Yeast metaplasm with anti-allergic effect as well as preparation method and application of yeast metaplasm

Abstract

The invention relates to the technical field of microorganisms, in particular to a yeast metagen with an anti-allergic effect as well as a preparation method and application of the yeast metagen. According to the invention, the metabolite is an inactivated saccharomyces boulardii strain and a fermentation metabolite thereof, wherein the preservation number of the saccharomyces boulardii strain is CCTCC (China Center for Type Culture Collection) NO: M 20251803. The metagen can be used for remarkably reducing the total IgE (Immunoglobulin E) level of serum and relieving anaphylactic reaction by adjusting the balance of Th1 / Th2, Th17 and Treg immune cells. Therefore, the metabiotics disclosed by the invention have a wide application prospect in preparation of foods, medicines or health-care products for relieving allergic symptoms.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a yeast postbiotic with anti-allergy effect and a preparation method and application thereof. BACKGROUND

[0002] Allergic diseases refer to the damage of the body to some substances, and the immune response caused by excessive immune response. The pathogenesis is the abnormal increase of IgE antibody after exposure to allergen and the immune response mediated by Th2 type cytokines. Allergic diseases mainly include atopic dermatitis, allergic rhinitis, food allergy and allergic asthma caused by oral intake, air inhalation and skin contact of sensitizing substances. Allergic diseases are the most common chronic diseases, and the prevalence rate continues to rise, which has become a global public health problem.

[0003] The imbalance of intestinal microorganisms is considered to be one of the key factors causing allergy. Existing research shows that intestinal flora and its metabolites can significantly affect the development of allergic diseases by activating immune cells, regulating inflammation, affecting intestinal barrier and immune homeostasis. Probiotics, as an important part of intestinal microbiome, can maintain intestinal microbial balance, enhance intestinal barrier function and regulate the immune system, thereby having a relieving effect on allergic symptoms.

[0004] Bullus yeast, as a kind of fungal probiotic, is different from bacteria, and is rich in protein, dietary fiber and other rich nutrients. In addition, inactivated yeast has safety, stability and activity retention characteristics, and has a significant advantage in postbiotic development. At present, the research of bullus yeast mainly focuses on the field of intestinal health, especially the significant effect of relieving various diarrhea. There is little research on yeast, especially postbiotic products, in anti-allergy, therefore, the development of a yeast postbiotic raw material with anti-allergy effect has wide application prospect.

[0005] The existing methods for regulating allergic diseases mainly include allergen-specific immunotherapy and non-specific immunotherapy. Allergen-specific immunotherapy has significant effect in desensitization, but it needs to identify allergen, has long treatment cycle and has the risk of inducing severe allergic reaction. Non-specific therapy mainly includes anti-allergic drug treatment such as antihistamines, glucocorticoids and leukotriene receptor antagonists. These drugs are mostly symptomatic treatment, and are easy to relapse after stopping, and long-term use can cause drowsiness, dry mouth and other side effects, and cannot fundamentally regulate immune imbalance. Probiotic therapy has gradually become a research hotspot in recent years, but the survival rate and colonization ability of live bacteria are challenging, and there is a potential risk of infection for immunodeficient and critically ill patients.

[0006] The mature product EPICOR® on the market is derived from Saccharomyces cerevisiae, and has no patent layout in the direction of allergy, but the strain source is unknown. Meanwhile, the immune regulation mechanism involved in EPICOR® is relatively single. SUMMARY

[0007] Therefore, the yeast metatonic agent has a wide application prospect in the preparation of food, medicine or health care products for relieving allergic symptoms.

[0008] In order to achieve the above-mentioned purposes of the application, the following technical solutions are provided by the present application:

[0009] In the first aspect, the present application provides a preparation method of a yeast metatonic agent, comprising the following steps:

[0010] Step 1, strain activation: taking Saccharomyces boulardii in an activation culture medium for activation to obtain an activated strain; the Saccharomyces boulardii is a Bld-3 strain of Saccharomyces boulardii, and the preservation number is CCTCC NO: M20251803; the activation culture medium comprises L-histidine;

[0011] Step 2, shake flask culture: taking the activated strain in step 1 in a shake flask culture medium for fermentation to obtain a shake flask culture solution; the shake flask culture medium comprises zinc ions;

[0012] Step 3, seed fermentation: taking the shake flask culture solution in step 2 in a seed culture medium for fermentation to obtain a seed solution;

[0013] Step 4, scale-up culture: taking the seed solution in step 3 in a fermentation culture medium for fermentation to obtain a fermentation product, inactivation to obtain an inactivated fermentation product; the fermentation culture medium comprises chitosan;

[0014] Step 5, spray drying: mixing the inactivated fermentation product and a protective agent, and spray drying to obtain the yeast metatonic agent; the protective agent comprises trehalose and vitamin C.

[0015] Preferably, the activation culture medium in step 1 comprises sucrose, yeast extract, L-histidine, KH2PO4 and MgSO4; or

[0016] The shake flask medium in Step 2 includes fructose, glucose, yeast extract, ZnSO4, KH2PO4, and MgSO4; or

[0017] The seed medium in Step 3 includes molasses, arabinose, MgSO4, ammonium sulfate, NH4H2PO4, and copper sulfate pentahydrate; or

[0018] The fermentation medium in Step 4 includes molasses, chitosan, ammonia water, NH4H2PO4, and copper sulfate pentahydrate.

[0019] Preferably, the activation medium in Step 1 includes sucrose 80-120 g / L, yeast extract 15-30 g / L, L-histidine 0.01-0.03 g / L, KH2PO4 0.5-1 g / L, and MgSO4 0-1 g / L; or

[0020] The shake flask medium in Step 2 includes fructose 40-80 g / L, glucose 30-50 g / L, yeast extract 15-30 g / L, ZnSO4 0.03-0.06 g / L, KH2PO4 0.5-1.0 g / L, and MgSO4 0-1 g / L; or

[0021] The seed medium in Step 3 includes molasses 80-150 g / L, arabinose 5-15 g / L, MgSO4 0.5-2.0 g / L, ammonium sulfate 10-30 g / L, NH4H2PO4 1-5 g / L, and copper sulfate pentahydrate 0.004-0.02 g / L; or

[0022] The fermentation medium in Step 4 includes molasses 80-200 g / L, chitosan 2-5 g / L, ammonia water with a mass concentration of 20% 5-30 mL / L, NH4H2PO4 1-5 g / L, and copper sulfate pentahydrate 0.004-0.02 g / L.

[0023] Preferably, the activation medium in Step 1 includes sucrose 100 g / L, yeast extract 20 g / L, L-histidine 0.01 g / L, KH2PO4 1 g / L, and MgSO4 0.5 g / L; or

[0024] The shake flask medium in Step 2 includes fructose 60 g / L, glucose 40 g / L, yeast extract 20 g / L, ZnSO4 0.05 g / L, KH2PO4 1.0 g / L, and MgSO4 0.5 g / L; or

[0025] The seed culture medium in step 3 comprises molasses 120 g / L, arabinose 10 g / L, MgSO4 1.2 g / L, ammonium sulfate 20 g / L, NH4H2PO4 2.5 g / L and copper sulfate pentahydrate 0.004 g / L; or

[0026] The fermentation medium in step 4 comprises molasses 130 g / L, chitosan 5 g / L, ammonia water with a mass concentration of 20% 12 mL / L, NH4H2PO4 1.5 g / L and copper sulfate pentahydrate 0.004 g / L.

[0027] Preferably, the conditions for the activation in step 1 comprise culturing at 26-32 ℃ for 24-48 h; or

[0028] The conditions for the fermentation in step 2 comprise culturing at 25-35 ℃ for 24 h; or

[0029] The conditions for the fermentation in step 3 comprise culturing at 30 ℃ for 24 h; or

[0030] The conditions for the fermentation in step 4 comprise fermenting at 31 ℃ for 5 h, reducing the temperature to 30 ℃, continuing to culture for 10 h, and then increasing the temperature to 34 ℃ for 9-15 h.

[0031] Preferably, the mass concentration of trehalose in step 5 comprises 1-2%, and the mass concentration of vitamin C comprises 0.05%-0.2%.

[0032] Preferably, the mass concentration of trehalose in step 5 comprises 2%, and the mass concentration of vitamin C comprises 0.1%.

[0033] Preferably, the inlet temperature of the spray drying in step 5 comprises 150-170 ℃, and the outlet temperature comprises 85-95 ℃.

[0034] In a second aspect, the present application further provides a yeast metagenomic product prepared by the preparation method.

[0035] Preferably, the yeast metagenomic product has a protein content of ≥25%.

[0036] In a third aspect, the present application further provides an application of the yeast metagenomic product in preparing an anti-allergy product.

[0037] Preferably, the application comprises any of the following:

[0038] (I) reducing the serum total IgE level;

[0039] (II) regulating the Th1 / Th2, Th17 / Treg immune cell balance;

[0040] (III) reducing the level of inflammatory factors and activating anti-inflammatory factors.

[0041] Preferably, the regulation of Th1 / Th2, Th17 / Treg immune cell balance comprises: increasing the percentage of CD4 + IFN-γ + T cell content, reducing the percentage of CD4 + IL-4 + T cell content, reducing the percentage of CD4 + IL-17a + T cell content or increasing CD25 + FOXP3 + T cell content.

[0042] Preferably, the inflammatory factors include TNF-α or IL-1β; and the anti-inflammatory factors include IL-10.

[0043] In a fourth aspect, the present application also provides a product comprising the yeast probiotic.

[0044] Preferably, the product comprises a medicine, a food or a health food.

[0045] The present application includes but is not limited to providing the following benefits:

[0046] The yeast probiotic provided by the present application can quickly enter the body through the intestinal mucosa, saving the time for the live bacteria to colonize in the intestine and quickly exerting the effect. In addition, the inactivated bacteria are stable, not only resistant to high temperature, gastric acid and bile salts, but also not limited in the later processing process, and can be applied in various fields and product types.

[0047] The probiotic related by the present application can significantly reduce the serum total IgE level by regulating the Th1 / Th2, Th17 / Treg immune cell balance, and relieve allergic reactions. The probiotic overcomes the shortcomings of live bacteria and can be used as an ideal way to prevent or treat allergic diseases.

[0048] Biological preservation instructions

[0049] Biological material: Saccharomyces boulardii Bld-3, classification name: Saccharomyces boulardii Bld-3, preserved in China Center for Type Culture Collection on August 8, 2025, located in Wuhan University, Wuhan, China, with the preservation number CCTCC NO: M 20251803. BRIEF DESCRIPTION OF DRAWINGS

[0050] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiment or prior art description will be briefly introduced as follows.

[0051] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiment or prior art description will be briefly introduced as follows.Figure 1 Total IgE levels in serum were shown;

[0052] Figure 2 CD4 + IFN-γ + Percentage of cells (Th1 cells);

[0053] Figure 3 CD4 + IL-4 + Percentage of cells (Th2 cells);

[0054] Figure 4 CD4 + IL-17a + Percentage of cells (Th17 cells);

[0055] Figure 5 CD25 + FOXP3 + Percentage of cells (Treg cells);

[0056] Figure 6 TNF-α mRNA cytokine expression;

[0057] Figure 7 TNF-γ mRNA cytokine expression;

[0058] Figure 8 IL-4 mRNA cytokine expression;

[0059] Figure 9 IL-6 mRNA cytokine expression;

[0060] Figure 10 IL-10 mRNA cytokine expression;

[0061] Figure 11 IL-12 mRNA cytokine expression. DETAILED DESCRIPTION

[0062] The present application discloses a yeast probiotics with anti-allergy efficacy, its preparation method and application. Those skilled in the art can refer to the content of the present application and improve the process parameters appropriately. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application described herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0063] The present application aims to solve the problems of limited application of live bacteria type raw materials (temperature, pressure, water activity, etc.; live bacteria type raw materials must be kept at low temperature, water activity <0.2, low survival rate, and difficult to colonize) and safety (live bacteria may cause bacteremia, and there is a potential risk of infection for immunodeficient and critically ill patients).

[0064] The present application aims to prove that the inactivation of specific yeast cells has a significant alleviating effect on allergic symptoms, can regulate Th1 / Th2, Th17 / Treg immune cell balance, significantly reduce serum total IgE level, and relieve allergic reaction.

[0065] The present application provides a preparation method of a postbiotic (including inactivated Blastomyces body and its fermentation metabolites), and the steps thereof include:

[0066] 1) Strain activation: pick the Blastomyces Bld-3 colony and inoculate it into 100 ml of liquid medium for activation, and the specific composition of the liquid medium is: sucrose 100 g / L (80-120 g / L), yeast extract 20 g / L (15-30 g / L), L-histidine 0.01 g / L (0.01-0.03 g / L), KH2PO4 1 g / L (0.5-1 g / L), MgSO4 0.5 g / L (0-1 g / L), at a temperature of 30℃ (26-32℃), 200 rpm, and the culture time is 30 h (24-48 h).

[0067] 2) Shake flask culture: inoculate 1 mL of the activated fermentation broth into a shake flask containing 1 L of liquid medium, and the specific composition of the liquid medium is: fructose 60 g / L (40-80 g / L), glucose 40 g / L (30-50 g / L), yeast extract 20 g / L (15-30 g / L), ZnSO4 0.05 g / L (0.03-0.06 g / L), KH2PO4 1.0 g / L (0.5-1.0 g / L), MgSO4 0.5 g / L (0-1 g / L), pH 4.5-5.0. The culture conditions are: 30℃ (25-35℃), 200 rpm, and the culture time is 24 h.

[0068] 3) Seed fermentation: 1 L of liquid medium after shake flask culture was inoculated into a fermenter containing 9 L of seed medium, and seed fermentation was continued. The specific composition of the liquid medium used for seed fermentation was: molasses (reducing sugar) 120 g / L (80-150 g / L), arabinose 10 g / L (5-15 g / L), magnesium sulfate 1.2 g / L (0.5-2.0 g / L), ammonium sulfate 20 g / L (10-30 g / L), NH4H2PO4 2.5 g / L (1-5 g / L), copper sulfate pentahydrate 0.004 g / L (0.004-0.02 g / L).

[0069] The initial air flow was 9 L / min, and the rotation speed was 200 rpm. Molasses, ammonium sulfate, and NH4H2PO4 were added, and the air flow eventually reached 30 L / min and the rotation speed reached 600 rpm as the seed fermentation proceeded. The fermentation time was 24 h, and the temperature during the entire fermentation process was 30°C. After the fermentation was completed, the seed fermentation liquid was separated using a centrifuge, and the yeast concentration in the heavy phase reached more than 500 g / L. At this time, the heavy phase solution was the seed liquid.

[0070] Yeast concentration measurement method: 10 mL of solution was taken into a centrifuge tube, and the weight was m0 (g). After 5000 rpm centrifugation for 4 min, the light phase solution was discarded, and the centrifuge tube was shaken three times to remove the light phase solution. The weight of the centrifuge tube was m1 (g). The yeast concentration in the solution (g / L) was (m0-m1) g / L.

[0071] 4) Commercial fermentation: The seed liquid was inoculated into a fermenter containing 9 L of commercial fermentation medium, and the yeast concentration of the fermentation liquid after inoculation was 50 g / L. The specific composition of the liquid medium used for commercial fermentation was: molasses 130 g / L (80-200 g / L), 5 g / L (2-5 g / L) of chitosan, 12 mL / L (5-30 mL / L) of 20% ammonia water, NH4H2PO4 1.5 g / L (1-5 g / L), and copper sulfate pentahydrate 0.004 g / L (0.004-0.02 g / L). The initial air flow was 15 L / min, the initial rotation speed was 400 rpm, and the initial temperature was 31°C. When the fermentation proceeded for 5 h, the temperature was reduced to 30°C, and the culture was continued for 10 h, and then the temperature was increased to 34°C. Molasses, ammonia water, and NH4H2PO4 were added, and the final air flow reached 30 L / min and the rotation speed reached 600 rpm as the commercial fermentation time proceeded. The fermentation time was 24-30 h, and the final fermentation temperature was 34°C. After the fermentation was completed, the fermentation liquid was sterilized at 90°C for 60 min.

[0072] 5) Spray drying: 2% (1-2%) trehalose (mass ratio of fermentation broth) and 0.1% (0.05%-0.2%) vitamin C are added to the inactivated fermentation broth, and then spray drying is carried out. The inlet temperature is 170℃ (150-170℃), the outlet temperature is 90℃ (85-95℃), the feeding speed is 0.8 L / h, and the dried powder is collected after drying to obtain the yeast postbiotic.

[0073] The postbiotic involved in the present application can quickly enter the body through the intestinal mucosa, saving the time for the live bacteria to colonize in the intestine and quickly playing a role. In addition, the inactivated bacteria are stable, not only resistant to high temperature, gastric acid and bile salts, but also not limited in the later processing process, and can be applied in various fields and product types.

[0074] The postbiotic involved in the present application can significantly reduce the serum total IgE level by adjusting the Th1 / Th2 and Th17 / Treg immune cell balance, and relieve allergic reactions. The postbiotic overcomes the shortcomings of live bacteria and can be used as an ideal way to prevent or treat allergic diseases.

[0075] The instruments and reagent sources used in the examples and comparative examples of the present application are shown in Table 1.

[0076] Table 1 Information of instrument and reagent sources

[0077]

[0078] Unless otherwise specified, the raw materials and reagents used in the yeast postbiotic with anti-allergic effect provided by the present application, the preparation method and the application thereof can be purchased from the market.

[0079] The present application will be further described below in combination with examples:

[0080] Preparation of postbiotic powder

[0081] 1) Strain activation: a colony of Saccharomyces boulardii Bld-3 is picked and inoculated into 100 mL of liquid medium for activation, and the liquid medium specifically comprises: sucrose 100 g / L, yeast extract 20 g / L, L-histidine 0.01 g / L, KH2PO41 g / L, and MgSO4 0.5 g / L. The culture is carried out at a temperature of 30℃, 200 rpm, and a culture time of 30 h.

[0082] 2) Shake flask culture: 1 mL of the activated fermentation broth was inoculated into a shake flask containing 1 L of liquid medium, wherein the liquid medium specifically consisted of: fructose 60 g / L, glucose 40 g / L, yeast extract 20 g / L, ZnSO4 0.05 g / L, KH2PO4 1.0 g / L, MgSO4 0.5 g / L, pH 4.5-5.0. The culture conditions were: 30°C, 200 rpm, and the culture was performed for 24 h.

[0083] 3) Seed fermentation: 1 L of the liquid medium after shake flask culture was inoculated into a fermenter containing 9 L of seed medium, and the seed culture was continued. The specific composition of the liquid medium used for seed culture was: molasses (calculated as reducing sugar) 120 g / L, arabinose 10 g / L, magnesium sulfate 1.2 g / L, ammonium sulfate 20 g / L, NH4H2PO4 2.5 g / L, copper sulfate pentahydrate 0.004 g / L.

[0084] The initial air flow was 9 L / min, and the rotation speed was 200 rpm. Molasses, ammonium sulfate, and NH4H2PO4 were added, and the air flow finally reached 30 L / min and the rotation speed reached 600 rpm as the seed fermentation proceeded. The fermentation time was 24 h, and the temperature during the whole fermentation process was 30°C. After the fermentation was completed, the seed fermentation broth was separated using a centrifuge, and the yeast concentration in the heavy phase reached more than 500 g / L. At this time, the heavy phase solution was the seed liquid.

[0085] Yeast concentration measurement method: 10 mL of the solution was taken into a centrifuge tube, and the weight was m0 (g). After centrifugation at 5000 rpm for 4 min, the light phase solution was removed, and the weight of the centrifuge tube was m1 (g). The yeast concentration (g / L) in the solution was (m0-m1) g / L.

[0086] 4) Commercial fermentation: inoculate the seed liquid into a fermenter containing 9 L of commercial fermentation medium, and the yeast concentration of the fermentation liquid is 50 g / L after inoculation. The specific composition of the liquid medium used for commercial fermentation is: molasses 130 g / L, 5 g / L of chitosan, 12 mL / L of ammonia water with a mass concentration of 20%, NH4H2PO4 1.5 g / L, and copper sulfate pentahydrate 0.004 g / L. The initial air volume is 15 L / min, the initial rotation speed is 400 rpm, the initial temperature is 31℃, when the fermentation is carried out for 5 h, the temperature is reduced to 30℃, and the culture is continued for 10 h, and then increased to 34℃. Molasses, ammonia water and NH4H2PO4 are added, and the final air volume reaches 30 L / min and the rotation speed reaches 600 rpm as the commercial fermentation time progresses. The fermentation time is 24-30 h, and the final fermentation temperature is 34℃. After the fermentation is completed, the fermentation liquid is heated to 90℃ and sterilized for 60 min.

[0087] 5) Spray drying: add 2% trehalose (mass ratio to fermentation liquid) and 0.1% vitamin C to the inactivated fermentation product, and then perform spray drying. The inlet temperature is 170℃, the outlet temperature is 90℃, the feeding speed is 0.8 L / h, and the dried powder is collected to obtain the yeast postbiotic.

[0088] Example Evaluation of the Anti-allergy activity of yeast postbiotics

[0089] I. Test design

[0090] 1. Animal grouping and intervention

[0091] 6-week-old BALB / c mice were used for in vivo experiments, and were divided into 5 groups, with 15 mice in each group. The specific grouping method is as follows:

[0092] 1) Blank group (group A): normal saline (NS) gavage + NS intraperitoneal injection.

[0093] 2) Model group (group B): NS gavage + adjuvant-OVA intraperitoneal injection.

[0094] 3) Postbiotic 1: Saccharomyces boulardii postbiotic (postbiotic obtained by the preparation example process of the present application) gavage (10 6 TFU / (mouse·day) - reference adult recommended dose 500 mg / 60 kg) + adjuvant-OVA intraperitoneal injection.

[0095] 4) Postbiotic 2: Saccharomyces boulardii postbiotic (postbiotic obtained from the process of example 1 in patent CN 118370776 B) gavage (10 6 TFU / (mouse·day) - reference adult recommended dose 500 mg / 60 kg) + adjuvant-OVA intraperitoneal injection.

[0096] 5) Postbiotic 3: Saccharomyces boulardii postbiotic (postbiotic obtained from inactivated L. rhamnosus, commercial strain, Ramen) gavage (10 6 TFU / (only day) - refer to the recommended adult dose of 500 mg / 60 kg) + adjuvant-OVA intraperitoneal injection.

[0097] From the 1st to the 42nd day, the postbiotic intervention group was gavaged with the corresponding dose of postbiotic solution, and the rest of the mice in the other groups were gavaged with the same volume of normal saline; during this period, on the 7th, 21st, 28th, and 35th days, all mice except those in the blank control group were intraperitoneally injected with 0.2 mL OVA to induce an allergic model, and the mice in the blank control group were injected with the same volume of normal saline.

[0098] 2. Sample collection

[0099] Mouse sacrifice at 43 d:

[0100] Sample collection: 6 fecal samples were collected before the mice were sacrificed on the 0th, 7th, 21st, 28th, 35th, and 43rd days and stored at -80°C; mouse blood was collected using the eyeball method at the time of sacrifice, and after standing and centrifugation, the serum was stored at -80°C; after blood collection, the mice were sacrificed by cervical dislocation, and ileum tissue was collected.

[0101] 3. Index determination

[0102] ELISA was used to determine the total IgE content in mouse serum; Luminex 200 and Magnetic Luminex® Assay Mouse Premixed Multi-Analyte Kit were used to determine the contents of cytokines such as IL-1β, IL-6, IL-10, TNF-α, and IFN-γ in serum; real-time fluorescent quantitative PCR was used to determine the expression of inflammatory cytokines such as IL-4, IL-6, IL-10, IL-12(P40), IL-17a, TNF-α, and IFN-γ in ileum tissue.

[0103] 4. Data statistics and analysis

[0104] Data were statistically analyzed using Excel and minitab software for single-factor or two-factor analysis of variance. All test results were expressed as mean ± SD. The significance level was set as: , #: P < 0.05 indicates a significant difference; , ##: P < 0.01 indicates a very significant difference. Among them indicates comparison with the blank group, and # indicates comparison with the model group.

[0105] Example 1: Serum total IgE level

[0106] The total IgE content in mouse serum was measured in the examples, and the results are as follows: Figure 1 As shown in Table 2, the serum total IgE level in the model group mice was significantly higher than that in the blank group, indicating that the adjuvant-OVA-induced mouse allergy model was successfully established. Compared with the model group, the serum total IgE level in the post-biotic 1 intervention group was significantly reduced, while there was no significant difference between the other two post-biotic intervention groups. This indicates that post-biotic 1 intervention can effectively alleviate allergic symptoms, while the other two post-biotic intervention groups had no significant effect.

[0107] Table 2 Serum total IgE levels

[0108]

[0109] Example 2: Detection of Ileal Immune Cell Subtypes

[0110] The subtypes of mouse ileal immune cells in the examples were detected, and the results are as follows: Figure 2 , 3 As shown in 4, 5 and Table 3. Among them Figure 2 Indicates CD4 + IFN-γ + Percentage of Th1 cells Figure 3 Indicates CD4 + IL-4 + Percentage of Th2 cells Figure 4 Indicates CD4 + IL-17a + Percentage of cells (Th17 cells) Figure 5 Indicates CD25 + FOXP3 + Percentage of cells (Treg cells).

[0111] according to Figure 2 As a result, compared with the blank group, the model group CD4 + IFN-γ + The percentage of T cells was significantly reduced, and the CD4 count in the post-genetic 1 intervention group was lower than that in the model group. + IFN-γ + The significantly increased percentage of T cells indicated a decrease in the percentage of IFN-γ cells in mice allergic to adjuvant-OVA. The post-biotic intervention group restored the percentage of IFN-γ cells to the level of the control group. There was no significant difference in post-biotic levels between the other two groups.

[0112] like Figure 3 As shown, compared with the blank group, the model group mice had higher CD4 counts. + IL-4 + The percentage of cells was significantly increased, and the CD4 count in the post-genetic 1 intervention group was significantly higher than that in the model group. + IL-4+ The percentage of cells was significantly reduced. This indicates that the post-genetic intervention group can inhibit Th2 cell levels, thereby reducing allergic reactions, while the other two post-genetic intervention groups showed no significant effect.

[0113] like Figure 4 As shown, model group CD4 + IL-17a + The percentage of cells was significantly higher in the control group than in the control group, and CD4 count was significantly higher after intervention with post-genetic 1. + IL-17a + The percentage of Th17 cells decreased and was significantly lower than that in the model group, returning to the level of the control group. There were no significant differences between the other two groups treated with post-biotic intervention. These results indicate that adjuvant-OVA induction significantly increased the content of Th17 cells in the mouse ileum. Post-biotic intervention effectively alleviated the allergic-induced increase in Th17 cells, restoring it to the level of the control group. The other two groups showed no significant improvement.

[0114] like Figure 5 As shown, compared with the blank group, CD25 levels in mice induced by adjuvant-OVA were significantly higher in each group. + FOXP3 + The percentage of cells was significantly reduced; compared with the model group, the CD25 content in the post-genetic 1 intervention group was significantly lower. + FOXP3 + The percentage of cells showed a significant upward trend. This indicates that Treg cell levels were generally suppressed in all groups after adjuvant-OVA intervention, with the post-biotic 1 intervention group showing a certain degree of effective recovery of Treg cell levels. The other two post-biotic intervention groups showed no significant differences compared to the model group.

[0115] Table 3 Ileal immune cell subtype detection

[0116]

[0117] Example 3: Serum inflammatory cytokines

[0118] As shown in Table 4, in the examples, the serum TNF-α and IL-β levels in the mouse model group were significantly higher than those in the control group, while the TNF-α and IL-β levels in the post-genetic 1 intervention group were significantly lower than those in the model group. There were no significant differences in IFN-γ and IL-6 levels among the three groups. Compared with the control group, the IL-10 level in the model group was significantly lower, while the IL-10 level after post-genetic 1 intervention was significantly higher, approaching the level of the control group. The TNF-α, IL-1β, and IL-10 levels in the post-genetic 2 and post-genetic 3 intervention groups were significantly different from those in the control group, but not significantly different from those in the model group.

[0119] The above results show that adjuvant-OVA induced allergy leads to an increase in serum TNF-α and IL-1β in mice, causing systemic inflammatory response, and the intervention of Postbiotic 1 can reduce the level of inflammatory factors, activate anti-inflammatory factors and thus inhibit systemic inflammatory response. The other two groups of Postbiotic intervention have no significant effect.

[0120] Table 4 Serum inflammatory cytokines

[0121]

[0122] Example 4 Relative expression amount of ileum cytokines

[0123] The results of the ileum cytokines of the mice in the example are shown in Table 5 and Table 6, wherein Figures 6-11 represents the expression amount of TNF-α mRNA cytokine, Figure 6 represents the expression amount of TNF-γ mRNA cytokine, Figure 7 represents the expression amount of IL-4 mRNA cytokine, Figure 8 represents the expression amount of IL-6 mRNA cytokine, Figure 9 represents the expression amount of IL-10 mRNA cytokine, Figure 10 represents the expression amount of IL-12 mRNA cytokine. Figure 11 The expression amount of TNF-α cytokine in the model group is significantly higher than that in the other groups; compared with the blank group, the expression amount of IL-6 cytokine in the Postbiotic 1 intervention group is significantly reduced, and the expression amount of IL-10 cytokine is significantly increased. The results show that the model group has an inflammatory trend, and the intervention of Postbiotic 1 can selectively inhibit the pro-inflammatory factor IL-6 and activate the anti-inflammatory factor IL-10 to relieve the inflammatory response. The other two groups of Postbiotic have no obvious relief effect.

[0124] Table 5 Relative expression amount of ileum cytokines

[0125]

[0126] Example 5 Protein content of Postbiotic

[0127] The Postbiotic powder prepared in the preparation example was taken to determine the protein content of Postbiotic (GB 5009.5-2016), and the protein content of Postbiotic is shown in Table 6.

[0128] Table 6 Protein content of Postbiotic

[0129]

[0130]

[0131] ​The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. A method for preparing a yeast postbiotic, characterized in that, Comprising the following steps: Step 1, strain activation: taking the Saccharomyces boulardii in the activation medium for activation to obtain the activated strain; the Saccharomyces boulardii is the Saccharomyces boulardii Bld-3 strain, the preservation number is CCTCC NO: M20251803; the activation medium comprises L-histidine; Step 2, shake flask culture: taking the activated strain in step 1 in the shake flask culture medium for fermentation to obtain a shake flask culture solution; the shake flask culture medium comprises zinc ions; Step 3, seed fermentation: taking the shake flask culture solution in step 2 in the seed culture medium for fermentation to obtain a seed solution; Step 4, scale-up culture: taking the seed solution in step 3 in the fermentation medium for fermentation to obtain a fermentation product, inactivation to obtain an inactivated fermentation product; the fermentation medium comprises chitosan; Step 5, spray drying: mixing the inactivated fermentation product and a protective agent, spray drying to obtain the yeast post-inoculum; the protective agent comprises trehalose and vitamin C.

2. The production method according to claim 1, wherein The activation medium in step 1 comprises sucrose, yeast extract, L-histidine, KH2PO4 and MgSO4; or The shake flask culture medium in step 2 comprises fructose, glucose, yeast extract, ZnSO4, KH2PO4 and MgSO4; or The seed culture medium in step 3 comprises molasses, arabinose, MgSO4, ammonium sulfate, NH4H2PO4 and copper sulfate pentahydrate; or The fermentation medium in step 4 comprises molasses, chitosan, ammonia, NH4H2PO4 and copper sulfate pentahydrate.

3. The production method according to claim 1, wherein The activation medium in step 1 comprises sucrose 80-120 g / L, yeast extract 15-30 g / L, L-histidine 0.01-0.03 g / L, KH2PO4 0.5-1 g / L and MgSO4 0-1 g / L; The shake flask culture medium in step 2 comprises fructose 40-80 g / L, glucose 30-50 g / L, yeast extract 15-30 g / L, ZnSO4 0.03-0.06 g / L, KH2PO4 0.5-1.0 g / L and MgSO4 0-1 g / L; The seed culture medium in step 3 comprises molasses 80-150 g / L, arabinose 5-15 g / L, MgSO4 0.5-2.0 g / L, ammonium sulfate 10-30 g / L, NH4H2PO4 1-5 g / L and copper sulfate pentahydrate 0.004-0.02 g / L; The fermentation medium in step 4 comprises molasses 80-200 g / L, chitosan 2-5 g / L, ammonia with a mass concentration of 20% 5-30 mL / L, NH4H2PO4 1-5 g / L and copper sulfate pentahydrate 0.004-0.02 g / L.

4. The production method according to claim 1, wherein The activation medium in step 1 comprises sucrose 100 g / L, yeast extract 20 g / L, L-histidine 0.01 g / L, KH2PO4 1 g / L and MgSO4 0.5 g / L; The shake flask medium in step 2 comprises fructose 60 g / L, glucose 40 g / L, yeast extract 20 g / L, ZnSO4 0.05 g / L, KH2PO4 1.0 g / L and MgSO4 0.5 g / L; The seed medium in step 3 comprises molasses 120 g / L, arabinose 10 g / L, MgSO4 1.2 g / L, ammonium sulfate 20 g / L, NH4H2PO4 2.5 g / L and copper sulfate pentahydrate 0.004 g / L; The fermentation medium in step 4 comprises molasses 130 g / L, chitosan 5 g / L, ammonia water with a mass concentration of 20% 12 mL / L, NH4H2PO4 1.5 g / L and copper sulfate pentahydrate 0.004 g / L.

5. The production method according to claim 1, wherein The activation condition in step 1 is 26-32℃ for 24-48 h; The fermentation condition in step 2 is 25-35℃ for 24 h; The fermentation condition in step 3 is 30℃ for 24 h; The fermentation condition in step 4 is 31℃ for 5 h, then the temperature is reduced to 30℃ for 10 h, and then the temperature is increased to 34℃ for 9-15 h.

6. The production method according to claim 1, wherein The mass concentration of trehalose in step 5 is 1-2%, and the mass concentration of vitamin C is 0.05%-0.2%.

7. The production method according to claim 1, wherein The mass concentration of trehalose in step 5 is 2%, and the mass concentration of vitamin C is 0.1%.

8. The production method according to claim 1, wherein The inlet temperature of the spray drying in step 5 is 150-170℃, and the outlet temperature is 85-95℃.

9. The yeast metagenome prepared by the method according to any one of claims 1 to 8.

10. The yeast postbiotic of claim 9, wherein, The protein content of the yeast metagenome is ≥25%.

11. The use of the yeast metagenome according to claim 9 or 10 in the preparation of a product for resisting allergy.

12. The use according to claim 11, wherein the compound is ###0002### Any of the following is included: (I) reducing the serum total IgE level; (II) regulating the Th1 / Th2, Th17 / Treg immune cell balance; (III) reducing the level of inflammatory factors and activating anti-inflammatory factors.

13. Use according to claim 12, wherein modulating the Th1 / Th2, Th17 / Treg immune cell balance comprises increasing CD4 + IFN-γ + T cell percentage, decreasing CD4 + IL-4 + T cell percentage, decreasing CD4 + IL-17a + T cell percentage or increasing CD25 + FOXP3 + T cell percentage.

14. The use according to claim 12, wherein the compound is ###00010### or a pharmaceutically acceptable salt thereof. The inflammatory factor is TNF-α or IL-1β, and the anti-inflammatory factor is IL-10.

15. A product characterised in that, The product comprises a medicine, a food or a health food.

16. The product of claim 15, wherein, The product comprises a medicine, a food or a health food.

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