Application of lactobacillus rhamnosus GOLDGUT-M520 or composition of lactobacillus rhamnosus GOLDGUT-M520 in preparation of product for enhancing organism immunity

Abstract

The invention provides application of lactobacillus rhamnosus GOLDGUT-M520 or a composition of the lactobacillus rhamnosus GOLDGUT-M520 in preparation of a product for enhancing the immunity of an organism. The lactobacillus rhamnosus GOLDGUT-M520 has the function of enhancing the immunity in both a viable form and an inactivated form of the lactobacillus rhamnosus GOLDGUT-M520. The lactobacillus rhamnosus GOLDGUT-M520 has a good antibacterial capability on escherichia coli and staphylococcus aureus which are pathogenic to intestinal tracts, and is beneficial to enhancing a chemical barrier in an innate immune barrier; the strain can improve the phagocytic activity of macrophages and enhance the innate immune response; the strain can improve the T cell proportion, the B cell proportion and the immune globulin level in mesenteric lymph nodes of immunocompromised mice, and can enhance adaptive immune response. The lactobacillus rhamnosus GOLDGUT-M520 can comprehensively play a role in enhancing immunity, and meanwhile, the inherent immune barrier, the inherent immune response and the adaptive immune response are enhanced.

Description

Technical Field

[0001] This application belongs to the field of microbial technology, and in particular relates to the application of Lactobacillus rhamnosus GOLDGUT-M520 or a combination thereof in the preparation of products that enhance the body's immunity. Background Technology

[0002] The innate immune barrier, innate immune response, and adaptive immune response are the three core components of the human immune system. The barrier structure formed by the skin, mucous membranes, and their appendages (the innate immune barrier) is the body's first line of defense against invading pathogens. The innate immune barrier consists of physical, chemical, and microbial barriers. The chemical barrier is composed of antimicrobial components in various body fluids. If the skin / mucous membranes are damaged, bacteria and viruses can easily invade. This triggers an innate immune response. If the innate immune response cannot completely eliminate these pathogens, a subsequent specific adaptive immune response will be triggered to clear these pathogens and protect the body. Immunity is a crucial defense mechanism for the human body to resist pathogen invasion and maintain its health. Studies have reported that *Lactobacillus rhamnosus* has immune-enhancing effects; however, the effectiveness and mechanism of action of this immune enhancement vary among different strains. Summary of the Invention

[0003] This application provides the use of Lactobacillus rhamnosus GOLDGUT-M520 or a combination thereof in the preparation of products that enhance the body's immunity. It can comprehensively enhance immunity, while strengthening the innate immune barrier, innate immune response, and adaptive immune response, thereby achieving a more comprehensive enhancement of immune function.

[0004] In a first aspect, this application provides the use of *Lactobacillus rhamnosus* GOLDGUT-M520 or a composition thereof in the preparation of products that enhance the body's immunity. The composition comprises at least one of *Lactobacillus rhamnosus* GOLDGUT-M520, its progeny, inactivated bacterial cells, metabolites, and bacterial components, and includes at least one of the following microorganisms: bacteria and fungi. The *Lactobacillus rhamnosus* GOLDGUT-M520 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26205. The bacteria include at least one specific strain selected from *Lactobacillus*, *Bifidobacterium*, *Bacillus*, *Propionibacterium*, *Streptococcus*, *Lactococcus*, and *Pediococcus*. The fungi include at least one specific strain selected from *Saccharomyces cerevisiae*, *Saccharomyces boulardii*, and *Kluyveromyces marxianus*.

[0005] In some embodiments, the Lactobacillus rhamnosus GOLDGUT-M520 is used to produce antimicrobial substances to enhance the chemical barrier in the innate immune barrier; the antimicrobial substances are used to inhibit pathogenic microorganisms, including at least one of enteropathogenic Escherichia coli and Staphylococcus aureus.

[0006] In some embodiments, the Lactobacillus rhamnosus GOLDGUT-M520 is used to enhance the phagocytic activity of macrophages and strengthen the innate immune response.

[0007] In some embodiments, the Lactobacillus rhamnosus GOLDGUT-M520 is used to improve organ indices and enhance immune function in immunocompromised mice.

[0008] In some embodiments, the Lactobacillus rhamnosus GOLDGUT-M520 is used to increase at least one of the proportion of T cells, the proportion of B cells, and the level of immunoglobulins in the mesenteric lymph nodes of immunocompromised mice, thereby enhancing the adaptive immune response; wherein the immunoglobulins include at least one of immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin G (IgG), and secretory immunoglobulin A (sIgA).

[0009] In some embodiments, the product includes biological products, food, or pharmaceuticals.

[0010] In some embodiments, the biological product includes microbial inoculum of Lactobacillus rhamnosus GOLTGU-M520, its subcultured offspring, mutant strains, or fermentation agents.

[0011] In some embodiments, the food includes at least one of soy products, dairy products, fruit and vegetable products, or health foods, and the health foods include at least one of solid beverages and compressed candies.

[0012] Compared with the prior art, the present invention has the following beneficial effects: 1) Lactobacillus rhamnosus GOLDGUT-M520 can comprehensively enhance immunity, while strengthening the innate immune barrier, innate immune response, and adaptive immune response, thus achieving a more comprehensive enhancement of immune function. 2) Lactobacillus rhamnosus GOLDGUT-M520 can produce antimicrobial components, which have a good inhibitory effect on enteropathogenic Escherichia coli and Staphylococcus aureus, and help to enhance the chemical barrier in the innate immune barrier; 3) Lactobacillus rhamnosus GOLDGUT-M520 can enhance the phagocytic activity of macrophages, which helps to strengthen the innate immune response; 4) Lactobacillus rhamnosus GOLDGUT-M520 can increase the proportion of T cells, B cells, and the levels of IgA, IgM, IgG, and sIgA in the mesenteric lymph nodes of immunocompromised mice, which helps to enhance adaptive immunity. 5) Lactobacillus rhamnosus GOLDGUT-M520 can increase the thymus index and spleen index in immunocompromised mice, thus enhancing immunity. Attached Figure Description

[0013] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0014] Figure 1 The results of thymus index detection by GOLDGUT-M520 provided in the embodiments of this application; Figure 2 The results of spleen index detection by GOLDGUT-M520 provided in the embodiments of this application; Figure 3 The results of the detection of the proportion of T cells in mesenteric lymph nodes using GOLDGUT-M520 provided in the embodiments of this application; Figure 4 The results of the detection of the proportion of B cells in mesenteric lymph nodes using GOLDGUT-M520 provided in the embodiments of this application; Figure 5 The results of detecting IgA in mouse serum using GOLDGUT-M520 provided in the embodiments of this application; Figure 6 The results of detecting IgM in mouse serum using GOLDGUT-M520 provided in the embodiments of this application; Figure 7 The results of detecting IgG in mouse serum using GOLDGUT-M520 provided in the embodiments of this application; Figure 8 The results of the detection of sIgA in the mouse intestine by GOLDGUT-M520 provided in the embodiments of this application.

[0015] in, Figures 1-8 In comparison with the model group, This indicates that p < 0.05. This indicates that p < 0.01. This indicates that p < 0.001. # indicates p < 0.0001; compared with the normal control group, # indicates p < 0.05, ## indicates p < 0.01, ### indicates p < 0.001, and #### indicates p < 0.0001. Detailed Implementation

[0016] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0017] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0018] The innate immune barrier, innate immune response, and adaptive immune response are the three core components of the human immune system. The barrier structure formed by the skin, mucous membranes, and their appendages (the chemical barrier within the innate immune barrier) is the body's first line of defense against invading pathogens. If the skin / mucous membranes are damaged, bacteria and viruses can easily invade. This triggers an innate immune response. Phagocytes, cells with phagocytic and killing functions, are the main effector cells of the innate immune system. The adaptive immune response, also known as the specific immune response, is an antigen-specific immune functional response induced by antigens. If the innate immune response cannot completely eliminate these pathogens, a subsequent specific adaptive immune response will occur to clear these pathogens, protect the body, and form immune memory. When encountering the same pathogen again, a faster and stronger immune response will be generated. In this process, immunoglobulins, as key proteins that can directly react with antigens, are not only important products of the immune response but also core indicators of the strength of the body's immunity. Among them, IgG is the immunoglobulin that plays a major role when the body is attacked again by pathogens such as bacteria and viruses. It has high affinity, wide distribution, and is the "main force" of the body's fight against infection. IgM has membrane-bound and secretory forms. It is the earliest antibody synthesized and secreted during individual development and also the earliest antibody to appear in the primary humoral immune response. It plays an important immune defense role in early infection (primary immune response). IgA participates in local mucosal immunity and protects the mucosa. slgA (secretory immunoglobulin A) is an important response antibody in intestinal mucosal immunity. When secreted into the intestinal lumen, it can prevent the adhesion of pathogens to intestinal epithelial cells through immune rejection.

[0019] Probiotics can interact with the host's immune system to promote immune balance and enhance the host's immune defense capabilities. Probiotics have a regulatory effect on the host's innate and adaptive immune responses. They can activate and enhance the phagocytic activity of macrophages in different sites, thereby strengthening immunity through increasing immunoglobulin levels, promoting immune cell maturation, and regulating inflammatory responses.

[0020] Therefore, screening probiotics that enhance the innate immune barrier, improve the innate immune response, and improve the adaptive immune response is of great significance, as it can more comprehensively enhance the body's immunity.

[0021] The existing technologies for enhancing immunity have the following technical problems: 1) Existing Lactobacillus rhamnosus strains primarily target innate and adaptive immune responses to enhance immunity, with fewer strains simultaneously enhancing the innate immune barrier, innate immune response, and adaptive immune response. 2) Among the existing published patents, there are few Lactobacillus rhamnosus with the function of enhancing immunoglobulins, and most are compositions containing Lactobacillus rhamnosus.

[0022] To address the aforementioned technical problems, this application provides the use of Lactobacillus rhamnosus GOLDGUT-M520 or a combination thereof in the preparation of products that enhance the body's immunity, which can enhance the innate immune barrier, innate immune response, and adaptive immune response.

[0023] The Lacticaseibacillus rhamnosus strain GOLDGUT-M520 in the following examples has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCCNo.26205, on December 14, 2022. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0024] Example 1: Inhibitory effect of Lactobacillus rhamnosus GOLDGUT-M520 on intestinal pathogens I. Experimental Methods Lactobacillus rhamnosus GOLDGUT-M520 was activated by inoculation into MRS broth medium, and the supernatant was collected after centrifugation for analysis. Enteropathogenic Escherichia coli 340977 and Staphylococcus aureus 186335 were inoculated into LB liquid medium via glycerol tubes at a 3% inoculation rate and incubated at 37°C for 48 h. A suitable concentration of pathogenic bacterial culture was added to LB solid medium, poured into plates, and agar plates containing pathogenic bacteria were prepared. Wells were punched, and probiotic supernatant was added. After drying, the plates were incubated at 37°C, and inhibition zones were observed.

[0025] II. Experimental Results The inhibition zone diameters of Lactobacillus rhamnosus GOLDGUT-M520 against enteropathogenic Escherichia coli and Staphylococcus aureus were 1.2 cm and 1.1 cm, respectively.

[0026] III. Experimental Conclusions Lactobacillus rhamnosus GOLDGUT-M520 has a good inhibitory effect on enteropathogenic Escherichia coli and Staphylococcus aureus, indicating that it can produce antimicrobial components and help enhance the chemical barrier in the innate immune barrier.

[0027] Example 2: Enhancement of macrophage phagocytic activity by Lactobacillus rhamnosus GOLDGUT-M520 I. Experimental Methods Resuscitated RAW246.7 macrophages were seeded in DMEM complete medium containing 10% fetal bovine serum and 1% penicillin or streptomycin and cultured in a 37°C, 5% CO2 cell culture incubator. When the cells reached 80% confluence with the bottom of the culture flask, they were passaged, and cells in the logarithmic growth phase were used for experiments. Sample preparation: *Lactobacillus rhamnosus* GOLDGUT-M520 cells were heat-inactivated to obtain heat-inactivated bacterial cells. RAW264.7 cells were cultured at 2 × 10⁻⁶ cells / year. 4 Cells were inoculated per well into 96-well plates and incubated at 37°C for 24 hours. The culture medium was then discarded. Experimental groups were set up with culture medium containing *Lactobacillus rhamnosus* GOLDGUT-M520 cells (MOI 1:100), and a normal control group was set up with culture medium added. After incubation at 37°C for 24 hours, the culture medium was discarded. Cell phagocytic activity was measured using the neutral red assay according to the instructions of the neutral red blood cell proliferation and cytotoxicity assay kit.

[0028] II. Experimental Results The phagocytosis rate of neutral red by phagocytes in the normal control group was 100%, while the phagocytosis rate of neutral red by Lactobacillus rhamnosus GOLDGUT-M520 group was 116.14%.

[0029] III. Experimental Conclusions Lactobacillus rhamnosus GOLDGUT-M520 can enhance the phagocytic activity of macrophages, which helps to strengthen the innate immune response.

[0030] Example 3: Enhancement of immune function in immunocompromised mice by Lactobacillus rhamnosus GOLDGUT-M520 I. Experimental Methods SPF-grade male BALB / c mice were randomly divided into a normal control group, a model group, a positive control group (levamisole hydrochloride), and a probiotic group (GOLDGUT-M520). Except for the normal control group, all other groups received intraperitoneal injections of cyclophosphamide (50 mg / kg) for three consecutive days to establish an immunodeficient mouse model. After successful model establishment, the mice underwent a 4-week treatment regimen of probiotics / drugs, with the probiotic group receiving 2 × 10⁻⁶ mg / kg. 9 CFU / mouse / day, with the positive control group receiving levamisole hydrochloride (40 mg / kg) by gavage. To prevent the recovery of immune function, mice were supplemented with cyclophosphamide (50 mg / kg) every 7 days. After 4 weeks, peripheral blood, thymus, spleen, and mesenteric lymph nodes were collected from mice. Splenic cell culture, flow cytometry, and enzyme-linked immunosorbent assay (ELISA) were used to detect organ indices, the proportion of T cells and B cells in the mesenteric lymph nodes, and immunoglobulin content, evaluating the immune-enhancing effect of GOLDGUT-M520 on immunocompromised mice.

[0031] (1) Determination of organ indices in mice: The thymus and spleen of mice were dissected, the water was removed, and the weight was recorded. The thymus index and spleen index were calculated based on the body weight.

[0032] (2) Determination of the proportion of T cells and B cells in mesenteric lymph nodes: Mesenteric lymph node cell suspensions from each group of mice were placed in flow cytometry tubes, and flow cytometry antibodies were added to each tube. After mixing, the cells were incubated at room temperature in the dark for 30 min. Negative and monostained control tubes were also set up. Changes in cell proportions were detected using flow cytometry.

[0033] (3) Determination of immunoglobulin content: The levels of IgG, IgM, IgA and sIgA were detected using an ELISA kit.

[0034] II. Experimental Results (1) Effects of Lactobacillus rhamnosus GOLDGUT-M520 on organ indices in immunocompromised mice Organ index analysis was performed on the thymus and spleen of mice. The results of the thymus index and spleen index are as follows: Figure 1 , Figure 2 The results showed that, compared with the normal control group, the thymus and spleen of the model group mice were shrunken, and both the thymus index and spleen index were significantly decreased (p<0.0001). Compared with the model group, the Lactobacillus rhamnosus GOLDGUT-M520 group significantly increased the thymus index and spleen index (p<0.001), reversing the shrinkage of the thymus and spleen in mice, and the effect was comparable to that of the positive control group. The thymus and spleen indices are important indicators for measuring the function of the body's immune system. This indicates that Lactobacillus rhamnosus GOLDGUT-M520 can enhance immune function by increasing the thymus index and spleen index.

[0035] (2) Effects of Lactobacillus rhamnosus GOLDGUT-M520 on the proportion of T cells and B cells in the mesenteric lymph nodes of immunocompromised mice Flow cytometry was used to detect the proportions of T cells and B cells in the mesenteric lymph nodes of mice. The results are as follows: Figure 3 , Figure 4 As shown, compared with the normal control group, the proportions of T cells and B cells in the mesenteric lymph nodes of the model group mice were significantly decreased (p<0.0001). Compared with the model group, the proportions of T cells and B cells in the mesenteric lymph nodes of mice in the *Lactobacillus rhamnosus* GOLDGUT-M520 group were significantly increased. The recovery effect of GOLDGUT-M520 on the proportions of T cells and B cells was comparable to that of the positive control group. This indicates that *Lactobacillus rhamnosus* GOLDGUT-M520 can increase the proportions of T cells and B cells in the mesenteric lymph nodes of mice, which helps to enhance adaptive immunity.

[0036] (3) Effects of Lactobacillus rhamnosus GOLDGUT-M520 on immunoglobulins in immunocompromised mice The levels of IgA, IgM, and IgG immunoglobulins in mouse serum and sIgA immunoglobulin in the intestine were detected by ELISA. The results are as follows: Figures 5-8 As shown, compared with the normal control group, the serum IgA, IgM, and IgG levels in the model group were significantly decreased (p<0.001), and the intestinal sIgA level was also significantly decreased (p<0.001). Compared with the model group, the serum IgA, IgG, and IgM levels in the *Lactobacillus rhamnosus* GOLDT-M520 group were significantly increased, by 41.06%, 16.65%, and 16.24%, respectively, and the intestinal sIgA level was also significantly increased by 147.43%. Furthermore, the recovery effect of *Lactobacillus rhamnosus* GOLDT-M520 on serum IgA, IgG, and intestinal sIgA in mice was comparable to that in the positive control group. This indicates that *Lactobacillus rhamnosus* GOLDT-M520 can increase immunoglobulin levels, thereby promoting humoral immunity and contributing to enhanced adaptive immune responses.

[0037] III. Experimental Conclusions Lactobacillus rhamnosus GOLDGUT-M520 can significantly increase the thymus index and spleen index, the proportion of T cells, B cells and immunoglobulin levels in the mesenteric lymph nodes of mice, which helps to enhance the body's adaptive immunity and has a good immune-enhancing effect.

[0038] Compared with the prior art, the present invention has the following beneficial effects: 1) Lactobacillus rhamnosus GOLDGUT-M520 can comprehensively enhance immunity, while strengthening the innate immune barrier, innate immune response, and adaptive immune response, thus achieving a more comprehensive enhancement of immune function. 2) Lactobacillus rhamnosus GOLDGUT-M520 can produce antimicrobial components, which have a good inhibitory effect on enteropathogenic Escherichia coli and Staphylococcus aureus, and help to enhance the chemical barrier in the innate immune barrier; 3) Lactobacillus rhamnosus GOLDGUT-M520 can enhance the phagocytic activity of macrophages, which helps to strengthen the innate immune response; 4) Lactobacillus rhamnosus GOLDGUT-M520 can increase the proportion of T cells, B cells, and the levels of IgA, IgM, IgG, and sIgA in the mesenteric lymph nodes of immunocompromised mice, which helps to enhance adaptive immunity. 5) Lactobacillus rhamnosus GOLDGUT-M520 can increase the thymus index and spleen index in immunocompromised mice, thus enhancing immunity.

[0039] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. The application of Lactobacillus rhamnosus GOLDGUT-M520 or a combination thereof in the preparation of products that enhance the body's immunity, characterized in that, The composition comprises at least one of Lactobacillus rhamnosus GOLDGUT-M520, progeny of Lactobacillus rhamnosus GOLDGUT-M520, inactivated bacterial cells, metabolites, and bacterial cell components, and includes at least one of the following microorganisms: bacteria and fungi; Lactobacillus rhamnosus GOLDGUT-M520 has been deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 26205; The bacteria include at least one specific strain selected from the genera *Lactobacillus*, *Bifidobacterium*, *Bacillus*, *Propionibacterium*, *Streptococcus*, *Lactococcus*, and *Pediococcus*. The fungi include at least one specific strain of Saccharomyces cerevisiae, Saccharomyces boulardii, and Kluyveromyces marxianus.

2. The application according to claim 1, characterized in that, The Lactobacillus rhamnosus GOLDGUT-M520 or a combination thereof is used to produce antimicrobial substances to enhance the chemical barrier in the innate immune barrier; the antimicrobial substances are used to inhibit pathogenic microorganisms, including at least one of enteropathogenic Escherichia coli and Staphylococcus aureus.

3. The application according to claim 1, characterized in that, The Lactobacillus rhamnosus GOLDGUT-M520 or its composition is used to enhance the phagocytic activity of macrophages and strengthen the innate immune response.

4. The application according to claim 1, characterized in that, The Lactobacillus rhamnosus GOLDGUT-M520 or its composition is used to improve organ indices and enhance immune function in immunocompromised mice; the organ indices include at least one of the thymus index and spleen index.

5. The application according to claim 1, characterized in that, The Lactobacillus rhamnosus GOLDGUT-M520 or its composition is used to increase at least one of the proportion of T cells, B cells, and immunoglobulin levels in the mesenteric lymph nodes of immunocompromised mice, thereby enhancing the adaptive immune response. The immunoglobulins include at least one of immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin G (IgG), and secretory immunoglobulin A (sIgA).

6. The application according to claim 1, characterized in that, The products include biological products, food, or pharmaceuticals.

7. The application according to claim 6, characterized in that, The biological products include microbial agents, subcultured progeny, mutant strains, or fermentation agents of Lactobacillus rhamnosus GOLTGU-M520.

8. The application according to claim 6, characterized in that, The food includes at least one of soy products, dairy products, fruit and vegetable products, or health foods, and the health foods include at least one of solid beverages and compressed candies.

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