Application of Lactobacillus kefir in improvement of immune aging and functional food containing Lactobacillus kefir

Abstract

The invention relates to the technical field of microorganisms, in particular to an application of Kefir Lactobacillus brachyphyllum in improvement of immune aging and functional food containing the Kefir Lactobacillus brachyphyllum. The invention provides an application of lactobacillus kefir Fanghua in preparation of an anti-aging product, and the preservation number of the lactobacillus kefir Fanghua is CGMCC (China General Microbiological Culture Collection Center) NO.27733. The invention further provides a preparation method of the lactobacillus kefir Fanghua. Research finds that edible Lactobacillus kefir Fanghua or a product containing the Lactobacillus kefir Fanghua can increase the weight of animal immune organs, change immune cell composition, regulate inflammation-related gene expression, improve cell senescence phenotypes, improve inflammation-related expression and improve inflammation-related expression. The method can be used for producing functional food products such as fermented milk, lactic acid bacteria beverages, probiotic powder, viable bacteria water, milk powder and cheese.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to use of a strain of Kefir Lactobacillus in improving immune aging and functional food containing the strain. Background Art

[0002] The global population is entering an aging stage. According to the 2019 World Population Prospects report released by the United Nations, by 2050, one in every six people in the world will be 65 years old or older (16%). In addition, the population aged 80 or above is expected to increase from 143 million in 2019 to 426 million in 2050. With the continuous increase in the number and proportion of elderly people, a large number of chronic diseases of the elderly, such as cardiovascular disease, osteoporosis and tumors, will appear in the next 20 years, which will greatly increase medical expenses and social burdens. Therefore, delaying aging is not only a research topic in biomedicine, but also an important issue that needs to be solved in the current social development process.

[0003] Aging is the functional decline of an organism accumulated over time, which is characterized by the gradual loss of physiological integrity, resulting in impaired physiological functions and increased susceptibility to death. Cells are the basic units that make up organs and organisms. Cell senescence is the driving force of organ and organism aging. The detection of cell senescence biomarkers can not only reflect the changes at the cellular level during the aging process, but also be used to indicate the biological age of the entire organ or organism. In addition, according to existing research, immunosenescence is one of the most important causes of human aging. Immunosenescence is an immune dysfunction process that occurs with age, which is mainly manifested by the body's decreased immune response to endogenous and exogenous antigens, impaired immune response to malignant tumors and vaccination, resulting in an individual's defense against infectious diseases, anti-tumor ability, and ability to clear senescent cells. The process of immunosenescence is regulated by factors such as aging, chronic inflammation, and changes in the microenvironment. Its important feature is that the thymus gradually degenerates with age, which is manifested by weight loss, reduced T cell output, and reduced thymus-dependent immune response.

[0004] Although immunosenescence has been identified as one of the key drivers of solid organ aging, solutions aimed at improving immune function and thereby delaying aging are often limited to the medical treatment of a few diseases. For example, it has been reported that blocking the PD-1 / PD-L1 signaling pathway with PD-L1 antibodies can significantly enhance the activity of immune cells, thus delaying the aging of individual organs. However, PD-L1 antibodies are highly toxic tumor treatment drugs, and their clinical application in oncology remains limited. This means that these drugs cannot serve as the mainstream and widely accepted solutions for delaying aging. In contrast, the concept of targeting the gut microbiota and improving health through probiotics has been increasingly accepted by more people and is also a widely recognized strategy in nutritional health research. 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 as early as 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. Lentilactobacillus kefiri Fanghua is a probiotic strain isolated from Tibetan kefir yogurt. There is currently no report on the improvement of immunosenescence by Lentilactobacillus kefiri Fanghua, nor is there any report on the preparation of related functional products using this strain, including but not limited to fermented milk, lactic acid bacteria beverages, probiotic powders, live bacteria water, milk powder, and cheese. Therefore, the development of new probiotics and functional products with the effect of improving immunosenescence is one of the promising methods in the field of anti-aging research and is also the problem that the present invention hopes to solve. Summary of the Invention

[0005] Based on the above technical problems, the present invention provides the use of a strain of Lentilactobacillus kefiri (synonym: Lactobacillus kefiri) Fanghua in improving immunosenescence and in the production of related functional foods or health products.

[0006] The first aspect of the present application provides the use of Lentilactobacillus kefiri Fanghua in the preparation of anti-aging products, and the preservation number of Lentilactobacillus kefiri Fanghua is CGMCC NO. 27733.

[0007] The second aspect of the present application provides a mixed fermented dairy product, which includes Lactobacillus kefiranofaciens Fanghua, and also includes one or a combination of more than one of Lactobacillus kefiri, Kluyveromyces marxianus, Bifidobacterium, Lactobacillus, Lactobacillus casei, and Lactiplantibacillus plantarum.

[0008] The third aspect of the present application provides the use of the aforementioned mixed fermented dairy product in the preparation of an anti-aging product.

[0009] The fourth aspect of the present application provides a lactic acid bacteria beverage product, which uses the aforementioned mixed fermented dairy product as a fermented milk base material, and also includes one or a combination of more than one of granulated sugar, stabilizer, and water.

[0010] The fifth aspect of the present application provides a probiotic agent, the raw materials of which include Lactobacillus kefiranofaciens Fanghua; optionally, it also includes one or more strains of Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactiplantibacillus plantarum, and Kluyveromyces.

[0011] The sixth aspect of the present application provides a probiotic powder product, which is obtained by adding a dietary supplement, vitamins, fructooligosaccharide, maltodextrin, and pectin to the aforementioned probiotic agent, mixing evenly, and then freeze-drying.

[0012] The seventh aspect of the present application provides a live bacteria aquatic product, which is prepared by adding vitamin C, a stabilizer, and water to the aforementioned probiotic agent. Based on the mass of the live bacteria aquatic product, the concentration of the probiotic agent is 10 6 ~10 11 CFU / g.

[0013] The eighth aspect of the present application provides a milk powder product, the raw materials of which include Lactobacillus kefiranofaciens Fanghua, and also includes one or a combination of more than one of whole milk powder, skim milk powder, demineralized whey powder, isomaltooligosaccharide, concentrated whey protein powder, inulin, and compound nutrients.

[0014] The ninth aspect of the present application provides a cheese product, the raw materials of which include Lactobacillus kefiranofaciens Fanghua, and also includes one or a combination of more than one of raw cow milk, starter culture, and rennet.

[0015] Compared with the prior art, the present application uses Lactobacillus kefiranofaciens (synonym: Lactobacillus kefiri) Fanghua (CGMCC NO. 27733), and for the first time discloses its use in restoring the function of animal immune organs and improving immune senescence, broadening the application scope of Lactobacillus kefiranofaciens as a probiotic. Description of the Drawings

[0016] 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.

[0017] Figure 2 . Whole genome sequence map of Lactobacillus kefiranofaciens Fanghua.

[0018] Figure 3 . Lactobacillus kefiranofaciens Fanghua has the effect of improving immune senescence in aged mice. (A) Observation of internal structural changes in spleen tissue by H&E staining. GC represents germinal center, bar represents 50 μm; (B) Expression of different genes in the spleen and thymus of mice; (C) Immunohistochemical analysis of spleen frozen sections with PD-1 antibody (Alexa Fluor 488, green) and DAPI (blue), bar represents 50 μm. *P<0.05; **P<0.01; ns is not statistically significant.

[0019] Figure 4 . Fermented milk products containing Lactobacillus kefiranofaciens Fanghua have the effect of improving immune senescence in mice. (A) Spleen weight; (B) Thymus weight; (C) White blood cell level; (D) Expression of immune-related genes in the spleen. *P<0.05; **P<0.01; ns is not statistically significant. Detailed implementation mode

[0020] In order to make the invention purpose, technical solution and beneficial technical effects of the present invention clearer, the present invention will be further described in detail below with reference to the 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.

[0021] On the one hand, the present application provides the use of Lactobacillus kefiranofaciens Fanghua in the preparation of anti-aging products.

[0022] In the purposes of this application, anti-aging includes improving immunosenescence. Immunosenescence is an immune dysfunction process that occurs with age, which is mainly manifested by a decrease in the body's immune response to endogenous and exogenous antigens, and an impaired immune response to malignant tumors and vaccinations, resulting in a decrease in the individual's defense against infectious diseases, anti-tumor ability, and ability to remove senescent cells. The process of immunosenescence is regulated by factors such as aging, chronic inflammation, and microenvironmental changes. Its important feature is that the thymus gradually degenerates with age, which is manifested by weight loss, reduced T cell output, and reduced thymus-dependent immune response.

[0023] Kefir Lactobacillus Fanghua is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration Committee, with the deposit number CGMCC NO.27733. The whole genome sequence data of the strain has been submitted to the NCBI online database (https: / / www.ncbi.nlm.nih.gov / ), with the sequence number: GCF_022810745.1. Kefir Lactobacillus Fanghua is a probiotic strain isolated from Tibetan mushrooms.

[0024] This application adopts kefir slow-growing lactobacillus Fanghua (CGMCC NO.27733), and discloses for the first time its uses in restoring the functions of animal immune organs, regulating immunity, and improving immune aging, thus broadening the application scope of kefir slow-growing lactobacillus as a probiotic.

[0025] In the uses provided in this application, products include but are not limited to food, health products, and medicines.

[0026] On the other hand, the present application provides a mixed fermented dairy product, including kefir lactobacillus Fanghua, and also includes a combination of one or more of kefir-like lactobacillus, Kluyveromyces marxianus, bifidobacterium, lactobacillus, lactobacillus casei, and lactobacillus plantarum. The mixed fermented dairy product of the present application has the function of improving immune aging, which is specifically embodied in that consuming fermented milk products containing kefir lactobacillus Fanghua can significantly increase the weight of the spleen and thymus, increase the white blood cell content in the blood, activate the function of immune organs, and significantly improve chronic inflammation and aging-related gene expression. The present invention discloses for the first time that consuming kefir lactobacillus Fanghua or products containing the strain can improve the symptoms of animal immune aging, broadens the application range of kefir lactobacillus as a probiotic, and proposes a way to delay aging by taking probiotic products.

[0027] In the mixed fermented dairy product provided by the present application, in a specific embodiment, the mixed fermented dairy product is Lactobacillus kefiranofaciens Fanghua and Lactobacillus kefiri; based on the total mass of the mixed fermented milk, the concentration ratio of Lactobacillus kefiranofaciens Fanghua to Lactobacillus kefiri is (1-100):(1-100); specifically, it can be (1-20):1, (20-50):1, (50-100):1, 1:(10-20), 1:(20-50), 1:(50-80), or 1:(80-20), etc.

[0028] In some embodiments, the mixed fermented dairy product further includes Kluyveromyces marxianus; based on the total mass of the mixed fermented milk, the concentration ratio of Lactobacillus kefiranofaciens Fanghua, Lactobacillus kefiri, and Kluyveromyces marxianus is (1-100):(1-100):1. In a specific embodiment, the concentration ratio of Lactobacillus kefiranofaciens Fanghua, Lactobacillus kefiri, and Kluyveromyces marxianus is 1:1:1.

[0029] In some embodiments, the preparation method of the mixed fermented dairy product is as follows:

[0030] 1. Mix the activated Lactobacillus kefiranofaciens Fanghua, Lactobacillus kefiri, and Kluyveromyces marxianus according to appropriate concentrations to obtain a mixed culture, inoculate it into skim milk at 3-5% (volume percentage), and culture it under appropriate conditions to prepare a starter.

[0031] 2. Heat the skim milk to an appropriate temperature, add 4-7% sucrose (mass percentage) and an appropriate amount of thickener, stir to dissolve and mix evenly, heat up to about 60-70 °C, and then homogenize it under a pressure of 18-22 MPa, where the primary pressure is 16-18 MPa and the secondary pressure is 2-4 MPa. After cooling to 35-42 °C, add the starter in step 1 at an inoculation amount of 3-5% (volume percentage), ferment at 35-42 °C until the milk curdles, and the final acidity at the end of fermentation is 70-90 °T. After cooling, store it at 4 °C - 6 °C to obtain a mixed fermented milk product with the function of improving immune senescence.

[0032] In the above preparation method of the mixed fermented dairy product, the activation in step 1 is a strain activation step well-known to those skilled in the art. For example, it can be to pick a single colony with an inoculation loop and streak it on an MRS ( Merck KGaA, Germany, product number: 1106610500) solid plate, culture it in a 37 °C constant temperature anaerobic incubator for 72 hours, and repeat three times. Finally, pick a single colony into an MRS liquid medium and culture it overnight in a 37 °C constant temperature incubator for 72 hours. After the culture is completed, obtain the bacterial cells by centrifugation, wash them twice with PBS, resuspend them in skim milk, and store them in a 4 °C refrigerator for standby.

[0033] In the preparation method of the above-mentioned mixed fermented dairy product, after adding the starter in step 2, optionally, Lactobacillus bulgaricus powder and Streptococcus thermophilus powder can be added according to experimental needs. Based on the quality of the mixed fermented milk product, the concentration of Lactobacillus bulgaricus powder is 0.01 - 0.02 g / kg; the concentration of Streptococcus thermophilus powder is 0.015 - 0.03 g / kg. The function of Lactobacillus bulgaricus powder and Streptococcus thermophilus powder here is to act as a starter, shorten the fermentation time, and improve the flavor of the fermented milk.

[0034] In the preparation method of the above-mentioned mixed fermented dairy product, the viable count of the coagulated live bacteria in step 2 is about 10 6 ~10 9 CFU / mL; specifically, it can be 10 6 ~10 7 CFU / mL, 10 7 ~10 8 CFU / mL, or 10 8 ~10 9 CFU / mL, etc.

[0035] On the other hand, the present application provides the use of the aforementioned mixed fermented dairy product in the preparation of anti-aging products. The products herein include but are not limited to foods, health products, and pharmaceuticals.

[0036] In the use provided by the present application, the product has any one or more of the following functions:

[0037] a) Increasing the germinal center of the spleen;

[0038] b) Reducing the expression level of senescence-associated secretory phenotype or chronic inflammation-related genes;

[0039] c) Increasing the weights of the spleen and thymus;

[0040] d) Increasing the white blood cell level;

[0041] e) Activating the functions of immune organs;

[0042] f) Reducing the expression of immune cell exhaustion-related genes;

[0043] g) Reducing the expression of cell senescence-related genes;

[0044] h) Down-regulating the expression of DNA damage-related genes.

[0045] In the uses provided by the present application, the products for improving immune senescence are targeted at mammals. In some embodiments, the mammals are 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.

[0046] In the uses provided by the present application, the germinal center (GC) of the spleen is a structure transiently formed in the B cell area (follicle) of the secondary lymphoid organ, where mature B cells are activated, proliferated, differentiated and mutate their antibody genes, which is one of the important processes for B cell maturation.

[0047] In the uses provided by the present application, the senescence-associated secretory phenotype (SASP) is considered to be one of the key hallmarks of senescence. Cells undergoing senescence show significant changes in their secretome, thereby altering the level of cell-cell communication, leading to chronic low-grade inflammation and diseases in the body, and can act on senescent cells and their neighboring cells to accelerate their senescence process. In some embodiments, the senescence-associated secretory phenotype genes or chronic inflammation-related genes are IL6 and / or IL1β. IL6 and IL1β are key genes of the senescence-associated secretory phenotype (SASP), and their expression is usually associated with chronic inflammation and senescence.

[0048] In the uses provided by the present application, the spleen and thymus, as important immune organs, an increase in their weights means the activation of immune organs, thereby enhancing cellular immune capacity and strengthening the efficacy of the in vivo immune system, and effectively resisting immune senescence.

[0049] In the uses provided by the present application, the 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, etc. An increase in the white blood cell level can be considered as an increase in the level of immune cells in the body.

[0050] In the uses provided by the present application, the immune organs are the spleen and / or thymus. The present application's research found that after intragastric administration of the mixed fermented dairy product to mice, the weights of the spleen and thymus were significantly restored, and there was no significant change in the white blood cell level, indicating that the mixed fermented milk product containing Lentilactobacillus kefiri Fanghua can activate the immune organs of immune senescence model mice to a certain extent.

[0051] In the use provided by the present application, the immune cell exhaustion-related genes are one or more combinations of FOXP3, PD-1, and CD44. FOXP3 is a marker gene of regulatory T cells (Tregs, which 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 (Tm).

[0052] In some embodiments, the injection of hydrocortisone altered the composition of mouse immune cells, including regulatory T cells and memory T cells, and promoted the expression of senescence-associated secretory phenotype (SASP) and chronic inflammation-related genes (IL6 and IL1β). The intervention of the fermented milk product could significantly reduce the expression levels of FOXP3, PD-1, CD44, IL6, and IL1β, and reduce the expression of immune cell exhaustion and inflammation-related genes induced by hydrocortisone.

[0053] In the use provided by the present application, the genes for cell senescence are P21 and P16. The proteins encoded by P21 and P16 are both cell cycle-related factors and important members of the cyclin-dependent kinase inhibitors (CDKI) family. The P21 protein acts as a cell cycle inhibitor by binding to cyclin-dependent kinase 2 (CDK2) to block the cell cycle from entering the gap 1 phase (G1 phase) / synthesis phase (S phase). As an inhibitor of CDK4 / 6, the P16 protein arrests the cell cycle in the G1 phase, affects the telomere length of cells, and participates in the negative regulation of cell proliferation.

[0054] In the use provided by this application, the gene with DNA damage is γ-H2AX. H2AX, whose full name is H2A histone family member X, can also be abbreviated as H2A.X, and is one of the variants of chromosomal histone H2A. When double-stranded DNA in cells breaks, ATM (Ataxia-telangiectasia mutated protein), ATR (ATM and RAD3-related), etc., which are members of the phosphatidylinositol 3-kinase (PI3K)-related kinases (PIKKs) family, phosphorylate the 139th serine on H2AX to form phosphorylated H2AX, that is, γ-H2AX. The content level of γ-H2AX produced by H2AX phosphorylation can clearly reflect the degree of DNA damage and repair, and is widely used in the research of DNA damage and apoptosis, becoming an important DNA damage marker.

[0055] In the use provided by this application, the product dosage form is solid, liquid, gel, semi-liquid, aerosol or powder.

[0056] In some embodiments, the product is selected from lactic acid bacteria beverage products, probiotic agents, probiotic powder products, live aquatic products, milk powder products or cheese products.

[0057] On the other hand, this application provides a lactic acid bacteria beverage product, which uses the aforementioned mixed fermented dairy product as the fermented milk base material, and also includes a combination of one or more of granulated sugar, stabilizer and water.

[0058] In some embodiments, the stabilizer is pectin.

[0059] In some embodiments, the preparation method of the lactic acid bacteria beverage product is as follows:

[0060] 1. Mix and stir granulated sugar, stabilizer and water evenly, cool after high-pressure homogenization and sterilization;

[0061] 2. Add the aforementioned mixed fermented dairy product to step 1, stir evenly, adjust the acidity, preheat to 55-60 °C, and then perform aseptic homogenization under a pressure of 18-22 MPa. After cooling to about 10-15 °C, perform aseptic filling to obtain a lactic acid bacteria beverage product with the function of improving immune senescence.

[0062] On the other hand, this application provides a probiotic agent, whose raw materials include Lactobacillus kefiranofaciens Fanghua; optionally, it also includes one or more strains of Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactiplantibacillus plantarum, Kluyveromyces. In some embodiments, the probiotic agent is obtained by fermenting and culturing the strains and adding a freeze-drying protectant for freeze-drying treatment.

[0063] In some embodiments, the fermentation culture of the strain is a method well-known to those skilled in the art. For example, the strain can be first activated, then inoculated into MRS liquid medium, and subjected to scale-up culture under appropriate temperature conditions, and then inoculated into a fermenter of the fermentation medium for scale-up fermentation and continued fermentation to obtain.

[0064] In some embodiments, the lyophilization protectant can be an acid regulator, a protein protectant, a saccharide protectant, a surfactant, vitamin C, and sodium glutamate, etc. Further, the acid regulator can be phosphate buffer, cysteine hydrochloride, etc. The protein protectant can be skim milk powder, whey protein powder, etc. The saccharide protectant can be sucrose, maltodextrin, lactose, trehalose, etc. The surfactant can be glycerol, polyethylene glycol, etc. Those skilled in the art can add appropriate lyophilization protectants according to experimental needs.

[0065] On the other hand, the present application provides a probiotic powder product, which is obtained by adding a dietary supplement, vitamins, fructooligosaccharide, maltodextrin, and pectin to the aforementioned probiotic agent, mixing them evenly, and then freeze-drying.

[0066] In some embodiments, the preparation method of the probiotic powder product is as follows:

[0067] Dissolve the aforementioned probiotic agent in water, heat it to 20-30 °C, and add 10-12% wt of dietary supplement, 8-10% wt of vitamins, 10-15% wt of fructooligosaccharide, 18-47% wt of maltodextrin, and 10-15% wt of pectin. After mixing evenly, perform freeze-drying to obtain a probiotic powder product with the function of improving immune senescence.

[0068] In some embodiments, the viable count of the probiotic powder product is 10 8 ~10 11 CFU / g; specifically, it can be 10 8 ~10 9 CFU / g, 10 9 ~10 10 CFU / g, or 10 10 ~10 11 CFU / g, etc.

[0069] On the other hand, the present application provides a viable probiotic aquatic product, which is obtained by adding vitamin C, a stabilizer, and water to the aforementioned probiotic agent.

[0070] In some embodiments, based on the mass of the viable probiotic aquatic product, the concentration of the probiotic agent is 10 6 ~10 11 CFU / g; specifically, it can be 10 6 ~107 CFU / g, 10 7 ~10 8 CFU / g, or 10 8 ~10 11 CFU / g, etc.

[0071] In some embodiments, the preparation method of the live bacteria aquatic product is as follows:

[0072] Dissolve the aforementioned probiotic agent in water, which also contains 100 - 300 mg / 100 ml of vitamin C and 0.1 - 0.2% wt of pectin stabilizer. After mixing evenly, the live bacteria aquatic product is prepared.

[0073] On the other hand, the present application provides a milk powder product, the raw materials of which include Lactobacillus kefiranofaciens Fanghua, and also include one or more combinations of whole milk powder, skim milk powder, demineralized whey powder, isomaltooligosaccharide, concentrated whey protein powder, inulin, and compound nutrients.

[0074] In some embodiments, the compound nutrients can be one or more combinations of ultramicro calcium carbonate, vitamin B6, vitamin C, vitamin E, vitamin A, vitamin D, ferric pyrophosphate, zinc oxide, sodium selenite, taurine, folic acid, arachidonic acid, docosahexaenoic acid, and nicotinic acid.

[0075] In some embodiments, optionally, the milk powder product further includes one or more strains of Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactobacillus plantarum.

[0076] In some embodiments, the preparation method of the milk powder product is as follows:

[0077] 1. Mix whole milk powder, demineralized whey powder, isomaltooligosaccharide, concentrated whey protein powder, inulin, and compound nutrients evenly, add water and then perform homogenization treatment, high-temperature sterilization, stir evenly, and spray dry to obtain milk powder;

[0078] 2. Add Lactobacillus kefiranofaciens Fanghua to the milk powder before packaging to obtain the milk powder product with the function of improving immune senescence.

[0079] On the other hand, the present application provides a cheese product, the raw materials of which include Lactobacillus kefiranofaciens Fanghua, and optionally, also include one or more combinations of raw milk, starter culture, and rennet.

[0080] In some embodiments, the preparation method of the cheese product is as follows:

[0081] 1. Standardize the raw milk so that the weight ratio of fat to protein in it is 1.5 - 1.8:1 to obtain the raw material milk;

[0082] 2. Pasteurize the raw milk and then cool it; inoculate the raw milk with the commercial starter culture CHOOZITTM TM (Danisco) and Lactobacillus kefiranofaciens Fanghua, add rennet (Chr. Hansen, Fromase 750XLG), stir, and ferment the curd at a constant temperature;

[0083] 3. Cut the fermented curd into curd blocks, stir slowly, wash the curd blocks with water, and keep stirring, then drain the whey, add square molds, press for a certain period of time, then salt-cure for several days, and ripen at a low temperature such as 10 - 12 °C for 3 - 6 months to obtain a cheese product with the function of improving immune senescence.

[0084] In some embodiments, in step 2 of the method for preparing the cheese product, the pasteurization is carried out at 73 °C for 15 s.

[0085] The following illustrates the embodiments of the present invention through specific examples. 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. 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.

[0086] 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 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.

[0087] When the embodiments give a numerical range, it should be understood that unless otherwise specified in the present invention, any value between the two endpoints of each numerical range and either endpoint 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 similar or equivalent to the methods, devices, and materials described in the embodiments of the present invention can also be used to implement the present invention.

[0088] This application first uses aged mice to evaluate the effect of consuming Lactobacillus kefiranofaciens Fanghua on immune senescence. Compared with aged mice, consuming Lactobacillus kefiranofaciens Fanghua can significantly downregulate the expression of genes related to immune senescence in the spleen and thymus of mice. Secondly, an immune senescence mouse model constructed with hydrocortisone is used to evaluate the effect of consuming fermented milk products containing Lactobacillus kefiranofaciens Fanghua on immune senescence. Compared with the normal control group, the immune organ function of mice injected with hydrocortisone was significantly reduced, the composition of immune cells was changed, the inflammatory response increased, and the indicators related to organ function decline in the serum were significantly elevated. Consuming fermented milk products containing Lactobacillus kefiranofaciens Fanghua can significantly increase the weights of the spleen and thymus, increase the white blood cell content in the blood, activate the function of immune organs, and significantly improve chronic inflammation and the expression of genes related to aging. The present invention first discloses that consuming Lactobacillus kefiranofaciens Fanghua or products containing this strain can improve the symptoms of animal immune senescence, broaden the application scope of Lactobacillus kefiranofaciens as a probiotic, and propose a way to delay aging by taking probiotic products.

[0089] 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 post hoc. P < 0.05 was considered statistically significant. Significant differences are indicated by *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. "ns" indicates no significant difference.

[0090] The following further illustrates this application through examples, but does not limit the scope of this application accordingly.

[0091] Example 1

[0092] Isolation, screening and identification of candidate strains:

[0093] Candidate samples considered to have immune - enhancing effects were collected from different regions, including Tibetan kefir, 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, and appropriate dilution gradients were selected for spreading on TPY (Qingdao Haibo Biotechnology Co., Ltd., HB8570) and MRS (Merck, 1106610500) agar plates, followed by anaerobic culture at 37°C for 48 - 72 hours. Suspected single colonies were picked by observing their colony morphology with the naked eye, and then examined under a microscope and subjected to 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% (v / v) glycerol aqueous solution.

[0094] 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 inspection, 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.

[0095] Table 1 Identification of microbial strains from different collected samples

[0096]

[0097]

[0098] 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 culturing, according to their sources, each strain was mixed in equal volume in 10% wt skim milk (for strains cultured with MRS or TPY, after culturing, 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. They were stored at - 80°C for future use as intervention samples.

[0099] Example 2

[0100] Lactobacillus kefiranofaciens Fanghua in Tibetan kefir is the key strain for regulating immunity:

[0101] 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, 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.

[0102] 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 decreased significantly, and the level of white blood cells (WBC) in the blood also decreased significantly. Compared with the other 4 sample groups, the weights of the spleen and thymus of the mice in the Tibetan kefir group recovered significantly, 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.

[0103] Next, in order to determine the key strains that are effective among the 4 candidate strains, using the same animal model and experimental method, 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.

[0104] 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 sequence number: GCF_022810745.1.

[0105] Example 3

[0106] Preparation of the intervention sample of Lentilactobacillus kefiri Fanghua

[0107] Use an inoculation loop to pick a single colony of Lentilactobacillus kefiri Fanghua 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, repeating three times. Finally, pick a single colony into an MRS liquid medium and culture it in a 37°C constant - temperature incubator overnight for 72 hours to obtain a bacterial solution (the viable 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 for standby in a 4°C refrigerator.

[0108] Example 4

[0109] Lentilactobacillus kefiri Fanghua improves immune senescence in aged mice:

[0110] Select 16 - month - old aged C57BL / 6J wild - type mice. The mice are raised under 12 - hour day - night cycles and at a constant temperature and humidity of 22°C, and provided with sufficient feed and water. Randomly divide all mice into 2 groups: the aged control group (Aged mice); the Lentilactobacillus kefiri Fanghua group (L.kefiri - FH). The aged control group mice are gavaged with skim milk; the L.kefiri - FH group mice are gavaged with Lentilactobacillus kefiri Fanghua resuspended in skim milk (Example 3), and the intervention dose is 1×108 CFU / kg / d. The gavage was conducted for 4 weeks. After the intervention, spleen and thymus tissue samples of the mice were collected for subsequent tests.

[0111] H&E staining: Staining was performed using an H&E staining kit (Solarbio, product number: G1120). Use sufficient hematoxylin to completely cover the tissue section for 2 - 20 minutes. Rinse the glass slide twice with tap water to remove excess stain. Differentiate with the differentiation solution for 3 minutes, and wash twice with tap water, 2 minutes each time. Counterstain with eosin Y aqueous solution for 10 seconds to 2 minutes. Dehydrate in alcohol (75%, 85%, 95%, 100% alcohol), 2 - 3 seconds each time, and rinse in 100% alcohol for 1 minute. Clear with xylene and mount with neutral gum.

[0112] Quantitative PCR: Total RNA was extracted from the tissue 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 (QIAGEN, product number: 208054). The reaction system is 20 μL, and the amplification procedure is shown in Table 3, with 40 cycles.

[0113] Table 2 Primer Sequences

[0114]

[0115] Table 3 Real - time Cycling Conditions

[0116]

[0117] Immunofluorescence staining: The spleen stored in 4% paraformaldehyde was cryosectioned and stained with PD - 1 antibody (Anti - PD1 antibody [NAT105], Abcam, product number: ab52587) (diluted 1:500) and DAPI. The secondary antibody (goat anti - mouse IgG H&L (Alexa 488), Abcam, product number: ab150113) was diluted 1:1000. The stained tissue sections were observed using a Leica THUNDER system.

[0118] Histopathological analysis of the spleen showed that the spleens of the mice in the L. kefiri - FH group had more germinal centers ( Figure 3A). The germinal center (GC) of the spleen is a transiently formed structure within the B-cell area (follicle) of secondary lymphoid organs. Mature B cells are activated, proliferated, differentiated, and mutate their antibody genes within it, which is one of the important processes for B-cell maturation. Quantitative PCR results showed that in spleen tissues, the intervention of Lactobacillus kefiri Fanghua could significantly reduce the expression levels of FOXP3, PD-1, IL6, IL1β, P21, and P16; in thymus tissues, the intervention of Lactobacillus kefiri Fanghua could significantly reduce the expression levels of FOXP3, PD-1, CD44, P21, and P16 ( Figure 3 B). In situ hybridization of PD-1 (labeled with Alexa Flour 488) showed that compared with L. kefiri-FH mice, PD-1 was enriched in the spleen of aged mice. Figure 3 C). FOXP3 is a marker gene of regulatory T cells (Tregs, which is one of the important factors for immune tolerance and inhibits the activation and proliferation of potentially autoreactive T cells existing in normal organisms through an active regulation method), PD-1 is a marker gene of T-cell apoptosis, CD44 is a marker gene of memory T cells (Tm); P21 and P16 are marker genes of cellular senescence; while IL6 and IL1β are key genes of the senescence-associated secretory phenotype (SASP), and their expression is usually related to chronic inflammation and aging. The senescence-associated secretory phenotype (SASP) is considered one of the key markers of aging. Cells undergoing senescence show significant changes in their secretome, thereby altering the level of cell-cell communication, leading to chronic low-grade inflammation and diseases in the body, and can act on senescent cells and their neighboring cells to accelerate their aging process. The above results indicate that consuming Lactobacillus kefiri Fanghua can regulate the immune function of aged mice and delay cellular senescence.

[0119] Example 5

[0120] Construction of a mouse immune senescence model:

[0121] 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 feed and water. All mice were randomly divided into three groups according to the standard of 10 mice per group: a normal control group (Control), a hydrocortisone group (Hydrocortisone), and a fermented milk product group (Fermented milk). The experiment lasted for 4 weeks (during which the intervention samples were given to each group separately), and at the 3rd week, the latter two groups of mice were injected intramuscularly with 40 mg / kg of hydrocortisone (injected once every other day, for a total of 5 times).

[0122] Example 6

[0123] A mixed fermented milk product containing Lactobacillus kefiranofaciens Fanghua with immunomodulatory and anti-aging effects:

[0124] The concentrations of the activated Lactobacillus kefiranofaciens Fanghua, Lactobacillus kefiranofaciens, and Kluyveromyces marxianus cultures were adjusted to 5×10 8 CFU / mL and mixed in a ratio of 1:1:1. Then, the mixed culture was inoculated into skim milk (heat-treated at 95 °C for 5 min) at 3% (v / v), and cultured at 37 °C for about 48 h. After cooling to 4 °C, it was used as a starter. The skim milk was heated to about 55 °C, 4% sucrose and an appropriate amount of thickener were added, and stirred for 15 min to fully dissolve and mix evenly. After heating to about 65 °C, homogenization was carried out at a pressure of 20 MPa. After cooling to 38 °C, the above-mentioned starter was added at an inoculation amount of 5% (v / v), and fermented at 37 °C until curdled (the viable count was about 10 6 ~10 9 CFU / mL), the acidity was 70 °T, and after cooling, it was stored at 4 °C - 6 °C to obtain a mixed fermented milk product with the function of improving immune senescence.

[0125] The following intervention samples were given to the immune senescence model mice in Example 5: The mice in the normal control group and the hydrocortisone group were gavaged with skim milk; the mice in the fermented milk product group were gavaged with the above-mentioned mixed fermented milk containing Lactobacillus kefiranofaciens Fanghua, and the intervention dose was 1×10 8 CFU / kg / d. The gavage time was 4 weeks. And at the 3rd week, hydrocortisone was injected intramuscularly into the latter two groups of mice according to the method in Example 4. After the intervention, the immune organs and blood samples of the mice were collected for subsequent detection.

[0126] Quantitative PCR: Refer to Example 4. The primer sequences (γ-H2AX) used for PCR amplification are shown in Table 4.

[0127] Table 4 Primer Sequences

[0128]

[0129] Hydrocortisone is a common immunosuppressant and is widely used to establish an immunosuppressive model in mice. The results showed that compared with the normal control group, the weights of the spleen and thymus of mice in the immunosuppressive model induced by hydrocortisone decreased significantly, and the level of white blood cells (WBC) in the blood also decreased significantly. After intragastric administration of the mixed fermented milk to mice, the weights of the spleen and thymus recovered significantly, and there was no significant change in the white blood cell level ( Figure 4 A-C). These results indicate that the mixed fermented milk product containing Lactobacillus kefiranofaciens Fanghua can activate the immune organs of immunosenescence model mice to a certain extent. Immunosuppression can affect the composition of immune cells and the expression of some genes related to aging. The quantitative PCR results of spleen tissue showed that the injection of hydrocortisone changed the composition of immune cells in mice, including regulatory T cells and memory T cells, and promoted the expression of senescence-associated secretory phenotype (SASP) and chronic inflammation-related genes (IL6 and IL1β). However, the intervention of the fermented milk product could significantly reduce the expression levels of FOXP3, PD-1, CD44, IL6, and IL1β, and reduce the expression of immune cell exhaustion and inflammation-related genes induced by hydrocortisone. In addition, compared with the mice in the hydrocortisone group, the marker genes of cellular senescence, P21 and P16, and the marker gene of DNA damage, γ-H2AX, were also significantly downregulated in the fermented milk group ( Figure 4 D). The above results indicate that the mixed fermented milk product containing Lactobacillus kefiranofaciens Fanghua has the function of improving immunosenescence in mice.

[0130] Example 7

[0131] A lactic acid bacteria beverage product containing Lactobacillus kefiranofaciens Fanghua and having the functions of regulating immunity and delaying aging:

[0132] Using the fermented milk in Example 6 as the fermented milk base material. Mix 10% wt of granulated sugar, 45% wt of water, and 0.5% wt of a stabilizer (pectin) evenly by stirring, cool after high-pressure homogenization and sterilization. Add 40% of the fermented milk base material, stir and mix evenly, and adjust the acidity to pH 4.4. After preheating to 60 °C, perform aseptic homogenization under a pressure of 20 MPa, and fill aseptically after cooling to about 15 °C, thus obtaining a lactic acid bacteria beverage product with the function of improving immunosenescence (the viable count is about 10 6 ~10 9 CFU / g).

[0133] Example 8

[0134] Probiotic powder product containing Lactobacillus kefiranofaciens Fanghua with immune regulation and anti-aging effects:

[0135] After activating Lactobacillus kefiranofaciens Fanghua according to the method in Example 3, inoculate it into MRS liquid medium and perform enlarged cultivation at 37°C for 72 hours. Inoculate the enlarged culture solution of the strain into the fermentation tank of the fermentation medium and stir-cultivate at 37°C for 48 hours (pH value controlled at about 5.5). Add freeze-drying protectant (10% wt soluble starch, 3% wt sodium salt, 12% wt skim milk powder, 14% wt fructooligosaccharide), mix well and then obtain the probiotic agent after freeze-drying treatment. Take 18% wt of the probiotic agent and add it to distilled water, heat to 25°C, and add 12% wt dietary supplement, 8% wt vitamin, 12% wt fructooligosaccharide, 38% wt maltodextrin and 12% wt pectin, mix well and then perform freeze-drying to obtain the probiotic powder product with the function of improving immune aging (the viable count is about 10 8 ~10 11 CFU / g).

[0136] Example 9

[0137] Mixed probiotic powder product containing Lactobacillus kefiranofaciens Fanghua with immune regulation and anti-aging effects:

[0138] After activating Lactobacillus kefiranofaciens Fanghua according to the method in Example 3, inoculate it into MRS liquid medium and perform enlarged cultivation at 37°C for 60 hours. Activate and enlarge the cultivation of Lactobacillus animalis subsp. lactis BB-12, Lactobacillus casei BDII, Lactobacillus rhamnosus B6, Lactobacillus paracasei BD5115, Lactiplantibacillus plantarum ST-III, Kluyveromyces marxianus KM3 respectively. Inoculate the enlarged culture solution of the strain into the fermentation tank of the fermentation medium and stir-cultivate at 37°C for 20 hours (pH value controlled at about 5.5). Add freeze-drying protectant, mix well and then obtain the probiotic agent after freeze-drying treatment.

[0139] Take 18% wt of the probiotic agent and add it to distilled water, heat to 25°C, and add 12% wt dietary supplement, 8% wt vitamin, 12% wt fructooligosaccharide, 38% wt maltodextrin and 12% wt pectin, mix well and then perform freeze-drying to obtain the mixed probiotic powder product with the function of improving immune aging (the viable count is about 10 8 ~10 11 CFU / g).

[0140] Example 10

[0141] Live probiotic aquatic product containing Lactobacillus kefiranofaciens Fanghua with immunomodulatory and anti-aging effects:

[0142] After the enlarged cultivation of Lactobacillus kefiranofaciens Fanghua according to the method in Example 8, the probiotic agent was obtained after freeze-drying treatment. It was diluted and added to pure water containing 200 mg / 100 ml of vitamin C and 0.2% wt of pectin stabilizer to prepare a live probiotic aquatic product with a final concentration of 10 6 ~10 9 CFU / g with the function of improving immune senescence.

[0143] Example 11

[0144] Milk powder product containing Lactobacillus kefiranofaciens Fanghua with immunomodulatory and anti-aging effects:

[0145] The milk powder formula includes: 74.96% wt of whole milk powder, 10% wt of demineralized whey powder, 8% wt of isomaltooligosaccharide, 3.5% wt of concentrated whey protein powder, 2.5% wt of inulin, 1% wt of compound nutrients (1000 parts of ultramicro calcium carbonate, 150 g of vitamin B6, 8.5 g of vitamin C, 14.2 g of vitamin E, 10 g of vitamin A, 1 g of vitamin D, 18 g of ferric pyrophosphate, 10 g of zinc oxide, 0.3 g of sodium selenite, 6.6 g of taurine, 0.5 g of folic acid, 0.5 g of arachidonic acid, 0.5 g of docosahexaenoic acid, 2 g of nicotinic acid), 0.04% wt of Lactobacillus kefiranofaciens Fanghua, and the addition of Bifidobacterium animalis subsp. lactis BB-12, Lactobacillus casei BDII, Lactobacillus rhamnosus B6, Lactobacillus casei BD5115, Lactobacillus plantarum ST-III.

[0146] Mix the whole milk powder, demineralized whey powder, isomaltooligosaccharide, concentrated whey protein powder, inulin, and compound nutrients evenly, add water and then homogenize, sterilize at high temperature, stir evenly, and spray-dry to obtain milk powder. Before packaging, add 0.04% wt of Lactobacillus kefiranofaciens Fanghua to the milk powder to obtain a milk powder product with the function of improving immune senescence.

[0147] Example 12

[0148] Cheese product containing Lactobacillus kefiranofaciens Fanghua with immunomodulatory and anti-aging effects:

[0149] Standardize raw milk so that the weight ratio of fat to protein is 1.8:1 to obtain raw milk; pasteurize the raw milk (15 s at 73 °C) and then cool it to 30 °C; inoculate commercial starter culture CHOOZITTM in the raw milkTM (Danisco) and Lactobacillus kefiranofaciens Fanghua. Add 0.006 g / L of rennet (Chr. Hansen, Fromase 750XLG), stir for 3 min, and ferment the curd at a constant temperature of 32 °C; cut the fermented curd into curds with a volume of 1.4 cm 3 of clots, and stir slowly for 20 min. Wash the curds with water at 35 °C and keep stirring for 25 min. Then drain the whey, add a square module, and press for 75 min. Cure with 2% wt of salt for 4 days. Age at 12 °C for 6 months to obtain a cheese product with the function of improving immune senescence.

[0150] In summary, the present invention effectively overcomes various disadvantages in the prior art and has high industrial utilization value.

[0151] 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. Use of Lactobacillus kefiranofaciens Fanghua in the preparation of anti-aging products, wherein the preservation number of Lactobacillus kefiranofaciens Fanghua is CGMCC NO. 27733.

2. A mixed fermented dairy product, characterized in that It includes Lactobacillus kefiranofaciens Fanghua, and optionally, also includes one or more combinations of Lactobacillus kefiri, Kluyveromyces marxianus, Bifidobacterium, Lactobacillus, Lactobacillus casei, and Lactiplantibacillus plantarum.

3. The mixed fermented dairy product according to claim 2, characterized in that The mixed fermented dairy product includes Lactobacillus kefiranofaciens Fanghua and Lactobacillus kefiri; based on the total mass of the mixed fermented milk, the concentration ratio of Lactobacillus kefiranofaciens Fanghua to Lactobacillus kefiri is (1-100):(1-100); Preferably, the mixed fermented dairy product also includes Kluyveromyces marxianus; based on the total mass of the mixed fermented milk, the concentration ratio of Lactobacillus kefiranofaciens Fanghua, Lactobacillus kefiri, and Kluyveromyces marxianus is (1-100): (1～100)：1。 4. Use of the mixed fermented dairy product according to any one of claims 2 to 3 in the preparation of antioxidant products; the products are foods, health products, or drugs.

5. The use according to claim 1 or the use according to claim 4, characterized in that The product has any one or more of the following functions: a) Increasing the germinal center of the spleen; b) Reducing the expression levels of senescence-associated secretory phenotype or chronic inflammation-related genes; c) Increasing the weights of the spleen and thymus; d) Increasing the white blood cell level; e) Activating the functions of immune organs; f) Reducing the expression of immune cell exhaustion-related genes; g) Reducing the expression of cell senescence-related genes; h) Downregulating the expression of DNA damage-related genes.

6. The use according to claim 5, characterized in that The senescence-associated secretory phenotype gene or chronic inflammation-related gene is IL6 and / or IL1β; and / or, the immune organ is the spleen and / or thymus; and / or, the immune cell exhaustion-related gene is one or more combinations of FOXP3, PD-1, and CD44; and / or, the cell senescence-related gene is P21 and P16; and / or, the DNA damage-related gene is γ-H2AX.

7. A lactic acid bacteria beverage product, using the mixed fermented dairy product according to any one of claims 2 to 3 as a fermented milk base material, and further comprising one or more combinations of granulated sugar, stabilizer, and water.

8. A probiotic agent, the raw materials of which include Lactobacillus kefiranofaciens Fanghua; optionally, it further includes one or more strains of Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactobacillus plantarum, Kluyveromyces.

9. A probiotic powder product, obtained by adding dietary supplements, vitamins, fructooligosaccharides, maltodextrin, and pectin to the probiotic agent according to claim 8, mixing evenly, and then freeze-drying.

10. A live bacteria aquatic product is obtained by adding vitamin C, a stabilizer, and water to the probiotic agent as described in claim 8. Based on the mass of the live bacteria aquatic product, the concentration of the probiotic agent is 10 6 ~10 11 CFU / g.

11. A milk powder product, the raw materials of which include Lactobacillus kefiranofaciens Fanghua. Optionally, it further includes one or a combination of whole milk powder, skim milk powder, demineralized whey powder, isomaltooligosaccharide, concentrated whey protein powder, inulin, and compound nutrients.

12. A cheese product, the raw materials of which include Lactobacillus kefiranofaciens Fanghua. Optionally, it further includes one or a combination of raw milk, a starter, and rennet.

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