Application of lactobacillus rhamnosus KF7 in anti-aging and anti-oxidation aspects and functional food containing lactobacillus rhamnosus KF7

Abstract

The invention relates to the technical field of microorganisms, in particular to application of lactobacillus rhamnosus KF7 in anti-aging and anti-oxidation aspects and functional food containing the lactobacillus rhamnosus KF7. The invention provides an application of lactobacillus rhamnosus KF7 in preparation of a product for resisting aging, resisting oxidation or improving exercise ability. The preservation number of the lactobacillus rhamnosus KF7 is CGMCC (China General Microbiological Culture Collection Center) NO.6430. Researches in the invention find that the lactobacillus rhamnosus KF7 can improve caenorhabditis elegans in-vivo cell senescence and oxidative stress caused by rhythm disorder and improve the exercise ability of the caenorhabditis elegans. Compared with rhythm disorder nematodes, when the lactobacillus rhamnosus KF7 is eaten, accumulation of lipofuscin and cell apoptosis in nematode bodies can be reduced, the oxidative stress level in the nematode bodies can be reduced, the head swing and body bending frequency of the nematodes can be increased, the exercise ability of the nematodes can be improved, and the nematodes can be prevented from being damaged. The method can be used for producing foods such as fermented milk, lactic acid bacteria beverages, probiotic powder, viable bacteria water, milk powder and cheese and functional food products.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, in particular to the use of Lactobacillus casei rhamnosus KF7 in anti-aging and antioxidant aspects and a functional food containing this strain. Background Art

[0002] The problem of population aging has become one of the social issues that have attracted widespread attention in China and even the world. According to the results of the seventh national population census, in 2020, the elderly population aged 65 and above in China increased to 191 million, and the aging rate was 13.52%. With the continuous increase in the number and proportion of the elderly, anti-aging has not only attracted more and more attention from people but is also considered one of the key directions in healthcare.

[0003] Aging refers to the irreversible basic life phenomenon that, under normal circumstances, after an organism develops and matures, its own functions decline with age, the ability to maintain internal homeostasis and stress resistance decreases, and it gradually tends to death. However, diseases or abnormal factors can cause pathological aging, making aging appear earlier. The circadian rhythm is the variation of life activities with a cycle of about 24 hours generated by organisms to synchronize with the solar day. The rhythm system is crucial for maintaining the synchronization between internal physiology, behavior, and external environmental cues (such as sunlight, temperature, humidity). When this synchronization is lost, circadian rhythm disorders occur. Research shows that the disruption of the biological clock function can cause a series of physiological and behavioral rhythm changes in the body, leading to symptoms such as decreased immune ability, endocrine disorders, and internal environmental disorders, accelerating the aging process. The busy lifestyle of modern people, including exposure to artificial light such as electric lights, computer screens, and mobile phone screens at night, shift work, and reduced time in sunlight during the day, may all lead to circadian rhythm disorders.

[0004] Reactive oxygen species (ROS) refers to the general term for oxygen-containing free radicals related to oxygen metabolism in organisms and peroxides that are prone to form free radicals. The imbalance between the production of reactive oxygen species and the scavenging by the antioxidant system is called oxidative stress. Multiple studies on shift workers have found that circadian rhythm disorders can lead to increased levels of hydrogen peroxide, lipid peroxidation, and oxidized low-density lipoproteins in the body. Therefore, oxidative stress is considered one of the candidate mechanisms by which circadian rhythm disorders increase the risk of disease. Among the various theories of aging, the free radical theory has been widely recognized. Free radicals produced in the body attack unsaturated fatty acids in cell membranes, causing lipid peroxidation reactions, and then destroying the lipid structure of biological membranes, resulting in organelle dysfunction. The degradation product of lipid peroxide, malondialdehyde, can also react with free amino groups to form lipofuscin, causing replication errors or inability to divide in the double helix chain of DNA, and protein cross-linking to form amorphous precipitates, leading to damage to the membrane structure and function. In addition, free radicals can directly oxidize and damage nucleic acids and proteins, ultimately leading to cell aging and death.

[0005] Probiotics are considered to be live microorganisms that confer health benefits on the host when consumed in sufficient amounts. Lacticaseibacillus rhamnosus (synonym: Lactobacillus rhamnosus) belongs to the genus Lactobacillus and is an important probiotic that has various beneficial effects on the health of the body and is widely used in fermented dairy products. Lacticaseibacillus rhamnosus KF7 is a probiotic strain isolated from Chinese Yunnan kefir. There is currently no report on the effect of Lacticaseibacillus rhamnosus KF7 in improving in vivo cell aging and oxidative stress caused by rhythm disorders, 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. Summary of the Invention

[0006] Based on the above technical problems, the present invention provides the use of a strain of Lacticaseibacillus rhamnosus (synonym: Lactobacillus rhamnosus) KF7 in improving in vivo cell aging and oxidative stress in animals and in the production of related functional foods.

[0007] The first aspect of the present application provides the use of Lacticaseibacillus rhamnosus KF7 in the preparation of products for anti-aging, antioxidant, or improving exercise ability, and the preservation number of the Lacticaseibacillus rhamnosus KF7 is CGMCC NO. 6430.

[0008] The second aspect of the present application provides a fermented milk product, the raw materials of which include Lactobacillus rhamnosus KF7; optionally, it further includes one or more combinations of animal milk, plant milk, sucrose, thickener, stabilizer, Lactobacillus bulgaricus, Streptococcus thermophilus, Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactiplantibacillus plantarum.

[0009] The third aspect of the present application provides a lactic acid bacteria beverage product, the raw materials of which include Lactobacillus rhamnosus KF7; optionally, it further includes one or more combinations of animal milk, plant milk, sucrose, thickener, stabilizer, Lactobacillus bulgaricus, Streptococcus thermophilus, Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactiplantibacillus plantarum.

[0010] The fourth aspect of the present application provides a probiotic agent, which is obtained by freeze-drying a fermentation culture strain after adding a freeze-drying protectant. The strain includes Lactobacillus rhamnosus KF7; optionally, it further includes one or more strains of Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactiplantibacillus plantarum, Kluyveromyces.

[0011] The fifth 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 them evenly and then freeze-drying.

[0012] The sixth aspect of the present application provides a live bacteria aquatic product, which is prepared by adding vitamin C and a stabilizer to the aforementioned probiotic agent. Based on the quality of the live bacteria aquatic product, the concentration of the probiotic agent is 10 6 ~10 9 CFU / g.

[0013] The seventh aspect of the present application provides a milk powder product, the raw materials of which include Lactobacillus rhamnosus KF7; optionally, it further includes one or more combinations of whole milk powder, skim milk powder, demineralized whey powder, isomaltooligosaccharide, concentrated whey protein powder, inulin and compound nutrients.

[0014] The eighth aspect of the present application provides a cheese product, the raw materials of which include Lactobacillus rhamnosus KF7; optionally, it further includes one or more combinations of raw cow milk, commercial fermenting agent and rennet.

[0015] Compared with the prior art, the present invention uses Lactobacillus rhamnosus (synonym: Lactobacillus rhamnosus) KF7 (CGMCC NO.6430), and for the first time discloses its new use in improving cell senescence and oxidative stress in animals caused by circadian rhythm disorders and enhancing exercise ability, broadening the application scope of Lactobacillus rhamnosus as a probiotic. Description of the Drawings

[0016] Figure 1. Effects of Lactobacillus rhamnosus KF7 on aging indicators of circadian rhythm-disrupted nematodes. (A) Fluorescence images of lipofuscin in nematodes; (B) Analysis of lipofuscin fluorescence intensity in nematodes; (C) Fluorescence images of apoptosis in nematodes; (D) Analysis of apoptosis fluorescence intensity in nematodes. *P<0.05; ns indicates no statistical significance.

[0017] Figure 2 . Effects of Lactobacillus rhamnosus KF7 on oxidative stress of circadian rhythm-disrupted nematodes. (A) Fluorescence images of ROS content in nematodes; (B) Analysis of ROS fluorescence intensity in nematodes; (C) MDA content in nematodes; (D) SOD activity in nematodes; (E) CAT activity in nematodes. *P<0.05; **P<0.01; ***P<0.001; ns indicates no statistical significance.

[0018] Figure 3 . Effects of Lactobacillus rhamnosus KF7 on locomotor ability of circadian rhythm-disrupted nematodes. (A) Body bending; (B) Head swinging. *P<0.05; **P<0.01; ***P<0.001; ns indicates no statistical significance. Detailed implementation manners

[0019] In order to make the invention purpose, technical solutions and beneficial technical effects of the present invention clearer, the present invention will be further described in detail below with reference to 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.

[0020] On the one hand, the present application provides the use of Lactobacillus rhamnosus KF7 in the preparation of products for anti-aging, antioxidant or improving locomotor ability. The preservation number of Lactobacillus rhamnosus KF7 is CGMCC NO.6430.

[0021] Lactobacillus rhamnosus KF7 is a probiotic strain isolated from Chinese Yunnan kefir. Lactobacillus rhamnosus (synonym: Lactobacillus rhamnosus) belongs to the genus Lactobacillus.

[0022] In the use provided by the present application, the product has any one or more of the following functions:

[0023] 1) Improving cell aging;

[0024] 2) Improving the level of oxidative stress;

[0025] 3) Improving locomotor ability.

[0026] In the uses provided by the present application, improving cellular senescence includes reducing the accumulation of lipofuscin and / or reducing apoptosis of cells. Among them, both the accumulation of lipofuscin and apoptosis of cells are important characteristics of senescence.

[0027] In some embodiments, cellular senescence is caused by circadian rhythm disorders; specifically, it is caused by circadian rhythm disruptions. The circadian rhythm is the variation of life activities with a period of about 24 hours that organisms generate to synchronize with the solar day. The circadian rhythm system is crucial for maintaining the synchronization between internal physiology, behavior, and external environmental cues (such as sunlight, temperature, humidity). When this synchronization is lost, circadian rhythm dysregulation occurs. Research has shown that dysfunction of the biological clock can cause a series of physiological and behavioral rhythm changes in the body, leading to symptoms such as decreased immune capacity, endocrine disorders, and internal environmental disorders, accelerating the aging process.

[0028] In the uses provided by the present application, improving the level of oxidative stress includes reducing the level of reactive oxygen species, reducing the level of malondialdehyde of lipid oxidation, increasing the level of total superoxide dismutase, increasing the level of catalase, or a combination of one or more of them.

[0029] Reactive oxygen species (ROS) refers to the general term for oxygen-containing free radicals and peroxides that are prone to form free radicals related to oxygen metabolism in living organisms. The imbalance between the generation of reactive oxygen species and the scavenging by the antioxidant system is called oxidative stress. Among the various theories of aging, the free radical theory has been widely recognized. Free radicals generated in the body attack unsaturated fatty acids in cell membranes, causing lipid peroxidation reactions, and then destroying the lipid structure of biological membranes, resulting in organelle dysfunction. The degradation product of lipid peroxide, malondialdehyde, can also react with free amino groups to form lipofuscin, causing replication errors or inability to divide in the double helix chain of DNA, and protein cross-linking to form amorphous precipitates, leading to damage to the membrane structure and function. In addition, free radicals can directly oxidize and damage nucleic acids and proteins, ultimately leading to the aging and death of cells. Excessive reactive oxygen species in the body not only cause cell damage but also lead to the occurrence of lipid peroxidation.

[0030] Malondialdehyde (MDA) of lipid oxidation is a natural product generated by lipid oxidation in living organisms. As the final product of lipid peroxidation, it can cause cross-linking and polymerization of macromolecules such as proteins and nucleic acids, further invade cells to induce lipofuscin, and lipofuscin will deposit on cells and further accelerate aging. Therefore, MDA is widely used as an indicator for qualitative oxidation and antioxidant defense systems.

[0031] The body's antioxidant system is mainly composed of antioxidant enzymes, including catalase (CAT), total superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and glutathione-S-transferase (GST), etc.

[0032] In some embodiments, the change in oxidative stress level is caused by circadian rhythm disorders; specifically, it is caused by circadian rhythm disorders.

[0033] The present invention uses Lactobacillus rhamnosus (synonym: Lactobacillus rhamnosus) KF7 (CGMCC NO. 6430), and for the first time discloses its new use in improving cellular senescence and oxidative stress in animals caused by circadian rhythm disorders and enhancing exercise ability, broadening the application scope of Lactobacillus rhamnosus as a probiotic.

[0034] In some embodiments, the subject of the above research is Caenorhabditis elegans, a simple multicellular organism that lives in soil and feeds on microorganisms. 40% of its genome is homologous to human genes, and the homology with human disease-related genes is as high as 65%. Therefore, Caenorhabditis elegans is widely used to simulate complex human diseases. In previous studies, it has been found that the movement, metabolism, protein activity, and transcriptional regulation of C. elegans all exhibit circadian rhythms. Whole-genome analysis found that a large number of genes in C. elegans exhibit rhythmic characteristics under light or temperature entrainment, and compared with light, temperature has a greater impact on the circadian rhythm of C. elegans. This may be due to the fact that C. elegans usually lives in soil, and the direct impact of the sun on it is reflected in the change of temperature.

[0035] In the use provided by the present application, the product dosage form is solid, liquid, gel, semi-liquid, aerosol or powder. In the use provided by the present application, the products include but are not limited to foods, health products, drugs, etc.

[0036] In some embodiments, the products are selected from fermented milk products, lactic acid bacteria beverage products, probiotic agents, probiotic powder products, live aquatic products, milk powder products or cheese products.

[0037] On the other hand, the present application provides a fermented milk product, the raw materials of which include one or a combination of Lactobacillus rhamnosus KF7, animal milk, plant milk, sucrose, thickener, stabilizer, Lactobacillus bulgaricus, Streptococcus thermophilus, Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactiplantibacillus plantarum.

[0038] In some embodiments, the stabilizer is pectin.

[0039] In some embodiments, the thickener is a conventional thickener well-known to those skilled in the art, for example, it can be pectin, agar, carrageenan, locust bean gum, gelatin, sodium caseinate, xanthan gum, modified starch, etc.

[0040] In some embodiments, the preparation method of the fermented milk product is:

[0041] 1. Inoculate the activated Lactobacillus casei rhamnosus KF7 at 3% - 5% (mass percentage) into whole milk and culture it under suitable conditions to prepare a starter culture.

[0042] 2. Heat the whole milk to about 55 - 60 °C, add 4 - 7% sucrose (mass percentage) and an appropriate amount of thickener, stir to fully dissolve and mix evenly, then heat to about 60 - 70 °C and perform homogenization 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 culture obtained in step 1 at an inoculation amount of 3 - 5% (volume percentage), ferment at 35 - 42 °C until coagulation, and the final acidity at the end of fermentation is 70 - 90 °T. After cooling, store at 4 - 6 °C to obtain a fermented milk product with anti - aging and antioxidant effects.

[0043] In the preparation method of the above - mentioned fermented milk 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 of Lactobacillus casei rhamnosus KF7 with an inoculation loop and streak it on an MRS solid plate, culture it in a 37 °C constant - temperature anaerobic incubator for two days, and repeat three times. Finally, pick a single colony and culture it overnight in an MRS liquid medium to prepare an activated Lactobacillus casei rhamnosus KF7 bacterial solution.

[0044] In the preparation method of the above - mentioned fermented milk product, in step 1, a suitable culture environment can be, for example: inoculate the activated bacterial solution at 3% - 5% (mass percentage) into whole milk (heat - treated at 95 °C for 5 min), culture at 37 °C for 24 h, and after cooling to 4 °C, use it as a starter culture (the viable cell count is about 10 8 CFU / mL) for standby.

[0045] In the preparation method of the above - mentioned fermented milk product, after adding the starter culture in step 2, optionally, Lactobacillus bulgaricus powder and Streptococcus thermophilus powder can be added according to experimental needs. Based on the mass 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 functions of Lactobacillus bulgaricus powder and Streptococcus thermophilus powder here are to act as a starter culture, shorten the fermentation time, and improve the flavor of the fermented milk.

[0046] On the other hand, the present application provides a lactic acid bacteria beverage product, the raw materials of which include Lactobacillus casei rhamnosus KF7; optionally, it also includes one or a combination of more of animal milk, plant milk, sucrose, thickener, stabilizer, Lactobacillus bulgaricus, Streptococcus thermophilus, Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactiplantibacillus plantarum.

[0047] In some embodiments, the product obtained according to the preparation method of the aforementioned fermented milk product is used as a fermented milk base material.

[0048] In some embodiments, the preparation method of the lactic acid bacteria beverage product is as follows:

[0049] 1. Mix white granulated sugar, stabilizer and water evenly, cool after high-pressure homogenization sterilization;

[0050] 2. Add the aforementioned fermented milk base material to step 1, stir evenly, adjust the acidity, preheat to 55 - 60 °C, then carry out aseptic homogenization under a pressure of 18 - 22 MPa, cool to about 10 - 15 °C and then carry out aseptic filling to obtain the lactic acid bacteria beverage product with anti-aging and antioxidant effects.

[0051] On the other hand, the present application provides a probiotic agent, which is obtained by freeze-drying a fermented and cultured strain after adding a freeze-drying protectant. The strain includes Lactobacillus rhamnosus KF7; optionally, it further includes one or more strains selected from Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactiplantibacillus plantarum, Kluyveromyces.

[0052] In some embodiments, the strain is Lactobacillus rhamnosus KF7. The fermentation and culture of the strain is a method well-known to those skilled in the art. For example, the strain can be activated first, then inoculated into an MRS liquid medium, expanded cultured under appropriate temperature conditions, and then inoculated into a fermentation tank of a fermentation medium for further expanded fermentation to obtain the product.

[0053] In some embodiments, the freeze-drying protectant can be an acid regulator, a protein protectant, a sugar 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 sugar protectant can be sucrose, maltodextrin, lactose, trehalose, etc. The surfactant can be glycerol, polyethylene glycol, etc. Those skilled in the art can add appropriate freeze-drying protectants according to experimental needs.

[0054] On the other hand, the present application provides a probiotic powder product, which is obtained by mixing and freeze-drying the aforementioned probiotic agent with a dietary supplement, vitamins, fructooligosaccharide, maltodextrin and pectin.

[0055] In some embodiments, the preparation method of the probiotic powder product is as follows:

[0056] Dissolve 15 - 30% wt of the aforementioned probiotic agent in distilled water, heat 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, carry out freeze-drying to obtain the probiotic powder product with anti-aging and antioxidant effects.

[0057] 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.

[0058] In some embodiments, the probiotic powder product is a mixed probiotic powder product, and the fermented milk raw material therein is Lactobacillus rhamnosus KF7 and other strains, and the other strains include one or more strains selected from Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactobacillus plantarum, Kluyveromyces. The fermentation and culture of the other strains are methods well known to those skilled in the art. In a specific embodiment of the present application, the other strains are activated and then expanded in culture, and the culture conditions are a temperature of 37°C and a culture time of 15 h. The expanded culture solution of the strains is inoculated into a fermentation tank of a fermentation medium and stirred and cultured at 37°C for 12 h, and the pH value is controlled at about 5.5.

[0059] On the other hand, the present application provides a viable bacteria aquatic product, which is obtained by adding vitamin C and a stabilizer to the aforementioned probiotic agent.

[0060] In some embodiments, based on the mass of the viable bacteria aquatic product, the concentration of the probiotic agent is 10 6 ~10 9 CFU / g; specifically, it can be 10 6 ~10 7 CFU / g, 10 7 ~10 8 CFU / g, or 10 8 ~10 9 CFU / g, etc.

[0061] In some embodiments, the preparation method of the viable bacteria aquatic product is:

[0062] Dissolve the aforementioned probiotic agent in water, and the water also contains 100 - 300 mg / 100 ml of vitamin C and 0.1 - 0.2% wt of pectin stabilizer. After mixing evenly, a viable bacteria aquatic product with anti-aging and antioxidant effects is obtained.

[0063] On the other hand, the present application provides a milk powder product, the raw materials of which include Lactobacillus rhamnosus KF7, 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.

[0064] In some embodiments, the compound nutrient can be a combination of one or more 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 niacin.

[0065] In some embodiments, optionally, the milk powder product further includes one or more strains of Bifidobacterium, Lactobacillus, Lactobacillus casei, and Lactiplantibacillus plantarum.

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

[0067] 1. Mix full-fat milk powder, demineralized whey powder, isomaltooligosaccharide, concentrated whey protein powder, inulin, and compound nutrient evenly, add water and then perform homogenization treatment, high-temperature sterilization, stir evenly, and spray-dry to obtain milk powder;

[0068] 2. Add 0.04 - 0.08% wt of Lactobacillus casei rhamnosus KF7 to the milk powder before packaging to obtain a milk powder product with anti-aging and antioxidant effects.

[0069] On the other hand, the present application provides a cheese product, the raw materials of which include Lactobacillus casei rhamnosus KF7, and also include a combination of one or more of raw milk, commercial starter, and rennet.

[0070] In some embodiments, the preparation method of the cheese product is as follows:

[0071] 1. Standardize the raw milk so that the weight ratio of fat to protein in it is 1.8:1 to obtain raw material milk;

[0072] 2. Perform pasteurization on the raw material milk and cool it to 30 - 32°C; inoculate with commercial starter and Lactobacillus casei rhamnosus KF7, add 0.002 - 0.006 g / L of rennet, stir, and ferment to obtain curd;

[0073] 3. Cut the fermented curd into curd blocks with a volume of 1.2 - 1.5 cm 3 and stir slowly for 10 - 30 min. Wash the curd blocks with water and keep stirring for 20 - 30 min. Drain the whey, add square modules, and press for 60 - 100 min;

[0074] 4. Cure with 1 - 3% wt of salt and ripen at 10 - 15°C for 5 - 6 months to obtain a cheese product with anti-aging and antioxidant effects.

[0075] In some embodiments, in step 2 of the preparation method of the cheese product, the pasteurization is carried out at 73°C for 15 s.

[0076] Compared with the prior art, the beneficial effects of the present application are:

[0077] This application uses Lactobacillus rhamnosus (synonym: Lactobacillus rhamnosus) KF7 (CGMCC NO. 6430), and for the first time discloses its new use in improving cell senescence and oxidative stress in animals caused by circadian rhythm disorders and enhancing exercise ability, broadening the application scope of Lactobacillus rhamnosus as a probiotic.

[0078] The following specific examples illustrate the embodiments of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0079] 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 for 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 plural forms.

[0080] When the embodiments give a numerical range, it should be understood that unless otherwise specified in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art of this technology. In addition to the specific methods, devices, and materials used in the embodiments, according to the knowledge of those skilled in the art of this technology and the description of the present invention, any methods, devices, and materials similar to 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.

[0081] This application induces circadian rhythm disorders in Caenorhabditis elegans through cyclic temperature changes. Intervention with Lactobacillus rhamnosus KF7 helps reduce the accumulation of lipofuscin and apoptosis of cells in the nematodes, lower the level of oxidative stress in the nematodes, and improve their exercise ability. The present invention broadens the application scope of Lactobacillus rhamnosus as a probiotic and provides a new method for improving cell senescence and oxidative stress caused by circadian rhythm disorders.

[0082] Statistical analysis was performed using SPSS 20.0 software, and various statistical charts were made using GraphPad Prism 7. All data were expressed as mean ± standard deviation. Unpaired t-test (Students test) was used for comparison between two groups, and one-way analysis of variance (ANOVA) was used for comparison among multiple groups (three groups and above). P<0.05 was considered statistically significant. Significant differences were indicated by *P<0.05, **P<0.01, ***P<0.001.

[0083] The present application will be further illustrated by the following examples, but the scope of the present application is not limited thereby.

[0084] Example 1

[0085] Culture of Escherichia coli OP50:

[0086] Using an inoculation loop, pick a single colony of Escherichia coli OP50 into LB liquid medium (tryptone 1 g, yeast extract 0.5 g, NaCl 1 g, add distilled water to a volume of 100 mL, autoclave at 121 °C for 15 min), and incubate at 37 °C with constant shaking at 100 - 200 rpm for 18 - 20 h. The viable cell count was measured to be 1.43×10 9 CFU / mL. Store at 4 °C in the refrigerator for later use.

[0087] Example 2

[0088] Culture of Lactobacillus rhamnosus KF7 and preparation of probiotic bacterial liquid for intervention:

[0089] Using an inoculation loop, pick a single colony of Lactobacillus rhamnosus KF7 (preservation number: CGMCC NO.6430) 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 two days, repeating three times. Finally, pick a single colony into MRS liquid medium and culture it overnight at 37 °C to prepare Lactobacillus rhamnosus KF7 bacterial liquid. Take the above bacterial liquid and add it to a new MRS liquid medium at a concentration of 3% (volume percentage), and culture it in a 37 °C incubator for 20 - 24 h. The viable cell count was measured to be 1.73×10 8 CFU / mL. Store at 4 °C in the refrigerator for later use.

[0090] Concentrate the above Lactobacillus rhamnosus KF7 bacterial liquid by 10 times, resuspend it in LB liquid medium, and mix it with the Escherichia coli OP50 bacterial liquid prepared in Example 1 at a volume ratio of 1:1 to obtain OP50+KF7 bacterial liquid, which is used to feed the nematodes in the probiotic intervention group.

[0091] Example 3

[0092] Preparation of nematode growth medium (NGM):

[0093] Weigh 0.3 g of NaCl, 2 g of agar powder, and 0.25 g of bacteriological peptone, add deionized water and make up the volume to 100 mL. After shaking well, autoclave at 121 °C for 15 min. After autoclaving is completed, cool to 55 °C, and add 0.1 mL of 0.45 μM ion solution CaCl2 (1 mol / L) filtered and sterilized by microporous membrane, 0.1 mL of MgSO4 (1 mol / L), 2.5 mL of K3PO4 (1 mol / L), and 0.32 mL of cholesterol solution (5 mg / mL absolute ethanol), and mix well. Pour the above solution into a petri dish with a diameter of 6 cm, and place it at room temperature for 2 days after solidification to volatilize water and check for contamination by miscellaneous bacteria. Store it in a refrigerator at 4 °C for later use.

[0094] Aspirate 200 μL of the Escherichia coli OP50 bacterial solution prepared in Example 1, drop it onto the center of the NGM plate and spread it evenly, and place it in an incubator at 37 °C for 8 h. Prepare the NGM plate coated with Escherichia coli OP50 and store it in a refrigerator at 4 °C for later use.

[0095] Aspirate 200 μL of the OP50+KF7 bacterial solution prepared in Example 2, drop it onto the center of the NGM plate and spread it evenly, and place it in an incubator at 37 °C for 8 h. Prepare the NGM plate coated with OP50+KF7 and store it in a refrigerator at 4 °C for later use.

[0096] Example 4

[0097] Culture and synchronization of Caenorhabditis elegans:

[0098] Use a pick to transfer a single nematode in the egg-laying period to a new NGM plate, or use a sterile scalpel to cut an agar block with nematodes of appropriate size on the old NGM plate, invert it face down on the surface of the new NGM plate, make a mark, and culture it in a biochemical incubator at 20 °C. Passage once every 3 - 4 days to maintain the activity of the nematodes.

[0099] Select a plate with a large number of adult worms in the reproductive stage for nematode synchronization. Add 2 mL of M9 buffer (0.5 g of NaCl, 0.6 g of Na2HPO4, 0.3 g of KH2PO4, make up to 100 mL with distilled water, add 0.1 mL of 1 mol / L MgSO4 sterilized by 0.45 μM microporous filtration after sterilization) to the plate, pipette with a sterile pipette to wash off the nematodes in the egg-laying stage from the NGM plate, transfer them to a 1.5 mL centrifuge tube, centrifuge at 800×g for 1 min, and discard the supernatant. Add 1 mL of M9 buffer, centrifuge again at 800×g for 1 min to wash the worms, and discard the supernatant. Add 1 ml of fresh nematode lysis solution (1 mL of 8 mol / L NaOH, 0.6 mL of 9% sodium hypochlorite, 3.4 mL of distilled water), vortex immediately for 10 s, centrifuge at 800×g for 1 min, and discard the supernatant. Immediately add 1 mL of M9 buffer to wash the eggs, centrifuge at 800×g for 1 min, and discard the supernatant. Repeat the washing 2 times. Finally, about 50 μL of suspension remains in the tube. Use a sterile pipette to drop the eggs onto the NGM plate coated with OP50 in Example 3, culture at 20 °C, and a large number of L1-stage larvae can be seen under the microscope after about 48 h, confirming successful synchronization.

[0100] Example 5

[0101] Experimental grouping of Caenorhabditis elegans:

[0102] Control group (Control): Transfer the synchronized L1-stage nematodes to the NGM plate coated with Escherichia coli OP50 in Example 3 and culture for 7 days under the temperature cycle conditions of dark, 20 °C for 12 h; 18.5 °C for 12 h.

[0103] Rhythm disorder model group (Model): Transfer the synchronized L1-stage nematodes to the NGM plate coated with Escherichia coli OP50 in Example 3 and culture for 4 days under the temperature cycle conditions of dark, 20 °C for 12 h; 18.5 °C for 12 h, and then transfer to a constant temperature environment of 20 °C for 3 days.

[0104] Probiotic intervention group (KF7): Transfer the synchronized L1-stage nematodes to the NGM plate coated with Escherichia coli OP50 in Example 3 and culture for 4 days under the temperature cycle conditions of dark, 20 °C for 12 h; 18.5 °C for 12 h; then transfer the nematodes to the NGM plate coated with OP50 + KF7 in Example 3 and culture for 3 days in a constant temperature environment of 20 °C.

[0105] Example 6

[0106] Effect of Lactobacillus rhamnosus KF7 on the aging index of rhythm-disordered nematodes:

[0107] At the end of the 7-day culture, nematodes in the control group, the rhythm disorder model group, and the probiotic intervention group of Example 5 were collected for the detection of lipofuscin levels. The nematodes were washed three times with M9 buffer, anesthetized with 60 μM levamisole hydrochloride solution (Shanghai Aladdin Biochemical Technology Co., Ltd., product number: L118865), and placed on a 2% wt agarose pad. Laser scanning confocal microscopy was used for photography, and the average fluorescence intensity of each nematode was calculated using ImageJ software to determine the lipofuscin level in vivo. At least 10 nematodes were detected in each group, with 3 biological replicates.

[0108] Lipofuscin accumulation is an important feature of aging. At a wavelength of 525 nm, lipofuscin can spontaneously emit a blue fluorescence signal. The results are as Figure 1 shown in A and B. The fluorescence images showed that the fluorescence intensity of nematodes in the rhythm disorder model group was stronger than that in the control group and the probiotic intervention group. Through fluorescence intensity analysis, although there was no significant difference in the lipofuscin levels between the model group and the control group of nematodes, there was an increasing trend; while the lipofuscin level of nematodes in the probiotic intervention group was significantly lower than that of nematodes in the model group (P < 0.05).

[0109] Acridine orange dye was used to stain nematodes to detect the level of apoptosis in nematode cells. Nematodes in each group after 7 days of treatment were collected, washed three times with M9 buffer, and transferred to 1 mL (25 μg / mL) acridine orange solution (MCE, USA, product number: HY-101879), and stained in the dark at 20 °C for 2 h in an incubator. After staining, the nematodes were transferred to NGM medium to recover for 10 min, and then the nematodes were placed on an agarose pad dropped with 60 μM levamisole hydrochloride solution, and the apoptosis of Caenorhabditis elegans was observed under a fluorescence microscope. The excitation wavelength was 488 nm, and the emission wavelength was 515 nm.

[0110] Under blue light excitation of the fluorescence microscope, if light green appears, it indicates that the cells have not undergone apoptosis. If it is bright green or orange-yellow, it indicates that the cells have undergone apoptosis. The results are as Figure 1 shown in C and D. The fluorescence images showed that the fluorescence intensity of nematodes in the model group was stronger than that in the control group and the probiotic intervention group. Through fluorescence intensity analysis, although there was no significant difference in the apoptosis level of nematodes in the rhythm disorder model group compared with the control group, there was a certain increasing trend; while the apoptosis level of nematodes in the probiotic intervention group was significantly lower than that of nematodes in the model group (P < 0.05).

[0111] The above results indicate that consuming Lactobacillus rhamnosus KF7 can significantly reduce the accumulation of lipofuscin and apoptosis of cells in nematodes caused by rhythm disorder.

[0112] Example 7

[0113] Effect of Lactobacillus rhamnosus KF7 on oxidative stress in nematodes with rhythm disorder:

[0114] Reactive oxygen species (ROS): The level of reactive oxygen free radicals in nematodes was determined using the H2DCFDA fluorescence probe method. The nematodes in the control group, the circadian rhythm disorder model group, and the probiotic intervention group of Example 5 were collected, washed 3 times with M9 buffer, and transferred to 1.5 mL EP tubes containing 50 μM H2DCFDA solution (product number: HY-D0940, MCE, USA). They were placed in a constant temperature incubator and incubated at 20 °C for 2 h. Subsequently, they were washed 3 times with M9 buffer, anesthetized with levamisole hydrochloride solution, placed on a 2% wt agarose pad, and photographed using a fluorescence confocal microscope (excitation wavelength 488 nm, emission wavelength 525 nm). The average fluorescence intensity of each nematode was calculated using ImageJ software for the fluorescence photos to determine the ROS level in vivo. 10 nematodes were detected in each group, with 3 biological replicates.

[0115] Malondialdehyde (MDA) of lipid oxidation: At the end of the 7-day culture, 500 nematodes from each group of Example 5 were collected, washed 3 times with M9 buffer, and the excess M9 buffer was discarded. 500 μL of M9 buffer was pipetted into each EP tube, and the nematodes were ultrasonically disrupted in an ice-water bath (ultrasonic conditions: power 380 W, ultrasonic for 6 s and pause for 3 s, ultrasonic for 20 min). After ultrasonic treatment, they were centrifuged at 4 °C and 12,000×g for 15 min, and the supernatant was transferred to a new EP tube. The protein concentration was measured and briefly stored at 4 °C for later use. The BCA protein content was determined with reference to the instructions of the BCA protein assay kit (product number: P0011, Beyotime Biotechnology Co., Ltd., Shanghai). The processed samples were taken out of the 4 °C refrigerator, and the MDA content in nematodes was determined with reference to the instructions of the MDA content assay kit (product number: S0131S, Beyotime Biotechnology Co., Ltd., Shanghai). The experiment was repeated three times.

[0116] Total superoxide dismutase (SOD): At the end of the 7-day culture, the nematodes in each group of Example 5 were collected for SOD activity detection. The sample treatment and BCA protein content determination were the same as described above. The determination was carried out with reference to the instructions of the SOD activity kit (product number: S0101S, Beyotime Biotechnology Co., Ltd., Shanghai).

[0117] Catalase (CAT): At the end of the 7-day culture, the nematodes in each group of Example 5 were collected for catalase activity detection. The sample treatment and BCA protein content determination were the same as described above. The catalase activity in nematodes was determined according to the instructions of the catalase activity assay kit (product number: S0051, Beyotime Biotechnology Co., Ltd., Shanghai).

[0118] The ROS results are as Figure 2As shown in A and B, fluorescence images and fluorescence intensity analysis revealed that the fluorescence intensity of nematodes in the rhythm disorder model group was significantly higher than that in the control group (P < 0.05). After intervention with Lactobacillus rhamnosus KF7, the fluorescence intensity decreased significantly (P < 0.05). ROS is formed during metabolic processes associated with enzyme-catalyzed reactions that sustain life (such as aerobic respiration) or when organisms are exposed to biotic and abiotic stress factors. Excessive intracellular ROS levels can lead to damage to lipids, proteins, and DNA, and inhibit the endogenous antioxidant system, resulting in stagnation of energy production, mitochondrial dysfunction, and apoptosis. This process is thought to be related to a variety of human pathologies.

[0119] The results of MDA are as Figure 2 shown in C. Compared with nematodes in the control group, the MDA level of nematodes in the model group increased significantly (P < 0.001); while the MDA level of nematodes in the probiotic intervention group was significantly lower than that of nematodes in the rhythm disorder model group (P < 0.01), and there was no statistical difference compared with the control group (P = 0.052). Excessive reactive oxygen species in the body can not only cause cell damage but also lead to lipid peroxidation. MDA is a natural product produced by lipid oxidation in organisms, and as the final product of lipid peroxidation, it can cause cross-linking and polymerization of macromolecules such as proteins and nucleic acids, further invading cells to induce lipofuscin, which will deposit on cells and further accelerate aging. Therefore, MDA is widely used as an indicator for qualitative oxidation and antioxidant defense systems.

[0120] The results of SOD and CAT are as Figure 2 shown in D and E. Compared with nematodes in the control group, the levels of SOD (P < 0.05) and CAT (P < 0.01) in nematodes in the rhythm disorder model group decreased significantly; while the levels of SOD (P < 0.05) and CAT (P < 0.01) in nematodes in the probiotic intervention group were significantly higher than those in nematodes in the rhythm disorder model group, and there was no statistical difference compared with the control group. The in vivo antioxidant system is mainly composed of antioxidant enzymes, including CAT, SOD, glutathione peroxidase (GSH-Px), and glutathione-S-transferase (GST), etc.

[0121] The above results indicate that consuming Lactobacillus rhamnosus KF7 can significantly improve the oxidative stress level in nematodes caused by rhythm disorder.

[0122] Example 8

[0123] Effect of Lactobacillus rhamnosus KF7 on the locomotor ability of nematodes with rhythm disorder:

[0124] At the end of the 7-day culture, the nematodes in the control group, the rhythm disorder model group, and the probiotic intervention group of Example 5 were collected. 6-7 nematodes were randomly selected from each group and transferred to NGM plates coated with the OP50 bacterial solution of Example 3. After allowing the nematodes to adapt on the plates for 1 minute, the number of head swings and body bends of the nematodes were observed under a 40-fold microscope. For each nematode, a 20-second video was recorded during head swings and a 30-second video was recorded during body bends. One head swing was defined as the head of the nematode moving from one side of the body to the central axis, and one body bend was defined as a sinusoidal process in which the nematode moved forward with the S-shaped central axis formed by the body as the axis.

[0125] The locomotor ability of nematodes can reflect their survival status. Among them, the head swing frequency and body bend frequency are the most common indicators for evaluating the survival status. The results are as Figure 3 shown. Compared with the nematodes in the control group, the number of body bends of the nematodes in the rhythm disorder model group was significantly reduced (P < 0.05), and the head swing frequency was significantly increased (P < 0.001). After intervention with Lactobacillus rhamnosus KF7, the number of body bends showed an increasing trend compared with the model group (no significant difference), and the head swing frequency was significantly reduced compared with the model group (P < 0.001). The above results indicate that consuming Lactobacillus rhamnosus KF7 can improve the locomotor ability of nematodes.

[0126] Example 9

[0127] Fermented milk product containing Lactobacillus rhamnosus KF7 with anti-aging and antioxidant effects:

[0128] Use an inoculation loop to pick a single colony of Lactobacillus rhamnosus KF7 and streak it on an MRS solid plate. Culture it in a 37°C constant temperature anaerobic incubator for two days, and repeat three times. Finally, pick a single colony and culture it overnight in an MRS liquid medium to prepare an activated Lactobacillus rhamnosus KF7 bacterial solution. Take the above-activated bacterial solution and inoculate it into whole milk (heat-treated at 95°C for 5 minutes) at 3% - 5% (volume percentage), and culture it at 37°C for 24 hours. After cooling to 4°C, it is used as a starter (the viable count is about 10 8 CFU / mL) for standby. Heat the whole milk to about 55°C, add 4% - 7% (mass percentage) sucrose and an appropriate amount of thickener, and stir for 15 minutes to fully dissolve and mix evenly. After heating to about 65°C, homogenize it under a pressure of 20 MPa. After cooling to 38°C, add the above starter at an inoculation amount of 3% - 5% (volume percentage), and ferment it at 37°C until it curdles (the viable count is 10 6 ~10 9 CFU / mL), the acidity is 70°T, and after cooling, store it at 4°C - 6°C to obtain a fermented milk product with anti-aging and antioxidant functions.

[0129] Example 10

[0130] Lactic acid bacteria beverage product containing Lactobacillus rhamnosus KF7 with anti - aging and antioxidant effects:

[0131] Using the fermented milk product in Example 9 as the fermented milk base material. Mix 10% wt of granulated sugar, 45% wt of water, and 0.5% wt of stabilizer 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 pre - heating to 60 °C, perform aseptic homogenization under a pressure of 20 MPa, and then perform aseptic filling after cooling to about 15 °C, thus obtaining a lactic acid bacteria beverage product with anti - aging and antioxidant functions (the viable count is about 10 6 ~10 9 CFU / g).

[0132] Example 11

[0133] Probiotic powder product containing Lactobacillus rhamnosus KF7 with anti - aging and antioxidant effects:

[0134] After activating Lactobacillus rhamnosus KF7 according to the method in Example 9, inoculate it into MRS liquid medium and perform enlarged cultivation at 37 °C for 24 h. Inoculate the enlarged culture solution of the strain into the fermenter of the fermentation medium and stir - cultivate at 37 °C for 12 h (control the pH value 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 perform freeze - drying treatment to obtain the probiotic agent.

[0135] 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 evenly and then perform freeze - drying, thus obtaining a probiotic powder product with anti - aging and antioxidant functions (the viable count is about 10 8 ~10 11 CFU / g).

[0136] Example 12

[0137] Mixed probiotic powder product containing Lactobacillus rhamnosus KF7 with anti - aging and antioxidant effects:

[0138] After activating Lactobacillus rhamnosus KF7 according to the method in Example 9, it was inoculated into MRS liquid medium and cultured under expansion at 37 °C for 24 h. Lactobacillus animalis subsp. lactis BB-12, Lactobacillus casei BDII, Lactobacillus rhamnosus B6, Lactobacillus paracasei BD5115, Lactiplantibacillus plantarum ST-III, Kluyveromyces marxianus KM3 were respectively activated and cultured under expansion. The expanded culture solution of the strains was inoculated into a fermenter with a fermentation medium and stirred and cultured at 37 °C for 12 h (the pH value was controlled at about 5.5). After adding a freeze-drying protectant and mixing evenly, a probiotic agent was obtained through freeze-drying treatment.

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

[0140] Example 13

[0141] Live bacteria aquatic product containing Lactobacillus rhamnosus KF7 with anti-aging and antioxidant effects:

[0142] After expanding the culture of Lactobacillus rhamnosus KF7 according to the method in Example 11 and then performing freeze-drying treatment, a freeze-dried probiotic agent was obtained. 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 bacteria aquatic product with anti-aging and antioxidant functions at a final concentration of 10 6 ~10 9 CFU / g.

[0143] Example 14

[0144] Milk powder product containing Lactobacillus rhamnosus KF7 with anti-aging and antioxidant effects:

[0145] 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 niacin), 0.04% wt Lactobacillus rhamnosus KF7, and the addition of Bifidobacterium animalis subsp. lactis BB-12, Lactobacillus casei BDII, Lactobacillus rhamnosus B6, Lactobacillus paracasei BD5115, Lactiplantibacillus 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 perform homogenization treatment, high-temperature sterilization, stir evenly, and spray dry to obtain milk powder. Before packaging, add 0.04% wt of Lactobacillus rhamnosus KF7 to the milk powder to obtain a milk powder product with anti-aging and antioxidant functions.

[0147] Example 15

[0148] A cheese product with anti-aging and antioxidant effects containing Lactobacillus rhamnosus KF7:

[0149] Standardize raw milk to make the weight ratio of fat to protein 1.8:1 to obtain raw milk; after pasteurizing the raw milk (15 s at 73 °C), cool it to 30 °C; inoculate commercial starter culture CHOOZITTM TM (Danisco) and Lactobacillus rhamnosus KF7. 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 curd blocks with a volume of 1.4 cm 3 and slowly stir for 20 min. Wash the curd 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 salt for 4 days. Mature at 12 °C for 6 months to obtain a cheese product with anti-aging and antioxidant functions.

[0150] In summary, this application uses Lactobacillus rhamnosus (synonym: Lactobacillus rhamnosus) KF7 (CGMCC NO. 6430), and for the first time discloses its new use in improving cell senescence and oxidative stress in animals caused by circadian rhythm disorders and enhancing exercise ability, broadening the application scope of Lactobacillus rhamnosus as a probiotic.

[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 made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. Use of Lactobacillus rhamnosus KF7 in the preparation of a product for anti-aging, antioxidant or improving exercise ability, wherein the preservation number of Lactobacillus rhamnosus KF7 is CGMCC NO.6430.

2. The use according to claim 1, characterized in that, The product has any one or more of the following functions: 1) Improve cellular senescence; 2) Improve the level of oxidative stress; 3) Enhance exercise ability.

3. The use according to claim 2, characterized in that, The improvement of cellular senescence includes reducing the accumulation of lipofuscin and / or reducing apoptosis of cells; and / or, the cellular senescence is caused by circadian rhythm disorder.

4. The use according to claim 2, characterized in that, The improvement of the level of oxidative stress includes one or a combination of more of reducing the level of reactive oxygen species, reducing the level of malondialdehyde of lipid oxidation, increasing the level of total superoxide dismutase, and increasing the level of catalase.

5. The use according to claim 2, characterized in that, The product includes food, health products, or drugs; preferably, the product is selected from fermented milk products, lactic acid bacteria beverage products, probiotic agents, probiotic powder products, live aquatic product products, milk powder products, or cheese products.

6. A fermented milk product, the raw materials of which contain Lactobacillus rhamnosus KF7; optionally, it further includes one or a combination of more of animal milk, plant milk, sucrose, thickener, stabilizer, Lactobacillus bulgaricus, Streptococcus thermophilus, Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactobacillus plantarum.

7. A lactic acid bacteria beverage product, the raw materials of which contain Lactobacillus rhamnosus KF7; optionally, it further includes one or a combination of more of animal milk, plant milk, sucrose, thickener, stabilizer, Lactobacillus bulgaricus, Streptococcus thermophilus, Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactobacillus plantarum.

8. A probiotic agent, obtained by freeze-drying a fermentation culture strain after adding a freeze-drying protectant, wherein the strain includes Lactobacillus rhamnosus KF7; optionally, it further includes one or more strains of Bifidobacterium, Lactobacillus, Lactobacillus casei, Lactobacillus plantarum, Kluyveromyces.

9. A probiotic powder product, obtained by adding a dietary supplement, vitamin, fructooligosaccharide, maltodextrin and pectin to the probiotic agent according to claim 8, mixing evenly and then freeze-drying.

10. A live bacteria aquatic product, obtained by adding vitamin C and a stabilizer to the probiotic agent according to claim 8, and based on the quality of the live bacteria aquatic product, the concentration of the probiotic agent is 10 6 ~10 9 CFU / g.

11. A milk powder product, the raw materials of which include Lactobacillus rhamnosus KF7, optionally, it further includes one or a combination of more 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 rhamnosus KF7, and optionally, also include one or more combinations of raw cow milk, commercial starter cultures, and rennet.

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