly increased after compound fermentation. Soybean saponin II, soybean saponin IV, and soybean saponin γ-A were derived from the transformation of soybean saponins and other substances in soybean flour by the strain. The abundance of these substances was increased by more than 50% after compound fermentation compared to single-strain fermentation. Tryptophan metabolites such as indolelactic acid and 3-indolecarboxaldehyde, and lysine metabolites such as D-piperidinic acid were also significantly increased.

[0108] Table 8. Effects of compound fermentation on the accumulation of active ingredients based on metabolomics analysis.

[0109] Note: In the table, pos represents [M+H]+, and neg represents [MH]-. Level refers to the hierarchical score: Level 1 is retention time ±0.3 min (18 s) and fragmentation score ≥ 45; Level 2 is retention time ±0.3 min (18 s) and fragmentation score ≤ 0 < 45. The values ​​in the table are peak areas converted using log2. Missing values ​​(0 values) within a group are replaced with half of the minimum ion intensity of all samples before conversion. The converted value is 11.979.

[0110] Example 7: Evaluation of efficacy in relieving colitis AcidGut postbiotic was obtained by fermentation of three strains (preparation method is the same as in Example 5), and its efficacy was evaluated in DSS-induced colitis mice.

[0111] Eighteen SPF-grade, 6-week-old male C57BL / 6 rats (weighing 19-21 g) were housed in an animal barrier at a temperature of 20-24 ℃. All animal care and experimental procedures were approved by the Laboratory Animal Ethics Committee of Jiangzhong Pharmaceutical Co., Ltd. (Approval No.: 20240903).

[0112] After 7 days of acclimatization, mice were randomly divided into three groups (control group, model group, and AcidGut group), with 6 mice in each group. From day 0 to day 7, mice in the control and model groups were administered 1 mL of PBS by gavage, while mice in the AcidGut group were administered AcidGut followed by a prebiotic (1 g / kg bw) by gavage. All mice received pure water during this period. From day 8 to day 14, the gavage procedure remained the same, but the drinking water for the model and AcidGut groups was replaced with a 2.5% DSS solution.

[0113] During the period, mice were scored using the Disease Activity Index (DAI), which is calculated as (weight loss score + rectal bleeding score + fecal characteristics score) / 3. Weight loss score was calculated as follows: 0 = no improvement; 1 = 1%-5%; 2 = 5%-10%; 3 = 10%-20%; 4 = >20%. Rectal bleeding score was calculated as follows: 0 = no bleeding in feces; 1 = slightly bloody feces; 2 = some bloody feces; 3 = significant bloody feces; 4 = large amount of bloody feces. Fecal characteristics score was calculated as follows: 0 = normal feces; 1 = slightly loose stools; 2 = loose stools; 3 = watery feces; 4 = severe diarrhea.

[0114] After gavage on day 14, mice were kept on a restricted diet but allowed free access to water. On day 15, after enucleation and blood collection, the mice were immediately euthanized, and colonic tissue, cecal contents, and spleen were collected. Blood samples were centrifuged at 3500 rpm for 10 min and then aliquoted as serum. The levels of TNF-α (Shanghai ELISA Biotechnology Co., Ltd. ml8624335), IL-6 (Shanghai ELISA Biotechnology Co., Ltd. ml862345), and IL-10 (Shanghai ELISA Biotechnology Co., Ltd. ml8623244) in the mouse serum were determined according to the ELISA kit instructions. A standard curve was first plotted, and then the serum was diluted twice before detection. The units are pg / mL.

[0115] The results are as follows Figure 2 As shown, the model group mice exhibited typical symptoms of colitis, including weight loss ( Figure 2 The symptoms included A), diarrhea and bloody stools (elevated DAI score), and significantly shortened colon length (P<0.01) and splenomegaly compared to the control group. After AcidGut intervention, these symptoms were reversed to varying degrees, and the DAI score ( Figure 2Compared with DSS, the DAI score in the B group was significantly lower (P<0.01). The DAI score in the DSS group reached its highest level on day 14. After gavage administration of AcidGut, the degree of bloody and watery stools in mice was reduced, and the DAI score was significantly lowered.

[0116] Colonic shortening is a common symptom of colitis, mainly caused by colonic congestion, inflammation, and edema. Figure 2 As shown in C, after DSS modeling, the colon length of mice was significantly shortened, and the AcidGut post-sponge group significantly alleviated this symptom, restoring the colon length of mice to basically normal.

[0117] HE staining of distal colon tissue from mice revealed significant damage after DSS treatment, with diffuse ulceration of the epithelial surface, disappearance of goblet cells, deformed crypts, thickened edema of the muscularis propria, and enhanced inflammatory cell infiltration. Figure 2 (D in the text). Compared with the DSS group, the AcidGut post-biotic group showed significant improvement in mucosal ulcers and crypt damage.

[0118] Furthermore, AcidGut postbiotic intervention significantly alleviated DSS-induced splenomegaly and reduced the spleen index ( Figure 3 (A in the formula) restores the immune response in mice.

[0119] Compared with the control group, the model group (DSS group) showed significantly higher levels of IL-6 and TNF-α (p<0.01). Figure 3 B and Figure 3 The levels of inflammatory factors D and IL-10 increased by 2.83-fold and 0.32-fold, respectively. However, after AcidGut post-biotic intervention, the levels of these two inflammatory factors were significantly reduced compared to the model group, restoring their levels to those of the control group. Furthermore, AcidGut post-biotic intervention also significantly increased the level of the anti-inflammatory factor IL-10 (P<0.05). Figure 3 The presence of C in the formula indicates that AcidGut post-biotics have good anti-inflammatory effects.

[0120] Based on the above indicators, it can be concluded that AcidGut postbiotic can significantly alleviate DSS-induced colitis symptoms in mice and reduce the level of inflammation in vivo.

[0121] Example 8: Evaluation of the efficacy in relieving constipation AcidGut postbiotic was obtained by fermentation of three strains (preparation method is the same as in Example 5), and its efficacy was evaluated in a loperamide-induced constipation model in SD rats.

[0122] Forty-eight 6-week-old SPF-grade male SD rats (weighing 190g-210g) were housed in an animal barrier at a temperature of 20-24℃. After 7 days of acclimatization, they were randomly assigned to four groups (control group, model group, lactulose group, high-dose AcidGut group, medium-dose AcidGut group, and low-dose AcidGut group), with eight rats in each group.

[0123] From day 0 to day 7, rats in the control and model groups were administered 1 mL of PBS by gavage, while the lactulose group was administered 0.1 mL of lactulose oral solution (Fenlitai) by gavage. The high-dose, medium-dose, and low-dose AcidGut groups were administered 0.5 g / kg bw, 0.25 g / kg bw, and 0.125 g / kg bw of AcidGut postbiotics by gavage, respectively. All rats drank pure water during this period. From day 8 to day 14, postbiotic intervention was performed concurrently with model initiation. The intervention method and dosage were the same as from day 0 to day 7. The model initiation method involved intraperitoneal injection of 18.0 mg / kg loperamide (Aladdin F2117175) solution, 1 mL twice daily.

[0124] On day 12, record the rats' defecation status and fecal water content. On day 14, after the gavage was completed, administer ink to each animal and record the time when the first black feces were expelled.

[0125] After a 24-hour fasting period with no water restriction, intestinal propulsion rate was measured. Each rat was given 2 mL of Evans blue paste (8 g starch, 16 g milk powder, 8 g sugar, 5 g carboxymethyl cellulose, and 7 mL of 1% Evans blue solution dissolved in approximately 250 mL of distilled water, mixed well, and then brought to a final volume of 300 mL, resulting in a blue semi-solid paste) by gavage. After 15 minutes, the rats were sacrificed, and the entire intestinal segment was dissected and placed on a glass plate. The total length from the pylorus to the ileocecal junction and the distance from the pylorus to the leading edge of the Evans blue paste were measured. The propulsion percentage was calculated using the formula: Intestinal propulsion rate % = Distance between the leading edge of the blue paste and the pylorus / Total length of the small intestine × 100%.

[0126] Animal blood samples were centrifuged at 3500 rpm for 10 min and then aliquoted into serum. The levels of gastrin (Gas), motilin (MTL), rat somatostatin (SS), and vasoactive intestinal peptide (VIP) in the serum were measured according to the instructions using ELISA kits from Shanghai Enzyme-Linked Biotechnology Co., Ltd. (ml862256), Wuhan Elite Biotechnology Co., Ltd. (WK04T640B2835), Shanghai Enzyme-Linked Biotechnology Co., Ltd. (Ml003102), and Wuhan Elite Biotechnology Co., Ltd. (WK04T640B2563), respectively. Units are pg / mL.

[0127] from Figure 4It can be seen that the fecal water content, time to first black stool excretion, and intestinal propulsion rate of the model group rats were significantly different from those of the control group. Fecal water content was reduced, defecation frequency decreased, and intestinal propulsion rate was significantly reduced, with obvious constipation symptoms, indicating that the model was successfully established. After intervention with different doses of AcidGut postbiotics, these symptoms were reversed to varying degrees, including a significant increase in fecal water content (no significant difference between different dose groups), a significant reduction in time to first black stool excretion, and an increase in intestinal propulsion rate, which also showed a certain dose dependence.

[0128] Compared with the control group, the model group had lower serum Gas ( Figure 5 A in MTL Figure 5 The levels of B in the serum were significantly decreased (p<0.01), while SS was significantly increased. After intervention with different doses of AcidGut, serum levels of Gas, MTL, and VIP were significantly increased compared to the model group. Figure 5 The level of C in the control group was higher than that in the control group, and it could also significantly reduce SS ( Figure 5 The level of D in the sample (P<0.05) indicates that the postbiotic AcidGut can regulate digestion and the secretion of intestinal kinases, thereby improving intestinal peristalsis and relieving constipation.

[0129] Example 9: Efficacy evaluation in alleviating 5-fluorouracil (5-FU)-induced chemotherapy-induced diarrhea in mice AcidGut postbiotic was obtained by fermentation of three strains (preparation method is the same as in Example 5), and its efficacy was evaluated in a mouse model of chemotherapy-induced colitis induced by 5-fluorouracil (5-FU).

[0130] Forty SPF-grade 6-week-old BALB / c mice (19g-21g) were housed in an animal barrier at a temperature of 20-24 ℃. After 7 days of acclimatization, they were randomly assigned to four groups (control group, model group, Imodium group, and AcidGut group), with 10 mice in each group.

[0131] From day 0 to day 5, mice in the control group, Imodium group, and model group were administered 1 mL of PBS by gavage, while the AcidGut group was administered 1.0 g / kg bw of AcidGut postbiotic by gavage. All mice drank pure water during this period. Starting on day 6, mice were induced to develop the model by intraperitoneal injection of fluorouracil (50 mg / kg), and this process continued for 5 days. During this period, mice in the control group and model group were administered 1 mL of PBS by gavage, the Imodium group was administered 3 mg / kg of the positive control drug loperamide hydrochloride (Imodium), and the AcidGut group continued to be administered 1.0 g / kg bw of AcidGut postbiotic by gavage. All animals drank pure water during this period.

[0132] After the modeling process began, each mouse was removed and weighed. They were then placed in a clean container with a clean filter paper at the bottom. The mice were allowed to defecate, and the condition of their feces was observed. The diarrhea index score was recorded for each mouse. The severity of diarrhea was evaluated using the Bowen score system, which was divided into four levels based on the consistency of the stool: 0 points, normal stool; 1 point, slightly wet and soft stool, indicating mild diarrhea; 2 points, wet and unformed stool, indicating moderate diarrhea; 3 points, watery stool.

[0133] From the start of the experiment until one day before dissection, the mice's defecation and perianal condition were observed daily, and the diarrhea index was recorded. The mice were fasted for 12 hours before dissection. After dissection, the small intestine tissue was separated, and the length of the small intestine was measured. The spleen was removed and weighed using an analytical balance.

[0134] Spleen index calculation method: Spleen index (mg / g) = organ mass (mg) / body mass (g).

[0135] The results show that ( Figure 6 AcidGut postbiotics effectively alleviated symptoms of 5-fluorouracil (5-FU)-induced chemotherapy-induced colitis in mice, significantly reducing diarrhea scores (P<0.05), significantly increasing spleen index (P<0.05), and significantly increasing small intestinal length (P<0.01) compared to the model group. Furthermore, AcidGut postbiotics significantly regulated inflammatory factors in mouse jejunal tissue, significantly reducing pro-inflammatory factors such as interleukin-1β (IL-1β) and interferon-γ (IFN-γ) while significantly increasing the level of the anti-inflammatory factor interleukin-10 (IL-10) compared to the model group.

[0136] In summary, AcidGut postbiotic can alleviate the symptoms of 5-fluorouracil (5-FU)-induced chemotherapy-induced colitis in mice, and regulate inflammatory factors such as IL-1β, IFN-γ and IL-10 to reduce inflammation in the body, thereby exerting the effect of alleviating chemotherapy-induced colitis.

[0137] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing an epigenetic agent, characterized in that, Includes the following steps: S1. Inoculate Lactobacillus acidophilus, Bifidobacterium animalis subsp. lactis and Lactobacillus rhamnosus into the fermentation medium according to the inoculation ratio and carry out fermentation. S2. After fermentation, heat inactivation and drying are performed to obtain a compound post-biotic containing Lactobacillus acidophilus, Bifidobacterium animalis subsp. lactis, and Lactobacillus rhamnosus. Lactobacillus acidophilus includes Lactobacillus acidophilus LIHUO 1978, with accession number GDMCC No:65514; Bifidobacterium animalis subsp. lactis includes Bifidobacterium animalis subsp. lactis JZ-FJ01, with accession number CGMCC No.37148; Lactobacillus rhamnosus includes Lactobacillus rhamnosus JZ-FJ02, with accession number CGMCC No.37149.

2. The preparation method according to claim 1, characterized in that, The inoculation ratio of Lactobacillus acidophilus, Bifidobacterium animalis subsp. lactis and Lactobacillus rhamnosus in step S1 is 10:(2-20):(2-10).

3. The preparation method according to claim 1, characterized in that, The inoculation amount of the compound bacterial powder of Lactobacillus acidophilus, Bifidobacterium animalis subsp. lactis and Lactobacillus rhamnosus in step S1 is 0.025‰-0.75‰.

4. The preparation method according to claim 1, characterized in that, The fermentation medium described in step S1 includes skim milk powder and soybean flour; or; Including skim milk powder and soy protein isolate; or; Including skim milk powder.

5. The preparation method according to claim 4, characterized in that, When the fermentation medium in step S1 includes skim milk powder, the content of skim milk powder in the medium is 8-30% (w / w). When the fermentation medium includes skim milk powder and soybean flour, the content of skim milk powder in the medium is 8-30% (w / w), and the content of soybean flour is 1-5% (w / w). When the fermentation medium includes skim milk powder and soy protein isolate, the content of skim milk powder in the medium is 8-30% (w / w), and the content of soy protein isolate is 1-5% (w / w).

6. The preparation method according to claim 1, characterized in that, The preparation method of the fermentation medium in step S1 is as follows: add the corresponding components according to the formula of the medium, add water to make up the volume, and then add enzyme for enzymatic hydrolysis; the enzyme is selected from one or more of neutral protease, acidic protease, papain, bromelain, and flavor protease; the amount of enzyme added is 0.01% (w / w) to 0.01% (w / w).

7. The postgenetic agent prepared by the preparation method according to any one of claims 1-6.

8. The use of the post-genetic agent according to claim 7 in the preparation of a medicament for treating colitis.

9. The use of the post-biotic according to claim 7 in the preparation of a product for treating constipation.

10. The use of the post-biotic according to claim 7 in the preparation of a medicament for treating chemotherapy-induced diarrhea.

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