Animal bifidobacterium lactis BL-16 and astragalus fermentation and anti-aging product application

Abstract

This invention, entitled "Application of Bifidobacterium animalis subsp. lactis BL-16 and its Astragalus ferment and anti-aging products," belongs to the field of microbial technology. The technical problem to be solved is to provide an anti-aging Bifidobacterium animalis subsp. lactis and its BL-16 fermented Astragalus extract. The key technical point is the application of Bifidobacterium animalis subsp. lactis BL-16 or its fermented Astragalus extract in the preparation of anti-aging compositions. Bifidobacterium animalis subsp. lactis BL-16 and its fermented Astragalus extract can scavenge free radicals in vivo and in vitro and enhance anti-lipid peroxidation capacity, exhibiting good antioxidant and immunomodulatory effects. They can effectively improve survival rate, motility, and reproductive capacity, thus having a good effect on delaying aging.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to the application of Bifidobacterium animalis subsp. lactis BL-16 and its Astragalus ferment and anti-aging products. Background Technology

[0002] For understanding the technical content of this invention:

[0003] Aging is a complex biological process involving changes in multiple physiological systems, including the decline of cellular function, tissue degeneration, and weakening of the immune system. In recent years, increasing research has shown that oxidative stress and chronic inflammation play important roles in the aging process. Oxidative stress refers to the generation of free radicals in the body exceeding the scavenging capacity of antioxidant mechanisms, leading to damage to cells and tissues. Free radicals can damage not only DNA, lipids, and proteins, but also trigger a series of pathological changes, closely related to various diseases such as aging, cancer, cardiovascular disease, and neurodegenerative diseases. With increasing age, the body's immune system gradually declines, leading to a low-level state of chronic inflammation. This inflammation not only promotes the occurrence of many age-related diseases but also accelerates the aging process. Therefore, reducing oxidative stress and inflammatory responses is an important strategy for delaying aging.

[0004] Probiotics, as an important component of the gut microbiota, have received widespread attention in recent years for promoting health and delaying aging. Many studies have found that certain probiotics can combat age-related pathological changes by regulating the gut microbiota, enhancing immune function, and reducing inflammatory responses. In particular, probiotics of the Bifidobacterium genus are highly regarded for their good safety profile and health benefits.

[0005] Astragalus ( Astragalus membranaceus Herbs containing polysaccharides, flavonoids, and saponins are a well-known traditional Chinese medicine containing various bioactive components that enhance immunity, combat fatigue, and provide antioxidant effects. These components have been proven to improve the body's antioxidant capacity, reduce inflammation, and promote cell growth and repair. Fermentation of Chinese herbs using microorganisms can protect the active components from the damage caused by traditional processes such as decoction, boiling, boiling, refining, steaming, and soaking. Furthermore, microorganisms produce various enzymes during metabolism, which cleave the cell walls composed of cellulose and hemicellulose, promoting the release of effective components and even generating new active substances, thereby altering the medicinal properties and efficacy of the herbs.

[0006] Although research on the combination of probiotics and traditional Chinese medicine is gradually increasing, systematic research on the anti-aging effects of specific strains combined with plant extracts is still relatively limited.

[0007] Relevant patent documents retrieved: This document, published in China (CN116622585A) on August 22, 2023, discloses the application of *Bifidobacterium lactis* subsp. *lactamase* BL03 in the preparation of antioxidant and anti-aging products. The invention discloses that *Bifidobacterium lactis* subsp. *lactamase* BL03 has the potential to significantly reduce ROS levels and significantly increase SOD activity in zebrafish in an oxidative stress model. This provides a theoretical reference and guidance for developing antioxidant probiotic preparations using *Bifidobacterium lactis* subsp. *lactamase* BL03.

[0008] Relevant non-patent literature retrieved: The literature, "Huang Xiaodan, Feng Yuebiao, Yu Bidan, et al. Synergistic effect of Lactobacillus paracasei FABYIO® TDM-2 and Bifidobacterium animalis subsp. lactis CECE 8145 in improving mitochondrial redox homeostasis and energy metabolism [C], Chinese Nutrition Society. Proceedings of the Chinese Nutrition Society Prebiotics Science Conference. 2025-11-03," discloses that combined intervention with Lactobacillus paracasei FABYIO® TDM-2 and Bifidobacterium animalis subsp. lactis CECE 8145 can reduce the level of reactive oxygen species in nematodes mitochondria and significantly prolong the lifespan of nematodes.

[0009] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: The literature "Huang Xiaodan, Feng Yuebiao, Yu Bidan, et al. Synergistic effect of Lactobacillus paracasei FABYIO® TDM-2 and Bifidobacterium animalis subsp. lactis CECE 8145 in improving mitochondrial redox homeostasis and energy metabolism [C], Chinese Nutrition Society. Proceedings of the Chinese Nutrition Society Prebiotics Science Conference. 2025-11-03." describes a compound strain with antioxidant activity. Furthermore, existing technologies lack systematic research on the anti-aging effects of the combination of Bifidobacterium animalis subsp. lactis and Astragalus membranaceus fermentation broth, highlighting the urgent need for a suitable anti-aging formula of Bifidobacterium animalis subsp. lactis and its Astragalus membranaceus fermentation broth. Summary of the Invention

[0010] The purpose of this invention is to provide: The invention relates to the application of Bifidobacterium animalis subsp. lactis BL-16 and its Astragalus ferment and anti-aging products, and related technologies, in order to solve the technical problems of providing an anti-aging Bifidobacterium animalis subsp. lactis and its Astragalus ferment, or a combination thereof.

[0011] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0012] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0013] Definitions of standard chemical terms can be found in the references "Principles and Identification Techniques of Bacterial and Archaea Systematic Taxonomy, Higher Education Press, Chief Editors Li Wenjun, Liu Lan, Jiao Jianyu, and Fang Baozhu, 2025-01"; "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Science Press, 4th Edition, 2017; and "Microbiology Experiments", Higher Education Press, 4th Edition, 2016.

[0014] Unless otherwise specified, conventional methods within the scope of the art, such as strain activation, culture, centrifugation, fermentation, gavage, etc., shall be used.

[0015] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0016] The term "Bifidobacterium lactis subsp. animalis" as used in this article refers to: Bifidobacterium genus ( Bifidobacterium Animal Bifidobacterium species ( B. animalis It is a subspecies of probiotics and is one of the most widely used and studied strains in the field of probiotics. It has clear probiotic characteristics and safety properties, and is widely found in the human gut, breast milk and fermented dairy products. It is also a core functional strain in food, health food and microecological preparations.

[0017] The term "CFU" as used in this article refers to the original viable number of bacteria corresponding to a single or multiple viable colonies formed by the reproduction of the same microbial cells on a suitable solid culture medium.

[0018] The term "culture medium" as used in this article refers to an artificially prepared nutrient substrate used for the growth, reproduction, and metabolism of microorganisms, cells, or tissues.

[0019] In a first aspect, the present invention provides the use of Bifidobacterium animalis subsp. lactis BL-16 or fermented Astragalus membranaceus extract of Bifidobacterium animalis subsp. lactis BL-16 in the preparation of an anti-aging composition, wherein the preservation number of Bifidobacterium animalis subsp. lactis BL-16 is CGMCC No. 32050.

[0020] The 16S sequence of Bifidobacterium animalis subsp. lactis BL-16 is shown in SEQ ID NO:1; SEQ ID NO:1:

[0021] Among them, the colony characteristics of Bifidobacterium animalis subsp. lactis BL-16 on MRS medium are as follows: the colonies are milky white, round, about 1-2 mm in diameter, smooth and raised, with neat edges.

[0022] Among them, the colony characteristics of Bifidobacterium animalis subsp. lactis BL-16 on Columbia blood agar plates are: non-hemolytic, single colonies are round, milky white and translucent, with regular edges and raised colonies.

[0023] The composition described herein has any one or more of the following functions: (1) Improve the scavenging rate of DPPH free radicals and hydroxyl free radicals; (2) Enhances the ability to resist lipid peroxidation; (3) Alleviate lung tissue damage; (4) Reduce the level of inflammatory factors; (5) Improve survival rate, motor skills, and reproductive capacity; (6) Enhance the activity of SOD, CAT and GSH-Px.

[0024] Furthermore, the composition has any one or more of the following functions: (1) Enhance antioxidant capacity; (2) Enhances the immune response; (3) Enhance anti-inflammatory capabilities; (4) Improve survival rate and delay aging.

[0025] Furthermore, the method for preparing the Astragalus ferment of Bifidobacterium lactis subsp. BL-16 includes the following steps: (1) Add Astragalus membranaceus to water for water extraction and concentration to obtain Astragalus membranaceus water extract; (2) Mix Bifidobacterium animalis subsp. lactis BL-16 with Astragalus membranaceus water extract, incubate and culture, centrifuge and collect the supernatant to obtain Astragalus membranaceus ferment of Bifidobacterium animalis subsp. lactis BL-16.

[0026] Furthermore, in step (1), the amount of water added is 8-12 times the volume of water; preferably, the amount of water added is 10 times the volume of water.

[0027] Furthermore, the water extraction conditions in step (1) are: reflux extraction at 90-100℃ 2-3 times, each extraction lasting 1.5-2 hours.

[0028] Furthermore, the extraction conditions in step (1) are reflux extraction at 100°C twice.

[0029] Furthermore, in step (1), the concentration was carried out under reduced pressure at 60°C to obtain Astragalus membranaceus water extract, which was then concentrated to 1 / 5 of its original volume.

[0030] Furthermore, in step (2), the concentration of the Astragalus membranaceus water extract is 0.2 g / mL, and it is diluted using MRS liquid culture medium.

[0031] Furthermore, in step (2), the volume ratio of Bifidobacterium lactis subsp. BL-16 to Astragalus membranaceus aqueous extract is 1:(1-10); preferably, the volume ratio of Bifidobacterium lactis subsp. BL-16 to Astragalus membranaceus aqueous extract is 1:5.

[0032] Furthermore, the incubation conditions in step (2) are 35-38℃ for 36-48h; preferably, the incubation conditions are 37℃ for 48h.

[0033] Furthermore, in step (2), the centrifugation conditions are 4℃ and 10000r / min for 15min.

[0034] Furthermore, in step (2), Bifidobacterium animalis subsp. lactis BL-16 is a bacterial suspension of Bifidobacterium animalis subsp. lactis BL-16.

[0035] The method for preparing the bacterial suspension of Bifidobacterium lactis subsp. BL-16 is as follows: Bifidobacterium animalis subsp. lactis BL-16 was activated and inoculated into a culture medium for cultivation. The culture supernatant was obtained by centrifuging the live bacterial suspension and then resuspending the bacterial sludge in buffer to obtain a bacterial suspension of Bifidobacterium animalis subsp. lactis BL-16.

[0036] Among them, Bifidobacterium animalis subsp. lactis BL-16 was activated for 2 generations before inoculation.

[0037] The inoculation amount is 2-8%; preferably, the inoculation amount is 5%.

[0038] The culture medium is MRS liquid culture medium.

[0039] The culture time is 16 h-24 h; further, the culture time is 18 h.

[0040] The culture was carried out until the concentration of Bifidobacterium animalis subsp. lactis BL-16 was approximately 5%. 10 9 CFU / mL.

[0041] The centrifugation conditions are 6000 r / min, 10 min, and 4℃.

[0042] Composition: a product comprising an active ingredient and an inert component (pharmaceutically acceptable excipient) constituting a carrier, and any product obtained directly or indirectly from a combination, complexation or aggregation of two or more components, or from the decomposition of one or more components, or from other types of reactions or interactions of one or more components.

[0043] The active ingredient can be in solid and liquid dosage forms, such as capsules, tablets, lozenges, sugar lozenges, granules, and powders, and liquid dosage forms such as elixirs, syrups, emulsions, dispersions, and suspensions. Other dosage forms include ointments, creams, drops, transdermal patches, or powders; ophthalmic solutions or suspensions for use in the eyes, i.e., eye drops; and gelatin capsules containing the active ingredient and a powdered carrier, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. Similar diluents can be used to prepare compressed tablets. Both tablets and capsules can be formulated as sustained-release products for sustained release over several hours. Compressed tablets can be sugar-coated or film-coated to mask any unpleasant taste and protect the tablet from air, or they can be enteric-coated for selective disintegration in the gastrointestinal tract. Generally, water, suitable oils, saline solutions, aqueous solutions of dextrose (glucose), and related sugar solutions, as well as glycols such as propylene glycol or polyethylene glycol, are suitable carriers for parenteral solutions. The parenteral solution preferably contains a water-soluble salt of the active ingredient, a suitable stabilizer, and a buffer substance as needed. Antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid, alone or in combination, are suitable stabilizers. Citric acid and its salts, and sodium EDTA can also be used. Furthermore, the parenteral solution may contain preservatives such as benzalkonium chloride, methylparaben, or propylparaben, and chlorobutanol. The compositions of the present invention can be conveniently delivered in aerosol form from pressurized packaging or a sprayer.

[0044] Furthermore, the composition further includes excipients selected from any one or more of diluents, excipients, fillers, disintegrants, solubilizers, osmotic pressure regulators, surfactants, pH regulators, and antioxidants; the composition is in any one or more of the following forms: powder, tablet, emulsion, pill, ointment, powder, lyophilized powder for injection, gel, drops, tincture, capsule, granule, or aerosol.

[0045] Furthermore, the composition contains at least 10% Bifidobacterium lactis subsp. BL-16 bacteria. 8 CFU.

[0046] Furthermore, the composition contains 10% Bifidobacterium animalis subsp. lactis BL-16. 8 CFU-10 12 CFU, specifically, 10 8 CFU, 109 CFU, 10 10 CFU, 10 11 CFU, 10 12 The bacterial count of CFU (Bifidobacterium animalis) subsp. lactis BL-16 can be selected from any point value within the above range or a range between two point values.

[0047] Preferably, the composition contains 10% Bifidobacterium lactis subsp. BL-16. 8 CFU.

[0048] Secondly, the present invention provides a microbial agent comprising Bifidobacterium animalis subsp. lactis BL-16, the preservation number of which is CGMCC No. 32050.

[0049] Specifically, the bacterial agent includes one or more of the following: Bifidobacterium animalis subsp. lactis BL-16 cells, fermentation broth, fermentation broth supernatant, fermentation broth precipitate, and lyophilized powder.

[0050] Preferably, the bacterial cells are a liquid suspension or a solid bacterial powder.

[0051] Specifically, the microbial agent also includes nutritionally acceptable nutrient additives.

[0052] Preferably, the nutritional additives include any one or more of dietary fiber, prebiotics, protein, lipids, minerals, and vitamins.

[0053] Thirdly, the present invention provides a preparation of Bifidobacterium animalis subsp. lactis BL-16, comprising: fermentation broth, fermentation broth precipitate, fermentation broth supernatant, live bacteria, inactivated bacteria, lyophilized powder, lysate, lysate, secondary metabolites, exosomes, and Astragalus fermentation product of Bifidobacterium animalis subsp. lactis BL-16, wherein the preservation number of Bifidobacterium animalis subsp. lactis BL-16 is CGMCC No. 32050.

[0054] Specifically, the fermentation broth of Bifidobacterium animalis subsp. lactis BL-16 is a mixed liquid system obtained by culturing Bifidobacterium animalis subsp. lactis BL-16 in a culture medium under artificially controlled fermentation conditions. It contains the bacteria themselves, intracellular and extracellular metabolites, unused culture medium components, and fermentation byproducts.

[0055] Specifically, the precipitate of Bifidobacterium animalis subsp. lactis BL-16 fermentation broth is the solid phase component separated from the fermentation broth of the strain after treatment such as standing, centrifugation or filtration. It mainly includes live / dead cells of the strain, cell fragments and insoluble substances produced in the fermentation system.

[0056] Specifically, the supernatant of the fermentation broth of Bifidobacterium animalis subsp. lactis BL-16 is a clear liquid phase component containing extracellular metabolites of the strain, soluble culture medium residues, and soluble fermentation by-products obtained after the fermentation broth of the strain has been allowed to stand, centrifuged, or filtered to remove solid phase precipitates such as bacterial cells.

[0057] Specifically, Bifidobacterium animalis subsp. lactis BL-16 live bacteria are bacteria with normal physiological activity and capable of carrying out life activities such as metabolism and reproduction.

[0058] Specifically, inactivated Bifidobacterium animalis subsp. lactis BL-16 refers to bacteria that have lost their metabolic and reproductive activities but whose overall structure has been basically preserved after being treated by physical, chemical or other means.

[0059] Specifically, Bifidobacterium animalis subsp. lactis BL-16 freeze-dried powder refers to a solid powder that retains the original active components, obtained by removing moisture from liquid materials such as strain fermentation broth, fermentation supernatant, and bacterial suspension through a freeze-drying process.

[0060] Specifically, Bifidobacterium animalis subsp. lactis BL-16 lysate refers to the mixture of intracellular substances formed after live or inactivated Bifidobacterium animalis subsp. lactis BL-16 bacteria are broken down through physical, chemical or enzymatic methods, releasing intracellular substances.

[0061] Specifically, Bifidobacterium animalis subsp. lactis BL-16 lysate refers to a mixed system containing intracellular active components and fragmented bacterial cells formed after live or inactivated Bifidobacterium animalis subsp. lactis BL-16 bacteria are ruptured through physical, chemical, enzymatic, or biological lysation methods to release all intracellular substances.

[0062] Specifically, secondary metabolites of Bifidobacterium animalis subsp. lactis BL-16 refer to various compounds produced by microorganisms such as the strain during their stable growth phase that are not essential for their own growth and reproduction and often possess specific biological activities such as anti-inflammatory and metabolic regulation.

[0063] Specifically, Bifidobacterium animalis subsp. lactis BL-16 exosomes refer to extracellular vesicles encapsulated by nanoscale lipid bilayer membranes that are actively secreted or released by Bifidobacterium animalis subsp. lactis during its growth and metabolism.

[0064] Fourthly, the present invention provides a composition comprising Bifidobacterium animalis subsp. lactis BL-16 or the above-described bacterial agent or the above-described preparation.

[0065] The composition described herein has any one or more of the following functions: (1) Improve the scavenging rate of DPPH free radicals and hydroxyl free radicals; (2) Enhances the ability to resist lipid peroxidation; (3) Alleviate lung tissue damage; (4) Reduce the level of inflammatory factors; (5) Improve survival rate, motor skills, and reproductive capacity; (6) Enhance the activity of SOD, CAT and GSH-Px.

[0066] Furthermore, the composition has any one or more of the following functions: (1) Enhance antioxidant capacity; (2) Enhances the immune response; (3) Enhance anti-inflammatory capabilities; (4) Improve survival rate and delay aging.

[0067] Furthermore, the composition further includes excipients selected from any one or more of diluents, excipients, fillers, disintegrants, solubilizers, osmotic pressure regulators, surfactants, pH regulators, and antioxidants; the composition is in any one or more of the following forms: powder, tablet, emulsion, pill, ointment, powder, lyophilized powder for injection, gel, drops, tincture, capsule, granule, or aerosol.

[0068] The present invention has at least the following beneficial effects: This invention provides a *Bifidobacterium animalis* subsp. lactis BL-16 strain and its fermentation broth containing *Astragalus membranaceus*. Both strain BL-16 and the fermentation broth exhibit good antioxidant and immunomodulatory effects by scavenging free radicals (DPPH and hydroxyl radicals) both in vivo and in vitro, and by enhancing anti-lipid peroxidation capacity. They can effectively improve survival rate, motor function, and reproductive capacity, thus demonstrating a good effect on delaying aging. This provides a scientific basis and practical solution for delaying aging and improving quality of life. This research direction has broad application prospects, helps promote the development of the anti-aging industry, and contributes to human health.

[0069] Considering the possibility of this invention entering other countries, this invention also provides the following technical solutions: An anti-aging method involves administering to a subject an effective amount of Bifidobacterium animalis subsp. lactis BL-16 or the above-mentioned bacterial agent, preparation, or composition.

[0070] The subjects include living organisms (e.g., mammals) that can elicit an immune response. Examples of subjects include humans, primates, cattle, horses, goats, sheep, dogs, cats, mice, rats, rabbits, guinea pigs, pigs, and their transgenic species.

[0071] The composition described herein has any one or more of the following functions: (1) Improve the scavenging rate of DPPH free radicals and hydroxyl free radicals; (2) Enhances the ability to resist lipid peroxidation; (3) Alleviate lung tissue damage; (4) Reduce the level of inflammatory factors; (5) Improve survival rate, motor skills, and reproductive capacity; (6) Enhance the activity of SOD, CAT and GSH-Px.

[0072] Furthermore, the composition has any one or more of the following functions: (1) Enhance antioxidant capacity; (2) Enhances the immune response; (3) Enhance anti-inflammatory capabilities; (4) Improve survival rate and delay aging.

[0073] Preservation Instructions Preserved strain: Bifidobacterium animalis subsp. lactis BL-16; Classification and nomenclature: Bifidobacterium animalis subsp. lactis Bifidobacterium animalis subsp.lactis ; Accession number: CGMCC No. 32050; Preservation period: September 24, 2024; Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections; Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0074] Figure 1 To evaluate the in vitro antioxidant capacity of *Bifidobacterium animalis* subsp. *lactamase* BL-16 and its fermented extract of *Astragalus membranaceus*. (A) DPPH radical scavenging rate, (B) hydroxyl radical scavenging rate, (C) anti-lipid peroxidation rate. Compared with the control group, p<0.001.

[0075] Figure 2 The pathological scores of lung tissue in mice with an acute inflammation model were determined by Bifidobacterium lactis subsp. BL-16 and its fermented Astragalus membranaceus extract. p<0.05, p<0.001.

[0076] Figure 3Immunomodulatory effects of Bifidobacterium lactis subsp. BL-16 and its fermented Astragalus membranaceus extract on an acute inflammatory mouse model. (A) Tumor necrosis factor α (TNF-α) content, (B) Interleukin 1β (IL-1β) content, (C) Interleukin 6 (IL-6) content, (D) Nitric oxide (NO) content. p<0.001.

[0077] Figure 4 To evaluate the antioxidant activity of *Bifidobacterium animalis* subsp. *lactamase* BL-16 and its fermented extract of *Astragalus membranaceus* against *Nematodes*. (A) Relative activity of superoxide dismutase (SOD), (B) Relative activity of catalase (CAT), (C) Relative activity of glutathione peroxidase (GSH-Px), (D) Relative content of glutathione (GSH). ns, p>0.05, p<0.05, p<0.01, p<0.001. Detailed Implementation

[0078] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0079] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0080] Data analysis and statistical analysis were performed using professional data processing software. One-way ANOVA was used for significance analysis, and P<0.05 was considered to indicate a significant difference.

[0081] Example 1: Preparation of strain, Astragalus membranaceus aqueous extract and Astragalus membranaceus fermentation broth (1) Activation and culture of the strain Bifidobacterium animalis subsp. lactis BL-16 was isolated from breast milk of healthy individuals and identified as Bifidobacterium animalis subsp. lactis BL-16 by 16S rRNA gene sequence analysis. Bifidobacterium animalis Subsp. lactis), with the sequence shown in SEQ ID NO: 1, is currently deposited at the China General Microbiological Culture Collection Center, accession number: CGMCC No. 32050.

[0082] After two generations of continuous activation and subculturing of the frozen strain, it was inoculated into MRS liquid medium at a 5% inoculum and cultured continuously for 18 hours until the concentration reached approximately 5%. 10 9 CFU / mL was used for Astragalus fermentation experiments. Simultaneously, the above live bacterial suspension was centrifuged (6000 r / min, 10 min, 4℃) to obtain the culture supernatant of the strain. After washing the bacterial sludge sediment, the bacterial count was adjusted to a suitable concentration with sterile PBS solution for subsequent experiments.

[0083] (2) Preparation of Astragalus fermentation broth Take an appropriate amount of Astragalus powder (Astragalus was purchased from Beijing Tongrentang Co., Ltd.), use 10 times the volume of distilled water, and heat and reflux at 100℃ twice for 1.5 hours each time. Combine the two filtrates and concentrate them under reduced pressure at 60℃ using a rotary evaporator to obtain Astragalus water extract, and store it in a refrigerator at 4℃ for later use.

[0084] During the fermentation of Astragalus membranaceus, the water extract of Astragalus membranaceus was diluted with MRS liquid medium to the experimental concentration (0.2 g / mL). After being treated with high-pressure steam sterilization (121℃, 15-20 min), it was cooled to room temperature. Then, the cultured BL-16 live bacteria solution was mixed with the sterilized Astragalus membranaceus water at a ratio of 1:5 (v / v). The mixture was then added to a fermenter and incubated at 37℃ for 48 h. After the incubation, the supernatant was collected by centrifugation at 4℃ and 10000 r / min for 15 min to obtain the Astragalus membranaceus fermentation broth.

[0085] Example 2: In vitro antioxidant capacity assessment of BL-16 and Astragalus fermentation broth (1) Determination of DPPH free radical scavenging ability The DPPH free radical scavenging assay was used to evaluate the free radical scavenging ability of fermented Astragalus membranaceus.

[0086] DPPH is a stable free radical in organic solvents. Its alcoholic solution is purple and has maximum absorption at a wavelength of 517 nm. In the presence of free radical scavengers, the solution color lightens and the absorbance decreases, which can be used to indicate the antioxidant capacity of a sample.

[0087] In the experiment, 10 μL of each sample solution, including the blank control (ultrapure water), BL-16 supernatant, Astragalus membranaceus aqueous extract, Astragalus membranaceus fermentation broth, and positive control vitamin C (0.1 mg / mL), were added to 90 μL of DPPH solution (5 mM). After shaking and mixing, the solutions were incubated at room temperature in the dark for 30 min. Then, the supernatant was collected by centrifugation and placed in a 96-well plate. The absorbance was measured at 517 nm, and the scavenging rate of DPPH free radicals for each sample was calculated: Scavenging rate % = [(Absorbance of control group - Absorbance of experimental group) / Absorbance of control group] 100%.

[0088] according to Figure 1 As shown in Figure A, the culture supernatant of Bifidobacterium animalis subsp. lactis BL-16, the aqueous extract of Astragalus membranaceus, and the fermentation broth of Astragalus membranaceus in this invention can all effectively scavenge DPPH free radicals, with scavenging rates of 68.41%, 58.25%, and 79.18%, respectively. Among them, the Astragalus membranaceus fermentation broth group showed the highest effect and was significantly higher than the scavenging efficiency of vitamin C (Vc) in the positive group (76.75%). This shows that the combined application of BL-16 and Astragalus membranaceus can significantly improve the DPPH free radical scavenging ability of both BL-16 strain and Astragalus membranaceus itself, exhibiting better antioxidant effects.

[0089] (2) Determination of hydroxyl radical scavenging ability The antioxidant capacity of Bifidobacterium animalis subsp. lactis BL-16 after fermentation of Astragalus membranaceus was evaluated by detecting its ability to scavenge hydroxyl radicals (•OH).

[0090] Hydroxyl radicals are highly reactive free radicals in the body that can cause oxidative damage to cells and tissues. This experiment will use the Fenton reaction to generate hydroxyl radicals and 2',7'-dichlorofluorescein (DCFH-DA) as a probe to assess the scavenging ability of samples by observing changes in fluorescence intensity.

[0091] Five experimental groups were set up in a 96-well plate: ① control group (no treatment), ② BL-16 group (10 μL of culture supernatant with BL-16 bacteria added), ③ Astragalus water extract group (0.2 g / mL, 10 μL), ④ Astragalus fermentation broth group (10 μL of supernatant after co-fermentation of strain and Astragalus), and ⑤ positive control Vc (2 mg / mL).

[0092] In the experiment, H₂O₂ (1 mM) and FeSO₄ (0.1 mM) were mixed to rapidly form hydroxyl radicals. This mixture was then added to all treatment groups. DCFH-DA (final concentration 10 µM) was added to each group, mixed thoroughly, and incubated at 37°C for 30 minutes to allow for complete reaction. After the reaction, the fluorescence intensity was measured using a fluorescence spectrophotometer. The excitation wavelength was set to 488 nm, and the emission wavelength to 525 nm. The hydroxyl radical scavenging rate was calculated based on the fluorescence intensity: Scavenging rate % = [(Fluorescence intensity of control group - Fluorescence intensity of experimental group) / Fluorescence intensity of control group] 100%.

[0093] according to Figure 1 As shown in Figure B, compared with the blank control, the culture supernatant of Bifidobacterium animalis subsp. lactis BL-16, the aqueous extract of Astragalus membranaceus, and the fermentation broth of Astragalus membranaceus all have the ability to scavenge hydroxyl radicals. The scavenging rates of •OH are 63.37%, 56.82%, and 77.40%, respectively. This indicates that the hydroxyl radical scavenging ability of Astragalus membranaceus aqueous extract after fermentation with Bifidobacterium animalis subsp. lactis BL-16 is significantly higher than that of the two treatments alone, thus improving their respective antioxidant effects. Furthermore, the scavenging rate of the Astragalus membranaceus fermentation broth group is comparable to that of the positive vitamin C group (77.40% vs 76.87%).

[0094] (3) Determination of anti-lipid peroxidation capacity The in vitro antioxidant activity assay aimed to evaluate the inhibitory effects of Bifidobacterium lactis subsp. BL-16 and Astragalus membranaceus aqueous extract on lipid peroxidation.

[0095] Lipid peroxidation is an important process of free radical-induced lipid oxidation in cell membranes, and its extent is assessed by measuring the production of malondialdehyde (MDA). MDA is a product of lipid peroxidation and can react with a variety of biomolecules, causing cell damage. Under high temperature and acidic conditions, MDA can react with thiobarbituric acid (TBA) to produce a purple-red compound (532 nm).

[0096] In the experiment, 50 μL of PBS, 100 μL of linoleic acid emulsion, 100 μL of 1% FeSO4, and 30 μL of each sample (including the supernatant of Bifidobacterium lactis subsp. BL-16 culture, Astragalus membranaceus water extract, and Astragalus membranaceus fermentation broth) were added, respectively. After shaking, the mixture was placed in a 37℃ water bath for 1.5 h. After removal, the mixture was vortexed and mixed, and 20 μL of 4% trichloroacetic acid and 200 μL of 0.8% thiobarbituric acid (TBA) were added. The mixture was then placed in a 100℃ water bath for 30 min. After cooling in cold water, the supernatant was collected by centrifugation at 5000 r / min for 5 min, and its absorbance at 532 nm was measured. The positive control group used butylated hydroxyanisole (BHA, 0.01 mg / mL), and the blank control was ultrapure water.

[0097] Statistical analysis showed that the anti-lipid peroxidation rate of Bifidobacterium lactis subsp. BL-16 in this invention was 27.60%, the anti-lipid peroxidation rate of Astragalus membranaceus water extract was 21.37%, and the anti-lipid peroxidation rate of Astragalus membranaceus fermentation broth was 31.02%. This result is comparable to the anti-lipid peroxidation rate of the positive control BHA (32.80%). Figure 1 (C in the middle).

[0098] Example 3: Evaluation of the immunomodulatory effects of Bifidobacterium animalis subsp. lactis BL-16 and Astragalus membranaceus fermentation broth This embodiment utilizes a lipopolysaccharide (LPS)-induced inflammation model to evaluate the immunomodulatory effects of Bifidobacterium lactis subsp. BL-16 and Astragalus membranaceus fermentation broth.

[0099] Purchase 10-12 week old male C57BL / 6 mice (Vitamin B), weighing 25-30 grams. The animals are housed under standard conditions with a 12 / 12-hour light / dark cycle, a temperature of 22±2℃, and a humidity of 50±10%. They are fed normal food and have free access to water.

[0100] After one week of acclimatization, mice were randomly divided into five groups: normal group, LPS control group, BL-16 group, Astragalus aqueous extract group, and Astragalus fermentation broth group, with 10 mice in each group. The control group and LPS group were given an equal volume of physiological saline. The BL-16 group, Astragalus aqueous extract group, and Astragalus fermentation broth group were administered 0.2 mL of BL-16 live bacterial suspension (10^8 CFU / mL), Astragalus aqueous extract (100 mg / kg), and Astragalus fermentation broth extract (100 mg / kg) by gavage daily for 7 days. On the last day, LPS was injected 1 hour after gavage to establish an inflammation model. Mice in the normal group were injected with an equal volume of physiological saline, while mice in the other groups were injected intraperitoneally with LPS (5 mg / kg).

[0101] Two hours after mouse modeling, blood was collected from the eyes, allowed to stand for 30 minutes, and then centrifuged to collect serum. The levels of major inflammatory factors (including TNF-α, IL-1β, IL-6, and NO) in the serum were measured. The levels of TNF-α, IL-1β, and IL-6 in mouse serum were detected using the corresponding ELISA kits from R&D Systems (catalog numbers DY410-05, DY401-05, and DY406-05, respectively), and the procedure was strictly performed according to the kit instructions. NO levels were detected using the nitrate reductase method kit from Beyotime Biotechnology (catalog number S0021), and the total nitrate / nitrite ratio reflected the amount of NO produced. Mice were then sacrificed and dissected, and lung tissue was collected. The lung tissue was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and routinely stained with hematoxylin and eosin (HE). Under an optical microscope, the pathological damage of mouse lung tissue was observed, and pathological scoring was performed based on pulmonary capillary dilation and congestion, inflammatory cell infiltration, and the extent of lesions. Semi-quantitative pathological scoring was conducted according to the method of Mitzner et al. (2000). [0 points: no damage or essentially normal; 1 point: minor damage, lesion area <25%; 2 points: moderate damage, lesion area 25%–50%; 3 points: severe damage, lesion area 50%–75%; 4 points: extremely severe damage, lesion area >75%].

[0102] according to Figure 2 The average pathological score of mice in the normal control group was approximately 4.22, while the pathological score of mice in the LPS group (the inflammation model group) was approximately 8.31. After treatment with BL-16 live bacterial suspension and Astragalus membranaceus (water extract, fermented Astragalus membranaceus liquid), the pathological scores of mice decreased, with the pathological score of the Astragalus membranaceus fermented liquid decreasing to 7.37. This indicates that the Bifidobacterium lactis subsp. BL-16 and Astragalus membranaceus fermented liquid in this invention can alleviate lung tissue damage in inflammatory mice.

[0103] Furthermore, by detecting inflammatory factors in serum samples, the results were as follows: Figure 3 As shown, 2 hours after LPS modeling, compared with the control group, the serum levels of TNF-α, IL-1β, IL-6, and NO in the other groups of mice increased significantly, indicating that the inflammation model was successfully induced. The levels of the above-mentioned inflammatory factors significantly decreased after treatment with BL-16 live bacteria, Astragalus membranaceus aqueous extract, and Astragalus membranaceus fermentation broth, and were significantly lower than those in the LPS group. (p<0.001) indicates that the systemic inflammatory response in the treated model mice was improved. These results demonstrate that the water extract of Astragalus membranaceus fermented with Bifidobacterium lactis subsp. BL-16 has good anti-inflammatory effects, and the combined application of the two can effectively enhance the immune response and exert immunomodulatory effects.

[0104] Example 4 Evaluation of the anti-aging function of Bifidobacterium animalis subsp. lactis BL-16 and Astragalus membranaceus ferment broth in nematodes Wild-type *C. elegans* N2 was selected for the experiment to ensure that the obtained nematodes were synchronously developing individuals of the same age. Nematode growth medium (1% agar, 2.5 g NaCl, 1 g yeast aqueous extract, 1 mL 1 M phosphate buffer, double-distilled water to 500 mL) was prepared, sterilized, poured into petri dishes, and stored after solidification. Experiments were designed with the following groups: control group, BL-16 group, *Astragalus membranaceus* aqueous extract group, and *Astragalus membranaceus* fermentation broth group. The control group nematodes were fed only standard NGM without BL-16. The BL-16 group had BL-16 bacterial suspension (10^8 CFU / mL) added to the NGM medium. The *Astragalus membranaceus* aqueous extract group had an appropriate amount of *Astragalus membranaceus* aqueous extract (0.5 mg / mL) added to the NGM medium. The *Astragalus membranaceus* fermentation broth group had *Astragalus membranaceus* extract fermented by BL-16 strain (0.5% v / v) added to the NGM medium. The experimental treatment began with the larval stage (L1 stage) and adult stage (L4 stage) of the nematodes. The culture medium was replaced every few days throughout the culture period to prevent bacterial overgrowth and nutrient depletion.

[0105] The following physiological indicators of nematodes were evaluated: ① Survival rate determination: The number of surviving individuals in each group of nematodes was recorded weekly, and the survival rate was calculated; ② Motility test: A touch test was used: the nematodes were gently stimulated with a small tool, and their reaction time and movement speed were observed; ③ Reproductive capacity: Each female nematode was placed in a separate culture dish after reaching adulthood, and the number of eggs laid within 3 days was recorded. The average number of eggs laid in each group was calculated; ④ Lifespan test: Starting from the adult stage, the condition of the nematodes was observed daily, and the time of death was recorded. The average lifespan of each group of nematodes was calculated; ⑤ Antioxidant enzyme activity: Water samples of nematodes were extracted, and the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), as well as the content of glutathione (GSH), were determined using a Solarbio reagent kit according to the manufacturer's instructions.

[0106] The statistical results of nematode survival indicators are shown in Table 1. Compared with the control group, nematodes treated with Bifidobacterium animalis subsp. lactis BL-16, Astragalus membranaceus aqueous extract, and Astragalus membranaceus fermentation broth showed improvements in survival rate, motility, reproductive capacity, and lifespan. Among them, the improvement effect of Astragalus membranaceus fermentation broth was the most significant, with the survival rates of L1 larvae and L4 adults increasing from 70% and 50% to 83% and 71%, respectively; the average movement speed increased from 0.5 mm / s to 1.0 mm / s; the average number of eggs laid increased to 64 per larva; and the average survival days increased from 15 days to 23.3 days.

[0107] Table 1. Statistics on nematode index detection

[0108] The results of the detection of antioxidant enzyme activity in nematodes showed that, compared with the blank control group, the SOD (superoxide dismutase) activity of nematodes treated with Astragalus fermentation extract was significantly higher. Figure 4 A in ( ), CAT ( Figure 4 B) and GSH-Px ( Figure 4 The activity of C in the nematodes increased by 26.8%, 27.1%, and 24.4%, respectively; meanwhile, the content of glutathione (GSH) in the nematodes showed ( Figure 4 In the D group, the GSH content in the Astragalus fermentation broth group was 1.73 times that of the control group, and the GSH content in the BL-16 group and the Astragalus water extract group also reached 1.42 times and 1.38 times, respectively. This indicates that the BL-16 strain and the Astragalus fermentation broth can effectively improve the activity of antioxidant enzymes and enhance the body's antioxidant capacity.

[0109] These results indicate that Bifidobacterium animalis subsp. lactis BL-16 and Astragalus membranaceus ferment broth can enhance the antioxidant capacity of nematodes, improve their survival rate, prolong their lifespan, and play a role in delaying aging.

[0110] Example 5: Comparison of in vitro antioxidant capacity of Astragalus fermentation broth and Astragalus extract from various strains. Referring to the experimental method of Example 2 of this invention, parallel measurements of three core antioxidant indicators—DPPH free radical scavenging rate, hydroxyl free radical scavenging rate, and anti-lipid peroxidation rate—were performed on the experimental group (BL-16 Astragalus fermentation broth), four commercially available probiotic strains, and laboratory-preserved Astragalus fermentation broth group and Astragalus extract group. A blank control group (ultrapure water) and a positive control group (Vc / DPPH & hydroxyl free radical, BHA / anti-lipid peroxidation) were also set up. Each experiment was repeated three times, and the average value was taken for single-factor ANOVA significance analysis. The results are shown in Table 2.

[0111] Commercially available experimental strains were purchased from the China Microbial Culture Collection Center / General Commercial Culture Bank, specifically: Control strain 1: Bifidobacterium animalis subsp. lactis HN019 (a classic antioxidant and immunomodulatory Bifidobacterium strain). Control strain 2: Lactobacillus rhamnosus GG (LGG, a classic probiotic lactic acid bacteria that can be used for both food and medicine). Control strain 3: Lactobacillus plantarum 299v (a commonly used probiotic lactic acid bacteria for intestinal regulation and anti-oxidation). Control strain 4: Bifidobacterium longum in Bifidobacterium triple live bacteria tablets (commercially available Bifidobacterium strain). Control strain 5: Bifidobacterium adolescentis LLA-5 (a Bifidobacterium that was isolated and preserved in the laboratory and has been systematically verified to have in vitro antioxidant and anti-inflammatory activities). Control strain 6: Bifidobacterium animalis subspecies BAW-3 (isolated and preserved in the laboratory, and systematically verified to have in vitro antioxidant and anti-inflammatory activities of the same species but a different subspecies); Control strain 7: Bifidobacterium animalis subsp. lactis BL-10 (a strain of the same subspecies that was isolated and preserved in the laboratory and has been systematically verified to have in vitro antioxidant and in vitro anti-inflammatory activities). Control strain 8: Bifidobacterium animalis subsp. S61 (isolated and preserved in the laboratory, and systematically verified to have in vitro antioxidant and in vivo anti-inflammatory activities).

[0112] All control strains were simultaneously activated and cultured according to the method in Example 1 of this invention, and Astragalus fermentation broth was prepared using the same process of mixing bacterial solution and Astragalus extract at a ratio of 1:5 (v / v) and incubating at 37°C for 48 hours. The Astragalus extract group was set up according to the stock solution prepared in Example 1. All experimental conditions, concentrations, and fermentation processes were completely unified to eliminate experimental variables and ensure the validity of the comparison results.

[0113] Experimental methods: Completely consistent with Example 2 of this invention, using DPPH free radical scavenging method, Fenton reaction-fluorescent probe method, and thiobarbituric acid (TBA) colorimetric method to determine the DPPH free radical scavenging rate, hydroxyl free radical scavenging rate, and anti-lipid peroxidation rate of each group of samples in turn, and calculating the results according to the corresponding formulas.

[0114] Experimental results: Table 2. Results of in vitro antioxidant index determination of Astragalus fermentation broth and Astragalus extract of various strains (%, x±s)

[0115] All probiotic strains of Astragalus fermentation broth showed significantly higher levels of three core antioxidant indicators compared to the Astragalus extract group (P<0.05), demonstrating that probiotic fermentation can effectively enhance the antioxidant activity of Astragalus, and verifying the universality of the synergistic effect of probiotics and Astragalus extract in anti-aging applications. The antioxidant capacity of the Astragalus fermentation broth from the eight probiotic strains varied, with control strain 1 (HN019) and BL-10 and S61 preserved in our laboratory showing relatively better effects, but still far lower than the BL-16 Astragalus fermentation broth of this invention. The BL-16 Astragalus fermentation broth exhibited the highest levels of all three antioxidant indicators among all test groups, with DPPH free radical scavenging rate and hydroxyl free radical scavenging rate significantly higher than the positive control group (P<0.05), and anti-lipid peroxidation rate on par with the positive control group, showing a significant difference compared to the Astragalus fermentation broth from the eight control strains (P<0.05), demonstrating that the BL-16 of this invention… The synergistic effect of the strain and Astragalus extract in fermentation is significantly better than that of commercially available Bifidobacterium and Lactobacillus probiotic strains, and its in vitro antioxidant capacity has outstanding and substantial advantages.

[0116] Example 6: Comparison of the anti-aging abilities of various strains of Astragalus fermentation broth and Astragalus extract in nematodes. Referring to the wild-type nematode aging model of C. elegans N2 in Example 4 of this invention, the core anti-aging indicators of the experimental group (BL-16 Astragalus fermentation broth), the Astragalus fermentation broth group of 8 control strains, and the Astragalus extract group were measured in parallel to verify the overall anti-aging superiority of the Astragalus fermentation broth of strain BL-16. All experimental operations, culture conditions, and detection methods were kept completely consistent with those in Example 4 of this invention to eliminate experimental variables.

[0117] 6.1 Experimental Design Experimental materials: C. elegans N2 wild-type nematodes, provided by the model organism resource platform; synchronized treatment to obtain L1 stage larvae of the same age for subsequent experiments; the nematode growth medium (NGM) formula is completely consistent with that in Example 4 of this invention.

[0118] Experimental Groups: Eleven experimental groups were set up, with three biological replicates in each group. The specific groupings are as follows: Blank control group (standard NGM medium only), Astragalus extract group; Control strain 1 (HN019) Astragalus fermentation broth group, Control strain 2 (LGG) Astragalus fermentation broth group, Control strain 3 (299v) Astragalus fermentation broth group, Control strain 4 (Bifidobacterium longum) Astragalus fermentation broth group; Control strain 5 (LLA-5) Astragalus fermentation broth group, Control strain 6 (BAW-3) Astragalus fermentation broth group, Control strain 7 (BL-10) Astragalus fermentation broth group, Control strain 8 (S61) Astragalus fermentation broth group; BL-16 Astragalus fermentation broth group (experimental group).

[0119] Administration method: Astragalus extract was added to NGM medium at a final concentration of 0.5 mg / mL, and Astragalus fermentation broth of each strain was added to NGM medium at a final concentration of 0.5% (v / v). The blank control group was added with an equal volume of sterile water. The experimental treatment started from the L1 larval stage of nematodes and continued until the end of the nematode life cycle. Fresh medium was replaced every 3 days during the culture period. All groups were cultured in the dark at a constant temperature of 20℃. The culture conditions were completely uniform.

[0120] Detection indicators: According to the method of Example 4 of the present invention, the L4 adult survival rate (%), reproductive capacity (average number of eggs laid / nematode), and average lifespan (days) of each group of nematodes were measured in sequence, and the relative activity of superoxide dismutase (SOD) in the nematodes was detected using the corresponding kit. The detection methods of all indicators are completely consistent with those of Example 4 of the present invention.

[0121] 6.2 Experimental Results All test results in this experiment are expressed as "mean ± standard deviation". One-way ANOVA was used to determine the statistical significance of differences between the experimental group and each control group, with P < 0.05 considered statistically significant. The specific results are as follows: L4 adult survival rate: The L4 adult survival rate of nematodes in the blank control group was 50.0±2.1%; the survival rate of L4 adult nematodes in the Astragalus extract group was increased to 59.0±2.2%, which was significantly higher than that in the blank control group (P<0.05). In the Astragalus fermentation broth group of the four commercially available control strains, the survival rate of L4 adult nematodes ranged from 60.3±2.2% to 66.2±0.8±0.03 mm / s, with only the HN019 group showing a significantly higher survival rate than the Astragalus extract group. Among the four Bifidobacterium strains preserved in the laboratory, the L4 adult nematode survival rates of Bifidobacterium longum subspecies LLA-5 and Bifidobacterium animalis subspecies BAW-3 were 67.5±1.9% and 68.3±1.9%, respectively, significantly better than the commercially available control strain group (P<0.05). The L4 adult nematode survival rates of the BL-10 and S61 groups, which are of the same subspecies as the strains of this invention, were 69.5±1.8% and 70.1±1.8%, respectively, while the adult nematode survival rate of BL-16 was 71.0±1.8%, the highest among all control groups, significantly better than the other six control strain groups (P<0.05).

[0122] Reproductive capacity (average number of eggs laid): The average number of eggs laid by nematodes in the blank control group was 40.0±2.5 eggs / nematode; the average number of eggs laid by nematodes in the Astragalus extract group was increased to 56.0±2.7 eggs / nematode, which was significantly higher than that in the blank control group (P<0.05). In the Astragalus fermentation broth group of the four commercially available control strains, the average number of nematode eggs laid ranged from 52.5±2.8 eggs / nematode to 58.8±2.6 eggs / nematode, all significantly higher than the blank control group. Among them, only the HN019 group was significantly higher than the Astragalus extract group. In the four Bifidobacterium strains preserved in the laboratory, the average number of nematode eggs laid in the LLA-5 and BAW-3 groups was 59.5±2.5 eggs / nematode and 60.3±2.4 eggs / nematode, respectively, significantly better than the commercially available control strain group (P<0.05). The average number of nematode eggs laid in the BL-10 and S61 groups of the same subspecies was 61.8±2.4 eggs / nematode and 62.5±2.3 eggs / nematode, respectively, the highest among all control groups, significantly better than the other six control strain groups (P<0.05). In the experimental group of Astragalus fermentation broth of this invention, the average number of nematodes laid reached 64.0±2.3 eggs / worm, which was the highest among all tested groups, 60% higher than the blank control group, and significantly higher than the Astragalus fermentation broth group of 8 control strains (P<0.05).

[0123] Average lifespan: The average lifespan of nematodes in the blank control group was 15.0±2.8 days; the Astragalus extract group extended the average lifespan of nematodes to 18.8±2.9 days, which was significantly longer than that of the blank control group (P<0.05). In the Astragalus fermentation broth groups of four commercially available control strains, the average lifespan of nematodes ranged from 18.1±2.8 days to 20.1±3.2 days; among the four Bifidobacterium strains preserved in the laboratory, the average lifespan of nematodes in the LLA-5 and BAW-3 groups was 19.5±2.7 days and 19.1±2.9 days, respectively; the average lifespan of nematodes in the BL-10 and S61 groups of the same subspecies was 19.4±2.8 days and 20.3±3.1 days, respectively. In the Astragalus fermentation broth experimental group of this invention, the average lifespan of nematodes reached 23.3±3.0 days, the longest among all tested groups, and significantly higher than that of the Astragalus fermentation broth groups of eight control strains.

[0124] Relative SOD activity in vivo: Compared with the blank control group, the SOD activity in nematodes was 1.00 times higher. The relative SOD activity in nematodes in the Astragalus extract group was increased to 1.38±0.05 times, which was significantly higher than that in the blank control group (P<0.05). In the Astragalus fermentation broth group of the four commercially available control strains, the relative SOD activity in nematodes ranged from 1.25±0.06 to 1.41±0.05, with only the HN019 group showing a slightly higher activity than the Astragalus extract group. Among the four Bifidobacterium strains preserved in the laboratory, the relative SOD activities in nematodes of the LLA-5 and BAW-3 groups were 1.45±0.05 and 1.50±0.05, respectively, significantly better than the commercially available control strains (P<0.05). The relative SOD activities in nematodes of the same subspecies, BL-10 and S61, were 1.56±0.04 and 1.61±0.04, respectively, the highest among all control groups, significantly better than the other six control strains (P<0.05). In the experimental group of Astragalus fermentation broth of this invention, the relative SOD activity in nematodes reached 1.73±0.04 times, which was the highest among all tested groups and significantly higher than that of the Astragalus fermentation broth group of 8 control strains (P<0.05).

[0125] Results analysis: Astragalus extract significantly improved the survival rate, reproductive capacity, and average lifespan of nematodes, while also increasing the activity of the antioxidant enzyme SOD in nematodes (P<0.05), demonstrating that Astragalus extract itself has clear in vivo anti-aging activity; the anti-aging indicators of Astragalus fermentation broth from all probiotic strains were significantly better than those of the Astragalus extract group (P<0.05), further verifying that the synergistic effect of fermentation between probiotics and Astragalus extract can significantly enhance the anti-aging effect of Astragalus. Among the four commercially available control strains, HN019, belonging to the same subspecies, showed the best anti-aging effect, but it was still significantly lower than the four Bifidobacterium strains preserved in the laboratory (P<0.05). Among the laboratory-preserved strains, LLA-5, belonging to the same genus but different species as the strain of this invention, and BAW-3, belonging to the same species but different subspecies, showed the next best effects. BL-10 and S61, belonging to the same subspecies as the strain of this invention, showed the most outstanding anti-aging effects, verifying that Bifidobacterium animalis subsp. lactis and Astragalus fermentation have an excellent synergistic anti-aging basis. However, the core anti-aging indicators of these two strains belonging to the same subspecies were still significantly lower than those of the BL-16 Astragalus fermentation broth group of this invention (P<0.05), and could not reach the anti-aging enhancement level of the BL-16 strain. The BL-16 Astragalus fermentation broth group showed the best anti-aging core indicators among all test groups. The survival rate of L4 adult nematodes increased to 73.2%, the average lifespan was extended to 23.3 days, the reproductive capacity increased the most, and the relative SOD activity in the body reached 1.73 times that of the blank control group. There were significant differences compared with the Astragalus fermentation broth of 8 control strains (P<0.05).

[0126] These results demonstrate, from the overall physiological level of the model organism, that the synergistic anti-aging effect of the fermentation of the BL-16 strain of this invention and Astragalus extract is not only significantly superior to the mainstream probiotic strains on the market, and Bifidobacterium strains of different species / subspecies within the same genus, but also significantly superior to strains within the same subspecies that have been verified to have high anti-aging activity. It has outstanding anti-aging activity and irreplaceability, providing a better technical solution for the development of anti-aging products.

[0127] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. The application of *Bifidobacterium animalis* subsp. *lactamase* BL-16 or *Bifidobacterium animalis* subsp. *lactamase* fermented astragalus extract in the preparation of anti-aging compositions, characterized in that... The preservation number of Bifidobacterium lactis subspecies BL-16 is CGMCC No. 32050.

2. The application according to claim 1, characterized in that, The preparation method of the fermented Astragalus membranaceus extract of Bifidobacterium lactis subsp. BL-16 includes the following steps: (1) Add Astragalus membranaceus to water for water extraction and concentration to obtain Astragalus membranaceus water extract; (2) Mix the water extract of Astragalus membranaceus with Bifidobacterium lactis subsp. BL-16, incubate and culture, and collect the supernatant by centrifugation to obtain the Astragalus membranaceus ferment of Bifidobacterium lactis subsp. BL-16.

3. The application according to claim 2, characterized in that, The conditions for water extraction in step (1) are: reflux extraction at 90-100℃ for 2-3 times, each extraction for 1.5-2 hours; the volume ratio of Bifidobacterium lactis subsp. BL-16 to Astragalus membranaceus water extract in step (2) is 1:(1-10).

4. The application according to claim 1, characterized in that, The composition has any one or more of the following functions: (1) Improve the scavenging rate of DPPH free radicals and hydroxyl free radicals; (2) Enhances the ability to resist lipid peroxidation; (3) Alleviate lung tissue damage; (4) Reduce the level of inflammatory factors; (5) Improve survival rate, motor skills, and reproductive capacity; (6) Enhance the activity of SOD, CAT and GSH-Px.

5. The application according to claim 4, characterized in that, The composition has any one or more of the following functions: (1) Enhance antioxidant capacity; (2) Enhances the immune response; (3) Enhance anti-inflammatory capabilities; (4) Improve survival rate and delay aging.

6. The application according to claim 1, characterized in that, The composition further includes excipients selected from any one or more of diluents, excipients, fillers, disintegrants, solubilizers, osmotic pressure regulators, surfactants, pH regulators, and antioxidants; the composition is in any one or more of the following forms: powder, tablet, emulsion, pill, ointment, powder, lyophilized powder for injection, gel, drops, tincture, capsule, granule, or aerosol.

7. The application according to claim 1, characterized in that, The composition contains no less than 10% Bifidobacterium lactis subsp. BL-16 bacteria. 8 CFU.

8. A microbial agent, characterized in that, The bacterial agent includes Bifidobacterium animalis subsp. lactis BL-16 or fermented Astragalus membranaceus extract of Bifidobacterium animalis subsp. lactis BL-16, with the preservation number CGMCC No. 32050.

9. A preparation of Bifidobacterium lactis subspecies BL-16, characterized in that, include: Fermentation broth, fermentation broth precipitate, fermentation broth supernatant, live bacteria, inactivated bacteria, lyophilized powder, lysate, lysate, secondary metabolites, exosomes, and Astragalus fermentation product of Bifidobacterium lactis subsp. lactis BL-16, wherein the preservation number of Bifidobacterium lactis subsp. lactis BL-16 is CGMCC No. 32050.

10. A composition comprising Bifidobacterium animalis subsp. lactis BL-16 or the bacterial agent of claim 8 or the preparation of claim 9, characterized in that, The composition described above has anti-aging properties.

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