Application of Lactobacillus kefir in preparation of product for delaying organ aging and composition containing Lactobacillus kefir

Abstract

The invention relates to the field of microbial products, in particular to application of lactobacillus kefir in preparation of medicines for delaying organ aging and a composition containing the lactobacillus kefir. The preservation number of the Kefir lactobacillus brachyphyllus provided by the invention is CGMCC (China General Microbiological Culture Collection Center) No.27733 According to the invention, the Lactobacillus kefir and the product containing the Lactobacillus kefir can obviously delay organ aging of an aging animal model and improve exercise activity and exercise coordination; after the excrement of the Kefir Lactobacillus brachygenesis group animal is transplanted to a receptor animal, indexes and inflammatory reactions related to organ function decline in the serum of the receptor animal can be obviously reduced. The Kefir Lactobacillus brachygenesis intervention measure can improve aging and decline of organ functions, and a way for improving organ decline by mediation of intestinal microenvironment through diet regulation is provided.

Description

Technical Field

[0001] The present invention relates to the field of microbial products, and particularly to the use of Lactobacillus kefiranofaciens in the preparation of products for delaying organ aging and a composition containing the same. Background Art

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

[0003] Aging is the decline of biological functions accumulated over time, characterized by the asymptotic loss of physiological integrity, which leads to impaired physiological functions and increased susceptibility to death. Organ aging is usually clinically manifested as a variety of aging-related diseases, seriously affecting the health and quality of life of the elderly population. By detecting the aging markers of different organs, it can provide an important reference basis for scientifically evaluating the degree of organ aging and warning aging-related degenerative diseases. In addition, according to existing research findings, the immune system plays an important role in the process of aging. With the increase in age, many types of immune cells show an increase in the expression of aging marker genes, and the immune system gradually loses its effective response ability to pathogens and cancer cells, and promotes the aging of other solid organs. Research has shown that the aging of immune cells not only reduces the immune function of ERCC1 gene knockout model mice but also promotes the aging of the heart, liver, kidneys, aorta, adipose tissue, brain, and skin, and further leads to a significant shortening of the lifespan of mice (Yousefzadeh MJ, et al. Nature. 2021 and Li Y, et al. Frontiers in Oncology. 2022).

[0004] Although it has been determined that immune senescence is one of the key drivers of solid organ aging, solutions starting from improving immune function and then 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, thereby delaying the aging of individual organs. However, PD-L1 antibodies are highly toxic tumor treatment drugs, and their clinical applications in oncology are still limited. This means that these drugs cannot be used 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 the Tibet Autonomous Region of 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 use of Lentilactobacillus kefiri Fanghua in delaying the aging and functional decline of animal organs, nor is there a 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, developing new probiotics with the effect of improving organ decline 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] In view of the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide the use of Lentilactobacillus kefiri (synonym: Lactobacillus kefiri) in the preparation of products for delaying organ aging and a composition containing the same, so as to solve the problems in the prior art.

[0006] To achieve the above purpose and other related purposes, the present invention provides the use of Lentilactobacillus kefiri in the preparation of products for delaying organ aging.

[0007] Preferably, the preservation number of the Lentilactobacillus kefiri is CGMCC No. 27733.

[0008] The present invention also provides the use of Lentilactobacillus kefiri in the preparation of a product for improving exercise ability.

[0009] The present invention also provides a composition for delaying organ aging or improving exercise ability, and the active ingredients of the composition include the aforementioned Lentilactobacillus kefiri and a pharmaceutically acceptable carrier and medium.

[0010] As described above, the use of Lentilactobacillus kefiri of the present invention in the preparation of a product for delaying organ aging and functional decline and the composition containing the same have the following beneficial effects:

[0011] 1) Delay the organ aging of the aging animal model, and improve exercise vitality and exercise coordination;

[0012] 2) Reduce the indexes related to organ function decline and inflammatory response in the animal serum.

[0013] The intervention measure of Lentilactobacillus kefiri in the present invention can improve the aging and decline of organ function, and provides a way to improve organ decline mediated by the intestinal microenvironment through dietary regulation. Description of the Drawings

[0014] Figure 1 It shows the detection result diagrams of serum α-HBDH, LDH, RFII and β2M of the immunosenescence model mice by Lentilactobacillus kefiri Fanghua of the present invention. Among them, * represents P<0.05; ** represents P<0.01; *** represents P<0.001; ns represents no statistical significance.

[0015] Figure 2 It shows the result diagrams of the effect of Lentilactobacillus kefiri Fanghua of the present invention on delaying organ aging in old mice.

[0016] Among them, (A) is the result of the open field test: Distance represents the total movement distance, Distance in edge represents the movement distance in the edge area, and Distance in center represents the movement distance in the center area; (B) is the result of the rotarod test: Time represents the time that the mouse adheres to the rotarod; (C) is the detection results of serum biochemical functions α-HBDH, LDH, RFII and β2M. * represents P<0.05; ** represents P<0.01; *** represents *P<0.0001; ns represents no statistical significance.

[0017] Figure 3 This is the result graph showing the effect of Lentilactobacillus kefiri Fanghua on the organ function of mice after fecal microbiota transplantation. * represents P < 0.05; ** represents P < 0.01; *** represents P < 0.001; ns represents no statistical significance.

[0018] Figure 4 This is the verification of the special effects of Lentilactobacillus kefiri Fanghua of the present invention. (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. Detailed implementation manners

[0019] The present invention provides the use of Lentilactobacillus kefiri in the preparation of products for delaying organ aging.

[0020] In some specific implementation manners, the Lentilactobacillus kefiri is deposited in the China General Microbiological Culture Collection Center (CGMCC), with the deposit number CGMCC No. 27733, the deposit date being June 29, 2023, and the deposit address being Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.

[0021] In some specific implementation manners, the products for delaying organ aging are selected from any of the following products:

[0022] 1) Products for preventing the increase or decreasing the expression of α-hydroxybutyric dehydrogenase;

[0023] 2) Products for preventing the increase or decreasing the expression of lactate dehydrogenase;

[0024] 3) Products for preventing the increase or decreasing the expression of rheumatoid factor RFII;

[0025] 4) Products for preventing the increase or decreasing the expression of β2-microglobulin;

[0026] 5) Products for regulating the intestinal microenvironment.

[0027] In some specific embodiments, in the product for delaying organ aging, based on the total mass of the product for delaying organ aging, the content of Lentilactobacillus kefiri is 10 6 -10 11 CFU / g. More specifically, the content of Lentilactobacillus kefiri is 10 6 -10 7 CFU / g, 10 7 -10 8 CFU / g, 10 8 -10 9 CFU / g, 10 9 -10 10 CFU / g or 10 10 -10 11 CFU / g.

[0028] In some specific embodiments, the product for delaying organ aging includes one or more of a product for delaying heart aging, a product for delaying liver aging, a product for delaying kidney aging, a product for delaying spleen aging, a product for delaying lung aging, a product for delaying small intestine aging, a product for delaying large intestine aging, a product for delaying muscle tissue aging, or a product for delaying nervous system aging.

[0029] The present invention also provides the use of Lentilactobacillus kefiri in the preparation of a product for improving exercise ability.

[0030] In some specific embodiments, the product for improving exercise ability is a product for improving exercise vitality or a product for improving exercise coordination.

[0031] The present invention also provides the use of Lentilactobacillus kefiri in the preparation of an anti-inflammatory product.

[0032] In some specific embodiments, the preparation of the anti-inflammatory product includes one or more of a product for regulating the expression of rheumatoid factor, a product for regulating the expression of histamine, a product for regulating the expression of kallikrein, or a product for regulating the expression of immunoglobulin.

[0033] The present invention also provides a composition for delaying organ aging or improving exercise ability, and the active ingredient of the composition includes the aforementioned Lentilactobacillus kefiri.

[0034] In some specific embodiments, the composition further comprises a pharmaceutically acceptable salt, ester, isomer, prodrug, polymorph or solvate, as well as a pharmaceutically acceptable carrier and medium.

[0035] Further, the acceptable carriers and media include, for example, sterile water or physiological saline, stabilizers, excipients, antioxidants (such as ascorbic acid), buffers (such as phosphoric acid, citric acid, and other organic acids), preservatives, surfactants (such as PEG, Tween, etc.), chelating agents (such as EDTA, etc.), binders, etc. Moreover, it may also contain other low-molecular-weight polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; amino acids such as glycine, glutamine, asparagine, arginine and lysine; saccharides or carbohydrates such as polysaccharides and monosaccharides; sugar alcohols such as mannitol or sorbitol. When preparing an aqueous solution for injection, such as physiological saline, an isotonic solution containing glucose or other auxiliary drugs, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, appropriate solubilizers such as alcohols (such as ethanol), polyols (such as propylene glycol, PEG, etc.), nonionic surfactants (such as Tween 80, HCO-50, etc.) can be used in combination.

[0036] As used herein, the dosage form of the composition is selected from: injections, tablets, pills, capsules, lozenges, spirits, powders, granules, syrups, solutions, tinctures, aerosols, powder inhalants, or suppositories. Those skilled in the art can select a suitable formulation form according to the administration mode. For example, the formulation forms suitable for oral administration can include, but are not limited to, pills, tablets, chewable tablets, capsules, granules, solutions, drops, syrups, aerosols or powder inhalants, etc. For another example, the formulation forms suitable for parenteral administration can include, but are not limited to, solutions, suspensions, reconstitutable dry formulations or sprays, etc. For another example, those suitable for rectal administration are usually suppositories.

[0037] Among them, tablets, lozenges, pills, capsules, etc. may also contain the following components: binders, such as gums, gum arabic, corn starch or gelatin; excipients, such as dibasic calcium phosphate; disintegrants, such as corn starch, potato starch, alginic acid, etc.; lubricants, such as magnesium stearate; sweeteners, such as sucrose, lactose or saccharin, or flavoring agents, such as peppermint, wintergreen oil or cherry flavoring agents, may be added. When the unit dosage form is a capsule, in addition to the above substances, it may contain a liquid carrier. Various other substances may exist in the form of coatings or be used to improve the physical form of the unit dosage form. For example, tablets, pills or capsules can be coated with shellac, sugar or both. Syrups or elixirs may contain the active compound, sucrose as a sweetener, methylparaben and propylparaben as preservatives, pigments and flavoring agents, such as cherry or orange flavoring agents. Any substance used to prepare any unit dosage form should be pharmaceutically pure and substantially non-toxic in the amounts used. In addition, the active compound can be incorporated into sustained-release products or formulations.

[0038] In this application, 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 Student's t-test was used for comparison 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 statistically significant. Significant differences were indicated by *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. "ns" indicates no significant difference.

[0039] The following specific examples illustrate the implementation modes 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 implementation modes. 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.

[0040] Before further describing the specific implementation modes of the present invention, it should be understood that the protection scope of the present invention is not limited to the specific implementation modes 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 implementation modes, 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.

[0041] When the embodiments give a numerical range, it should be understood that unless otherwise specified in the present invention, any value at both ends of each numerical range and any value between the two ends 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 to or equivalent to those described in the embodiments of the present invention can also be used to implement the present invention.

[0042] Example 1 Isolation, screening, and identification of candidate strains

[0043] Candidate samples thought 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 in a gradient manner with 0.85% normal saline under sterile conditions, 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% (volume percentage) glycerol aqueous solution.

[0044] 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 an - 80°C refrigerator for future use.

[0045] Table 1 Identification of microbial strains from different collected samples

[0046]

[0047] 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. They were stored at - 80°C for future use as intervention samples.

[0048] Example 2 Verification of the special effects of Lactobacillus kefiranofaciens Fanghua

[0049] 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 of 10 according to the standard: 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 gavaged with 10% wt skim milk for 4 weeks, while the other 5 sample groups were gavaged with 400 μL / day of the intervention sample in Example 1. 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 blood samples were detected.

[0050] Hydrocortisone is a common immunosuppressant and is widely used to construct an immunosuppressive model in mice. As Figure 4 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.

[0051] Next, in order to determine the key strain that takes effect 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 gavage 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 4As shown in Figures 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.

[0052] 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, Courtyard 1, Beichen West Road, Chaoyang District, Beijing), with the deposit number CGMCC No. 27733.

[0053] Example 3 Improvement of organ function decline in immune - senescent mice by Lentilactobacillus kefiri

[0054] Preparation of the Lentilactobacillus kefiri intervention sample: Use an inoculation loop to streak a single colony of Lentilactobacillus kefiri (named "Fanghua") on an MRS ( Merck, 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 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 for standby in a 4°C refrigerator.

[0055] Construction of a mouse immune - senescence model and grouped intervention: Select 8 - week - old C57BL / 6J wild - type mice for the experiment. 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. According to the standard of 10 mice per group, all mice are randomly grouped. The grouping is as follows: normal control group (Control); hydrocortisone group (Hydrocortisone); Lentilactobacillus kefiri group (L.kefiri). Among them, the normal control group and the hydrocortisone group are given intragastric intervention with 10% wt skim milk to the mice during the 4 - week intervention period, while the Lentilactobacillus kefiri group is given the above - mentioned Lentilactobacillus kefiri intragastric intervention, and the strain intervention dose is 1×10 8 CFU / kg / d. In the 3rd week of the intervention, mice in the hydrocortisone group and the Lentilactobacillus kefiri group are injected intramuscularly with 40 mg / kg of hydrocortisone (injected once every other day, for a total of 5 times).

[0056] Lactobacillus kefiri Fanghua Improves the Decline of Organ Function in Immunosenescent Mice: According to the method of constructing and grouping interventions for the mouse immunosenescence model described above, the mice were sacrificed on the 2nd day after the last injection of hydrocortisone. Blood was collected from the mice by cardiac puncture and left at room temperature for 15 minutes. It was centrifuged at 3400×g for 15 minutes at 4°C to obtain serum. When further analyzing α-hydroxybutyrate dehydrogenase (α-HBDH), lactate dehydrogenase (LDH), RFII, and β2-microglobulin (β2M), any serum samples with severe hemolysis were excluded. The levels of α-HBDH, LDH, RFII, and β2M in the serum were measured by a biochemical analyzer (Mindary). Among them, α-HBDH, LDH, and β2M are serum indicators used to detect the functions of the heart, liver, and kidneys, and RFII is a rheumatoid factor that can regulate the immune response in the body and can be used as an auxiliary judgment for autoimmune diseases.

[0057] The above results are as Figure 1 shown: Compared with the normal control group, the organ functions of the mice in the hydrocortisone group were significantly decreased, and the inflammatory response increased. On the contrary, in the mice of the Lactobacillus kefiri group, after injection of hydrocortisone, the organ functions in the body did not significantly decline, and the inflammatory response did not change significantly, that is, Lactobacillus kefiri Fanghua has the effect of delaying the decline of organ function in immunosenescence model mice.

[0058] Example 4 Lactobacillus kefiri Improves the Decline of Organ Function and Motor Ability in Aged Mice

[0059] Sixteen-month-old aged C57BL / 6J wild-type mice were selected. 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 the mice were randomly divided into 2 groups: the aged control group (Aged mice); the Lactobacillus kefiri Fanghua group (L.kefiri-FH). The mice in the aged control group were gavaged with 10% wt skim milk; the mice in the L.kefiri-FH group were gavaged with Lactobacillus kefiri Fanghua (Example 3) resuspended in skim milk, and the intervention dose was 1×10 8 CFU / kg / d. The gavage time was 4 weeks. Behavioral experiments were started in the 3rd week, and the feces of the two groups of aged mice were collected and stored at -80°C for later use. After the gavage ended in the 4th week, the mice were sacrificed, and the blood samples of the mice were collected and subjected to biochemical analysis and detection according to the method in Example 3.

[0060] Open field test: The test mice were placed in an open field of 60 cm × 60 cm × 60 cm and allowed to freely explore for 10 min to adapt to the environment. Subsequently, the camera system was turned on to record the total movement distance of the mice in the next 10 min, the movement distance in the central area (30 cm × 30 cm, 50% of the total area), and the movement distance in the marginal area (20% of the total area), and subsequent analysis was carried out.

[0061] Rotarod test: Before conducting the rotarod test on mice, the test mice were placed on a non-rotating rotarod for 5 min to adapt to the surface of the rotarod and the surrounding environment. The speed of the rotating rod was gradually increased, but not exceeding the range that the mice could bear, so that the mice could adapt to the feeling of the rotating rod and the changes in the environment, and the adaptation time was 10 min. At the start of the experiment, the mice were placed on a slowly rotating rotarod, and the speed of the rotarod was adjusted from 4 rpm / min to 40 rpm / min within 5 min. When the mice fell or lost their balance, the falling time was recorded. Four trials were repeated.

[0062] The test mice first underwent the open field test and then the rotarod test. After each test, the test mice were gently taken out and put back into the cage, and then the device was wiped with 75% alcohol. After drying, the next mouse was tested to prevent bias caused by olfactory cues. All behavioral experiments used TopScan Lite software (CleverSys, Inc.) to record, track, and analyze data. Among them, the total movement distance and the movement distance in the central area of the open field test can reflect the motor activity and anxiety state of the mice; the rotarod test can reflect the motor coordination and balance ability of the mice.

[0063] The results of the open field test are as Figure 2 shown in A. Compared with the old control group mice, the total movement distance of the L. kefiri-FH group mice was significantly increased. Although there was no obvious change in the movement distance in the central area, the proportion of activity in the marginal area decreased significantly.

[0064] The results of the rotarod test are as Figure 2 shown in B. The time that the L. kefiri-FH group mice persisted on the rotarod was significantly increased, indicating that their motor coordination and balance ability were higher than those of the old group mice.

[0065] The results of the serum biochemical analysis of the old mice are as Figure 2 shown in C. The results of the serum biochemical analysis of the old mice were consistent with the results of the immune senescence model in Example 3 above, that is, after the intervention of L. kefiri Fanghua, the levels of α-HBDH, LDH, β2M, and RFII in the serum of the old mice all decreased significantly.

[0066] Based on the above results, feeding Lactobacillus kefiranofaciens Fanghua can slow down the organ aging of aged mice and improve their motor ability.

[0067] Example 5 Fecal microbiota transplantation of the fecal microbiota of mice in the Lactobacillus kefiranofaciens Fanghua group can improve the organ aging of mice

[0068] 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 feed and water. Before fecal microbiota transplantation, the original intestinal 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 fecal microbiota of normal control group mice (8-week-old young mice) in Example 3, the FMT-SM group that transplanted the fecal microbiota of middle-aged control group mice (gavage with 10% wt skim milk) in Example 4, and the FMT-L.kefiri-FH group that transplanted the fecal microbiota of aged Fanghua group mice. 0.1 g of fecal pellets was added to 1 mL of sterile PBS and mixed well to make a fecal suspension. Each mouse was gavaged with 250 μL of the fecal suspension every day for 2 consecutive 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 blood samples of the mice were collected according to the method in Example 3 and subjected to biochemical analysis and detection.

[0069] The results of serum biochemical analysis of mice after fecal microbiota transplantation are shown in the figure, which are similar to the results of the immune senescence model and the aged mouse model in Examples 3 and 4 above. The levels of α-HBDH, LDH, and β2M in FMT-L.kefiri-FH mice were significantly lower than those in the FMT-SM group and were similar to those in the FMT-Young group. The level of RFII had no significant difference from that in the FMT-SM group. That is, the results of the fecal microbiota transplantation experiment showed that Lactobacillus kefiranofaciens Fanghua can delay the organ aging of mice through the mediation of the gut microbiota.

[0070] Example 6 Preparation of a composition containing Lactobacillus kefiranofaciens for delaying organ aging and functional decline

[0071] A mixed fermented milk product containing Lactobacillus kefiranofaciens Fanghua:

[0072] The concentrations of the activated Lactobacillus kefiranofaciens Fanghua, Lactobacillus kefiranofaciens, and Kluyveromyces marxianus cultures were each adjusted to 5×108 CFU / mL and mix them in a ratio of 1:1:1. Then, inoculate the mixed culture into skim milk (heat-treated at 95°C for 5 min) at 3% (volume percentage), incubate at 37°C for about 48 h, and cool to 4°C for use as a starter culture. Heat the skim milk to about 55°C, add 4% wt sucrose and an appropriate amount of thickener, and stir for 15 min to fully dissolve and mix evenly. After heating to about 65°C, homogenize at a pressure of 20 MPa. After cooling to 38°C, add the above-mentioned starter culture at an inoculation amount of 5% (volume percentage), with or without adding Lactobacillus bulgaricus powder (0.02 g / kg) and Streptococcus thermophilus powder (0.03 g / kg), and ferment at 37°C until coagulation (the viable cell count is about 10 6 ~10 9 CFU / mL), with an acidity of 70°T. After cooling, store at 4°C - 6°C to obtain a mixed fermented milk product with the effect of delaying organ aging and functional decline.

[0073] Lactic acid bacteria beverage product containing Lentilactobacillus kefiranofaciens Fanghua:

[0074] Use the above-mentioned fermented milk as the fermented milk base material. Mix 10% wt granulated sugar, 45% wt water, and 0.5% wt stabilizer (pectin) evenly, cool after high-pressure homogenization and sterilization. Add 40% wt fermented milk base material, stir and mix evenly, and adjust the acidity to pH 4.4. After preheating to 60°C, perform aseptic homogenization at a pressure of 20 MPa, and aseptically fill after cooling to about 15°C to obtain a lactic acid bacteria beverage product with the effect of delaying organ aging and functional decline (the viable cell count is about 10 6 ~10 9 CFU / g).

[0075] Probiotic powder product containing Lentilactobacillus kefiranofaciens Fanghua:

[0076] After activating Lentilactobacillus kefiranofaciens Fanghua according to the method in Example 3, inoculate it into MRS liquid medium and perform enlarged cultivation at 37°C for 72 h. Inoculate the enlarged culture solution of the strain into a fermentation tank of the fermentation medium and stir and cultivate at 37°C for 48 h (the pH value is 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 evenly, and then obtain a probiotic agent after freeze-drying treatment. Take 18% wt 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 evenly and then perform freeze-drying to obtain a probiotic powder product with the effect of delaying organ aging and functional decline (the viable cell count is about 10 8 ~1011 CFU / g).

[0077] Mixed probiotic powder product containing Lactobacillus kefiranofaciens Fanghua:

[0078] After activating Lactobacillus kefiranofaciens Fanghua according to the method in Example 3, inoculate it into MRS liquid medium and perform enlarged culture at 37 °C for 60 h. Activate and enlarge the culture (temperature 37 °C, culture time 15 h) for one or more strains among Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactiplantibacillus plantarum, and Kluyveromyces. Inoculate the enlarged culture solution of the strains into the fermenter of the fermentation medium and stir-culture at 37 °C for 20 h (pH value controlled at about 5.5). Add freeze-drying protectant, mix well, and then obtain the probiotic agent after freeze-drying. Take 18% wt of the probiotic agent, add it to distilled water, heat to 25 °C, and add 12% wt of dietary supplement, 8% wt of vitamins, 12% wt of fructooligosaccharide, 38% wt of maltodextrin, and 12% wt of pectin. Mix well and then perform freeze-drying to obtain the mixed probiotic powder product with the function of delaying organ aging and function decline (the viable count is about 10 8 ~10 11 CFU / g).

[0079] Live aquatic product containing Lactobacillus kefiranofaciens Fanghua:

[0080] After enlarging the culture of Lactobacillus kefiranofaciens Fanghua according to the method of the probiotic powder product in the above preparation and then performing freeze-drying treatment, obtain the probiotic agent. Dilute and add it to pure water containing 200 mg / 100 ml of vitamin C and 0.2% wt of pectin stabilizer to make a live aquatic product with the function of delaying organ aging and function decline and a final concentration of 10 6 ~10 9 CFU / g.

[0081] Milk powder product containing Lactobacillus kefiranofaciens Fanghua:

[0082] The milk powder formula includes: 74.96% wt whole milk powder, 10% wt demineralized whey powder, 8% wt isomaltooligosaccharide, 3.5% wt concentrated whey protein powder, 2.5% wt inulin, 1% wt 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 Lactobacillus kefiranofaciens Fanghua, and adding or not adding one or more strains of Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactobacillus plantarum. Mix the whole milk powder, demineralized whey powder, isomaltooligosaccharide, concentrated whey protein powder, inulin, and compound nutrients evenly, homogenize after adding water, sterilize at high temperature, stir evenly, and spray dry to obtain milk powder. Add 0.04% wt of Lactobacillus kefiranofaciens Fanghua to the milk powder before packaging to obtain a milk powder product with the effect of delaying organ aging and functional decline.

[0083] Cheese product containing Lactobacillus kefiranofaciens Fanghua:

[0084] Standardize raw milk to make the weight ratio of fat to protein 1.8:1 to obtain raw milk; pasteurize the raw milk (15 s at 73 °C) and then cool it to 30 °C; inoculate the raw milk with commercial starter culture CHOOZITTM TM (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 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 square modules, 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 effect of delaying organ aging and functional decline.

[0085] In summary, the present invention effectively overcomes various drawbacks in the prior art and has high industrial utilization value.

[0086] The above embodiments are intended to illustrate the implementation schemes disclosed by the present invention and should not be construed as limitations on the present invention. In addition, various modifications listed herein and changes in the methods of the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, all the obvious modifications to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. Use of Lentilactobacillus kefiri in the preparation of products for delaying organ aging.

2. The use according to claim 1, wherein , the preservation number of the Lentilactobacillus kefiri is CGMCC No. 27733.

3. The use according to claim 1, characterized in that, The product for delaying organ aging is selected from any one of the following products: 1) A product for preventing the increase in the expression of α-hydroxybutyric acid dehydrogenase or reducing the expression of α-hydroxybutyric acid dehydrogenase; 2) A product for preventing the increase in the expression of lactate dehydrogenase or reducing the expression of lactate dehydrogenase; 3) A product for preventing the increase in the expression of rheumatoid factor RFII or reducing the expression of rheumatoid factor RFII; 4) A product for preventing the increase in the expression of β2-microglobulin or reducing the expression of β2-microglobulin-like; 5) A product for regulating the intestinal microenvironment.

4. The use according to claim 1, characterized in that, In the organ aging delay product, based on the total mass of the organ aging delay product, the content of Lentilactobacillus kefiri is 10 6 -10 11 CFU / g.

5. The use according to claim 1, characterized in that, The product for delaying organ aging includes one or more of a product for delaying heart aging, a product for delaying liver aging, a product for delaying kidney aging, a product for delaying spleen aging, a product for delaying lung aging, a product for delaying small intestine aging, a product for delaying large intestine aging, a product for delaying muscle tissue aging, or a product for delaying nervous system aging.

6. Use of Lentilactobacillus kefiri in the preparation of products for improving exercise ability.

7. The use according to claim 6, wherein The product for improving exercise ability is a product for improving exercise vitality or a product for improving exercise coordination.

8. Use of Lentilactobacillus kefiri in the preparation of anti-inflammatory products.

9. The use according to claim 8, wherein The anti-inflammatory product includes one or more of a product for regulating the expression of rheumatoid factor, a product for regulating the expression of histamine, a product for regulating the expression of kallikrein, or a product for regulating the expression of immunoglobulin.

10. A composition for delaying organ aging or improving exercise ability, characterized in that, The active ingredient of the composition includes Lentilactobacillus kefiri as described in the use according to claim 1 or 2 and a pharmaceutically acceptable carrier and medium.

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