Lactobacillus helveticus KLDS1.1 1105 as well as method and application of lactobacillus helveticus KLDS1.1 1105 for preparing metabiotics

Abstract

The invention discloses a lactobacillus helveticus KLDS1.1105, a method for preparing a metagen by using the lactobacillus helveticus KLDS1.1105 and application of the lactobacillus helveticus KLDS1.1105, belongs to the technical field of microorganisms, and provides the lactobacillus helveticus KLDS1.1105 which is preserved in the China General Microbiological Culture Collection Center on September 12, 2024, the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.31947. The lactobacillus helveticus KLDS1.1105 can be used for preparing the metagen by using the lactobacillus helveticus KLDS1.1105. The method for preparing the metagen comprises the following steps: inoculating lactobacillus helveticus KLDS1.1. 1105 into an activation culture medium, performing activation culture, and repeating for three times to obtain activated lactobacillus helveticus KLDS1.1. 1105; and inoculating to a milk-based culture medium, culturing, and spray-drying to obtain the metabiotics. The prepared metagen has a good function of enhancing the immune regulation ability and enhancing the intestinal immune barrier.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms, and in particular relates to a Lactobacillus helveticus KLDS1.1105 and a method for preparing a postbiotic thereof and an application thereof. Background Art

[0002] The immune system is a vital component of the human body. It is an organization of cells and molecules with specialized roles in defending against infection. It destroys and eliminates antigens that enter the body, as well as necrotic and tumor cells produced by the body itself, to maintain normal physiological functions. The immune system has two distinct defense mechanisms against invading microorganisms: the innate immune system and the adaptive immune system. A decline in the body's immune function can have a variety of impacts on the body, including chronic inflammation, decreased resilience, and an increased risk of autoimmune diseases and cancer. Therefore, preventing and treating immunodeficiency is crucial to maintaining human health.

[0003] Lactobacillus helveticus is a lactic acid bacterium used in the production of fermented dairy products and is widely used in the food industry. Compared to most probiotic bacteria, such as Lactobacillus and Bifidobacterium, L. helveticus is more adaptable to industrial fermentation conditions because it can survive extreme conditions such as high temperatures or low pH and osmotic pressure. The health-promoting properties of L. helveticus and its metabolites make it a promising functional food additive for health promotion and disease prevention.

[0004] Postbiotics are a general term for the metabolites of probiotics that have been processed, including the bacterial cells and their metabolites. Research has demonstrated that selected postbiotics are superior to the original live bacteria, retaining high levels of physiological activity even after exposure to high temperatures or gastrointestinal digestive fluids. Evidence suggests that representative postbiotic components, such as lipoteichoic acid, are key to their tolerance to acid, alkali, and heat. Postbiotics are known to have numerous benefits for human health. Concerns regarding the safety of live microorganisms have fueled research and development of probiotic-derived bacterial components and their metabolites. Some scholars define the structural components of probiotics and their metabolites as parabiotics and postbiotics, respectively. Postbiotics refer to the complex mixture of metabolites secreted by probiotics in their cell-free supernatant, specifically enzymes, secreted proteins, short-chain fatty acids, vitamins, biosurfactants, amino acids, peptides, and organic acids. Parabiotics, on the other hand, refer to inactivated, intact or disrupted probiotic cells or crude cell extracts, also possessing a complex chemical composition. Postbiotics do not contain live bacteria, so they carry no risk of bacterial infection and are safer for people with weakened immune systems. The preparation of Lactobacillus helveticus postbiotics with good efficacy is still under research and development. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention proposes a Lactobacillus helveticus KLDS1.1105 and a method and application for preparing postbiotics thereof. The postbiotics prepared from Lactobacillus helveticus KLDS1.1105 include inactivated bacteria and their metabolites, which have good immune regulation enhancing ability, repairing immune organ damage function, enhancing humoral immune function and enhancing the intestinal immune barrier function.

[0006] To achieve the above object, the present invention provides a Lactobacillus helveticus KLDS1.1105, which was deposited on September 12, 2024 at the General Microbiology Center of the China Culture Collection Administration Committee, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being: CGMCC No. 31947.

[0007] The present invention also provides a method for preparing postbiotics using the Lactobacillus helveticus KLDS1.1105, comprising the following steps:

[0008] (1) inoculating the Lactobacillus helveticus KLDS1.1105 into an activation culture medium, performing activation culture, and repeating the activation culture three times to obtain activated Lactobacillus helveticus KLDS1.1105;

[0009] (2) inoculating the activated Lactobacillus helveticus KLDS1.1105 obtained in step (1) into a milk-based culture medium, culturing, and spray-drying the fermentation liquid to obtain postbiotics.

[0010] Preferably, the inoculation amount of Lactobacillus helveticus KLDS1.1105 in step (1) is calculated as 3% to 6% of the volume of the activation culture medium; and the activation culture medium in step (1) is MRS liquid culture medium.

[0011] Preferably, the temperature of the activation culture in step (1) is 36-38° C., and the time of the activation culture is 14-18 hours.

[0012] Preferably, the inoculation amount of the activated Lactobacillus helveticus KLDS1.1105 in step (2) is calculated as 4-6% of the volume of the milk-based culture medium; the milk-based culture medium in step (2) is skim milk with a fat content of ≤0.5%.

[0013] Preferably, the culture temperature in step (2) is 35-37°C, and the culture time is 16-24h; the inlet air temperature of the spray drying in step (2) is 140-185°C, the exhaust air temperature of the spray drying is 45-78°C, the feed flow rate of the spray drying is 7-8 mL / min, and the drying air flow rate of the spray drying is 30-35 m / min.3 / h; the concentration of Lactobacillus helveticus KLDS1.1105 in the fermentation broth was 4×10 9 ~7×10 9 CFU / mL.

[0014] The present invention also provides a method for preparing postbiotics by Lactobacillus helveticus KLDS1.1105, and the postbiotics obtained are prepared.

[0015] The present invention also provides the use of the postbiotics in preparing products with enhanced immune regulation ability.

[0016] The present invention also provides the use of the postbiotics in preparing products having the function of repairing immune organ damage and enhancing humoral immune function.

[0017] The present invention also provides the use of the postbiotics in preparing a product capable of enhancing the intestinal immune barrier function.

[0018] Compared with the prior art, the present invention has the following advantages and technical effects:

[0019] The Lactobacillus helveticus postbiotic of the present invention is prepared by fermenting and inactivating Lactobacillus helveticus KLDS1.1105. The postbiotic prepared from the provided Lactobacillus helveticus 1.1105 can regulate T cell differentiation in mice and restore the Th17 / Treg balance, significantly improve the immune organ index, spleen lymphocyte proliferation ability, NK cell killing activity, serum immunoglobulin level, and restore immune organ and intestinal damage, increase the percentage of Th17 cells and the level of related cytokine IL-17, and reduce the percentage of Treg cells and the level of related cytokines;

[0020] In summary, the postbiotics prepared from Lactobacillus helveticus 1.1105 showed significant effects in regulating T cell differentiation in mice, restoring the Th17 / Treg balance, improving cellular immunity and humoral immunity, repairing immune organ damage, and enhancing the intestinal immune barrier and immune function, and have great prospects in regulating immune function. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 The following are the results of immune organ index determination of mice in each group, where A is spleen index, B is thymus index, and lowercase letters represent significant differences.

[0023] Figure 2 The results of spleen lymphocyte proliferation rate and NK cell cytotoxicity assays of mice in each group are shown in Table 1. A represents spleen lymphocyte proliferation rate, B represents NK cell cytotoxicity, and lowercase letters represent significant differences.

[0024] Figure 3 Flow cytometry of mouse T cells in each group of mice;

[0025] Figure 4 The percentages of Th17 cells, Treg cells and Th17 / Treg ratios of mice in each group are measured, where A is the percentage of Th17 cells, B is the percentage of Treg cells, and C is the Th17 / Treg ratio. Lowercase letters represent significant differences.

[0026] Figure 5 The results of serum cytokines, serum immunoglobulins and intestinal tissue SIgA determination of mice in each group, among which A is IL-17, B is TGF-β, C is IgA, D is IgG, E is IgM, and F is SIgA. Lowercase letters represent significant differences.

[0027] Figure 6 Figures 2 and 3 are HE staining results of the spleen and ileum of mice in each group, wherein A is spleen staining of the normal group, B is ileum staining of the normal group, C is spleen staining of the model group, D is ileum staining of the model group, E is spleen staining of the control group, F is ileum staining of the control group, G is spleen staining of the low-dose postbiotic group prepared in Example 2, H is ileum staining of the low-dose postbiotic group prepared in Example 2, I is spleen staining of the high-dose postbiotic group prepared in Example 2, and J is ileum staining of the high-dose postbiotic group prepared in Example 2. The scale bar is 50 μm.

[0028] Figure 7 Flowchart for the isolation and identification of lactic acid bacteria from traditional fermented dairy products;

[0029] Figure 8 This is the electrophoresis diagram of the PCR amplification product of the 16S rRNA gene of strain KLDS1.1105;

[0030] Figure 9 Phylogenetic tree based on the 16S rRNA gene of strain KLDS1.1105. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] Sources of materials used in the present invention: traditional fermented dairy products are Inner Mongolia yogurt, MRS liquid culture medium is purchased from Qingdao Haibo Biotechnology Co., Ltd.; skim milk with a fat content of ≤0.5% is purchased from Fonterra Company of New Zealand.

[0037] Example 1

[0038] The process of screening and isolation of Lactobacillus helveticus KLDS1.1105:

[0039] Lactic acid bacteria in fermented milk are isolated using the bromocresol green (BCG) milk nutrient agar plate separation method. Bromocresol green, an indicator, appears yellow in acidic environments and blue in alkaline environments. Adding bromocresol green to the separation medium (pH 6.8) turns it blue-green. Lactic acid bacteria grow in this medium and break down lactose, producing lactic acid, which causes the colonies to appear yellow, and the surrounding medium also turns yellow. In medium containing CaCO₃, the lactic acid produced by lactic acid bacteria dissolves the CaCO₃ in the medium, creating a transparent zone around the colonies. After preliminary identification, lactic acid bacteria should undergo physiological and biochemical tests to determine their genus, and methods such as 16S rRNA sequence analysis should be combined to accurately identify the species.

[0040] like Figure 7 The figure shows the flow chart for isolating and identifying lactic acid bacteria in traditional fermented dairy products. The process includes the sample to be isolated, gradient dilution, plate isolation and culture, observation of colony characteristics, picking of single colonies, purification culture, microscopic examination of individual morphology and purity, H2O2 enzyme test, subculture, selection of curd tubes, lactic acid determination and strain preservation.

[0041] Sample dilution: Use a sterile pipette to draw 5 mL of the sample to be separated and transfer it into a flask containing 45 mL of sterile saline with glass beads. Shake thoroughly to mix. -1 Take another pipette from 10 -1 1 mL was drawn into the flask and 10 -2 Sterile saline solution was pipetted repeatedly to mix, and then diluted in 10-fold gradients to 10 -6 , 10 -7 Dilution.

[0042] Plate separation and culture: take the above 10 -6 , 10 -7 Pour 1 mL of each dilution into a sterile Petri dish, performing two replicates for each dilution. Aseptically add sterilized CaCO₃₃ to melted acidified MRS agar medium at a concentration of 20-30 g / L. Rapidly cool the medium to 50°C in tap water. While cooling, shake the CaCO₃ in the bottle to mix thoroughly (avoiding bubbles). Immediately pour the solution into the Petri dish and shake thoroughly (do not allow the CaCO₃ to settle at the bottom of the dish). Ensure that the sample dilution and CaCO₃ are evenly distributed throughout the medium. After the medium solidifies, invert and incubate in an incubator at 30°C (for kimchi juice samples) or 40°C (for yogurt samples) for 48 hours.

[0043] Observe the characteristics of the colonies: a CaCO3 dissolution zone is produced around the colonies, the colonies are 1 to 3 mm in diameter, the edges are irregular, white to grayish white, and the colony surface is relatively rough.

[0044] Purification culture: Pick 6 typical single colonies and inoculate them into MRS liquid culture tubes respectively, and culture them in a 40℃ incubator for 24 hours.

[0045] Microscopic examination of morphology: Take one loop of the above test tube liquid culture, perform smear and Gram staining, and observe the individual morphology and purity under an oil microscope. + The cells are approximately 2 μm wide and are rod-shaped, varying in length, and may grow singly, in pairs, or in filaments. At the same time, a loopful of culture medium was mixed with 3% H2O2 on a glass slide and the formation of bubbles was observed.

[0046] Lactic acid determination: The supernatant of the above test tube culture was taken and the production of lactic acid was detected by paper chromatography.

[0047] The results showed that the bacteria fermented fructose, galactose, sucrose, glucose, and lactose, but not arabinose or raffinose. The optimal pH was 5.5-6.2, and the optimal fermentation temperature was 37°C. Gram staining revealed long, solitary rods, and the bacteria produced lactobacilli.

[0048] Amplify the 16S rRNA gene Figure 8 As shown, the bands of the PCR products of the 16S rRNA gene of the strain KLDS1.1105 to be sequenced are clear and bright, the amplification specificity is strong, and the size of the PCR products is basically consistent with the size of the 16S rRNA gene fragment of the bacteria, which is about 1500 bp.

[0049]

[0050] The amplified 16s rRNA gene products were sequenced and the phylogenetic tree was constructed based on the results (e.g. Figure 9 The results showed that the type strain L. helveticus DSM 20075 was very closely related to L. acidophilus ATCC 4356 (100%), and the test strain KLDS1.1105 was the closest related to L. helveticus DSM 20075 (100%). Therefore, the strain KLDS1.1105 was inferred to be Lactobacillus helveticus from the phylogenetic tree.

[0051] Example 2

[0052] (1) Lactobacillus helveticus KLDS1.1105 (inoculation amount according to 5% of the volume of MRS liquid culture medium) was inoculated into MRS liquid culture medium, activated and cultured at 37°C for 16 hours, and repeated 3 times to obtain activated Lactobacillus helveticus KLDS1.1105;

[0053] (2) The activated Lactobacillus helveticus KLDS1.1105 (inoculation amount is 5% of the volume of skim milk with fat content ≤ 0.5%) was inoculated into skim milk with fat content ≤ 0.5%, and cultured at 37°C for 18 h to obtain a fermentation broth (the concentration of Lactobacillus helveticus KLDS1.1105 was 5.5 × 10 9 CFU / mL), with an inlet air temperature of 140-185°C, an exhaust air temperature of 68-78°C, a feed flow rate of 7.5 mL / min, and a drying air flow rate of 32.5 m 3 / h conditions to spray dry and obtain postbiotics.

[0054] Example 3

[0055] (1) Lactobacillus helveticus KLDS1.1105 (inoculation amount according to 3% of the volume of MRS liquid culture medium) was inoculated into MRS liquid culture medium, activated and cultured at 36°C for 14 h, and repeated 3 times to obtain activated Lactobacillus helveticus KLDS1.1105;

[0056] (2) The activated Lactobacillus helveticus KLDS1.1105 (inoculation amount is 4% of the volume of skim milk with fat content ≤ 0.5%) was inoculated into skim milk with fat content ≤ 0.5%, and cultured at 35°C for 16 h to obtain a fermentation broth (the concentration of Lactobacillus helveticus KLDS1.1105 was 4 × 10 9 CFU / mL), with an inlet air temperature of 140-185°C, an exhaust air temperature of 45-50°C, a feed flow rate of 7 mL / min, and a drying air flow rate of 30 m 3 / h conditions to spray dry and obtain postbiotics.

[0057] Example 4

[0058] (1) Lactobacillus helveticus KLDS1.1105 (inoculation amount according to 6% of the volume of MRS liquid culture medium) was inoculated into MRS liquid culture medium, activated and cultured at 38°C for 18 hours, and repeated 3 times to obtain activated Lactobacillus helveticus KLDS1.1105;

[0059] (2) The activated Lactobacillus helveticus KLDS1.1105 (inoculation amount is 5% of the volume of skim milk with fat content ≤ 0.5%) was inoculated into skim milk with fat content ≤ 0.5%, and cultured at 37°C for 24 h to obtain a fermentation broth (the concentration of Lactobacillus helveticus KLDS1.1105 was 7 × 10 9 CFU / mL), with an inlet air temperature of 140-185°C, an exhaust air temperature of 50-68°C, a feed flow rate of 8 mL / min, and a drying air flow rate of 35 m 3 / h conditions to spray dry and obtain postbiotics.

[0060] Experimental Example 1

[0061] (1) Animal experiment design:

[0062] SPF-grade male BALB / c mice, 6–8 weeks old, weighing 18–22 g, were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. (license number SCXK(Beijing)2019-0010). They were housed in a clean laboratory animal room at 20–23°C, with alternating dark and light cycles of 12 h each day, and had free access to water and food.

[0063] Thirty BALB / c mice were randomly divided into five groups after 7 days of adaptive feeding. The experimental design is shown in Table 1. Except for the normal group, the remaining groups were intraperitoneally injected with 80 mg / kg bw cyclophosphamide (CTX) every day, and the normal group was intraperitoneally injected with an equal amount of normal saline for 3 consecutive days to establish an immunocompromised mouse model. Starting from the 4th day, the low-dose group and the high-dose group were gavaged with 600 and 1200 mg / kg bw of the postbiotics prepared in Example 2, respectively, the control group was gavaged with 30 mg / kg bw levamisole hydrochloride (LEV), and the normal group and the model group were gavaged with an equal amount of normal saline for 17 consecutive days. The mice were weighed every day during the experiment. After the last dose, the mice were fasted but not watered for 12 hours and killed, and serum, thymus, spleen, etc. were taken for subsequent experiments.

[0064] Table 1 Experimental design

[0065]

[0066] (2) Immune organ index:

[0067] The immune organ index is an important indicator of the body's immune status. The absolute weight and relative weight of the immune organs reflect the strength of the body's cellular and humoral immune functions.

[0068] Immune organ index = immune organ mass (g) / body mass (g).

[0069] Depend on Figure 1 China A and Figure 1 As shown in Figure B, intraperitoneal injection of CTX damaged the immune organs of mice, resulting in significantly lower spleen and thymus indexes in the model group than in the normal group (P<0.05). Oral administration of the postbiotics prepared in Example 2 to the low-dose and high-dose groups repaired the immune organs of mice and significantly increased the immune organ indexes (P<0.05). This indicates that the postbiotics prepared in Example 2 can repair immune organ damage and maintain normal immune organ function.

[0070] (3) Splenic lymphocyte proliferation rate:

[0071] Splenic lymphocyte proliferation rate is the proliferation rate or activity of lymphocytes in the spleen. Lymphocytes are key members of the immune system and they play a vital role in the human immune response.

[0072] The spleens of mice in the normal, model, control, low-dose, and high-dose groups were isolated and single-cell suspensions were prepared and plated in 96-well plates at 3,000 to 10,000 cells per well. Splenocytes from each group were divided into two wells, 7.5 μg / mL ConA solution (purchased from Beijing Solebow Technology Co., Ltd.) was added to one well, and the same volume of RPMI 1640 complete medium (purchased from Beijing Bio-Toda Technology Co., Ltd.) was added to the other well. Each group was repeated three times and cultured in a 37°C, 5% CO2 incubator for 72 h. Four hours before the end of the culture, the supernatant was carefully aspirated, 90 μL RPMI 1640 complete medium was added, and then 10 μL MTT solution (purchased from Beijing Solebow Technology Co., Ltd.) was added. The culture was continued for another 4 h, the supernatant was aspirated, and 110 μL DMSO (purchased from Beijing Solebow Technology Co., Ltd.) was added to each well. The plates were shaken at low speed for 10 min to fully dissolve the crystals. The absorbance of each well was measured at 490 nm using a microplate reader. The proliferation rate of mouse spleen lymphocytes was calculated as follows:

[0073] Splenic lymphocyte proliferation rate (%) = (OD A490nm -OD of group without ConA A490nm ) / OD of group without ConA A490nm .

[0074] Depend on Figure 2As shown in Figure A, the spleen lymphocyte proliferation rate of the model group was significantly lower than that of the normal group (P<0.05). After treatment with the postbiotics prepared in Example 2, the spleen lymphocyte proliferation rate of mice in the low-dose group and the high-dose group increased significantly (P<0.05). This indicates that the postbiotics prepared in Example 2 can repair the immune damage caused by CTX by improving the proliferation ability of spleen lymphocytes and improve the adaptive immune system, which is consistent with the results of the spleen index experiment.

[0075] (4) NK cell killing activity assay:

[0076] NK cells are cytotoxic lymphocytes of the innate immune system that can kill virally infected and / or cancerous cells. The strength of NK cell killing activity is of great significance in immune regulation and anti-tumor treatment.

[0077] Target cells (YAC-1) were subcultured 24 h before the experiment, washed three times with 1× PBS before use, and the cell concentration was adjusted to 4×10 5 The spleen cell concentration was adjusted to 2×10 cells / mL using RPMI1640 complete culture medium. 7 100 μL of target cells and effector cells (50:1 effector-target ratio) were taken and added to a 96-well culture plate. 100 μL of target cells and culture medium were added to the wells with natural target cell release, and 100 μL of target cells and 2.5% Triton X-100 were added to the wells with maximum target cell release. Three replicates were set up for each of the above items and cultured in a 37°C, 5% CO2 incubator for 4 h. 100 μL of supernatant was aspirated from each well and placed in a 96-well culture plate. 100 μL of LDH matrix solution (purchased from Tianjin Maiji Biotechnology Co., Ltd.) was added at the same time. The reaction was carried out for 10 min. 30 μL of 1 mol / L HCl was added to each well, and the absorbance of each well was measured at 490 nm on a microplate reader.

[0078] NK cell activity (%) = (OD A490nm -Natural release hole OD A490nm ) / (maximum release hole OD A490nm -Natural release hole OD A490nm )×100%.

[0079] Depend on Figure 2 As shown in Figure B, the NK cell killing activity in the model group was significantly lower than that in the normal group (P < 0.05). After treatment with the postbiotics prepared in Example 2, the NK cell killing activity of mice in the low-dose and high-dose groups significantly increased (P < 0.05). This indicates that the postbiotics prepared in Example 2 can repair the immune damage caused by CTX by increasing the NK cell killing activity and enhance the body's immunity.

[0080] (5) Splenic T lymphocyte subset differentiation:

[0081] Th17 and Treg cells are two different types of CD4 + T cell subsets have opposite functions in immune responses. Under normal conditions, Th17 and Treg cells coexist and maintain a balance in the body. However, once the Th17 / Treg balance is disrupted, chronic inflammation and autoimmune diseases may occur.

[0082] Adjust the splenocyte cell density to 1×10 cells / mL using RPMI 1640 complete medium. 7 / mL, fluorescent antibody staining was performed using a mouse flow cytometry staining kit (purchased from Hangzhou Lianke Biotechnology Co., Ltd.) according to the kit instructions, and then the ratio of mouse Th17 / Treg cells was analyzed by flow cytometry.

[0083] Depend on Figure 3 and Figure 4 Middle A, Figure 4 Middle B and Figure 4 As shown in Figure C, compared with the normal group, the percentage of Th17 cells in the model group was significantly reduced and the percentage of Treg cells was significantly increased (P<0.05). The percentage of Th17 cells in the low-dose group and the high-dose group of mice after treatment with the postbiotics prepared in Example 2 was significantly increased, and the percentage of Treg cells was significantly decreased (P<0.05). The ratio of Th17 cells to Treg cells was further calculated, and it was found that the Th17 / Treg ratio in the model group was significantly lower than that in the normal group (P<0.05). The Th17 / Treg ratio in the low-dose group and the high-dose group after treatment with the postbiotics prepared in Example 2 was significantly increased (P<0.05).

[0084] (6) Serum cytokines:

[0085] IL-17 is a key pro-inflammatory cytokine that promotes the recruitment and activation of inflammatory cells and increases local and systemic inflammatory responses by promoting the production of cytokines, chemokines, and pro-inflammatory mediators. TGF-β plays a key role in the immune system, inhibiting the activation of T and B cells and regulating macrophage function, thereby maintaining immune tolerance and preventing autoimmune reactions.

[0086] Mouse blood was collected and centrifuged at 300 g for 10 min to separate serum, which was then tested using mouse IgM, IgG, and IgA Elisa kits (purchased from Jiangsu Enzyme Immunity Industry Co., Ltd.).

[0087] Depend on Figure 5 China A and Figure 5As shown in Figure B, compared with the normal group, the serum Th17 cytokine (IL-17) in the model group was significantly reduced, and the Treg cytokine (TGF-β) was significantly increased, with statistically significant differences (P < 0.05). In the low-dose and high-dose groups treated with the postbiotics prepared in Example 2, the Th17 cytokine (IL-17) was significantly increased, and the Treg cytokine was significantly decreased.

[0088] (7) Serum immunoglobulins:

[0089] Immunoglobulins are antibodies synthesized by B lymphocytes. They are a major component of humoral immunity and an important parameter reflecting the body's immune capacity. Blood was collected from mice and centrifuged at 300g for 10 minutes to separate serum. Mouse IL-17 and TGF-β ELISA kits (purchased from Jiangsu Enzyme Immunity Industry Co., Ltd.) were used for detection.

[0090] Depend on Figure 5 Middle C, Figure 5 D and Figure 5 As shown in Figure E, compared with the normal group, the serum IgA, IgG, and IgM levels in the model group were significantly reduced (P < 0.05). After treatment with the postbiotics prepared in Example 2, the serum IgA, IgG, and IgM levels of mice in the low-dose and high-dose groups recovered to varying degrees (P < 0.05). This indicates that the postbiotics prepared in Example 2 can restore the decrease in immunoglobulin content caused by CTX, thereby enhancing the body's humoral immune function.

[0091] (8) Intestinal tissue SIgA:

[0092] SIgA is the predominant antibody class in intestinal mucosal secretions, and the majority of IgA-producing plasma cells are located within the mucosal membrane lining the intestine. SIgA provides protection by preventing pathogens from adhering to and penetrating the intestinal barrier.

[0093] The frozen intestinal tissue was homogenized at 4°C with PBS (pH 7.4), and the supernatant was separated at 300 g for 10 min. The mouse SIgAElisa kit (purchased from Jiangsu Enzyme Immunity Industry Co., Ltd.) was used for detection.

[0094] Depend on Figure 5 As shown in Figure 5, CTX induced intestinal mucosal damage in the model group mice and significantly inhibited the secretion of SIgA (P < 0.05). The intestinal SIgA levels in the low-dose and high-dose groups treated with the postbiotics prepared in Example 2 were significantly increased (P < 0.05). This shows that the postbiotics prepared in Example 2 can enhance intestinal SIgA secretion, protect the intestinal immune barrier, and improve immunity.

[0095] (9) Organizational Observation:

[0096] The spleen and thymus of mice were collected, fixed with 4% paraformaldehyde and embedded in paraffin. Pathological sections (4 μm) were then made using a freezing microtome. The sample sections were stained with hematoxylin and eosin (H&E) and observed under an optical microscope. Images were taken from different fields of view to observe the morphological changes of the mouse spleen and thymus.

[0097] Depend on Figure 6 Middle A~ Figure 6 As can be seen from Figure 5, the white pulp and red pulp structures in the spleen of the normal group are obvious, with clear boundaries and no necrotic tissue. Obvious lesions appeared in the spleen tissue of the mice in the model group, characterized by incomplete structures of the white pulp and red pulp, and the lack of a clear boundary between the two. After treatment with the postbiotics prepared in Example 2, the spleen integrity of the mice in the low-dose and high-dose groups was restored, the area of ​​the white pulp increased, and the boundary between the white pulp and red pulp gradually became clear. The small intestine of the mice in the normal group had good structural organization, and the villi were slender, complete, and closely connected. The small intestinal villi of the mice in the model group were incomplete, and their integrity was destroyed. After treatment with the postbiotics prepared in Example 2, the length of the small intestinal villi increased and the small intestinal structure was restored. This indicates that the postbiotics prepared in Example 2 have the potential to repair immune organ damage, strengthen the intestinal immune barrier, and enhance overall immunity.

[0098] In summary, the postbiotics prepared using Lactobacillus helveticus KLDS1.1105 in this study have the ability to regulate T cell differentiation, restore the Th17 / Treg balance, increase levels of the pro-inflammatory cytokine IL-17, and reduce levels of the anti-inflammatory cytokine TGF-β. These effects help balance cellular and humoral immunity in mice, repair immune organ damage, strengthen the intestinal immune barrier, and enhance immune function.

[0099] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A Lactobacillus helveticus KLDS1.1105, characterized in that The Lactobacillus helveticus KLDS1.1105 was deposited in the General Microbiology Center of the China Culture Collection Administration on September 12, 2024, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being: CGMCC No. 31947.

2. A method for preparing postbiotics using the Lactobacillus helveticus KLDS1.1105 of claim 1, characterized in that: The following steps are involved: (1) inoculating the Lactobacillus helveticus KLDS1.1105 into an activation culture medium, performing activation culture, and repeating the activation culture three times to obtain activated Lactobacillus helveticus KLDS1.1105; (2) inoculating the activated Lactobacillus helveticus KLDS1.1105 obtained in step (1) into a milk-based culture medium, culturing, and spray-drying the fermentation liquid to obtain postbiotics.

3. The method for preparing postbiotics according to claim 2, characterized in that: The inoculation amount of Lactobacillus helveticus KLDS1.1105 in step (1) is calculated as 3% to 6% of the volume of the activation culture medium; the activation culture medium in step (1) is MRS liquid culture medium.

4. The method for preparing postbiotics according to claim 2, characterized in that: The temperature of the activation culture in step (1) is 36-38° C., and the time of the activation culture is 14-18 hours.

5. The method for preparing postbiotics according to claim 2, characterized in that: The inoculation amount of the activated Lactobacillus helveticus KLDS1.1105 in step (2) is calculated as 4-6% of the volume of the milk-based culture medium; the milk-based culture medium in step (2) is skim milk with a fat content of ≤0.5%.

6. The method for preparing postbiotics according to claim 2, characterized in that: The culture temperature in step (2) is 35-37°C, and the culture time is 16-24h; the inlet air temperature of the spray drying in step (2) is 140-185°C, the exhaust air temperature of the spray drying is 45-78°C, the feed flow rate of the spray drying is 7-8 mL / min, and the drying air flow rate of the spray drying is 30-35 m / min. 3 / h; the concentration of Lactobacillus helveticus KLDS1.1105 in the fermentation broth was 4×10 9 ~7×10 9 CFU / mL.

7. The postbiotics prepared by the method for preparing postbiotics using Lactobacillus helveticus KLDS1.1105 according to any one of claims 2 to 6.

8. Use of the postbiotic according to claim 7 in preparing a product having enhanced immunomodulatory ability.

9. Use of the postbiotic according to claim 7 in preparing a product having the function of repairing immune organ damage and enhancing humoral immunity.

10. Use of the postbiotic according to claim 7 in preparing a product capable of enhancing intestinal immune barrier function.

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