Preparation method and application of plant extract fermentation product for promoting aging resistance of dogs

Abstract

The invention discloses a preparation method and application of a plant extract fermentation product for promoting aging resistance of dogs, and belongs to the technical field of animal health care product preparation. Comprising a metabolite (LA20k9) of a brown algae extract which has an anti-aging function and is fermented by bifidobacterium adolescentis LifeAge20-k9 and other raw materials, and the metabolite is prepared from one or more of avenine, fucosterol, 3 ', 4', 5, 7-tetrahydroxyflavone, ciprofibrate, nicotinamide, epigallocatechin, leucine, syringic acid and glutathione. The metabolite of the brown algae extract fermented by the bifidobacterium adolescentis LifeAge20-k9 disclosed by the invention has a remarkable effect in the anti-aging aspect, and can improve the content of nicotinamide adenine dinucleotide in vascular endothelial cells of dogs, the angiogenesis capability and the antioxidant capability; the activity of beta-galactosidase in cells and the content of senescence phenotype markers are reduced, and a multi-channel anti-aging effect is realized. LA20k9 and additives are added to prepare the health-care agent, so that the health-care agent is more beneficial to aging resistance of pets and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of animal health product preparation technology, and in particular relates to a method for preparing and applying fermented metabolites of plant extracts with anti-aging effects for pets. Background Technology

[0002] The aging of vascular endothelial cells intensifies significantly with age and plays a crucial role in the occurrence and development of vascular aging and age-related vascular diseases. With the increasing prominence of pet aging, vascular health management and disease prevention in pets have become important topics in the field of nutrition and health. In recent years, with the improvement of pet food quality and pet nutrition levels, coupled with continuous advancements in medical care, pet lifespans have increased; statistics from the past 60 years show that the average lifespan of pets has doubled. Since the beginning of the 21st century, investment in research resources for pet health products has been continuously increasing, resulting in a wide variety of pet health products on the market, among which those related to extending pet lifespan are the most popular.

[0003] Bifidobacterium adolescentis is an anaerobic, Gram-positive probiotic. Due to its therapeutic effects on diarrhea and anti-aging properties, this probiotic has been added to health supplements and probiotic products with good results. Brown algae extract, primarily derived from seaweed, possesses strong antioxidant, anti-inflammatory, and immunomodulatory functions.

[0004] Currently, there are already some products on the market with anti-aging functions. CN119235997B discloses a product that can... A traditional Chinese medicine composition for prolonging the lifespan of mice, its preparation method, and its application. The formula includes: 9-30 parts of Codonopsis pilosula, 9-30 parts of Poria cocos, 6-20 parts of Angelica sinensis, 6-20 parts of stir-fried Ziziphus jujuba var. spinosa, 6-15 parts of Ophiopogon japonicus, 3-15 parts of processed Polygala tenuifolia, 10-60 parts of longan pulp, 10-30 parts of Rehmannia glutinosa, 9-30 parts of Scrophularia ningpoensis, 3-20 parts of Acorus tatarinowii, 9-30 parts of Platycladus orientalis, 15-30 parts of processed tortoise shell, 15-30 parts of calcined dragon bone, 6-30 parts of lotus flower, and 3-60 parts of licorice root. In addition, CN116850091B discloses a compound fermentation product with anti-aging effects, its preparation method, and its application. This compound fermentation product is composed of Candida albicans fermentation product, glucose fermentation product, and rapeseed oil methyl ester fermentation product. Similarly, CN116875516B discloses the application of human-derived Bifidobacterium adolescentis BA-3 in prolonging the lifespan of Caenorhabditis elegans. The above patents are mainly applied to animals such as mice and Caenorhabditis elegans, and there are no anti-aging products for companion animals such as dogs.

[0005] However, current patents all utilize Bifidobacterium adolescentis derived from humans, which limits its application in pet anti-aging. Furthermore, existing patents have not yet identified the key biological pathways by which functional ingredients promote pet anti-aging. Given the current lack of anti-aging products for dogs on the market, this invention proposes a novel strategy for promoting canine anti-aging based on precise nutritional metabolite regulation. Through in-depth research into the metabolic mechanisms of anti-aging, we have discovered several key biological metabolic pathways. The regulation of metabolites in these pathways provides a more comprehensive solution for promoting canine anti-aging, and a novel formulation has been developed. Summary of the Invention

[0006] In view of this, the present invention aims to provide a fermentation metabolite of a plant extract with anti-aging effects for pets, its preparation method and application. The fermentation metabolite can increase the content of nicotinamide adenine dinucleotide (NAD+), angiogenesis capacity and antioxidant capacity in canine vascular endothelial cells, reduce the activity of β-galactosidase and the content of aging phenotypic markers in cells, and achieve multi-pathway anti-aging effects.

[0007] To achieve the above objectives, the technical solution of the present invention is implemented as follows: A method for preparing a fermentation metabolite of a plant extract with anti-aging effects, comprising the following steps: Step (1) Strain activation: Bifidobacterium adolescentis LifeAge20-k9 strain (accession number: CCTCCM2025638) was inoculated into slant culture medium for activation culture; Step (2) Fermentation: The activated cultured Bifidobacterium adolescentis LifeAge20-k9 was inoculated into a fermentation medium of Lactobacillus delbrueckii containing brown algae extract and fermented to obtain Bifidobacterium adolescentis LifeAge20-k9 fermentation broth; Step (3) Freeze-drying: The fermentation broth was centrifuged and freeze-dried to obtain the freeze-dried powder of the metabolite of brown algae extract fermented by Bifidobacterium adolescentis LifeAge20-k9 (hereinafter referred to as LA20k9).

[0008] The slant culture medium described in step (1) is composed of the following raw materials in parts by weight: 10-30 parts glucose, 5-10 parts tryptone, 5-10 parts beef peptone, 1-5 parts yeast extract, 5-10 parts sodium acetate trihydrate, 1-2 parts citrate, 1-2 parts dipotassium hydrogen phosphate, 0.5-2 parts magnesium sulfate heptahydrate, 0.05-0.1 parts manganese sulfate tetrahydrate, 1-2 parts sorbitol monooleate, 18 parts agar, and water to make up to 1000 parts.

[0009] The activation conditions for the Bifidobacterium adolescentis LifeAge20-k9 strain described in step (1) are as follows: inoculate one loop of the strain into an agar slant culture medium, fermentation temperature: 37℃, fermentation time: 48h, and anaerobic culture.

[0010] The fermentation medium for Lactobacillus delbrueckii extract described in step (2) consists of the following raw materials in parts by weight: 10-30 parts of brown algae extract, 10-30 parts of glucose, 5-10 parts of tryptone, 5-10 parts of beef peptone, 1-5 parts of yeast extract, 5-10 parts of sodium acetate trihydrate, 1-2 parts of citrate, 1-2 parts of dipotassium hydrogen phosphate, 0.5-2 parts of magnesium sulfate heptahydrate, 0.05-0.1 parts of manganese sulfate tetrahydrate, 1-2 parts of sorbitol monooleate, and water to make up to 1000 parts.

[0011] The brown algae extract mentioned above is prepared according to the following steps: Step (1): Wash and dry the fresh brown algae, weigh the dried brown algae, crush it, add 60% ethanol at a ratio of 1:10 (g / mL), reflux extract twice at 55℃ for 1 hour each time, filter to obtain brown algae extract; Step (2): The above brown algae extract is concentrated to dryness using a vacuum concentrator to obtain brown algae extract.

[0012] The fermentation process conditions described in step (2) are: inoculum amount 10%, fermentation temperature 37℃, fermentation time 48h, and anaerobic culture.

[0013] After the fermentation described in step (3) is completed, the fermentation broth is centrifuged at 4200 rpm at room temperature for 10 min, and the supernatant is collected. The supernatant is mixed and then dispensed into freeze-drying trays and pre-frozen at -70℃ for 5 h. After pre-freezing, it is placed in a freeze dryer and freeze-dried for 48 h. After freeze-drying, LA20k9 freeze-dried powder is obtained.

[0014] This invention relates to a health supplement with anti-aging effects, which is composed of the following raw materials in parts by weight: The above-mentioned plant extracts with anti-aging effects contain 3-70 parts of the freeze-dried powder of fermented metabolites, 1-2 parts of styracin, 1-2 parts of fucosterol, 1-2 parts of 3',4',5,7-tetrahydroxyflavone, 2-4 parts of cyclopropofol, 2-4 parts of nicotinamide, 2-4 parts of epigallocatechin, 2-4 parts of leucine, 1-2 parts of syringic acid, and 5-10 parts of glutathione.

[0015] The preparation method of the health supplement with anti-aging effects of the present invention is carried out according to the following steps: Step (1): Weigh 3-70 parts by weight of the freeze-dried powder of the fermented metabolites of plant extracts with anti-aging effects; Step (2): Mix the freeze-dried powder of the fermented metabolites of the plant extract with anti-aging effect weighed in step (1) with the additives, and add water until the mixture is submerged. Step (3): After stirring evenly, granulate and air dry to obtain granular health care agent.

[0016] Preferably, the additive in step (2) is one or more of the following: styraxin, fucosterol, 3',4',5,7-tetrahydroxyflavone, cyclopropofol, nicotinamide, epigallocatechin, leucine, syringic acid, and glutathione.

[0017] Preferably, the additives in step (2) are 1-2 parts of styrax, 1-2 parts of fucosterol, 1-2 parts of 3',4',5,7-tetrahydroxyflavone, 2-4 parts of cyclopropofol, 2-4 parts of nicotinamide, 2-4 parts of epigallocatechin, 2-4 parts of leucine, 1-2 parts of syringic acid, and 5-10 parts of glutathione.

[0018] The application of LA20k9 described in this invention can significantly increase the intracellular nicotinamide adenine dinucleotide (NAD+) content, angiogenesis capacity, and antioxidant capacity.

[0019] The application of LA20k9 described in this invention can significantly reduce intracellular β-galactosidase activity and the content of aging phenotypic markers.

[0020] Compared with existing technologies, the method for preparing fermentation metabolites of plant extracts with anti-aging effects described in this invention has the following beneficial effects: 1. This invention focuses on cellular-level research in dogs, and based on this, delves into the metabolic mechanisms of canine aging, discovering the influence of factors such as the regulation of lipolysis in adipocytes, flavonoid biosynthesis, bile acid metabolism, amino acid metabolism, retinol metabolism, and glutathione metabolism. Through the use of key components such as stigmosiderin, fucosterol, 3',4',5,7-tetrahydroxyflavone, cyclopropofol, nicotinamide, epigallocatechin gallate, leucine, syringic acid, and glutathione, it is more beneficial to address the problems of canine aging.

[0021] 2. The LA20k9 of this invention effectively regulates key biochemical pathways in the aging process by supplementing various nutritional metabolism regulators, promoting cell metabolism, and providing energy to cells. This comprehensive nutritional support approach precisely optimizes and enhances the effects of traditional methods.

[0022] 3. The Bifidobacterium adolescentis used in this invention is derived from the feces of long-lived dogs, which is more conducive to solving the problem of anti-aging in dogs. Attached Figure Description

[0023] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Canine vascular endothelial cells were grouped as follows: NC was the blank control group; doxorubicin (hereinafter referred to as DOX) was the 0.2 μmol / LDOX treatment group; and LA20k9+DOX was the intervention group treated with 1280 g / mL LA20k9+0.2 μmol / LDOX.

[0024] Figure 1 Effects of LA20k9 intervention on the content of nicotinamide adenine dinucleotide (NAD+) in canine vascular endothelial cells.

[0025] Figure 2 Effects of LA20k9 intervention on the levels of (A) vascular endothelial growth factor (VEGF) and (B) basic fibroblast growth factor (bFGF) in canine vascular endothelial cells.

[0026] Figure 3 Effects of LA20k9 intervention on intracellular oxidative stress markers in canine vascular endothelial cells. (A) Superoxide dismutase (SOD) activity in canine vascular endothelial cells; (B) Malondialdehyde (MDA) content in canine vascular endothelial cells; (C) Glutathione (GSH) content in canine vascular endothelial cells.

[0027] Figure 4 Effects of LA20k9 intervention on β-galactosidase activity in canine vascular endothelial cells. (A) β-galactosidase activity in canine vascular endothelial cells; (B) Blue infiltration of senescent canine vascular endothelial cells.

[0028] Figure 5 Effects of LA20k9 intervention on the levels of aging phenotypic markers in canine vascular endothelial cells. (A) Matrix metalloproteinase-1 (MMP-1) concentration; (B) Matrix metalloproteinase-3 (MMP-3) concentration; (C) Interleukin-1β (IL-1β) concentration; (D) Interleukin-6 (IL-6) concentration; (E) Interleukin-8 (IL-8) concentration; (F) Monocyte chemoattractant protein-1 (MCP-1) concentration; (G) Tumor necrosis factor-α (TNF-α) concentration; (H) Granulocyte-macrophage colony-stimulating factor (GM-CSF) concentration. NC: Blank control group; DOX: 0.2 μmol / LDOX treatment group; LA20k9+DOX: Intervention group treated with 1280 g / mL LA20k9 + 0.2 μmol / LDOX. P indicates statistical significance between different groups (P < 0.05).

[0029] Figure 6Volcano plots of the overall distribution of differentially expressed metabolites and heatmaps of differentially expressed metabolites showing significant changes among the groups. NC: Blank control group; DOX: 0.2 μmol / LDOX treatment group; LA20k9+DOX: Intervention group treated with 1280 g / mL LA20k9+0.2 μmol / LDOX. (A) Volcano plot of differentially expressed metabolites showing significant changes compared to the blank control group in the 0.2 μmol / LDOX treatment group; (B) Volcano plot of differentially expressed metabolites showing significant changes compared to the 0.2 μmol / LDOX treatment group in the intervention group treated with 1280 g / mL LA20k9+0.2 μmol / LDOX; (C) Heatmaps of differentially expressed metabolites among the groups. P-value refers to the statistical significance of the t-test; FoldChange refers to the ratio of the quantitative values ​​of the experimental substances in the two groups; VIP refers to the projected importance of the variable. Detailed Implementation

[0030] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0031] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0032] A method for preparing a fermentation metabolite of a plant extract with anti-aging effects, comprising the following steps: Step (1) Strain activation: Bifidobacterium adolescentis LifeAge20-k9 strain (accession number: CCTCCM2025638) was inoculated into slant culture medium for activation culture; The slant culture medium mentioned in step (1) has the following formula: 30g glucose, 10g tryptone, 10g beef peptone, 5g yeast extract, 10g sodium acetate trihydrate, 2g citrate, 2g dipotassium hydrogen phosphate, 2g magnesium sulfate heptahydrate, 0.1g manganese sulfate tetrahydrate, 2mL sorbitol monooleate, 18g agar, add water to a final volume of 1000mL, pH 6.2, sterilize at 121℃ for 20min; The activation conditions for the Bifidobacterium adolescentis LifeAge20-k9 strain described in step (1) are as follows: inoculate one loop of the strain into the slant culture medium, fermentation temperature: 37℃, fermentation time: 48h, and static anaerobic culture.

[0033] Step (2) Fermentation: The activated cultured Bifidobacterium adolescentis LifeAge20-k9 was inoculated into a fermentation medium of Lactobacillus delbrueckii containing brown algae extract and fermented to obtain Bifidobacterium adolescentis LifeAge20-k9 fermentation broth; The fermentation medium containing brown algae extract of Lactobacillus delbrueckii in step (2) has the following formula: Brown algae extract 30g, glucose 30g, tryptone 10g, beef peptone 10g, yeast extract 5g, sodium acetate trihydrate 10g, citrate 1g, dipotassium hydrogen phosphate 1g, magnesium sulfate heptahydrate 1g, manganese sulfate tetrahydrate 0.1g, sorbitol monooleate 2mL, add water to make up to 1000mL, pH 6.2, sterilize at 121℃ for 20min; The brown algae extract is prepared according to the following steps: fresh brown algae are washed and dried, 300g of dried brown algae are weighed, crushed, 3000mL of 60% ethanol is added, and the mixture is refluxed at 55℃ twice for 1 hour each time. The mixture is then filtered to obtain the brown algae extract. The above brown algae extract is concentrated to dryness using a vacuum concentrator to obtain 30g of brown algae extract.

[0034] The fermentation process conditions described in step (2) are as follows: inoculum amount 10%, fermentation temperature 37℃, fermentation time 48h, and static anaerobic culture.

[0035] Step (3) Freeze-drying: After the fermentation described in step (2) is completed, the fermentation broth is centrifuged at 4200 rpm at room temperature for 10 min, and the supernatant is collected. The supernatant is mixed and dispensed into freeze-drying trays, pre-frozen at -70℃ for 5 h; after pre-freezing, it is placed in a freeze dryer and freeze-dried for 48 h. After freeze-drying, the freeze-dried powder of the metabolite of brown algae extract fermented by Bifidobacterium adolescentis LifeAge20-k9 (hereinafter referred to as LA20k9) is obtained.

[0036] The *Bifidobacterium adolescentis* strain LifeAge20-k9 used in this invention is currently deposited at the China Center for Type Culture Collection (CCTCC) of Wuhan University, Wuhan, Hubei Province, China, on March 31, 2025, with accession number CCTCCNO: M2025638, and the accession name is *Bifidobacterium adolescentis* LifeAge20-k9Bifidobacterium adolescentis, *Bifidobacterium LifeAge20-k9*. The suggested classification name is *Bifidobacterium adolescentis*.

[0037] 1. Test Methods (1) Determination of nicotinamide adenine dinucleotide (NAD+) content PETCC200 canine vascular endothelial cells were cultured in M-3 medium containing 10% fetal bovine serum and incubated at 37°C in a 5% CO2 incubator. Cell passage and experiments were performed when the cells reached 80% confluence. Cells were then digested with trypsin, and the cell concentration was adjusted to 1×10⁶ cells / mL with added medium. 5 Cells were seeded at a density of 1 mL / well in 6-well plates and incubated for 24 h. Then, 1 mL of 0.2 μmol / L DOX was added for 24 h of intervention. Cells were washed three times with phosphate-buffered saline (PBS, pH 7.4), and 1 mL of LA20k9 (1280 μg / mL) solution was added as the experimental group. A negative control containing only DOX was used. Untreated 6-well plates served as blank controls. Cells were lysed with lysis buffer after another 24 h, and NAD+ levels were detected using a kit.

[0038] (2) Detection of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) levels Cells were treated according to method (1), and the supernatant was collected after culturing for 24 hours. The concentrations of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were detected by enzyme-linked immunosorbent assay (ELISA). The specific operation was performed according to the instructions of the ELISA test kit, and the absorbance value at a wavelength of 450 nm was measured using an enzyme-linked immunosorbent assay reader.

[0039] (3) Detection of oxidative stress index content Cells were treated according to method (1), cultured for 24 hours, and then lysed with lysis buffer. The SOD, MDA and GSH indicators were detected using a kit.

[0040] (4) Detection of β-galactosidase activity Cells were treated according to method (1), cultured for 24 hours, and then lysed with lysis buffer. The β-galactosidase activity assay kit was used to detect the indices. Cells were treated according to method (1), cultured for 24 hours, and then the supernatant was discarded. The β-galactosidase staining assay kit was used to detect the indices.

[0041] (5) Detection of aging phenotypic markers Cells were treated according to method (1), and the supernatant was collected after culturing for 24 h. The concentrations of matrix metalloproteinase 1 (MMP-1), matrix metalloproteinase 3 (MMP-3), interleukin 1β (IL-1β), interleukin 6 (IL-6), interleukin 8 (IL-8), monocyte chemoattractant protein 1 (MCP-1), tumor necrosis factor α (TNF-α), and granulocyte-macrophage colony-stimulating factor (GM-CSF) were detected by enzyme-linked immunosorbent assay (ELISA). The specific operation was performed according to the instructions of the ELISA kit, and the absorbance value at a wavelength of 450 nm was measured using an enzyme-linked immunosorbent assay reader.

[0042] 2. Test Results (1) LA20k9 can effectively alleviate the decrease in NAD+ levels in canine vascular endothelial cells induced by DOX. For example... Figure 1 As shown, the NAD+ level decreased in the DOX group compared to the NC group. This indicates a decline in DOX-induced metabolic function, DNA repair, and immune cell function in canine vascular endothelial cells. In contrast to the DOX group, NAD+ levels recovered after intervention with 1280 mg / mL LA20k9, demonstrating that LA20k9 can effectively alleviate DOX-induced declines in cellular metabolism, DNA repair, and immune cell function.

[0043] (2) LA20k9 can effectively slow down DOX-induced vascular damage. For example... Figure 2 As shown, compared with the NC group, the levels of VEGF and bFGF in the DOX group were significantly reduced. This indicates that vascular endothelial cells underwent significant damage after DOX induction, which may lead to vascular dysfunction and increase the risk of sclerosis and thrombosis. In contrast, the levels of VEGF and bFGF were significantly increased under 1280 mg / mL LA20k9 intervention compared to the DOX group. This indicates that LA20k9 can effectively slow down DOX-induced vascular endothelial cell damage, thereby reducing the risk of thrombosis.

[0044] (3) LA20k9 can effectively alleviate the level of oxidative stress in canine vascular endothelial cells induced by DOX. For example... Figure 3 As shown in the figure, compared with the NC group, the DOX group showed decreased SOD and GSH levels and increased intracellular MDA levels. This indicates that DOX-induced canine vascular endothelial cells underwent oxidative stress. Compared with the DOX group, intervention with 1280 mg / mL LA20k9 promoted SOD and GSH levels and inhibited MDA levels, indicating that LA20k9 can effectively alleviate DOX-induced oxidative stress in cells and enhance antioxidant capacity.

[0045] (4) LA20k9 can effectively slow down the senescence of canine vascular endothelial cells induced by DOX. For example... Figure 4 As shown, compared with the NC group, the DOX group exhibited significantly increased β-galactosidase activity and a significantly larger blue infiltration area in senescent cells. This indicates that vascular endothelial cells underwent significant senescence after DOX induction. In contrast, under 1280 mg / mL LA20k9 intervention, β-galactosidase activity was significantly reduced, and the blue infiltration area in senescent cells was significantly smaller. This suggests that LA20k9 can effectively slow down DOX-induced senescence of vascular endothelial cells.

[0046] (5) LA20k9 can effectively slow down the increase in the content of aging phenotypic markers in blood vessels induced by DOX. For example... Figure 5As shown, compared with the NC group, the levels of MMP-1, MMP-3, IL-1β, IL-6, IL-8, MCP-1, TNF-α, and GM-CSF were significantly increased in the DOX group. This indicates that vascular endothelial cells underwent significant senescence after DOX induction. Compared with the DOX group, the levels of MMP-1, MMP-3, IL-1β, IL-6, IL-8, MCP-1, TNF-α, and GM-CSF were significantly decreased under 1280 mg / mL LA20k9 intervention. This indicates that LA20k9 can effectively slow down DOX-induced vascular endothelial cell senescence.

[0047] II. Metabolomics Analysis of LA20k9 in Canine Vascular Endothelial Cells 1. Test Methods To gain a deeper understanding of the metabolic differences of LA20k9 in canine vascular endothelial cells, untargeted metabolomics analysis was performed using high-performance liquid chromatography-tandem mass spectrometry (LC-MS). Untreated cells served as the control group (NC), DOX-treated cells as the negative control group, and LA20k9-treated cells as the experimental group. An equal volume of each experimental sample was mixed and used as a quality control group. A 53% methanol aqueous solution was used as a blank control instead of the experimental sample. Cell samples were placed in EP tubes, and 300 mL of 80% methanol aqueous solution was added; the tubes were then rapidly frozen in liquid nitrogen for 5 min; after thawing on ice, the tubes were vortexed for 30 s and sonicated for 6 min; the tubes were centrifuged at 5000 rpm and 4℃ for 1 min, and the supernatant was transferred to a new centrifuge tube and lyophilized into a dry powder; the powder was then dissolved in the corresponding 10% methanol solution according to the sample volume and analyzed by LC-MS.

[0048] Samples were separated and analyzed using a Vanquish UHPLC (ThermoFisher) and QExactive™ HF mass spectrometer (ThermoFisher), with a scan range of m / z 100-1500. The ESI source settings were as follows: Spray Voltage: 3.5 kV; Sheath gas flow rate: 35 psi; Aux Gas flow rate: 10 L / min; Capillary Temp: 320 °C; S-lens RF level: 60 °C; Aux Gas Heater Temp: 350 °C; Polarity: positive, negative. MS / MS secondary scans were data-dependent scans. Raw data were filtered by retention time and mass-to-charge ratio and normalized to the initial QC sample to obtain peak area accuracy. Peak extraction was performed with a 5 ppm mass tolerance and a 30% signal intensity bias. Metabolites were identified by comparing molecular and fragment ion data with the mzCloud, mzVault, and Masslist databases. Background ions were removed using blank samples. Quantification results were standardized based on the ratio of metabolite concentration in the sample to that in the QC sample. Compounds with a coefficient of variation (CV) exceeding 30% in the QC sample were excluded to finalize metabolite identification and relative quantification.

[0049] 2. Test Results Figure 6 This invention presents volcano plots and cluster heatmaps of differentially expressed metabolites among the groups. Results showed that compared to the NC control group, 55 metabolites were upregulated and 132 metabolites were downregulated in the DOX-treated group; compared to the DOX control group, 292 metabolites were upregulated and 382 metabolites were downregulated in the LA20k9-treated group. Specifically, the DOX-treated group showed decreased levels of 3',4',5,7-tetrahydroxyflavone, associated with flavonoid biosynthesis, and glutathione, associated with glutathione metabolism. After LA20k9 treatment, the metabolite changes were the opposite of those under DOX treatment, showing significant differences. Differentially expressed metabolites were annotated in the Kyoto Encyclopedia of Genetics and Genomes (KEGG) database to identify the involved metabolic pathways, and the number of annotated differentially expressed metabolites in a given pathway was enriched. The results showed that pathways such as the regulation of lipolysis, flavonoid biosynthesis, bile acid metabolism, amino acid metabolism, retinol metabolism, and glutathione metabolism in adipocytes were significantly enriched. Therefore, LA20k9 inhibits canine vascular endothelial cell senescence by participating in these pathways.

[0050] Based on the metabolomics analysis of canine vascular endothelial cells, additional substances should be supplemented, such as styraxine (a polyphenol compound with antioxidant and anti-inflammatory properties, which can also promote weight gain in malnourished children), fucosterol (which regulates biological metabolism, stress response, and lowers cholesterol), 3',4',5,7-tetrahydroxyflavone (which has multiple effects including anti-inflammatory, anti-tumor, anti-fibrotic, antioxidant, antimicrobial, antidiabetic, neuroprotective, and anti-allergic properties, and also has the function of regulating chronic inflammation), and ciprofibrate (which has a lipid-lowering effect and can significantly reduce very low density and low density cholesterol). It lowers lipoprotein levels and increases high-density lipoprotein. By improving cholesterol distribution, it can dissolve fibrin and prevent platelet aggregation. Other components include nicotinamide (antioxidant, enhancing cellular energy metabolism), epigallocatechin (antibacterial, antiviral, antioxidant, anti-atherosclerotic, antithrombotic, anti-angiogenic, anti-inflammatory, and anti-tumor effects), leucine (maintaining normal nervous system function), syringic acid (anti-glycation, anti-lipid, and anti-inflammatory effects), and glutathione (participating in the metabolism of various intracellular compounds, drugs, and hormones, and preventing oxidative stress).

[0051] Table 1. Differential metabolites and metabolic pathways in canine vascular endothelial cells treated with LA20k9 Example 1: The health supplement with anti-aging effects of this invention is prepared by the following steps: Step (1): Weigh 70g of LA20k9 freeze-dried powder prepared by the aforementioned method for preparing fermented metabolites of a plant extract with anti-aging effects. Step (2): Mix the LA20k9 freeze-dried powder weighed in step (1) with the additives, and add water until the mixture is submerged; Step (3): After stirring evenly, granulate and air dry to obtain granular health care agent.

[0052] The additives mentioned in step (2) are 3g of styraxanone, 3g of fucosterol, 3',4',5,7-tetrahydroxyflavone, 3g of cyclopropofol, 3g of nicotinamide, 3g of epigallocatechin, 3g of leucine, 3g of syringic acid, and 6g of glutathione.

[0053] The granular health supplement mentioned above is prepared by the following steps: Step (1): Send the mixed material into a low-temperature cold press pellet mill and extrude capsule granules through the low-temperature cold press pellet mill.

[0054] Step (2): Air dry the capsule granules, package them into finished products, seal them, and obtain granular health supplements.

[0055] Example 2: The health supplement with anti-aging effects of this invention is prepared by the following steps: Step (1): Weigh 40g of LA20k9 lyophilized powder according to weight; Step (2): Mix the LA20k9 freeze-dried powder weighed in step (1) with the additives, and add water until the mixture is submerged; Step (3): After stirring evenly, granulate and air dry to obtain granular health care agent.

[0056] The additives mentioned in step (2) are 6g of styrax, 6g of fucosterol, 6g of 3',4',5,7-tetrahydroxyflavone, 6g of cyclopropofol, 6g of nicotinamide, 6g of epigallocatechin, 6g of leucine, 6g of syringic acid, and 12g of glutathione.

[0057] The granular health supplement mentioned above is prepared by the following steps: Step (1): Send the mixed material into a low-temperature cold press pellet mill and extrude capsule granules through the low-temperature cold press pellet mill.

[0058] Step (2): Air dry the capsule granules, package them into finished products, seal them, and obtain granular health supplements.

[0059] Example 3: The health supplement with anti-aging effects of this invention is prepared by the following steps: Step (1): Weigh 10g of LA20k9 lyophilized powder according to weight; Step (2): Mix the LA20k9 freeze-dried powder weighed in step (1) with the additives, and add water until the mixture is submerged; Step (3): After stirring evenly, granulate and air dry to obtain granular health care agent.

[0060] The additives mentioned in step (2) are 9g of styrax, 9g of fucosterol, 9g of 3',4',5,7-tetrahydroxyflavone, 9g of cyclopropofol, 9g of nicotinamide, 9g of epigallocatechin, 9g of leucine, 9g of syringic acid, and 18g of glutathione.

[0061] The granular health supplement mentioned above is prepared by the following steps: Step (1): Send the mixed material into a low-temperature cold press pellet mill and extrude capsule granules through the low-temperature cold press pellet mill.

[0062] Step (2): Air dry the capsule granules, package them into finished products, seal them, and obtain granular health supplements.

[0063] Comparative Example 1 The difference from Example 1 is that LA20k9 freeze-dried powder is not added, but only 9g of styraxin, 9g of fucosterol, 9g of 3',4',5,7-tetrahydroxyflavone, 9g of cyclopropofol, 9g of nicotinamide, 9g of epigallocatechin, 9g of leucine, 9g of syringic acid, and 18g of glutathione are added.

[0064] Comparative Example 2 The difference from Example 1 is that it does not contain styrax, fucosterol, 3',4',5,7-tetrahydroxyflavone, cyclopropofol, nicotinamide, epigallocatechin, or leucine, but only 70g of LA20k9 lyophilized powder, 12g of syringic acid, and 18g of glutathione.

[0065] Effect Experiment 1: 1. Test Methods (1) Experimental animals and grouping: All mice were selected from strain 8, which is susceptible to accelerated aging. All mice were 6 months old, with 30 male and 30 female mice. They were randomly divided into 5 groups of 12 mice each.

[0066] 2. Intervention Program: Groups 1-3 were experimental groups (Groups 1-3), fed a basal diet daily (composed of Northeast corn, wheat, imported fish meal, chicken meal, soybean meal, soybean oil, sodium chloride, limestone powder, choline chloride, methionine, vitamin A, vitamin D3, D-biotin, dL-α-tocopherol acetate, yeast selenium, etc.), and simultaneously fed the granular health supplement prepared in Examples 1-3 daily. Groups 4-5 were control groups, fed a basal diet daily, and simultaneously fed the granular health supplement prepared in Comparative Examples 1-2 daily. The dosage of the granular health supplement for each group was 2 capsules once daily for 3 months. Activity was observed, and after 3 months, rotator drop time was measured, blood samples were collected to measure glutathione peroxidase (GPX) activity and NAD+ content, and Doppler ultrasound was used to observe vascular wall thickness.

[0067] 3. Test Results The effects on mice in each group are shown in Table 2. Compared with the control group, feeding mice with Examples 1-3 increased the rotarod drop time, glutathione peroxidase (GPX) activity and NAD+ content, and thinned the blood vessel walls.

[0068] Table 2 Results of aging index measurement in mice of each group Effect Experiment 2: 1. Test Methods (1) Experimental animals and grouping: 20 male and 20 female senior dogs of all breeds were selected and randomly divided into 5 groups of 8 dogs each.

[0069] 2. Intervention Program: Groups 1-3 were experimental groups (Groups 1-3), fed a basal diet daily (composed of fresh chicken, frozen turkey, whole herring, whole cod, fresh chicken liver, sardine meal, dried carrot cubes, cranberries, dried kelp, protein zinc, natural vitamin E, dL-α-tocopherol acetate, vitamin D3, citric acid, etc.), and simultaneously fed the granular health supplement prepared in Examples 1-3 daily. Groups 4-5 were control groups, fed a basal diet daily, and simultaneously fed the granular health supplement prepared in Comparative Examples 1-2 daily. The dosage of the granular health supplement for each group was 2 capsules once daily for 3 months. Activity levels were observed. After 3 months, blood samples were collected to measure interleukin-6 (IL-6), NAD+, and SOD levels, and Doppler ultrasound was used to observe vascular wall thickness.

[0070] 3. Test Results The effects on each group of dogs are shown in Table 3. Compared with the control group, feeding dogs with Examples 1-3 increased NAD+ and SOD levels, decreased interleukin-6 (IL-6) levels, and thinned blood vessel walls.

[0071] Table 3 Results of aging index measurement in each group of dogs

Claims

1. A method for preparing a fermented metabolite of a plant extract having an anti-aging effect, characterized in that The following steps are taken: Step (1) strain activation: Bifidobacterium adolescentis LifeAge20-k9 strain is inoculated into the slant medium for activation culture, and the preservation number of Bifidobacterium adolescentis LifeAge20-k9 strain is CCTCCM2025638; Step (2) fermentation: the activated Bifidobacterium adolescentis LifeAge20-k9 is inoculated into the Lactobacillus debrueckii fermentation medium containing brown algae extract for fermentation, and Bifidobacterium adolescentis LifeAge20-k9 fermentation liquor is obtained; Step (3) freeze-drying: the fermentation liquor is centrifuged and freeze-dried to obtain the freeze-dried powder of the metabolic product of the brown algae extract fermented by Bifidobacterium adolescentis LifeAge20-k9.

2. The method of claim 1, wherein the fermentation metabolite of the plant extract having an anti-aging effect is prepared by the following steps: (a) extracting a plant extract from a plant; (b) fermenting the plant extract; and (c) drying the fermented plant extract. The slant medium in step (1) is composed of the following raw materials in parts by weight: glucose 10-30 parts, tryptone 5-10 parts, beef protein peptone 5-10 parts, yeast extract 1-5 parts, sodium acetate trihydrate 5-10 parts, citrate 1-2 parts, potassium phosphate dibasic 1-2 parts, magnesium sulfate heptahydrate 0.5-2 parts, manganese sulfate tetrahydrate 0.05-0.1 parts, sorbitan monooleate 1-2 parts, agar 18 parts, and water is added to 1000 parts; The activation conditions of Bifidobacterium adolescentis LifeAge20-k9 strain in step (1) are as follows: 1 ring of strain is inoculated into the slant medium, the fermentation temperature is 37℃, and the fermentation time is 48h, and anaerobic culture is carried out.

3. The method of claim 1, wherein the fermentation metabolite of the plant extract having an anti-aging effect is prepared by the following steps: (a) extracting a plant extract from a plant; (b) fermenting the plant extract; and (c) drying the fermented plant extract. The Lactobacillus debrueckii fermentation medium of brown algae extract in step (2) is composed of the following raw materials in parts by weight: brown algae extract 10-30 parts, glucose 10-30 parts, tryptone 5-10 parts, beef protein peptone 5-10 parts, yeast extract 1-5 parts, sodium acetate trihydrate 5-10 parts, citrate 1-2 parts, potassium phosphate dibasic 1-2 parts, magnesium sulfate heptahydrate 0.5-2 parts, manganese sulfate tetrahydrate 0.05-0.1 parts, sorbitan monooleate 1-2 parts, and water is added to 1000 parts; The preparation method of the brown algae extract is as follows: Step (1): Fresh brown algae is washed and dried, and the dried brown algae is weighed, crushed, and added with 60% ethanol at a ratio of 1:10 (g / mL), and refluxed at 55℃ for 2 times, 1 hour each time, filtered to obtain brown algae extract solution; Step (2): The brown algae extract solution is concentrated to dryness by vacuum concentration pump under reduced pressure to obtain brown algae extract. The fermentation process conditions in step (2) are as follows: the inoculation amount is 10%, the fermentation temperature is 37℃, the fermentation time is 48h, and anaerobic culture is carried out.

4. The method of claim 1, wherein the fermentation metabolite of the plant extract having an anti-aging effect is prepared by the following steps: (a) extracting a plant extract from a plant; (b) fermenting the plant extract; and (c) drying the fermented plant extract. After the fermentation in step (3) is completed, the fermentation liquor is centrifuged at 4200rpm at room temperature for 10min, and the supernatant is collected. The supernatant is mixed and then divided into freeze-drying trays, pre-frozen at-70℃ for 5h; after pre-freezing, it is placed in a freeze dryer and freeze-dried for 48h. After freeze-drying, LA20k9 freeze-dried powder is obtained.

5. A health agent having anti-aging efficacy of a fermented metabolite containing the plant extract prepared by the method according to claims 1 to 4, characterized in that consists of the following raw materials by weight: 3~70 parts of freeze-dried powder of metabolic products of brown algae extract fermented by Bifidobacterium adolescentis LifeAge20-k9, 1~2 parts of Prosopis juliflora, 1~2 parts of fucosterol, 1~2 parts of 3', 4', 5, 7-tetrahydroxyflavone, 2~4 parts of ciprofibrate, 2~4 parts of nicotinamide, 2~4 parts of epigallocatechin, 2~4 parts of leucine, 1~2 parts of syringic acid, and 5~10 parts of glutathione.

6. A method for preparing a health agent having an anti-aging effect of a fermented metabolite containing the plant extract prepared by the method according to claims 1 to 4, characterized in that The following steps are taken: Step (1): 3~70 parts of freeze-dried powder of metabolic products of brown algae extract fermented by Bifidobacterium adolescentis LifeAge20-k9 are weighed by weight; Step (2): the freeze-dried powder of metabolic products of brown algae extract fermented by Bifidobacterium adolescentis LifeAge20-k9 weighed in step (1) is mixed with additives, and water is added to immerse the mixed raw materials; Step (3): after uniform stirring, granulation and air drying are performed to obtain granular health care agent.

7. The method of claim 6, wherein the health care agent having an anti-aging effect is prepared by adding 0.01 to 10% by weight of the extract of the plant of the genus Rosmarinus to 90 to 99.99% by weight of the carrier. The additives in step (2) are one or more of Prosopis juliflora, fucosterol, 3', 4', 5, 7-tetrahydroxyflavone, ciprofibrate, nicotinamide, epigallocatechin, leucine, syringic acid, and glutathione.

8. The method for preparing the health supplement with anti-aging effects according to claim 6, characterized in that, The additives in step (2) are 1~2 parts of Prosopis juliflora, 1~2 parts of fucosterol, 1~2 parts of 3', 4', 5, 7-tetrahydroxyflavone, 2~4 parts of ciprofibrate, 2~4 parts of nicotinamide, 2~4 parts of epigallocatechin, 2~4 parts of leucine, 1~2 parts of syringic acid, and 5~10 parts of glutathione.

9. Use of the fermented metabolic products of plant extracts prepared by the method of claims 1-4 or the health care agent with anti-aging efficacy of claim 5 for increasing the content of nicotinamide adenine dinucleotide (NAD+) in cells, angiogenic capacity, and antioxidant capacity.

10. Use of the fermented metabolic products of plant extracts prepared by the method of claims 1-4 or the health care agent with anti-aging efficacy of claim 5 for reducing the activity of β-galactosidase and the content of senescence markers in cells.

Images