Lactobacillus kefir and application of lactobacillus kefir in aspect of regulating immune function

Abstract

The invention relates to the technical field of microorganisms, in particular to lactobacillus kefir and application thereof in the aspect of regulating animal immune functions. The invention provides Kefir lactobacillus brachyphyllum Fanghua, and the preservation number of the Kefir lactobacillus brachyphyllum Fanghua is CGMCC (China General Microbiological Culture Collection Center) NO.27733 Research finds that compared with an immunosuppression model mouse constructed by using hydrocortisone, the weight of immune organs thymus and spleen can be increased, the number of peripheral blood leucocytes can be increased, and immune cell composition and expression of inflammation-related genes can be changed when the mouse is fed with the Lactobacillus kefir Fanghua. Besides, after the excrement of the aged mice fed with the lactobacillus kefir Fanghua is transplanted to the sterile mice, compared with excrement only transplanted to the aged mice, the content of dendritic cells in the spleen of the receptor mice can be reduced, differentiation of helper T cells can be inhibited, expression of inflammation-related genes can be remarkably reduced, and the inflammation-related effects can be improved. Further, the immune inflammatory response of the receptor mouse is regulated.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly to a Lactobacillus kefiranofaciens and its use in regulating immune function. Background Art

[0002] Immunity is a complex physiological protection function by which the body recognizes "self" and "non-self" components, and thus reacts to foreign, allogeneic and self-substances to maintain human health. It is closely related to a series of life processes such as human embryonic development, disease occurrence, and aging. The immune system is composed of immune organs, immune cells and immune active substances, and has main functions including defense, stability and immune surveillance. These functions work together to maintain the stability of the human internal environment, and once dysregulated, immune pathological reactions will occur. With the increase of age, the human immune system may gradually lose its effective response ability to pathogens and cancer cells. Its characteristics include changes in the proportion of memory T cells and the CD4:CD8 ratio, impaired calcium-mediated signal transduction, high expression of Programmed cell death protein 1 (PD-1), and thymic atrophy. The aging of immune cells weakens the individual's own immune response ability.

[0003] The intestine is not only a digestive organ but also an important immune organ. A large number of studies have confirmed that improving the intestinal microenvironment can regulate immune function. With the application of high-throughput sequencing technology and fecal microbiota transplantation (FMT) technology in the study of the gut microbiota in recent years, the key role of the gut microbiota in regulating the body's immunity has been revealed. The gut microbiota is considered to be widely involved in various disease processes, such as type II diabetes, autoimmune diseases, and colorectal cancer. The concept of targeting the gut microbiota to improve health has been accepted by more and more people. Probiotics are a group of bacteria that have beneficial effects on human health, especially on digestive system health, and their interaction with the host may affect the regulation of the human immune system function. Lentilactobacillus kefiri (synonym: Lactobacillus kefiri) is a strain widely present in yogurt, and its consumption history can be traced back to the Russian Caucasus region and Tibet, China, 3,600 years ago. In the traditional knowledge of local herdsmen, yogurt rich in various lactobacilli is widely used to treat various diseases, such as gastrointestinal diseases, constipation, abnormal metabolic diseases, hypertension, anemia, heart disease, allergies, obesity, and hyperlipidemia and hyperglycemia. However, there is currently no report on the study of Lentilactobacillus kefiri in regulating immune function. Developing new probiotics with immune-regulating functions is one of the promising methods in the field of improving human immunity research and is also the problem that the present invention hopes to solve. Summary of the Invention

[0004] Based on the above technical problems, in the first aspect of the present application, Lentilactobacillus kefiri Fanghua, isolated from Tibetan kefir grains (Tibetan kefir grains, a starter used to make Tibetan kefir fermented milk), with a preservation number of CGMCC NO. 27733, is provided.

[0005] In the second aspect of the present application, the use of the aforementioned Lentilactobacillus kefiri Fanghua in the preparation of a product for regulating immune function is provided.

[0006] In the third aspect of the present application, a product for regulating immune function is provided, and the product includes the aforementioned Lentilactobacillus kefiri Fanghua.

[0007] Compared with the prior art, the present invention provides a strain of Lentilactobacillus kefiri Fanghua (CGMCC NO. 27733), and for the first time discloses the new use of Lentilactobacillus kefiri Fanghua in regulating animal immune function, increasing the weight of immune organs, changing the composition of immune cells, and regulating the expression of immune-related genes. Brief Description of the Drawings

[0008] Figure 1 . Lactobacillus kefiranofaciens Fanghua in Tibetan kefir is a key strain for regulating immunity. (A) Effects of strain mixtures from different sample sources on the spleen weight of mice; (B) Effects of strain mixtures from different sample sources on the thymus weight of mice; (C) Effects of strain mixtures from different sample sources on the white blood cell level in the blood of mice; (D) Effects of 4 candidate strains on the spleen weight of mice; (E) Effects of 4 candidate strains on the thymus weight of mice; (F) Effects of 4 candidate strains on the white blood cell level in the blood of mice. *P<0.05; **P<0.01; ****P<0.0001.

[0009] Figure 2 . Whole genome sequence map of Lactobacillus kefiranofaciens Fanghua.

[0010] Figure 3 . Immunomodulatory effect of Lactobacillus kefiranofaciens Fanghua on immunosuppressed model mice induced by hydrocortisone. (A) Spleen weight; (B) Thymus weight; (C) White blood cell level; (D) Expression of immune-related genes in the spleen; (E) Expression of immune-related genes in the thymus. *P<0.05; **P<0.01.

[0011] Figure 4 . Transplanting the fecal microbiota of mice in the Lactobacillus kefiranofaciens Fanghua group through fecal microbiota transplantation can change the composition of immune cells in the spleen of recipient mice. (A) Schematic diagram of the fecal microbiota transplantation experiment; (B) Content of dendritic cells in the spleen; (C) Content of Th1 and Th2 cells in the spleen. *P<0.05.

[0012] Figure 5 . Observe the immunomodulatory effect of Lactobacillus kefiranofaciens Fanghua on the immune function of mice after fecal microbiota transplantation. (A) Differential expression of immune-related genes in the spleen and thymus; (B) Expression level of the IL6 gene in different organ tissues of mice; (C) Immunohistochemical analysis of mouse spleen tissue sections (PD-1 antibody Alexa Fluor 488, green; DAPI blue; scale bar length 50 μm). *P<0.05; **P<0.01; ns no statistical significance. Detailed implementation manners

[0013] To make the invention objectives, technical solutions and beneficial technical effects of the present invention clearer, the present invention will be further described in detail below in conjunction with embodiments. Those familiar with this technology can easily understand other advantages and effects of the invention of this application from the content disclosed in this specification.

[0014] On the one hand, the present application provides a Lactobacillus kefiranofaciens Fanghua, which is deposited in the China General Microbiological Culture Collection Center, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC NO. 27733. The whole genome sequence data of this strain has been submitted to the NCBI online database (https: / / www.ncbi.nlm.nih.gov / ), sequence number: GCF_022810745.1. Lactobacillus kefiranofaciens Fanghua is a probiotic strain isolated from Tibetan kefir grains. Through a large number of screening and research, the inventors of the present application found that Lactobacillus kefiranofaciens Fanghua has a new use in regulating the immune function of animals, activating immune organs, changing the composition of immune cells, and regulating the expression of immune-related genes.

[0015] In some embodiments, Lactobacillus kefiranofaciens Fanghua is a Gram-positive bacterium and rod-shaped.

[0016] On the other hand, the present application provides the use of the aforementioned Lactobacillus kefiranofaciens Fanghua in the preparation of a product for regulating immune function.

[0017] In the use provided by the present application, the product for regulating immune function has any one or more of the following functions:

[0018] a) Improving immunosuppressive function;

[0019] b) Increasing the weights of the immune organs spleen and thymus;

[0020] c) Increasing the white blood cell level;

[0021] d) Activating the function of immune organs under immunosuppression;

[0022] e) Reducing the expression of chronic inflammation-related genes;

[0023] f) Reducing the expression of immune cell exhaustion-related genes;

[0024] g) Changing the composition of immune cells;

[0025] h) Reducing the content of dendritic cells and inhibiting the differentiation of helper T cells.

[0026] In the use provided by the present application, the object targeted by the product for regulating immune function is a mammal. In some embodiments, the mammal is selected from rodents, artiodactyls, perissodactyls, lagomorphs, primates, etc. In a specific embodiment of the present application, the rodent is selected from mice. The primate is preferably a monkey, an ape or a human.

[0027] In the uses provided by this application, immunosuppression can be, for example, hydrocortisone-induced immunosuppression. Hydrocortisone is a common immunosuppressant and is widely used to establish an immunosuppression model in mice.

[0028] In the uses provided by this application, the spleen and thymus, as important immune organs, an increase in their weights means the activation of the functions of immune organs, thus enhancing cellular immune capacity and strengthening the efficacy of the in vivo immune system.

[0029] In the uses provided by this application, white blood cells are white blood cells in the blood. Blood components include white blood cells, red blood cells, platelets, and plasma. Among them, red blood cells mainly transport oxygen, platelets are mainly responsible for blood clotting, and white blood cells are an important part of the body's immune system. White blood cells mainly include various immune cells, such as lymphocytes, neutrophils, monocytes, eosinophils, basophils, and so on. An increase in the white blood cell level can be considered an increase in the level of immune cells in the body.

[0030] In the uses provided by this application, the immune organs are the spleen and / or thymus. It was found in the research of this application that injecting hydrocortisone can cause immunosuppression in mice. After intragastric administration of Lactobacillus kefiranofaciens Fanghua, the weights of the spleens and thymuses of mice increased significantly, and the white blood cell level in the blood increased significantly. The results showed that Lactobacillus kefiranofaciens Fanghua has the function of improving immunosuppression in hydrocortisone model mice.

[0031] In the uses provided by this application, the chronic inflammation-related genes are IL6 and / or IL1β. The expression of IL6 and IL1β genes is usually related to chronic inflammation.

[0032] In the uses provided by this application, the immune cell exhaustion-related genes are one or a combination of more of FOXP3, PD-1, and CD44. FOXP3 is a marker gene of regulatory T cells (Regulatory T cells, Tregs) (Tregs are one of the important factors for immune tolerance and inhibit the activation and proliferation of potential autoreactive T cells existing in the normal body through an active regulation method), PD-1 is a marker gene of T cell apoptosis, and CD44 is a marker gene of memory T cells (memory T cell, Tm).

[0033] In the immunosuppression model group, hydrocortisone increased the expression levels of FOXP3, PD-1, and CD44 and promoted the expression of chronic inflammation-related genes (IL6 and IL1β). In the uses provided by this application, the intervention of Lactobacillus kefiranofaciens Fanghua can significantly reduce the expression of immune cell exhaustion-related genes such as FOXP3, PD-1, and CD44 and inflammation-related genes such as IL6 and IL1β induced by hydrocortisone.

[0034] In the uses provided by the present application, the products for regulating immune function include, but are not limited to, drugs, health products or foods.

[0035] In some embodiments, the above-mentioned Lactobacillus kefiranofaciens Fanghua can be prepared into a bacterial powder product, and the preparation process is to let Lactobacillus kefiranofaciens Fanghua grow in MRS, then centrifuge to obtain the bacterial cells, resuspend the bacterial cells in skim milk, and freeze-dry to form the bacterial powder. Lactobacillus kefiranofaciens Fanghua can also be made into fermented dairy products, and the preparation process is as follows: select other bacteria (such as Lactobacillus bulgaricus + Streptococcus thermophilus) as the starter, and then add Lactobacillus kefiranofaciens Fanghua after fermentation is completed to obtain fermented dairy products. The above-mentioned bacterial powder products or fermented dairy products can be used as health foods for improving immune function, especially health foods for restoring the weight of immune organs and the level of peripheral blood leukocytes, changing the composition of immune cells in the thymus, and regulating the expression of immune-related genes. These fermented dairy products can usually be fermented milk, fermented milk powder, cheese, etc.

[0036] The above-mentioned bacterial powder products or fermented dairy products can be used as health foods for improving immune function, especially health foods for restoring the weight of immune organs and the level of peripheral blood leukocytes, changing the composition of immune cells in the thymus, and regulating the expression of immune-related genes. These fermented dairy products can usually be fermented milk, fermented milk powder, cheese, etc.

[0037] In some embodiments, the preparation method of the bacterial powder product is as follows: Lactobacillus kefiranofaciens Fanghua is first activated, after the cultivation is completed, the bacterial cells are obtained by centrifugation, washed twice with PBS, resuspended in skim milk, and freeze-dried to obtain the bacterial powder.

[0038] Furthermore, the activation is a strain activation step well-known to those skilled in the art. For example, it can be to pick a single colony of Lactobacillus kefiranofaciens Fanghua with an inoculation loop and streak it on a solid plate of MRS ( Merck KGaA, Germany, product number: 1106610500) at 37 °C in a constant temperature anaerobic incubator for 72 hours, and purified three times. Finally, pick a single colony into an MRS liquid medium and culture it overnight at 37 °C in a constant temperature incubator for 72 hours.

[0039] In the uses provided by the present application, the product for regulating immune function is a drug, and the drug also includes a pharmaceutically acceptable carrier or excipient.

[0040] "Pharmaceutically acceptable" means that when drugs are properly administered to animals or humans, they do not produce adverse, allergic, or other untoward reactions. "Pharmaceutically acceptable carriers or excipients" should be compatible with the active ingredient, i.e., they can be blended with it without significantly reducing the effectiveness of the drug under normal circumstances. Specific examples of some substances that can be used as pharmaceutically acceptable carriers or excipients are sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethyl cellulose, and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; coloring agents; flavoring agents; tabletting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions; and phosphate buffer solutions, etc. These substances are used as needed to help with the stability of the formulation or to enhance the activity or its bioavailability or to produce an acceptable taste or odor in the case of oral administration.

[0041] In the uses provided by this application, the product dosage forms for regulating immune function are solids, liquids, gels, semi-liquids, aerosols, or powders. In certain embodiments of the present invention, the product dosage form is a powder, i.e., bacterial powder. The concentration of Lactobacillus kefiranofaciens Fanghua in the powder is 1×10 6 CFU / g to 1×10 11 CFU / g.

[0042] On the other hand, this application provides a product for regulating immune function, including the aforementioned Lactobacillus kefiranofaciens Fanghua. The product of this application can be used to regulate the immune function of animals, activate immune organs, change the composition of immune cells, and regulate the expression of immune-related genes. In a specific embodiment of this application, the product can increase the weights of the thymus and spleen, increase the number of peripheral blood leukocytes, and change the composition of immune cells and the expression of related genes.

[0043] In some embodiments, the products of this application include, but are not limited to, drugs, health products, or foods.

[0044] In some embodiments, the above-mentioned Lactobacillus kefiranofaciens Fanghua can be prepared into a bacterial powder product, and the preparation process is to grow Lactobacillus kefiranofaciens Fanghua in MRS, then centrifuge to obtain the bacterial cells, resuspend the bacterial cells in skim milk, and freeze-dry to form the bacterial powder. Lactobacillus kefiranofaciens Fanghua can also be made into fermented dairy products, and the preparation process is as follows: select other bacteria (such as Lactobacillus bulgaricus + Streptococcus thermophilus) as the starter, and then add Lactobacillus kefiranofaciens Fanghua after fermentation is completed to obtain the fermented dairy products. The above-mentioned bacterial powder products or fermented dairy products can be used as health foods for improving immune function, especially health foods for restoring the weight of immune organs and the level of peripheral blood leukocytes, changing the composition of immune cells in the thymus, and regulating the expression of immune-related genes. These fermented dairy products can usually be fermented milk, fermented milk powder, cheese, etc.

[0045] The present invention provides Lactobacillus kefiranofaciens Fanghua, and further provides its new uses in regulating the immune function of animals, activating immune organs, changing the composition of immune cells, and regulating the expression of immune-related genes, which are specifically manifested as increasing the weights of the spleen and thymus, raising the leukocyte level, reducing the expression of chronic inflammation-related genes, and reducing the expression of immune cell exhaustion-related genes, and having good industrialization prospects.

[0046] The following specific examples illustrate the embodiments of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0047] Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are for the purpose of describing specific embodiments, rather than limiting the protection scope of the present invention; in the specification and claims of the present invention, unless otherwise clearly indicated in the text, the singular forms "a", "an", and "the" include the plural forms.

[0048] When numerical ranges are given in the embodiments, it should be understood that unless otherwise specified in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art of this technology. In addition to the specific methods, devices, and materials used in the embodiments, according to the knowledge of those skilled in the art of this technology and the description of the present invention, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention can also be used to implement the present invention.

[0049] In this application, a mouse immunosuppression model was constructed using hydrocortisone, and it was found that consuming Lactobacillus kefiranofaciens (synonym: Lactobacillus kefir) could significantly restore the weights of the immune organs and the levels of peripheral blood leukocytes in the model mice. At the same time, it changed the composition of immune cells in the thymus and regulated the expression of immune-related genes. Further, after transplanting the intestinal microbiota of different groups of mice (old group, young group, and Lactobacillus kefiranofaciens group) into germ-free mice through fecal microbiota transplantation technology, it was found that Lactobacillus kefiranofaciens could significantly reduce the expression of inflammatory genes in mice. The present invention first discloses the use of Lactobacillus kefiranofaciens as a probiotic with immune-regulating functions and proposes a way to improve immune-related diseases by regulating the intestinal microenvironment through diet.

[0050] Statistical analysis and graphing were performed using GraphPad Prism 9 software. Unless otherwise specified, all data are expressed as mean ± standard error. Unpaired two-tailed t-tests were used for comparisons between two groups. One-way ANOVA analysis was used for multiple comparisons of univariate data, and Tukey's test was used afterwards. P < 0.05 was considered to be statistically significant. Significant differences were indicated by *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. "ns" indicates no significant difference.

[0051] The present application is further illustrated by the following examples, but the scope of the present application is not limited thereby.

[0052] Example 1

[0053] Isolation, screening, and identification of candidate strains:

[0054] Candidate samples considered to have immune - enhancing effects were collected from different regions, including Tibetan kefir grains, Xinjiang camel milk, Xinjiang camel yogurt, Sichuan enzyme, and Inner Mongolia milk curd, and placed in sterile sampling tubes for transportation in an ice box. They were diluted with 0.85% normal saline in a sterile condition in a gradient manner. Appropriate dilution gradients were selected and spread on TPY (Qingdao Haibo Biotechnology Co., Ltd., HB8570) and MRS (Merck, 1106610500) agar plates, and anaerobically cultured at 37°C for 48 - 72 hours. Suspected single colonies were picked by observing their colony morphology with the naked eye, and then observed under a microscope for preliminary screening and purification culture. After purification, TPY and MRS liquid media were used for anaerobic culture at 37°C for 48 - 72 hours. After centrifugation to remove the supernatant, the cells were resuspended in a sterile 30% (volume percentage) glycerol aqueous solution.

[0055] The screened strains were cultured in liquid. The cells were collected, genomic DNA was extracted, and PCR amplification reactions were carried out using universal primers (27F / 1492R). The PCR amplification program was as follows: pre - denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 90 seconds, for a total of 35 cycles, and finally extension at 72°C for 10 minutes. Subsequently, the content and purity of the PCR amplification products were detected. After passing the detection, they were sent to Shanghai Jieli Biotechnology Co., Ltd. for first - generation sequencing. According to the sequencing results, sequence homology alignment was performed using BLAST in the NCBI database, and a total of 20 strains were identified, as shown in Table 1. Different strains were stored in a - 80°C refrigerator for future use.

[0056] Table 1 Identification of microbial strains from different collected samples

[0057]

[0058]

[0059] The above - obtained strains were separately cultured in liquid at 37°C for 72 hours (for strains that can grow in skim milk, 10% wt skim milk was used for culture; for strains that cannot grow in skim milk, MRS or TPY was used for culture). After the culture was completed, according to their sources, each strain was mixed in equal volume in 10% wt skim milk (for strains cultured with MRS or TPY, after the culture was completed, the cells were obtained by centrifugation, washed twice with PBS, and then resuspended in an equal volume of 10% wt skim milk) to simulate the microbial composition of different collected samples. It was stored at - 80°C for future use as an intervention sample.

[0060] Example 2

[0061] Lactobacillus kefiranofaciens Fanghua in Tibetan kefir grains is the key strain for regulating immunity:

[0062] Eight-week-old C57BL / 6J wild-type mice were selected for the experiment. The mice were housed under a 12-hour light-dark cycle and at a constant temperature and humidity of 22 °C, and provided with sufficient food and water. All mice were randomly divided into groups according to the standard of 10 mice per group: normal control group (Control), hydrocortisone group (Hydrocortisone), Tibetan kefir group (Tibetankefir), Xinjiang camel milk group (Xinjiang camel milk), Xinjiang camel yogurt group (Xinjiang camelyogurt), Sichuan enzyme group (Sichuan enzyme), and Inner Mongolia dried milk group (Inner Mongolia dried milk). Among them, the normal control group and the hydrocortisone group were given intragastric administration of 10% wt skim milk to the mice during the 4-week intervention period, while the other 5 sample groups were given 400 μL / day of the intervention sample in Example 1 to the mice by intragastric administration. In the 3rd week of the intervention, the mice in the hydrocortisone group and the 5 sample groups were injected intramuscularly with 40 mg / kg of hydrocortisone (injected once every other day, for a total of 5 times). After the experiment, the mice were sacrificed, and the immune organs were obtained after dissection for weighing, and the blood samples were detected.

[0063] Hydrocortisone is a common immunosuppressant and is widely used to construct an immunosuppressive model in mice. As Figure 1 shown in A-C, compared with the control group, the weights of the spleen and thymus of the immunosuppressive model mice induced by hydrocortisone were significantly decreased, and the level of white blood cells (WBC) in the blood was also significantly reduced. Compared with the other 4 sample groups, the weights of the spleen and thymus of the mice in the Tibetan kefir group were significantly restored, and there was no significant change in the white blood cell level. These results indicate that the 4 candidate strains isolated from Tibetan kefir may have the effect of improving immunity.

[0064] Next, in order to determine the key strains that take effect among the 4 candidate strains, using the same animal model and experimental method, the mice were respectively given 10% wt skim milk containing a single strain, and the intragastric administration dose was 1×10 8 CFU / kg / d, including 2 strains of Lactobacillus kefiranofaciens, 1 strain of Lentilactobacillus kefiri, and 1 strain of Kluyveromyces marxianus. As Figure 1As shown in D - F, through the detection of mouse immune organs and blood samples, it was found that compared with the hydrocortisone group, the spleen and thymus weights of mice in the Lentilactobacillus kefiri group were significantly increased, and the white blood cell level in the blood was significantly elevated. These results indicate that Lentilactobacillus kefiri among the candidate strains is the key strain for regulating immunity.

[0065] The finally screened strain of Lentilactobacillus kefiri (synonym: Lactobacillus kefiri) was named Fanghua and deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms (Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), with the deposit number CGMCC No. 27733. The whole - genome sequence of this strain is as Figure 2 shown, and the relevant data have been submitted to the NCBI online database (https: / / www.ncbi.nlm.nih.gov / ), with the accession number: GCF_022810745.1.

[0066] Example 3

[0067] Preparation of products containing Lentilactobacillus kefiri Fanghua

[0068] Preparation of Lentilactobacillus kefiri Fanghua: Use an inoculation loop to pick a single colony of Lentilactobacillus kefiri Fanghua (CGMCC NO.27733) and streak it on an MRS ( Merck KGaA, Germany, product number: 1106610500) solid plate, and culture it in a 37°C constant - temperature anaerobic incubator for 72 hours, and purify it three times. Finally, pick a single colony and culture it overnight for 72 hours in an MRS liquid medium at 37°C to obtain a bacterial solution (the viable cell count is 5.0×10 8 CFU / mL). After culturing, obtain the bacterial cells by centrifugation, wash them twice with PBS, and resuspend them in 10% wt skim milk or 3% wt maltodextrin solution, and then freeze - dry to form bacterial powder.

[0069] Preparation of products containing Lentilactobacillus kefiri Fanghua: Add Lentilactobacillus kefiri Fanghua as an original ingredient or starter to probiotic powder, fermented milk, lactic acid bacteria beverage, cheese, milk powder, live - bacteria water, probiotic drugs and other foods, health products or drugs, so that the final concentration of this strain reaches 1.0×10 6 ~1.0×10 11 CFU / g.

[0070] Example 4

[0071] Preparation of samples intervened by Lentilactobacillus kefiri Fanghua:

[0072] The bacterial cells obtained by centrifugation in Example 3 were washed twice with PBS and then resuspended in 10% wt skim milk, and stored in a refrigerator at 4°C for later use. Or directly use the bacterial powder product obtained by freeze-drying in Example 3, resuspend it in 10% wt skim milk, and store it in a refrigerator at 4°C for later use.

[0073] Example 5

[0074] Construction and grouped intervention of the mouse immunosuppression model:

[0075] Eight-week-old C57BL / 6J wild-type mice were selected for the experiment. The mice were housed under a 12-hour day-night cycle and at a constant temperature and humidity of 22°C, and provided with sufficient feed and water. All mice were randomly grouped according to the standard of 10 mice per group: normal control group (Control), hydrocortisone group (Hydrocortisone), and Lactobacillus kefiri group (L. kefiri). Among them, the normal control group and the hydrocortisone group were given intragastric intervention with 10% wt skim milk for 4 weeks, and the Lactobacillus kefiri group was given intragastric intervention with Lactobacillus kefiri in Example 4, and the strain intervention dose was 1×10 8 CFU / kg / d. In the 3rd week of the intervention, mice in the hydrocortisone group and the Lactobacillus kefiri group were injected intramuscularly with 40 mg / kg of hydrocortisone (injected once every other day, for a total of 5 times). Hydrocortisone is a common immunosuppressant and is widely used to construct the mouse immunosuppression model.

[0076] Example 6

[0077] Lactobacillus kefiri Fanghua has the function of improving immunosuppression

[0078] On the 2nd day after the last injection of hydrocortisone in the mice of Example 5, the mice were sacrificed, and immune organs and blood samples were obtained after dissection, and relevant indicators were detected.

[0079] Total RNA was extracted from different tissues using an RNA extraction kit (TAKARA, product number: 9767), and cDNA was synthesized using a reverse transcription kit (TIANGEN, product number: KR123). The primer sequences used for PCR amplification are shown in Table 2, where GAPDH is the internal reference gene. The PCR amplification system refers to the instructions of the QuantiNova SYBR Green PCR Kit kit (QIAGEN, product number: 208054), the reaction system is 20 μL (Table 3), the amplification process is shown in Table 4, and the number of cycles is 40 times.

[0080] Table 2 Primer sequences

[0081]

[0082] Table 3 Real-time fluorescence quantitative PCR reaction system

[0083]

[0084] Table 4 Real-time cycling conditions

[0085]

[0086] Detection of mouse immune organs and blood samples revealed that, compared with the hydrocortisone group, the spleen and thymus weights of mice in the L. kefiri Fanghua group were significantly increased, and the white blood cell level in the blood was significantly elevated ( Figure 3 A-C). Quantitative PCR results of spleen and thymus tissues showed that the injection of hydrocortisone changed the composition of mouse immune cells, including regulatory T cells and memory T cells, and promoted the expression of chronic inflammation-related genes (IL6 and IL1β). While the intervention of L. kefiri Fanghua could significantly reduce the expression levels of FOXP3, PD-1, CD44, IL6 and IL1β, that is, reduce the immune cell exhaustion and inflammation-related gene expression induced by hydrocortisone. Notably, this improvement was more significant in the thymus than in the spleen ( Figure 3 D, E). FOXP3 is a marker gene of regulatory T cells (Regulatory T cells, Tregs, which is one of the important factors for immune tolerance and inhibits the activation and proliferation of potential autoreactive T cells existing in normal organisms through active regulation), PD-1 is a marker gene of T cell apoptosis, and CD44 is a marker gene of memory T cells (memory T cell, Tm); while the expression of IL6 and IL1β genes is usually related to chronic inflammation. The above results indicate that L. kefiri Fanghua has the function of improving the immune suppression of hydrocortisone on mice.

[0087] Example 7

[0088] Transplanting the fecal microbiota of mice in the L. kefiri Fanghua group by fecal microbiota transplantation can regulate the immune function of recipient mice:

[0089] Sixteen-month-old C57BL / 6J wild-type mice were selected. The mice were housed under a 12-hour light-dark cycle and at a constant temperature and humidity of 22 °C, and provided with sufficient food and water. According to the standard of 10 mice per group, all mice were randomly divided into 2 groups: the L. kefiri-FH group and the old group. Mice in the L. kefiri-FH group were gavaged with L. kefiri Fanghua resuspended in 10% skim milk, and the intervention dose was 1×108 CFU / kg / d. The old - group mice were gavaged with 10% wt skim milk. After 3 weeks of gavage, the feces of the two groups of old mice were collected and stored at - 80 °C for later use.

[0090] Eight - week - old C57BL / 6J wild - type mice were selected for fecal microbiota transplantation (FMT) experiments. The mice were housed under a 12 - hour light - dark cycle and at a constant temperature and humidity of 22 °C, and provided with sufficient food and water. Before fecal transplantation, the original gut microbiota in the mice was eliminated by drinking drinking water containing antibiotics (50 mg / kg vancomycin, 100 mg / kg neomycin, 100 mg / kg metronidazole) for 3 weeks. Then, all the mice were randomly divided into 3 groups: the FMT - Young group that transplanted the gut microbiota of the normal control group mice (8 - week - old young mice) in Example 5, the FMT - SM group that transplanted the gut microbiota of the old - group mice (gavaged with 10% skim milk), and the FMT - L.kefiri - FH group that transplanted the gut microbiota of the old L.kefiri - FH group mice. 0.1 g of fecal pellets was added to 1 mL of sterile PBS, and after mixing, a fecal suspension was prepared. Each mouse was gavaged with 250 μL of the fecal suspension every day for 2 weeks according to the group to achieve the purpose of fecal microbiota transplantation. Two weeks after fecal microbiota transplantation, the mice were sacrificed and dissected, and the mouse tissue organs were collected and related indexes were detected ( Figure 4 A).

[0091] Flow cytometry sorting: After obtaining the mouse spleen tissue, the spleen cells were separated using a cell strainer (40 μm) (Guangzhou Jet, CSS013040), and then the red blood cells were removed using a red blood cell lysis buffer (Solarbio, R1010). The remaining cells were resuspended in PBS. Subsequently, under light - protected conditions, the cells were incubated with antibodies of PE - MHC II (Thermo Fisher, 17 - 5321 - 81), APC - CD11c (Thermo Fisher, 17 - 0114 - 82), FITC - CD3 (Thermo Fisher, 11 - 0032 - 82), PE - CD4 (Thermo Fisher, 79 - 0042 - 80), APC - IFNγ (Thermo Fisher, 17 - 7311 - 81), and PE - Cy7 - IL17a (Thermo Fisher, 25 - 7177 - 80) at a concentration of 1 μg / test at room temperature for 20 minutes. The fluorescence signals were analyzed using an Agilent Novocyte flow cytometer (Agilent), and the obtained data were analyzed by FlowJo software.

[0092] The method of real - time fluorescence quantitative PCR is shown in Example 6.

[0093] Immunofluorescence staining: The collected mouse spleen tissues were preserved in 4% paraformaldehyde and cryosectioned. The primary antibody against PD-1 (Anti-PD1 antibody [NAT105], Abcam, catalog number: ab52587) was diluted at 1:500, and the secondary antibody (Goat anti-mouse IgG H&L (Alexa 488), Abcam, catalog number: ab150113) was diluted at 1:1000 and then used for staining. The stained tissue sections were observed using a Leica THUNDER high-resolution fluorescence microscope.

[0094] We chose to use PE-MHC class II antibody and APC-CD11c antibody as markers to perform flow sorting of dendritic cells (DC) (CD11 + MHC II high ). The results showed that the content of dendritic cells in the spleens of mice in the FMT-L.kefiri-FH group (38.8%) was lower than that in the FMT-SM group (52.3%) ( Figure 4 B). We chose CD3, CD4, IFNγ, and IL17a antibodies as analysis markers for helper T cells (including Th1 and Th17 cells) and found that in the spleens of mice in the FMT-L.kefiri-FH group, the contents of IFNγ + Th1 and IL17a + Th17 cells were significantly reduced ( Figure 4C). Dendritic cells are key antigen-presenting cells in the immune system, which present antigens to different receptors on different immune cells to activate innate and adaptive immune responses. Dendritic cells are not only responsible for eliciting immune responses in T cells but also involved in the differentiation of helper T cells. Helper T cells are a type of immune cells that can play an important role and are activated by reacting with polypeptide antigens presented by major histocompatibility complex (MHC) II, secreting various cytokines to regulate or assist immune responses. Among them, both Th1 and Th17 cells can produce a variety of pro-inflammatory cytokines, which are related to the occurrence and development of many inflammatory reactions and autoimmune diseases. The above results indicate that the fecal microbiota of Lactobacillus kefiri Fanghua group mice by fecal microbiota transplantation can change the composition of immune cells in the spleen of recipient mice, specifically including reducing the content of dendritic cells and inhibiting the differentiation of Th1 and Th17 cells, thereby regulating inflammatory reactions. By real-time fluorescence quantitative PCR, we found that compared with the FMT-Young group transplanted with the intestinal microbiota of young mice, the expression levels of FOXP3, PD-1, CD44, IL6, and IL1β in the spleen and thymus tissues of the FMT-SM group transplanted with the intestinal microbiota of old mice increased, while there was no significant change in the FMT-L.kefiri-FH group ( Figure 5 A). The qPCR results of IL6 in other different organs also found that the inflammatory level of mice in the FMT-L.kefiri-FH group was significantly lower than that in the FMT-SM group and was similar to that in the FMT-Young group ( Figure 5 B). The immunofluorescence staining results also confirmed that compared with the FMT-SM group, the protein expression content of PD-1 in the spleen of mice in the FMT-L.kefiri-FH group was lower ( Figure 5 C). The results of the above fecal microbiota transplantation experiments indicate that Lactobacillus kefiri Fanghua may regulate the immune function of mice through the mediation of the gut microbiota.

[0095] In summary, the present invention effectively overcomes various disadvantages in the prior art and has high industrial utilization value.

[0096] The above embodiments are only illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes completed by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A Lactobacillus kefiranofaciens Fanghua, with the preservation number of CGMCC NO. 27733.

2. Use of the Lactobacillus kefiranofaciens Fanghua according to claim 1 in the preparation of a product for regulating immune function.

3. The use according to claim 2, characterized in that, The product for regulating immune function has any one or more of the following functions: a) Improving immunosuppressive function; b) Increasing the weights of immune organs, the spleen and thymus; c) Raising the white blood cell level; d) Activating the functions of immune organs; e) Reducing the expression of chronic inflammation-related genes; f) Decreasing the expression of immune cell exhaustion-related genes; g) Altering the immune cell composition; h) Reducing the content of dendritic cells and inhibiting the differentiation of helper T cells.

4. The use according to claim 3, characterized in that, The chronic inflammation-related genes are IL6 and / or IL1β.

5. The use according to claim 3, characterized in that, The immune cell exhaustion-related genes are a combination of one or more of FOXP3, PD-1 and CD44; and / or, the immune organs are the spleen and / or thymus. The use according to claim 3, wherein the helper T cells are Th1 and / or Th17 cells.

6. The use according to claim 3, characterized in that, The product for regulating immune function is a drug, a health product or a food.

7. The use according to claim 3, characterized in that, The product is a drug, and the drug further comprises a pharmaceutically acceptable carrier or excipient.

8. The use according to claim 3, characterized in that, The dosage form of the product for regulating immune function is solid, liquid, gel, semi-liquid, aerosol or powder.

9. The use according to claim 8, characterized in that, The concentration of Lactobacillus kefiranofaciens Fanghua in the powder is 1×10 6 CFU / g to 1×10 11 CFU / g.

10. A product for regulating immune function, characterized in that, The product comprises the slow-growing Lactobacillus kefiranofaciens Fanghua as described in claim 1.

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