A high nucleotide saccharomyces cerevisiae extract for improving the health of aging dogs, and a preparation method and application thereof

Abstract

The application discloses a high-nucleotide saccharomyces cerevisiae extract for improving health of aging dogs and a preparation method and application thereof, and belongs to the technical field of biotechnology. In view of the technical problems of low nucleotide enrichment efficiency of the existing saccharomyces cerevisiae extract, lack of systematic efficacy verification of aging dogs and difficulty in simultaneously improving multiple systems of aging, the application provides a high-nucleotide saccharomyces cerevisiae extract prepared by using saccharomyces cerevisiae (CGMCC No. 38790). Test results show that the extract can effectively alleviate aging-related symptoms of the aging dogs, such as weight loss, muscle attenuation, reduction of bone density, reduction of liver and kidney functions and aging damage of the fur, regulate intestinal flora structure and metabolic function, regulate aging pathways through epigenetic modification, maintain telomere homeostasis, enhance the antioxidant capacity of the body and inhibit chronic low-grade inflammation, has high biological safety, can be fed for a long time and is suitable for development of functional feed raw materials for the aging dogs.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a high-nucleotide Saccharomyces cerevisiae extract for improving the health of aging dogs, its preparation method, and its application. Background Technology

[0002] With the development of the pet breeding industry, the health care of aging dogs has become a research focus in the field of pet nutrition. Dogs aged 7 years and older are prone to aging-related problems such as weight loss, muscle atrophy, decreased bone density, weakened liver and kidney function, intestinal microecological imbalance, and aging and damage to the skin and coat. At the same time, they are accompanied by cellular aging characteristics such as telomere shortening, DNA damage accumulation, and abnormal epigenetic regulation, which seriously affect the quality of life of aging dogs.

[0003] Saccharomyces cerevisiae extract is made from Saccharomyces cerevisiae (Saccharom Saccharomyces cerevisiae It is a water-soluble natural extract obtained by culturing, fermenting, autolyzing or hydrolyzing by exogenous enzymes, followed by separation, concentration and drying. It is a complex nutrient mixture, with core active ingredients such as amino acids, small peptides, 5'-nucleotides and B vitamins. It is recognized as a natural and safe raw material by most food safety certification agencies around the world. Different preparation processes will directly affect its active ingredient content and biological efficacy.

[0004] Nucleotides, as the basic monomers constituting DNA and RNA in organisms, are core substances for cell structure, metabolism, and energy regulation. Exogenous supplementation of nucleotides possesses clear and important physiological functions in the field of anti-aging, which have been verified in multiple animal studies: First, they can regulate DNA methylation levels, improve insulin sensitivity, and play a positive role in muscle and fat composition. Second, they promote cell repair and proliferation; nucleotides can support the replication and division of metabolically active cells such as intestinal epithelial cells, hepatocytes, and immune cells, accelerate tissue damage repair, improve intestinal barrier function and liver and kidney function, while enhancing the proliferation capacity of immune cells and maintaining immune homeostasis during aging. Third, they improve skin aging; nucleotides can increase the thickness of the epithelial and dermal layers of the skin in aging animals, increase the content of hydroxyproline in the skin, alleviate skin structural degeneration, and reduce the expression of skin aging-related markers. Fourth, they regulate the intestinal microecology; nucleotides can act as prebiotics to promote the proliferation of beneficial intestinal bacteria, optimize the metabolic activity of intestinal flora, promote the synthesis of beneficial metabolites such as short-chain fatty acids, and indirectly alleviate age-related metabolic disorders by regulating the host's overall metabolism through gut-axis interactions.

[0005] In the pet food industry, brewer's yeast extract has been applied to some extent, but its application directions still have obvious limitations: First, it is mainly used as a natural palatability enhancer, utilizing its glutamic acid, nucleotides, and other components to enhance the flavor and aroma of feed, improve pet palatability, and solve the problem of picky eating in dogs and cats. Some products can increase pets' food intake by more than 9% through yeast extract coating technology, while replacing non-natural palatability enhancers and improving feed safety. Second, it is used as a basic nutritional supplement to provide pets with high-quality small molecule peptides, amino acids, and B vitamins to make up for the nutritional gaps in conventional feed. Third, it is used in small quantities for intestinal health and immune regulation. Related studies have shown that it can increase the IgG content in cat serum, increase the content of short-chain fatty acids in feces, stabilize the intestinal flora structure, and promote the proliferation of beneficial bacteria.

[0006] Natural 5'-nucleotides are mainly derived from non-GMO Saccharomyces cerevisiae, but there are currently few reports of nucleotides being used as a primary functional ingredient in pets. The main reasons for this are: conventional Saccharomyces cerevisiae extracts have low 5'-nucleotide content, most are not specifically converted and contain almost no 5'-nucleotides, with a few products containing only 2%-10%; simultaneously, existing Saccharomyces cerevisiae extract products lack systematic efficacy verification for the specific physiological state of aging dogs. Their development goals are mostly general nutritional supplements or palatability enhancers, without conducting targeted animal efficacy trials focusing on core aging issues such as muscle loss, decreased bone density, weakened liver and kidney metabolism, and aging fur, making it difficult to meet industry demands as a functional feed ingredient specifically for aging dogs. This invention creatively combines the function of 5'-nucleotides with Saccharomyces cerevisiae extract, obtaining a high-nucleotide-content extract through a specific preparation process, and systematically verifying its comprehensive improvement effect on multiple aging-related indicators in aging dogs. Developing a dedicated functional feed ingredient for aging dogs is of great significance for maintaining their health. Summary of the Invention

[0007] The purpose of this invention is to provide: This invention provides a preparation process for a high-nucleotide Saccharomyces cerevisiae extract, the resulting product, and its application in improving the health of aging dogs. This addresses the technical problems of low nucleotide enrichment efficiency and the lack of systematic functional studies of Saccharomyces cerevisiae extract preparation processes in aging dogs. The high-nucleotide Saccharomyces cerevisiae extract prepared by this invention achieves highly efficient nucleotide enrichment, significantly increasing the total nucleotide content while retaining active components such as polysaccharides and functional peptides from the yeast. This extract can specifically improve age-related health problems in aging dogs, such as weight loss, decreased bone density, weakened liver and kidney function, intestinal microecological imbalance, and aging and damage to the skin and coat. It achieves comprehensive improvement of multi-system aging in aging dogs through multiple pathways, including regulating intestinal microecological function, epigenetic modification, and maintaining telomere homeostasis. This fills the gap in existing technologies for natural extracts that simultaneously regulate multi-system aging in aging dogs, and it poses no biosafety risks, allowing for long-term feeding.

[0008] 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.

[0009] 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.

[0010] Unless otherwise stated, conventional methods within the scope of the art shall be used.

[0011] 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.

[0012] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0013] The term "senile dog" or "middle-aged dog" used in this article refers to dogs that are 7 years of age or older. At this stage, dogs enter the physiological aging period and are prone to aging-related characteristics such as muscle atrophy, decreased bone density, weakened liver and kidney metabolism, intestinal microecological imbalance, aging of skin and fur, shortening of telomeres, and accumulation of DNA damage.

[0014] The term "Body Condition Score" or "BCS" used in this article refers to a 9-point scale used to assess a dog's body fat reserves. 1 point indicates extreme emaciation, 9 points indicate extreme obesity, and 3-5 points indicate healthy body condition. A lower score suggests age-related loss of body fat and abnormal nutrient metabolism.

[0015] The term “muscle score” or “MS” used in this article refers to a 5-point scale used to assess muscle mass and firmness in dogs. A score of 1 indicates severe muscle loss, while a score of 5 indicates excellent muscle condition. A decrease in score is the core indicator for diagnosing sarcopenia in aging dogs.

[0016] The term "bone mineral density" or "BMD" used in this article refers to the volumetric bone mineral density (vBMD, unit: mg / cc) of the lumbar spine (L1-L4) measured by quantitative computed tomography (QCT) in this invention. It directly reflects the mineral content of cancellous bone, and a decrease in the value indicates osteoporosis and bone loss.

[0017] The term "intestinal microecology" used in this article refers to the sum of the microbial community and metabolic environment of bacteria, fungi, archaea and other microorganisms in the canine intestine; Alpha diversity (Shannon index, Simpson index) reflects the richness and stability of the microbial community, while Beta diversity reflects the differences in community structure. Bacteroidetes, Bifidobacterium, and Parasartella are the core beneficial microbial communities in the intestine.

[0018] The term “fecal untargeted metabolomics” used in this article refers to the unbiased detection of small molecule metabolites (molecular weight less than 1000 Da) in feces using the LC-MS / MS platform, followed by differential metabolite screening and KEGG pathway enrichment to elucidate the metabolic regulatory mechanisms of gut microbiota-host interactions.

[0019] The term "relative telomere length" used in this article refers to the ratio of the length of peripheral blood cells telomeres to the internal reference gene, as detected by qPCR. Telomere shortening is a core marker of cellular senescence, and the telomere length of aging dogs is significantly lower than that of young dogs.

[0020] The term “whole-genome methylation sequencing” or “WGBS” used in this article refers to the detection of methylation status of CpG, CHH, and CHG sites at the whole genome level based on bisulfite conversion technology. Differentially methylated regions (DMRs) refer to genomic fragments where the methylation levels of the experimental group and the control group are significantly different. Promoter region DMRs can regulate gene transcription and expression and are directly related to aging and the activation or inhibition of metabolic pathways.

[0021] The term "plasma proteomics" used in this article refers to the high-throughput identification of proteins expressed in peripheral plasma based on mass spectrometry. Differentially expressed proteins are those whose expression levels differ statistically between groups (P < 0.05). This is used to analyze the regulatory effects of extracts on lipid metabolism, protein repair, and immune homeostasis in aging dogs.

[0022] The term "oxidative stress and antioxidant indicators" used in this article refers to malondialdehyde (MDA) as a marker of lipid peroxidation damage; superoxide dismutase (SOD), catalase (CAT), and reduced glutathione (GSH) as endogenous antioxidant enzymes; and total antioxidant capacity (T-AOC) and coenzyme NAD+ as core indicators of the body's overall antioxidant level. Decreased levels of the above indicators suggest the accumulation of age-related oxidative damage.

[0023] The term "inflammatory markers" used in this article refers to C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) as pro-inflammatory markers, and interleukin-10 (IL-10) and interferon-γ (IFN-γ) as anti-inflammatory markers. Chronic low-grade inflammation in aging dogs is characterized by elevated pro-inflammatory factors and decreased anti-inflammatory factors.

[0024] The term "liver and kidney biochemical indicators" used in this article refers to alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase (GGT) as core indicators of liver function; and creatinine (CREA) and uric acid (UA) as core indicators of kidney function. Abnormally elevated levels of these indicators suggest liver and kidney metabolic dysfunction.

[0025] The technical solution of this invention is as follows: In a first aspect, the present invention provides a strain of Saccharomyces cerevisiae, the strain having the accession number CGMCC No. 38790.

[0026] Based on a further solution to the technical problem of the present invention, a preferred embodiment of the technical solution provided in the first aspect of the present invention includes: The first preferred option is a strain of *Saccharomyces cerevisiae*, with accession number CGMCC No. 38790. This option solves the technical problem of providing a new strain of *Saccharomyces cerevisiae* capable of efficiently producing high-nucleotide extracts.

[0027] In a second aspect, the present invention provides: a high-nucleotide Saccharomyces cerevisiae extract, said extract being prepared from the Saccharomyces cerevisiae strain described in the first aspect.

[0028] Based on a further solution to the technical problem of the present invention, a preferred embodiment of the technical solution provided in the second aspect of the present invention includes: The first preferred solution is a high-nucleotide Saccharomyces cerevisiae extract, prepared from a Saccharomyces cerevisiae strain with accession number CGMCC No. 38790. This solution solves the technical problem of obtaining a high-nucleotide content extract from a specific source with stable performance.

[0029] Thirdly, the present invention provides: a method for preparing a high-nucleotide Saccharomyces cerevisiae extract, comprising the following steps: (a) Cultivate the brewing yeast strain described in the first aspect, and ferment it to obtain yeast slurry; (b) Inactivate the yeast slurry; (c) Add nuclease to enzymatically hydrolyze the inactivated yeast slurry, so that the yeast nucleic acid is degraded into nucleotides; (d) The enzymatic hydrolysate is separated, washed, concentrated and dried to obtain the high nucleotide Saccharomyces cerevisiae extract.

[0030] The fermentation method is selected from: continuous fermentation only, batch fermentation only, continuous fermentation followed by batch fermentation, and batch fermentation followed by continuous fermentation; preferably: continuous fermentation followed by batch fermentation; further preferably: continuous fermentation is performed first to enrich RNA, followed by batch fermentation to enrich glutathione; even more preferably: continuous fermentation is performed first, and after the nucleic acid content reaches the predetermined index, batch fermentation is performed, and an amino acid precursor solution containing L-glutamic acid, L-cysteine ​​and glycine is added during the batch fermentation process.

[0031] The conditions for the batch fermentation include: a ventilation rate of 5000-10000 m³ / h for the first hour. 3 / h, the second hour is 8000-15000 m 3 / h; pH 4.0-6.0; temperature 27-33℃; carbon source flow rate 500-900 kg / h; amino acid precursor solution flow rate 15-182 L / h; total fermentation time 4-10 hours.

[0032] The inactivation conditions are selected from: heat inactivation, chemical inactivation, and ultraviolet inactivation; preferably: heat inactivation; further preferably: heating the yeast slurry to 55-65℃ and 85-95℃ in sequence, and then cooling it to 50-60℃; even more preferably: passing it through a plate heat exchanger in sequence, with heat exchanger #1 heating to 55-65℃, heat exchanger #2 heating to 85-95℃, and heat exchanger #3 cooling to 50-60℃.

[0033] The nuclease used in the enzymatic hydrolysis conditions is selected from 5'-phosphodiesterase and other nucleases; preferably 5'-phosphodiesterase; the amount added is selected from 0.3%-1.0% of the yeast slurry mass and other proportions; preferably 0.3%-1.0%; the hydrolysis temperature is selected from 58-62℃ and other temperatures; preferably 58-62℃; the hydrolysis pH is selected from 4.8-5.5 and other pH values; preferably 4.8-5.5; the hydrolysis time is selected from 8-15 hours and other durations; preferably 8-15 hours.

[0034] The separation and washing in the post-processing method are selected from: retaining 0%-5%, retaining 5%-15%, and retaining 15%-30% of the enzymatic hydrolysate without separation; preferably: retaining 5%-15% of the enzymatic hydrolysate without separation; more preferably: retaining 5%-15% of the enzymatic hydrolysate without separation, the ratio of enzymatic hydrolysate to washing water during separation is 1:0.3-1:0.8, and the separated heavy phase is washed 1-3 times; even more preferably: retaining 5%-15% of the enzymatic hydrolysate without separation, to control the dextran content to be stable.

[0035] The concentration in the post-treatment method is selected from: single-effect evaporation, triple-effect evaporation, and membrane concentration; preferably: triple-effect evaporation; more preferably: controlling the temperature of the first effect to 75-85℃, the temperature of the second effect to 65-75℃, and the temperature of the third effect to 50-69℃, and concentrating to a solid content of 25%-45%.

[0036] The drying method in the post-treatment process is selected from: spray drying, freeze drying, fluidized bed drying; preferably: spray drying; more preferably: controlling the inlet air temperature to 170-185℃ and the outlet air temperature to 70-90℃.

[0037] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the third aspect of the present invention includes: The first preferred option: the fermentation in step (a) includes: first continuous fermentation, then batch fermentation. This technical solution, while solving the technical problem of how to prepare high-nucleotide extracts, further solves the technical problem of simultaneously enriching nucleotides and glutathione in the same process.

[0038] The second preferred option: The batch fermentation conditions described in step (a) are as follows: ventilation rate of 5000-10000 m³ / h for the first hour. 3 / h, the second hour is 8000-15000 m 3 / h; pH 4.0-6.0; temperature 27-33℃; carbon source flow rate 500-900 kg / h; amino acid precursor solution flow rate 15-182 L / h; total fermentation time 4-10 hours. This technical solution, building upon the existing solutions for preparing high-nucleotide extracts, further addresses the challenge of efficiently accumulating glutathione during batch fermentation.

[0039] The third preferred option: The enzymatic hydrolysis conditions described in step (c) are: temperature 58-62℃, pH 4.8-5.5, time 8-15 hours, and addition of 0.3%-1.0% 5'-phosphodiesterase. This technical solution, in addition to solving the technical problem of how to prepare high-nucleotide extracts, further solves the technical problem of efficiently degrading yeast nucleic acids into 5'-nucleotides.

[0040] The fourth preferred option: the separation and washing in step (d) includes retaining 5%-15% of the enzymatic hydrolysate without separation. This technical solution, while addressing the technical problem of how to prepare high-nucleotide extracts, further solves the technical problem of stabilizing the β-glucan content in the extract to optimize its prebiotic effects.

[0041] The fifth preferred option: The concentration in step (d) employs triple-effect evaporation, controlling the first-effect temperature at 75-85℃, the second-effect temperature at 65-75℃, and the third-effect temperature at 50-69℃, concentrating to a solids content of 25%-45%. This technical solution, while solving the technical problem of how to prepare high-nucleotide extracts, further addresses the technical problem of low-temperature, high-efficiency concentration to protect heat-sensitive components.

[0042] The sixth preferred option: the drying in step (d) is spray drying, with an inlet air temperature of 170-185℃ and an outlet air temperature of 70-90℃. This technical solution, while solving the technical problem of how to prepare high-nucleotide extracts, further solves the technical problem of obtaining low-moisture, high-flowability powder while maintaining product activity.

[0043] Fourthly, the present invention provides the use of the high-nucleotide Saccharomyces cerevisiae extract described in the second aspect or the high-nucleotide Saccharomyces cerevisiae extract prepared by the method described in the third aspect in the preparation of functional feeds, feed additives or drugs for improving the health of aging dogs.

[0044] The target of application is selected from: aging dogs, dogs aged 7 years or older, and other aging pets; preferably: aging dogs; more preferably: dogs aged 7 years or older.

[0045] The product type is selected from: functional feed, feed additives, drugs, and health products; preferably: functional feed or feed additives; and even more preferably: functional feed additives.

[0046] Based on a further solution to the technical problem of the present invention, in the technical solution provided in the fourth aspect of the present invention, a preferred embodiment includes: The first preferred solution is the application of the high-nucleotide Saccharomyces cerevisiae extract described in the second aspect in the preparation of functional feeds or feed additives to improve the health of aging dogs. This solution, having addressed the technical issues of the extract's application method, further addresses the technical problem of converting it into a form acceptable for pets' daily diet.

[0047] The second preferred option: the aging dogs are those that are 7 years of age or older. This technical solution, by clarifying the target audience, further addresses the issue of precise application technology for dogs at specific physiological stages.

[0048] Fifthly, the present invention provides: a functional feed, feed additive or drug for improving the health of aging dogs, comprising the high nucleotide Saccharomyces cerevisiae extract described in the second aspect or the high nucleotide Saccharomyces cerevisiae extract prepared by the method described in the third aspect.

[0049] The product type is selected from: functional feed, feed additives, drugs, and health products; preferably: functional feed or feed additives; and even more preferably: functional feed additives.

[0050] The active ingredient is selected from: the extract described in the second aspect and the extract prepared by the method in the third aspect.

[0051] Based on a further solution to the technical problem of the present invention, a preferred embodiment of the technical solution provided in the fifth aspect of the present invention includes: The first preferred solution is a functional feed additive for improving the health of aging dogs, comprising the high-nucleotide Saccharomyces cerevisiae extract described in the second aspect. This solution, in addition to addressing the technical issues related to product form, further addresses the technical problems of convenient daily quantitative supplementation and improved canine compliance.

[0052] In a sixth aspect, the present invention provides: the high-nucleotide Saccharomyces cerevisiae extract of the second aspect or the high-nucleotide Saccharomyces cerevisiae extract prepared by the method of the third aspect, for the treatment or prevention of age-related diseases in aging dogs.

[0053] The age-related diseases mentioned are selected from one or more of the following: muscle atrophy, osteoporosis, liver and kidney dysfunction, chronic low-grade inflammation, and abnormal telomere shortening.

[0054] The term "senior dog" refers to a dog that is 7 years of age or older.

[0055] Based on a further solution to the technical problem of the present invention, a preferred embodiment of the technical solution provided in the sixth aspect of the present invention includes: The first preferred option: the high-nucleotide Saccharomyces cerevisiae extract described in the second aspect, used to treat or prevent osteoporosis in aging dogs. This technical solution, building upon the technical challenges of improving bone health, further addresses the technical challenge of treating or preventing osteoporosis by increasing bone density.

[0056] The second preferred option: the high-nucleotide Saccharomyces cerevisiae extract described in the second aspect, used to treat or prevent muscle loss in aging dogs. This technical solution, in addition to addressing the technical issues of improving muscle condition, further addresses the technical issues of reversing age-related muscle loss.

[0057] The third preferred option: the high-nucleotide Saccharomyces cerevisiae extract described in the second aspect, used to inhibit chronic low-grade inflammation in aging dogs. This technical solution, while addressing the technical issues of improving immune homeostasis, further addresses the technical issues of inhibiting age-related chronic inflammation.

[0058] The present invention has at least the following beneficial effects: 1. Nutritional status and body condition management: Significantly reverses age-related weight loss and decline in body condition. The experimental group showed significant improvement in nutritional status under the intervention of Saccharomyces cerevisiae extract. In terms of body weight, the experimental group increased from baseline 8.84±1.37 kg to 9.40±1.21 kg (+6.3%), while the control group decreased from 8.61±1.04 kg to 7.56±0.90 kg (-12.2%), with a difference of 24.3%. Regarding body condition score (BCS), the experimental group improved from 3.13±0.64 to 3.50±0.93 (+11.8%), while the control group decreased from 3.25±1.04 to 2.63±0.52 (-19.1%), with a significant difference between the groups. In terms of muscle mass score (MS), the experimental group maintained its baseline level (2.75→3.00), while the control group decreased by 18.2% (3.00→2.25). These results indicate that Saccharomyces cerevisiae extract can effectively improve the efficiency and utilization of nutrient intake in middle-aged and aging dogs, and combat age-related muscle loss and deterioration of body condition.

[0059] 2. Bone health maintenance: Significantly increases bone density and improves mineral metabolism. CT imaging data showed that the lumbar spine bone mineral density (BMD) in the experimental group reached 173.67±4.50 mg / cc, a significant increase of 85.4% compared to the control group (93.67±5.03 mg / cc). This improvement in imaging was correlated with blood biochemical indicators: at day 42, the inorganic phosphorus level in the experimental group was significantly higher than that in the control group by 11.6% (1.54±0.15 vs 1.38±0.14 mmol / L), suggesting improved intestinal phosphorus absorption efficiency and active bone mineral deposition. The significant increase in bone mineral density indicates that Saccharomyces cerevisiae extract has a significant preventive and ameliorative effect on osteoporosis in middle-aged and aging dogs, possibly through regulating calcium and phosphorus metabolism balance or inhibiting osteoclast activity.

[0060] 3. Liver and kidney function protection: Improves kidney filtration function and reduces the metabolic burden on the liver. Regarding renal function, creatinine (CREA) levels remained stable and showed a decreasing trend from baseline (significantly reduced by 15.8% at D42), while uric acid (UA) showed a critical decreasing trend (-25.6%, P=0.072). Combined with the dynamic balance of inorganic phosphorus, this reflects the synergistic optimization of glomerular filtration efficiency and nitrogen / purine metabolism homeostasis.

[0061] Regarding liver function, at D90, the ALT level in the experimental group was significantly lower than that in the control group by 22.3% (31.07±12.14 vs 39.98±9.82 U / L), AST showed a critical decrease (21.5%), and GGT also showed a decreasing trend (18.8%). This indicates that the antioxidant components in the Saccharomyces cerevisiae extract can alleviate oxidative stress in hepatocytes, protect cell membrane integrity, and promote liver metabolic homeostasis.

[0062] 4. Improved fur health: Protects hair cuticle structure and slows down hair aging and damage. Saccharomyces cerevisiae extract protected the cuticle ultrastructure of middle-aged and older Beagle dogs and delayed hair aging damage. After 90 days of intervention, the hair cuticles in the experimental group were neatly arranged in an imbricate pattern, with thin and regular edges, closely adhering to the hair shaft surface, and showing minimal surface damage. In contrast, the control group dogs showed significant aging damage characteristics of the hair cuticles, manifested as disordered scale arrangement, rough and raised edges, and numerous pores and cracks on the surface. These results indicate that Saccharomyces cerevisiae extract can effectively protect the integrity of the hair cuticle structure, significantly slow down the process of hair aging damage, and has a positive effect on improving the health of the coat in middle-aged and older dogs.

[0063] 5. Gut microbiota and metabolic function remodeling: precise functional regulation under structural homeostasis After 90 days of intervention with Saccharomyces cerevisiae extract, the gut microbiota of middle-aged and aging dogs exhibited a unique regulatory pattern of "structural homeostasis-functional remodeling." Microbial diversity analysis showed that the Shannon and Simpson indices in the experimental group were significantly higher than those in the control group, indicating a significant improvement in microbial richness and community stability. Although the overall community structure (Beta diversity) remained largely undisturbed, specific optimization of core genera occurred: Bacteroidetes (… Bacteroidota The relative abundance of [organism name] increased significantly, replacing Pseudomonas species enriched at baseline; Bifidobacterium animalis, [organism name] ... Parasutterella The significant enrichment of beneficial bacteria suggests a transition of the gut microbiota towards a functionally balanced state.

[0064] At the metabolomics level, 291 differentially expressed metabolites were identified between the experimental and control groups, exhibiting polar-specific regulation characterized by predominantly upregulation by positive ions and predominantly downregulation by negative ions. KEGG analysis showed significant enrichment of β-alanine metabolism, steroid hormone biosynthesis, and histidine metabolism pathways, accompanied by enhanced synthesis of muscle-protective metabolites such as carnosine and histidine derivatives (related to barrier function).

[0065] This "microbial community structure fine-tuning-metabolic function activation" model synergistically improves the body's condition through the gut-host axis: gut-muscle axis (β-alanine metabolism and carnosine enrichment support stable muscle scores, with a decrease of 18.2% in the control group), gut-bone axis (steroid hormone pathways are associated with an 85.4% increase in bone mineral density and an 11.6% increase in serum phosphorus), and gut-barrier axis (histidine metabolism is consistent with stable blood routine tests and the absence of inflammatory responses throughout the process). The results indicate that targeted activation of specific functional pathways without disrupting the gut microbiota homeostasis constitutes an important mechanism for its multi-target anti-aging effect.

[0066] 6. Epigenetic regulation: regulating core aging pathways through DNA methylation. Whole-genome methylation sequencing (WGBS) revealed the anti-aging mechanism of *Saccharomyces cerevisiae* extract at the epigenetic level. Among 3,820 DMR-related genes, promoter regions involved 58 key aging-related genes. IGF1R The promoter exhibits hypermethylation (downregulation), which is associated with reduced IGF-1 signaling and prolonged lifetime. FOXO4 Located in the DMR region of the promoter, it regulates oxidative stress resistance; MTOR It exhibits hypomethylation (upregulated expression), which finely regulates cellular metabolism; SIRT3 (mitochondrial deacetylase) and NDUFS7 Hypomethylation of (core subunits of the mitochondrial respiratory chain) suggests optimization of mitochondrial function; DDB1Hypomethylation of DNA damage repair proteins suggests enhanced DNA repair capacity. These changes are concentrated in the IGF-1 / insulin signaling pathway, mitochondrial function, and DNA repair mechanisms, indicating that *Saccharomyces cerevisiae* extract can delay cellular aging through epigenetic modification. Furthermore, the relative telomere length assay provides independent and crucial validation at the cellular level: after 90 days of *Saccharomyces cerevisiae* extract intervention, the relative telomere length in aged beagle dogs was significantly longer than in the control group, indicating that the extract can effectively inhibit telomere wear and maintain telomere homeostasis. This improvement in a core marker of cellular aging, synergistically with the aforementioned enhanced DNA damage repair capacity (DDB1 hypomethylation) and optimized mitochondrial function (SIRT3 / NDUFS7 hypomethylation) epigenetic regulatory mechanisms, together constitute a complete anti-aging evidence chain of "epigenetic modification - telomere maintenance - cellular homeostasis," further confirming that *Saccharomyces cerevisiae* extract, through an epigenetic-telomere synergistic regulatory network, plays a role in delaying systemic aging in middle-aged and older dogs at the molecular and cellular levels.

[0067] 7. Immune and oxidative homeostasis: Maintains immune system stability, enhances antioxidant defense, and has no adverse reactions. Complete blood count monitoring showed no significant differences in white blood cell count, red blood cell parameters, and platelet count between the two groups, suggesting that the Saccharomyces cerevisiae extract had no adverse effects on the immune system and hematopoietic function of middle-aged and aging dogs and did not induce pathological inflammatory responses. Further analysis of inflammatory and antioxidant indicators revealed that the experimental group exhibited significant dual anti-inflammatory and antioxidant effects: In terms of inflammatory indicators, C-reactive protein (CRP) decreased by 11.3% from D45 to D90, the pro-inflammatory factor TNF-α remained stable at a low level throughout, IL-6 decreased significantly from D0 to D90 (P<0.05), while the anti-inflammatory factor IL-10 was significantly higher than that in the control group (P<0.05), and IFN-γ was also significantly increased (P<0.05). This indicates that the Saccharomyces cerevisiae extract can help middle-aged and aging dogs establish a more balanced immune microenvironment and combat age-related chronic low-grade inflammation by increasing the level of anti-inflammatory factors and inhibiting the fluctuation of pro-inflammatory factors. Regarding antioxidant indicators, the lipid peroxidation marker MDA decreased by 11.0% in the experimental group, while catalase (CAT) activity was significantly increased (P<0.05). Total antioxidant capacity (T-AOC), superoxide dismutase (SOD), reduced glutathione (GSH), and coenzyme NAD+ levels were all increased, suggesting that *Saccharomyces cerevisiae* extract can enhance the endogenous antioxidant defense system and reduce oxidative stress damage. Plasma proteomics analysis revealed 66 candidate differentially expressed proteins involved in lipid transport (MTTP downregulation), protein repair (PCMT1 upregulation), and cell adhesion (PCDH1 upregulation), which synergistically enhanced the aforementioned improvements in inflammation and antioxidant indicators, suggesting that the intervention may exert its effects by mildly regulating protein homeostasis, membrane transport function, and the oxidative-anti-inflammatory balance.

[0068] 8. Safety verification: No adverse effects on multiple organ functions, good applicability. Regarding cardiac function and anesthesia tolerance, both groups of dogs successfully completed anesthesia and CT scans without respiratory depression, sudden changes in heart rate, or allergic reactions, and were in good condition after anesthesia recovery. Regarding electrolyte homeostasis, although sodium, potassium, and chloride ion levels fluctuated, they remained within physiologically acceptable ranges, and at day 90, sodium ion levels in the experimental group decreased by 4.6% (…). P The value <0.05 may reflect homeostatic regulation after long-term adaptation. Blood glucose, blood lipids, and major organ indicators were all normal, confirming that the *Saccharomyces cerevisiae* extract has good biocompatibility during the 12-week intervention period.

[0069] Preservation Instructions Preserved strain: Y37; Classification and naming: Brewing yeast ( Saccharomyces cerevisiae ); Accession number: CGMCC No. 38790; Preservation period: May 20, 2026; 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

[0070] Figure 1 This describes the dog's weight changes.

[0071] Figure 2 The dog's body condition and muscle score were assessed.

[0072] Figure 3 This refers to the bone density of the dog.

[0073] Figure 4 Blood biochemistry results after Nucleovive feeding (D90, control group VS experimental group).

[0074] Figure 5 Effects of Nucleovive feeding on peripheral blood inflammatory markers in Bigger dogs (D90, control group vs. experimental group).

[0075] Figure 6 Effects of Nucleovive feeding on peripheral blood antioxidant in dogs (D90, control group vs. experimental group).

[0076] Figure 7 The changes in hair scales in each group were observed using scanning electron microscopy. From top to bottom, they are: group D0, control group D90, and experimental group D90.

[0077] Figure 8Alpha diversity analysis of gut microbiota after Nucleovive feeding (D90, control group vs. experimental group).

[0078] Figure 9 PCoA plot based on Bray-Curtis distance (D0 group, D90 experimental group, D90 control group).

[0079] Figure 10 Heatmap of phylum-level bacterial abundance clusters (D90 control group, D90 experimental group, D0 group, young dog group).

[0080] Figure 11 Heatmap of genus-level bacterial abundance clusters (D90 control group, D90 experimental group, D0 group, young dog group).

[0081] Figure 12 Box plot of relative abundance differences of dominant bacterial groups at the phylum level between groups (D90 control group, D90 experimental group, D0 group).

[0082] Figure 13 Bar chart showing the comparison of relative abundance of genus-level differential bacterial communities among groups (D90 control group, D90 experimental group, D0 group).

[0083] Figure 14 Bar chart showing the size of the differential bacterial community LDA effect in LEfSe analysis (D90 control group, D90 experimental group, D0 group).

[0084] Figure 15 Evolutionary clade diagram of differentially expressed microbial communities analyzed by LEfSe (D90 control group, D90 experimental group, D0 group).

[0085] Figure 16 Statistical analysis of differences in fecal metabolites (D90, control group vs. experimental group).

[0086] Figure 17 Statistical analysis of differential pathways in fecal metabolite KEGG (D90, control group vs. experimental group).

[0087] Figure 18 The changes in the relative length of telomeres in dogs of different groups and over time.

[0088] Figure 19 The overall results of methylation detection are shown in D90 (control group vs. experimental group).

[0089] Figure 20 Analysis of the KEGG pathway for methylation genes (D90, control group vs. experimental group).

[0090] Figure 21 For plasma proteomic analysis (D90, control group VS experimental group).

[0091] Figure 22 Volcano plots for plasma proteomic analysis (D90, control group vs. experimental group) Detailed Implementation 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.

[0092] 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.

[0093] 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.

[0094] Example 1 1. Strains Saccharomyces cerevisiae was used as the production strain. Strain preservation number: CGMCC No. 38790.

[0095] 2. Seed culture (1) Culture medium preparation Test tube slant culture medium: containing glucose, yeast extract, peptone, and agar, natural pH, sterilized at 121°C for 20 minutes.

[0096] Liquid seed culture medium in Erlenmeyer flasks: containing glucose, yeast extract, peptone, and natural pH; the liquid volume is 50mL / 250mL or 1L / 5L Erlenmeyer flasks, and sterilized at 121℃ for 20 minutes.

[0097] (2) Activation and scale-up culture of strains Take two loops of *Saccharomyces cerevisiae* culture preserved on an agar slant and transfer it to fresh test tube agar slant culture medium. Activate and culture at 28-30℃ (30℃ in this example) for 48 hours. Inoculate the activated agar slant culture into small Erlenmeyer flasks (two loops per flask) of liquid culture medium and culture in a shake flask at 160-180 rpm and 20-40℃ (180 rpm and 30℃ in this example) for 24 hours. Then, transfer all the culture from the small Erlenmeyer flasks to large Erlenmeyer flasks and continue culturing at the same conditions for 24 hours to obtain qualified liquid seed culture.

[0098] 3. Continuous fermentation The 8L liquid seed culture medium, cultivated in Erlenmeyer flasks, was transferred to sterilized two-stage seed tanks for incremental expansion. The substrate in the seed tanks consisted primarily of molasses (COFCO Beihai Sugar Co., Ltd.), with an addition of 350kg (based on sugar content), 30kg ammonium sulfate, 2kg magnesium sulfate, 0.2kg zinc sulfate, and 2g copper sulfate. Ammonia and phosphoric acid were added during the continuous feed process in subsequent fermenters. The culture process was controlled at 30-32℃ and pH 4.2-4.5. When the wet bacterial count reached 50±5g / L, the culture was transferred to sterilized secondary seed tanks. Yeast fermentation in the seed tanks lasted approximately 12 hours, with ventilation controlled at 1000-2000m³ / h. 3 At a temperature of 30-32℃ and a pH of 4.2-4.5, when the wet bacterial concentration in the seed culture reaches 70±5 g / L, transfer it to a fermenter and continuously add molasses (800-1000 kg / h based on sugar content), ammonia, and phosphoric acid (nitrogen-to-phosphorus ratio of 20:1) and process water, maintaining an aeration rate of 10,000-12,000 m³ / h. 3 At a temperature of 30-32℃ and a pH of 4.2-4.5, when the wet bacterial concentration is 95±5g / L, the residual sugar is less than 0.3wt%, and the nucleic acid content reaches 18-22%, the fermentation mash is continuously discharged. After being centrifuged and concentrated by a separator, the fermentation mash is used to obtain continuously fermented yeast slurry.

[0099] 4. Batch fermentation (1) Preparation of amino acid precursors Prepare 1–5 mol / L aqueous solutions of L-glutamic acid, L-cysteine, and glycine respectively; wherein, the glutamic acid and glycine solutions are sterilized at 110–120℃ for 10–60 minutes, and then mixed evenly with the L-cysteine ​​solution under sterile conditions for later use.

[0100] (2) Fermentation control The 80m obtained from continuous fermentation 3 All yeast slurry was transferred to the batch fermentation tank, and the ventilation rate was controlled at 5000-10000 m³ / h for the first hour. 3 / h (10000m in this example)3 / h), adjust to 8000-15000m in the second hour. 3 / h (12000m in this example) 3 The pH was controlled at 4.0-6.0 and the temperature at 27-33℃ throughout the process (pH 5.0-5.5 and temperature 30±1℃ in this example); the carbon source flow rate was 500-900 kg / h (based on sugar) (700-900 kg / h in this example); starting from the second hour, a 5 mol / L amino acid precursor solution was added at a flow rate of 15-182 L / h (175 L / h in this example); the total fermentation time was 4-10 hours (6 hours in this example), and the fermentation broth was obtained.

[0101] 5. Separation and washing of fermentation broth Centrifugal separation was performed using a high-speed disc separator. The fermentation broth was transported to the separator via pipeline, where the light and heavy phases were separated by the action of the discs. The light phase waste liquid was discharged, and the heavy phase yeast slurry was retained. The obtained yeast slurry was washed with water at a material-to-water volume ratio of 1:0.5-1:0.8 (1:0.8 in this example). The mixture was then sent to a centrifuge via pipeline for mixing and separation. The light phase waste water was discharged, and the heavy phase was collected. The separation operation was repeated 1-3 times (3 times in this example) with the same material-to-water ratio to obtain clean yeast slurry for later use.

[0102] 6. Yeast inactivation Water was added to the obtained clean yeast slurry to adjust the solid content to 10%-18% (12±2% in this embodiment). The yeast slurry was then passed through plate heat exchangers #1, #2, and #3 in sequence to complete the inactivation and cooling: heat exchanger #1 used hot water at 90-95℃ (92±1℃ in this embodiment) to heat the yeast slurry to 55-65℃ (60±1℃ in this embodiment); heat exchanger #2 used steam to heat the yeast slurry to 85-95℃ (90±1℃ in this embodiment); and heat exchanger #3 used circulating water at 5-25℃ (20±1℃ in this embodiment) to cool the yeast slurry to 50-60℃ (55±1℃ in this embodiment).

[0103] 7. Nuclease digestion Adjust the temperature of the inactivated yeast slurry to 58-62℃ and the pH to 4.8-5.5 (in this example, the temperature is 60±1℃ and the pH is 5.0-5.2). Add nuclease (5'-phosphodiesterase, Amano Co., Ltd. (model RP-1)) at a rate of 0.3%-1.0% of the yeast slurry mass (0.5% in this example). Perform the enzymatic hydrolysis reaction at a constant temperature for 8-15 hours (10 hours in this example) to ensure complete degradation of the yeast nucleic acid.

[0104] 8. Separation and washing of enzymatic hydrolysate The enzymatic hydrolysate was separated using a high-speed disc centrifuge, retaining 5%-15% (v / v) (in this example, 10±1% of the enzymatic hydrolysate was not separated and was directly sent to the evaporation buffer tank; during the separation process, the ratio of enzymatic hydrolysate to washing water was 1:0.3-1:0.8 (1:0.6 in this example), and the separated heavy phase was washed 1-3 times (3 times in this example), with the ratio of heavy phase to washing water being 1:0.7-1:1.2 each time (1:1 in this example). All the supernatant and unseparated enzymatic hydrolysate were combined and set aside for later use.

[0105] 9. Triple-effect evaporation and concentration The combined enzymatic hydrolysis supernatant was fed into a triple-effect evaporator for concentration. The temperature of the first effect was controlled at 75-85℃, the temperature of the second effect at 65-75℃, and the temperature of the third effect at 50-69℃ (in this embodiment, the temperature of the first effect was 80±1℃, the temperature of the second effect was 68±1℃, and the temperature of the third effect was 55±1℃). The concentration was carried out until the solid content was 25%-45% (in this embodiment, it was 35-40%), and the concentrated solution was obtained.

[0106] 10. Spray drying After the concentrated liquid is dehumidified by the dehumidifier, it is sent to the spray dryer. The inlet air temperature is controlled at 170-185℃ and the outlet air temperature is controlled at 70-90℃ (in this embodiment, the inlet air temperature is 180±2℃ and the outlet air temperature is 80±2℃). The moisture content of the finished product after drying is ≤6%, which is the brewing yeast extract powder.

[0107] 11. Finished Product Packaging Within the clean production area, brewer's yeast extract powder is packaged and sealed according to market demand specifications, and stored in the warehouse after passing inspection.

[0108] Example 2 2.1 Experimental Design 2.1.1 Experimental Animals and Grouping Experimental subjects: The product used in this feeding trial was the brewer's yeast extract (i.e., Nucleovive) from Example 1. Eighteen female Beagles aged 7 years and older were used in this trial and were randomly divided into two groups (n=9 / group): a control group and an experimental group. Three younger dogs were additionally included as a young control group for middle-aged and older dogs. Basic information recorded: age, breed, sex, spayed / neutered status, health status, dog food type, etc. Feeding and management: Free access to food and water; daily cleaning of cages. Necessary immunizations and deworming were completed before the trial. Detailed information on the 18 Beagles is shown in Table 1. Table 1. Basic Information on Beagles

[0109] 2.1.2 Test Methods The control group dogs were fed a basic dog food (Zhenzhile complete dog food), while the experimental group dogs had a certain dose of brewer's yeast extract (1.3g / dog / day) added to their basic dog food. The experiment lasted for 90 days. During the experiment, all indicators were strictly monitored according to requirements. The specific monitoring content and time points are as follows: (1) Food intake, water intake, and weight: Recorded every two weeks for a total of 90 days; (2) Body condition score (BCS) and muscle score (MS): recorded once before the start of the test and once at the end of the test; (3) Feces, hair, and blood were collected from both groups of dogs a total of three times during the experimental period. Feces were used for intestinal flora detection, fecal assessment, and metabolomics. Hair was used for electron microscopy of hair cuticles. Blood samples were used for various blood parameters. (4) Anesthesia and CT scan: Considering the potential impact of anesthesia on dogs, CT scans were only performed on the two groups of dogs after anesthesia at the end of the experiment to observe bone-related imaging indicators; (5) Evaluation of fur quality: The coat along the back of the dogs was observed and scored, and the hair scales of the two groups of dogs were observed by scanning electron microscopy after 90 days of feeding to establish an evaluation of the ultrastructure. (6) Intestinal microecological balance analysis: Based on the assessment of microbial diversity by 16S rRNA sequencing, combined with the quantitative detection of fecal metabolites, the microecological balance status was systematically assessed. (7) Methylation gene sequencing: Whole genome bisulfite sequencing (WGBS) technology was used to analyze the changes in genes related to differentially methylated regions (DMRs) after formulation intervention; (8) Plasma proteomics detection: The effects of additives on plasma protein expression in middle-aged and aged dogs were evaluated by mass spectrometry proteomics technology.

[0110] 2.1.2 Reagents and Instruments Reagents: Saccharomyces cerevisiae extract, isoflurane, and DNA extraction kit from Example 1.

[0111] Instruments: X-ray computed tomography equipment, Insitum CT 768, electronic benchtop scale, blood routine five-part differential analyzer, blood biochemistry analyzer, fluorescence quantitative PCR instrument, protein mass spectrometer, metabolite mass spectrometer, 16S amplicon sequencer, and scanning electron microscope, etc.

[0112] 2.1.3 Blood index testing and instruments The blood index testing equipment is shown in Table 2: Table 2. Testing Items and Instruments

[0113] Sources of purchase for each kit: Canine Immunoglobulin A (IgA) ELISA Kit (Model GY-L7011A, Shanghai Guyan), Canine Immunoglobulin G (IgG) ELISA Kit (Model GY-L72301A, Shanghai Guyan), Canine Immunoglobulin M (IgM) ELISA Kit (Model GY-L73212, Shanghai Guyan), Canine Secretory Immunoglobulin A (sIgA) ELISA Kit (Model GY-L7562A, Shanghai Guyan), Canine Total Antioxidant Capacity (T-AOC) ELISA Kit (Model GY-L7210A, Shanghai Guyan), Canine Superoxide Dismutase (SOD) ELISA Kit (Model GY-L7357A, Shanghai Guyan), Canine Malondialdehyde (MDA) ELISA Kit (Model GY-L7350A, Shanghai Guyan), Canine Catalase (CAT) ELISA Kit Canine Glutathione (GSH) ELISA kit (GY-L74500A, Shanghai Guyan), Canine C-Reactive Protein (CRP) ELISA kit (GY-L7827A, Shanghai Guyan), Canine Tumor Necrosis Factor α (TNF-α) ELISA kit (GY-L7615A, Shanghai Guyan), Canine Interferon N-γ (IFN-γ) ELISA kit (GY-L7626A, Shanghai Guyan), Canine Interleukin 6 (IL-6) ELISA kit (GY-L7225A, Shanghai Guyan), Canine Interleukin 10 (IL-10) ELISA kit (GY-L7303A, Shanghai Guyan), and Canine Nicotinamide Adenine Dinucleotide NAD+ ELISA kit (GY-L7755A, Shanghai Guyan).

[0114] 2.1.3 Sample Detection Indicators Blood test indicators: Immunology: IgA, IgG, IgM, SIgA; Antioxidants: T-AOC, SOD, MDA, CAT, GSH; Inflammation: CRP, TNF-α, IFN-γ, IL-6, IL-10; Complete blood count: Five-part differential blood count; Biochemistry: 12 liver function tests, 4 kidney function tests, 4 blood glucose and lipid tests, 2 cardiovascular tests, 2 pancreatic function tests, 10 trace elements; Methylation genomics; Plasma proteomics.

[0115] Hair and skin indicators: Scanning electron micrographs of hair cuticles.

[0116] Intestinal microecological indicators: 16S rRNA sequencing of intestinal flora; fecal metabolomics detection.

[0117] 2.1.4 Data Processing and Analysis All data are presented as mean ± standard deviation (Mean ± SD). T-tests or one-way ANOVA were used to compare differences between groups. Omics data were analyzed separately according to their respective categories.

[0118] 2.2 Test Results 2.2.1 Results of food intake, water intake, and weight monitoring During the experiment, the food intake (120g / meal / 2 meals per day, fixed amount per meal, ensuring the dog food was completely consumed, and no additional dog food was given after the meal) and water intake (free access to water) of the two groups of middle-aged and older dogs were recorded every two weeks for 12 consecutive weeks. Weight changes were recorded as follows: Figure 1 As shown.

[0119] At the initial stage of the experiment, there was no significant difference in the weight of the dogs in the experimental group (8.84±1.37 kg) compared with that in the control group (8.61±1.04 kg). P >0.05). As the experiment progressed, the weight of the control group dogs showed a slow decreasing trend; while the weight of the experimental group dogs showed an increasing trend in the first 8 weeks, and the weight remained relatively stable at the end of the experiment. At week 12 of the experiment, the weight of the experimental group dogs (9.40±1.21 kg) was significantly higher than that of the control group (7.56±0.90 kg), and the difference was statistically significant. P <0.05).

[0120] 2.2.2 Results of Body Condition Score (BCS) and Muscle Score (MS) The Body Condition Score (BCS) uses a 9-point scoring system (1 point: extremely thin, 9 points: extremely obese), and the Muscle Score (MS) uses a 5-point scoring system (1 point: severe muscle loss, 5 points: excellent muscle condition).

[0121] The two groups of dogs were scored at the beginning and end of the experiment, and the specific results are as follows: Figure 2 As shown. At the start of the experiment, there was no significant difference in BCS and MS scores between the two groups of dogs ( P >0.05). After 90 days of Nucleovive intervention, the BCS and MS scores in the experimental group increased compared to the initial scores, while those in the control group decreased. Intergroup comparisons showed that at the end of the trial, the BCS score in the experimental group (6.30±0.93) was significantly higher than that in the control group (4.73±0.52), and the difference was statistically significant. P <0.05); The MS score in the experimental group showed an increasing trend compared to the control group, but there was no significant difference in MS scores between the two groups. P >0.05).

[0122] 2.3.3 Anesthesia and CT scan results 2.3.3.1 Anesthesia Both groups of dogs were anesthetized (propofol for induction and isoflurane for sustained anesthesia). Vital signs such as respiration, heart rate, and blood pressure were closely monitored during anesthesia. Anesthesia was successfully completed in both groups without any respiratory depression, sudden changes in heart rate, or allergic reactions. Upon recovery, the dogs were in good spirits and showed no obvious signs of discomfort.

[0123] 2.3.3.2 CT scan results Bone mineral density (BMD) of the L1-L4 lumbar vertebrae in two groups of dogs was measured using quantitative computed tomography (QCT). The scanning parameters were set as follows: tube voltage 120 kVp, tube current 180 mA (effective mAs=90), slice thickness 2 mm, pitch 0.5, rotation time 0.50 s, reconstruction matrix 512×512, and bone windowing algorithm (window width 1000, window level 200). Dogs were placed in the ventral recumbency position. Based on lateral and ventrodorsoventral positioning images, single-slice transverse section scans were performed in the mid-plane of the vertebral body (avoiding the endplates and transverse process origins). According to the standard QCT method, the BMD of the lumbar vertebrae in the experimental group (173.67 ± 4.50 mg / cc) was significantly higher than that in the control group (93.67 ± 5.03 mg / cc), and the difference was statistically significant. P <0.05)( Figure 3 This value is volumetric bone mineral density (vBMD), which directly reflects the mineral content of cancellous bone and is more sensitive to detecting bone loss than area bone mineral density (aBMD, such as DXA measurement).

[0124] 2.3.4 Analysis of blood routine and blood biochemistry results The blood routine tests of all 16 beagles were within the healthy range, meeting the inclusion criteria for the experiment (two dogs had insufficient samples and were not tested). Although there were slight deviations in the neutrophil percentage and platelet count in individual dogs—for example, in terms of neutrophils, dog #32 had a measured neutrophil percentage of 85.25%, which was 4.25% higher than the upper limit of 81%, and dog #34 had a measured neutrophil percentage of 85.13%, which was 4.13% higher—the average platelet count was 464.56 ± 108.75 × 10⁻¹⁰. 9 / L, slightly higher than the reference upper limit of 460 × 10 9 The baseline status was / L, but key hematopoietic and immune indicators remained stable and did not reach clinicopathological thresholds. This baseline status effectively ruled out interference from potential hematological diseases, ensuring that subsequent intergroup differences were attributable to intervention rather than individual health differences (Table 3).

[0125] The blood biochemical indicators of the 16 middle-aged and older beagle dogs were generally within the healthy range. The core parameters of liver and kidney function (ALT 47.42±10.02 U / L, AST 33.72±6.28 U / L, creatinine 63.52±10.99 μmol / L, urea 5.01±1.25 mmol / L) all met the reference standards for adult dogs. Although the mean values ​​of glucose (2.64±0.67 mmol / L) and potassium (6.16±0.91 mmol / L) deviated slightly from the normal range, considering the fasting and stress factors before sampling, and the consistent variation within the group, it did not affect the comparability between groups. The mean albumin-globulin ratio of 0.64±0.12 was slightly lower than the general reference lower limit, which may be related to the immune status of middle-aged and older dogs and breed specificity (Table 4).

[0126] Table 3. Blood routine test results of dogs (D0) (16 dogs)

[0127] Table 4. Blood biochemistry results of canine D0 blood

[0128] After 90 days of intervention with Nucleovive, a yeast extract, liver function indicators showed that alanine aminotransferase (ALT) in the experimental group decreased significantly by 22.3% (P<0.05), and aspartate aminotransferase (AST) and gamma-glutamyl transferase (GGT) also showed a synergistic decreasing trend, suggesting that hepatocyte membrane integrity was protected and the metabolic burden on the liver was reduced. Regarding kidney function indicators, creatinine (CREA) levels remained stable and showed a decreasing trend from baseline (a significant decrease of 15.8% was observed at D42), while uric acid (UA) showed a critical decreasing trend (-25.6%, P=0.072). Combined with inorganic phosphorus dynamic balance, this reflects a synergistic optimization of glomerular filtration efficiency and nitrogen / purine metabolic homeostasis. All indicators fluctuated within the physiological reference range without pathological elevation, confirming that this intervention exerted a mild hepatoprotective and renal protective effect while possessing good long-term biocompatibility.

[0129] Furthermore, the results of inflammatory marker detection showed that after 90 days of intervention with *Saccharomyces cerevisiae* extract *Nucleovive*, the experimental group exhibited a good anti-inflammatory effect. C-reactive protein (CRP) decreased by 11.3% from D45 to D90, indicating a reduction in systemic inflammation levels; the pro-inflammatory factor TNF-α remained stable at a low level throughout the experimental group, and IL-6 significantly decreased from D0 to D90. P <0.05, indicating that the pro-inflammatory response was effectively suppressed; at the same time, the anti-inflammatory factor IL-10 was significantly higher in the experimental group than in the control group at D90 ( P<0.05), IFN-γ was also significantly increased ( P <0.05 indicates enhanced anti-inflammatory ability of the body ( Figure 5 Overall, the Saccharomyces cerevisiae extract did not induce a systemic inflammatory response, and by increasing the levels of anti-inflammatory factors and inhibiting fluctuations in pro-inflammatory factors, it may help middle-aged and older dogs establish a more balanced immune microenvironment to combat age-related chronic low-grade inflammation.

[0130] Regarding antioxidant indicators, after 90 days of intervention with *Saccharomyces cerevisiae* extract *Nucleovive*, the experimental group showed a significant increase in antioxidant capacity. The lipid peroxidation marker MDA showed a clear decreasing trend in the experimental group (reduced by 11.0%), and the absolute value was lower than that in the control group, indicating that cell membrane oxidative damage was effectively inhibited; catalase (CAT) showed statistical significance in the comparison between the D90 groups (…). P <0.05, indicating that the Saccharomyces cerevisiae extract can specifically enhance the activity of key antioxidant enzymes; the total antioxidant capacity (T-AOC), superoxide dismutase (SOD), reduced glutathione (GSH), and coenzyme NAD+ levels were all slightly increased compared with the control group at D90, suggesting that the overall antioxidant capacity of the experimental group was improved. Figure 6 The above results indicate that Saccharomyces cerevisiae extract can exert an anti-aging effect by enhancing the endogenous antioxidant defense system and reducing oxidative stress damage in middle-aged and older dogs.

[0131] 2.3.5 Effects of feeding with Saccharomyces cerevisiae extract on fur health This invention systematically evaluates the effects of Nucleovive, a yeast extract, on the coat health of middle-aged and older beagle dogs through gross clinical observation combined with scanning electron microscopy (SEM) ultrastructural analysis.

[0132] Before the start of the trial (D0) and 90 days after the intervention (D90), the coat of the dogs along the thoracolumbar region was grossly observed and scored. The evaluation indicators included coat luster, fullness, skin condition, and coat color saturation.

[0133] The results showed that all middle-aged and older dogs in the baseline group (D0) generally exhibited moderate levels of coat aging, with moderate shine and density. After feeding with Nucleovive (a brewer's yeast extract) for 90 days, the experimental group (D90) showed a significant improvement in coat shine, a smoother feel, increased fullness of the coat, rapid skin rebound, no obvious dandruff, and maintained its bright original color. The middle-aged and older dogs in the control group (D90CON) showed obvious signs of coat aging, including fading and whitening of hair tips, sparse hair revealing skin texture, increased dandruff, and decreased skin elasticity (manifested as a prolonged time for the skin to spring back after being pulled up).

[0134] The surface morphology of the canine hair in D0 and D90 (experimental and control groups) was observed using scanning electron microscopy. The results are shown in Table 5. Figure 7 The experimental results showed that after 90 days of intervention with the Saccharomyces cerevisiae extract Nucleovive, it could improve the health of the fur in middle-aged and older dogs: protecting the hair cuticle structure and slowing down hair aging damage.

[0135] Table 5 Comparison of the observation results of hair scales in each group under scanning electron microscopy (D0 group, D90 control group, D90 experimental group)

[0136] 2.3.6 Effects of feeding canines with Nucleovive (a yeast extract) on intestinal flora and fecal metabolites. 2.3.6.1 Overview of Experimental Design This invention employs 16S rRNA gene sequencing (V3-V4 region) and untargeted metabolomics (LC-MS / MS platform) to systematically evaluate the effects of 90-day intervention with *Saccharomyces cerevisiae* extract Nucleovive on the gut microbiota of middle-aged and aging dogs. Through pairwise analysis, the intervention mechanism was revealed at both the level of microbiota structure diversity and metabolic function.

[0137] 2.3.6.2 Analysis of Gut Microbiota Diversity Alpha diversity analysis (Shannon and Simpson indices) showed that supplementation with Nucleovive, a yeast extract, was associated with improved gut microbiota diversity and structural stability, with better results than the control group based on a conventional diet.

[0138] The Shannon index comprehensively reflects species richness and evenness. The results showed that after 90 days of intervention, the Shannon index of the D90 group (Saccharomyces cerevisiae extract Nucleovive intervention group) was significantly higher than that of the D90 CON group (conventional diet control group). P <0.05. Compared with baseline (D0), the Shannon index of the D90 group was significantly increased ( P <0.01), indicating a significant increase in bacterial diversity; in contrast, although the D90CON group showed some fluctuations compared to D0, the overall improvement was relatively limited.

[0139] The Simpson index is more sensitive to changes in dominant bacterial communities and is often used to assess community structure stability. The results showed that the Simpson index of group D90 was significantly higher than that of group D90CON. P <0.01, and showed a highly significant increase compared to D0 ( P<0.001). This result suggests that supplementation with Saccharomyces cerevisiae extract Nucleovive may help promote the optimization of the dominant flora structure, thereby improving the stability of the gut microbiota. Figure 8 ).

[0140] Beta diversity analysis (PCoA based on Bray-Curtis distance) showed that, compared with the D90CON group, the D90 group samples exhibited a relatively more concentrated distribution trend, suggesting that the inter-individual differences in gut microbiota were reduced and the community structure tended to be relatively stable; however, the overall structural differences between the two groups did not reach statistical significance. P >0.05, Figure 9 ).

[0141] 2.3.6.3 Analysis of the core gut microbiota The gut microbiota of different groups of dogs showed significant structural differences at the phylum and genus levels. Figures 10-15 At the phylum level, the baseline group (D0) was predominantly Pseudomonas (Pseudomonas). Pseudomonadota Proteobacteria Proteobacteria The core characteristic was high enrichment of Bacteroidetes. After a 90-day trial period, compared with the control group (D90 CON) and the baseline group (D0), the intervention group (D90) showed a higher enrichment of Bacteroidetes (Bacteroidetes phylum). Bacteroidota The relative abundance of ) increased significantly ( P <0.05), and the improvement of the D90 group is better than that of the D90 CON group.

[0142] At the genus level, the baseline group (D0) was dominated by the genus *Psychrophilus* (…). Psychrobacter ), active rumen cocci ( Ruminococcus gnavus group ) and Collins spp. ( Collinsella The main characteristic was the enrichment of *Lactobacillus* spp., a Gram-negative opportunistic pathogen originating from the environment. The high abundance of *Psychrophilus* spp. in group D0 may reflect the initial state of decreased intestinal flora stability and microecological imbalance in older dogs. After a 90-day experimental period, the abundance of *Lactobacillus* spp. in the control group (D90 CON) was significantly higher. Lactobacillus Unidentified rodentaceae ( Unclassified_Muribaculaceae Enrichment of beneficial bacteria such as *Saccharomyces cerevisiae* extract. In contrast, the Nucleovive intervention group (D90) of *Saccharomyces cerevisiae* extract drove... Segatella Parasartella spp. Parasutterella ) and Zurich bacillus ( Turicibacter The specific upregulation of ) and the core probiotic species—Bifidobacterium animalis ( Bifidobacterium animalis Significant enrichment of ).

[0143] The results of the experiment suggest that, compared to the general adjustments made by a regular diet, supplementation with Nucleovive, a yeast extract, may help promote a more balanced and functionally diverse gut microbiota.

[0144] 2.3.6.4 Significant reshaping of fecal metabolite profile Untargeted metabolomics analysis revealed a significant reshaping of the fecal metabolite profile after intervention with *Saccharomyces cerevisiae* extract. Compared with the D90CON group, the D90 group screened 258 differentially expressed metabolites in positive ion mode, of which 202 were upregulated (78.3%) and 56 were downregulated (21.7%); in negative ion mode, 33 differentially expressed metabolites were screened, of which 7 were upregulated (21.2%) and 26 were downregulated (78.8%). Figure 16 This polarity-specific regulatory pattern, characterized by "predominantly upregulation in the positive ion mode and predominantly downregulation in the negative ion mode," suggests that Saccharomyces cerevisiae extract may have directionally altered the synthesis and secretion pathways of different polar metabolites.

[0145] KEGG pathway enrichment analysis showed that differentially metabolites were significantly enriched in the following key biological pathways ( Figure 17 ): β-Alanine metabolism P =0.024): This pathway was the most significantly enriched of differentially expressed metabolites, with carnosine as the core metabolite. Carnosine is a dipeptide with antioxidant and muscle-protective functions, and its enrichment suggests that Saccharomyces cerevisiae extract may improve host condition through the gut-muscle axis by promoting the synthesis of muscle-protective metabolites by gut microbiota.

[0146] Steroid hormone biosynthesis P =0.070): This pathway involves the metabolic regulation of steroid hormone precursors such as dehydroepiandrosterone sulfate (DHEA-S). Changes in the steroid hormone pathway corresponded to the phenotype of significantly increased bone mineral density (+85.4%) and improved body condition score (BCS +11.8%) in the experimental group, suggesting that gut microbiota-metabolite interactions may participate in the regulation of host mineral metabolism and bone health through the gut-endocrine-bone axis.

[0147] Histidine metabolism P =0.070): Histidine is a precursor amino acid for histamine synthesis and is closely related to intestinal barrier function and immune regulation. Enrichment of this pathway suggests that Saccharomyces cerevisiae extract may enhance intestinal mucosal barrier function and maintain immune homeostasis by regulating intestinal flora-mediated amino acid metabolism.

[0148] GSEA analysis further validated the overall regulatory trends of metabolic pathways, with "metabolic pathways" and "biosynthesis of cofactors" showing significant enrichment. P The value <0.05 indicates that the effect of Saccharomyces cerevisiae extract on intestinal metabolic function is systematic and multi-level.

[0149] 2.3.6.5 Microbial Community-Metabolite Interaction Patterns By integrating 16S rRNA gene sequencing and non-targeted metabolomics data, this study reveals the unique mechanism by which Saccharomyces cerevisiae extract regulates the gut microbiota through a "structural homeostasis-functional remodeling" model.

[0150] At the phylum level, changes in the bacterial community structure were observed in the D90 group compared to the D90CON group, with the D90 group exhibiting a higher proportion of Bacteroidetes phylum ( Bacteroidota A significant increase in the relative abundance of Bacteroidetes was observed. Bacteroidetes are important carbohydrate-degrading bacteria in the gut, and increased abundance is generally associated with a healthier gut microbiota. Notably, the baseline group (D0) was dominated by Pseudomonas (…). Pseudomonadota The high enrichment of *Proteobacteria* was a characteristic feature of the disease, and the relative dominance of this phylum decreased after intervention, suggesting that *Saccharomyces cerevisiae* extract may help correct the excessive proliferation of opportunistic pathogens in the intestines of aged dogs.

[0151] At the genus level, the D90 group drives... Segatella Parasartella spp. Parasutterella ), Zurich bacillus ( Turicibacter The specific upregulation of ) and the core probiotic species—Bifidobacterium animalis ( Bifidobacterium animalis Significant enrichment of [organism name missing]. Among them, Bifidobacterium animalis is an important probiotic with functions of regulating immunity and improving intestinal barrier function; Parasutterella It is closely related to the host's bile acid metabolism and steroid hormone metabolism; Turicibacter It participates in the metabolism and synthesis of various amino acids and vitamins.

[0152] Although Beta diversity analysis showed that the overall microbial community structure difference between the D90 group and the D90CON group did not reach statistical significance ( P >0.05), but the metabolomics showed significant differentiation. This "structural homeostasis-functional remodeling" pattern suggests that Saccharomyces cerevisiae extract may exert its effects through the following mechanisms: (1) Regulation of metabolic activity in specific genera: While the extract of *Saccharomyces cerevisiae* does not alter the proportion of dominant phyla, it significantly regulates the metabolic activity of specific functional genera. For example, ParasutterellaThe enrichment of these bacteria corresponds to the activation of the steroid hormone biosynthesis pathway. This genus is known to participate in the metabolic transformation of host bile acids and steroid hormones. The upregulation of Bifidobacterium animalis may promote the synthesis of protective metabolites such as carnosine in the β-alanine metabolic pathway.

[0153] (2) Metabolic regulation of the gut-host axis: There is a clear association between differential metabolite enrichment pathways and host phenotype improvement: Gut-muscle axis: The enrichment of carnosine in the β-alanine metabolic pathway was consistent with the stable muscle score in the experimental group (MS 2.75→3.00) and the decrease in the control group (3.00→2.25), suggesting that muscle-protective metabolites from gut microbiota may delay muscle loss in older dogs through the gut-muscle axis; Gut-bone axis: Activation of the steroid hormone biosynthesis pathway was associated with a significant increase in bone mineral density (+85.4%), suggesting that gut microbiota-metabolite interactions may participate in bone health maintenance by regulating calcium and phosphorus metabolism and bone mineral deposition; Gut-barrier axis: The enrichment of histidine metabolic pathway was associated with intestinal barrier function and immune regulation, consistent with the observation that blood routine indicators remained stable throughout the process and no inflammatory response was observed.

[0154] (3) Polarity-specific regulation of metabolic flow: The predominantly upregulated (78.3%) positive ion mode and predominantly downregulated (78.8%) negative ion mode suggest that Saccharomyces cerevisiae extract may specifically promote the synthesis of alkaline or neutral metabolites (such as carnosine and certain amino acid derivatives) while inhibiting the accumulation of acidic metabolites. This directional regulation of metabolic flow may be related to specific bacterial groups (such as Bifidobacteria, etc.). Turicibacter This is related to the optimization of metabolic pathways.

[0155] In summary, Saccharomyces cerevisiae extract, through a two-layer regulatory model of "microbial community structure fine-tuning - metabolic function remodeling", can directionally activate metabolic pathways related to muscle protection, bone health and intestinal barrier function without drastically disturbing the gut microbiota homeostasis. This precise functional regulation may be one of the important mechanisms by which it exerts its anti-aging effects.

[0156] 2.3.7 Detection of relative telomere length in dogs by feeding them with Nucleovive, a yeast extract. This study evaluated the effect of *Saccharomyces cerevisiae* extract (Nucleovive) on the relative telomere length of aged beagle dogs by feeding them. qPCR analysis of peripheral blood samples at D0 and D90 revealed that the baseline relative telomere length of middle-aged dogs (control group 211.77±28.67) was significantly lower than that of young dogs (1073.17±165.09), consistent with an aging model. P<0.001); After 90 days of intervention, the relative telomere length in the experimental group (284.26±51.09) was significantly higher than that in the control group (199.23±27.82). P <0.001)( Figure 18 This result suggests that the Saccharomyces cerevisiae extract Nucleovive may assist older beagle dogs in producing an anti-aging effect by delaying the shortening of the relative length of telomeres.

[0157] 2.3.8 Results of Methylation Gene Sequencing Analysis 2.3.8.1 Overview of DMR Gene Statistics This study evaluated the effect of feeding Nucleovive, a yeast extract, on improving the aging process of older beagle dogs. Whole-genome bisulfite sequencing (WGBS) was used to analyze changes in differentially methylated regions (DMRs) of peripheral blood cells after formula intervention, revealing the anti-aging mechanism of the formula from an epigenetic perspective.

[0158] The overall statistical results are as follows (Table 6 and Figure 19 There are a total of 3,820 DMR-related genes, of which 2,172 (56.9%) are hypermethylated genes (Hyper-silencing trend), 2,238 (58.6%) are hypomethylated genes (Hypo-activation trend), and 1,624 (42.5%) are DMR genes in promoter regions.

[0159] Table 6. Distribution of Sequence Context

[0160] 2.3.8.2 Analysis of key aging-related genes and key aging pathways (1) Senescence-related genes in the promoter region DMR (58 genes), with key genes including (Table 7): IGF1R (Insulin-like growth factor 1 receptor): Regulates growth and metabolism; FOXO4 (Forkhead box protein O4): A key transcription factor in the longevity pathway; MTOR (Target of Rapamycin): A center for cell growth and metabolic regulation; SIRT3 (Deacetylase 3): Regulation of mitochondrial function; NDUFS7 (NADH dehydrogenase): Mitochondrial respiratory chain complex I; DDB1 (DNA damage binding protein 1): DNA damage repair.

[0161] Table 7 Summary of methylation status of key genes

[0162] 2. The key pathways are as follows: The results are as follows Figure 20 As shown: IGF-1 / insulin signaling pathway: This pathway is one of the core pathways regulating aging. IGF1R It appears simultaneously in promoter DMR and hypermethylation, and may be subject to dual regulation. Its expression may be downregulated, and reduced IGF-1 signaling is associated with longevity. FOXO4 Located in the promoter region DMR, FOXO transcription factors are key targets for longevity research, regulating oxidative stress resistance and cell cycle.

[0163] Mitochondrial function-related genes: Mitochondrial dysfunction is one of the hallmarks of aging. NDUFS7 Located in the promoter DMR and exhibiting hypomethylation, it is a core subunit of mitochondrial complex I and is crucial for NADH oxidation and ATP synthesis. The MRPL family ( MRPL14 , MRPL21 , MRPL33 (etc.) are mitochondrial ribosomal large subunit proteins that regulate the synthesis of proteins encoded by mitochondrial DNA.

[0164] DNA repair-related genes: DNA damage accumulation is an important mechanism of aging. DDB1 Located in the promoter DMR and hypomethylated, it is a damage-specific DNA-binding protein that participates in nucleotide excision repair.

[0165] 2.3.9 Results of plasma proteomics analysis 2.3.9.1 Overview of Statistical Significance This invention utilizes peripheral blood plasma proteomics to evaluate the effects of *Saccharomyces cerevisiae* extract *Nucleovive* on protein expression in middle-aged and aging dogs (D90 experimental group) and a control group (D90 control group). A total of 1,919 plasma proteins were analyzed, with nine biological replicates in each group.

[0166] Differential analysis showed that 66 proteins (37 upregulated and 29 downregulated) were differentially expressed. P <0.05%, accounting for 3.44% of the total detected proteins. Among them, highly significant differentially expressed proteins ( P <0.001) 1 protein, highly significantly different ( P <0.01) 14, significantly different proteins ( P <0.05) 51 proteins. Looking at the overall regulatory trend, upregulated proteins (37) slightly outnumbered downregulated proteins (29), suggesting that the *Saccharomyces cerevisiae* extract Nucleovive may primarily activate specific biological pathways rather than exert inhibitory interventions (Table 8 and...). Figures 21-22 This mild protein remodeling, consistent with the stability of complete blood count indicators, suggests that the intervention did not trigger a severe systemic stress response.

[0167] Table 8 P Value distribution statistics

[0168] 2.3.9.2 Analysis of Differentially Expressed Proteins and Their Biological Functions according to P The top 10 differentially expressed proteins by value are shown in Table 9. These proteins are mainly involved in key biological processes such as lipid transport, endometrial system transport, protein repair, and cell adhesion.

[0169] Key differentially expressed protein functions: (1) Regulation of lipid metabolism (MTTP downregulation): Microsomal triglyceride transfer protein (MTTP) was significantly downregulated by approximately 2.9-fold in the experimental group. MTTP is a key enzyme in the assembly and secretion of very low-density lipoprotein (VLDL), and its downregulation may reflect reduced hepatic lipid output, forming a synergistic effect with the decreased ALT and AST levels (improved liver function) in the experimental group. This "throttling" pattern of lipid metabolism may be related to the antioxidant components (such as glutathione precursors) in Nucleovive, a Saccharomyces cerevisiae extract, which alleviate oxidative stress in hepatocytes and reduce lipid peroxidation damage.

[0170] (2) Protein homeostasis maintenance (PCMT1 upregulation): Protein-L-ispartate methyltransferase (PCMT1) was upregulated approximately 1.3-fold in the experimental group. PCMT1 is an important intracellular "molecular repairer" that can recognize and repair proteins damaged by ispartate, preventing abnormal protein aggregation. In middle-aged and aging dogs, the rate of protein damage increases due to accumulated oxidative stress. The upregulation of PCMT1 suggests that the Saccharomyces cerevisiae extract Nucleovive may activate the cell's protein quality control system and reduce age-related protein toxicity stress. This, together with the hypomethylation state of the DDB1 (DNA damage repair) gene in the WGBS results, constitutes a "nucleic acid-protein" bilayer damage repair network.

[0171] (3) Endometrial system and vesicle transport (LMAN2 upregulated): Lectin-manose-binding 2 (LMAN2) was upregulated by approximately 1.7-fold. LMAN2, located in the Golgi-Endoplasmic Reticulum Intermediate Compartment (ERGIC), is responsible for glycoprotein sorting and vesicle transport. Its upregulation may reflect enhanced function of the secretory pathway, promoting the processing and secretion of glycoproteins such as immunoglobulins (IgA, IgG), which is consistent with the stable immunoglobulin levels and absence of age-related immune decline in the experimental group.

[0172] (4) Cell adhesion and tissue integrity (PCDH1 upregulation): Protocadherin 1 (PCDH1) was significantly upregulated by approximately 3.3-fold. PCDH1 belongs to the cadherin superfamily and is expressed in epidermal cells and vascular endothelial cells, regulating intercellular adhesion junctions. Decreased skin barrier function and increased vascular permeability are common in middle-aged and aging dogs; the upregulation of PCDH1 may help maintain the integrity of the skin and vascular endothelium, consistent with improved BCS scores and well-maintained body condition.

[0173] Table 9 Top 10 Differentially Expressed Proteins

[0174] 2.3.9.3 Biological Explanation and Multi-omics Linkage (1) Mild protein differential expression: After strict FDR correction, although no proteins reached statistically significant differences at the methylation genomic level, 66 nominally significant ( P Proteins with a concentration <0.05 g / mL exhibit high functional enrichment (lipid metabolism, protein repair, vesicle transport), suggesting that the effect of Nucleovive, a Saccharomyces cerevisiae extract, on the plasma proteome is selective rather than broad. This "precision nutrition" approach helps avoid systemic metabolic disturbances.

[0175] (2) Activation of anti-aging related pathways: Differential protein functional clustering showed that upregulated proteins were mainly concentrated in "protein repair" (PCMT1), "vesicle transport" (LMAN2) and "cell adhesion" (PCDH1), while downregulated proteins were mainly involved in "lipid export" (MTTP). This protein expression profile of "enhanced repair, reduced damage, and optimized structure" forms a multi-omics evidence chain with epigenetic results (SIRT3, DDB1 hypomethylation) and phenotypic indicators (increased bone density, improved liver function), which all point to the anti-aging mechanism of maintaining cell homeostasis and reducing damage accumulation.

[0176] (3) Effect size and clinical relevance: The effect size of differentially expressed proteins was mostly between 1.4 and 3-fold (log2FC 0.4-3.0), which is considered a moderate biological effect. Considering that the metabolic rate of middle-aged and older dogs is slower than that of younger dogs, this mild and continuous adjustment of protein expression is more in line with physiological adaptive changes than with pharmacological interventions, which is consistent with the safety data that no adverse reactions were observed during the 12-week intervention period.

[0177] (4) Correlation analysis with methylation and blood indicators Synergistic effect of protein and DNA repair: The upregulation of PCMT1 (protein repair) corresponds to the hypomethylation state of the DDB1 (DNA damage repair gene) promoter, indicating that the Saccharomyces cerevisiae extract Nucleovive may simultaneously enhance genomic and proteomic stability, forming comprehensive maintenance at the "central dogma" level. Improved lipid metabolism and liver function: Downregulation of MTTP and a 22.3% reduction in ALT (… P The correlation between LMAN2 and vesicle transport and immune homeostasis (<0.05) suggests a reduced hepatic lipid load, which may be related to improved hepatocyte membrane fluidity and reduced oxidative stress. Upregulation of LMAN2 was associated with stable blood routine tests (no inflammatory response) and maintained immunoglobulin levels, suggesting that optimized secretion pathways may support mucosal immune barrier function.

[0178] (5) Potential reasons for no significant differences in proteins after FDR correction At the statistical level, the sample size of 9 samples per group may lead to insufficient test power, making it difficult to capture small to moderate effect sizes of protein expression changes. In addition, the large metabolic variability among middle-aged and aged dogs further reduces statistical sensitivity. In terms of time, a 90-day intervention may only be in the early stage of proteome remodeling, and more significant differences may require long-term intervention of 6 months or more to be fully manifested. Furthermore, tissue-specific limitations cannot be ignored. Plasma proteome can only reflect the average level of systemic secreted proteins, while the intracellular protein regulatory effect of Saccharomyces cerevisiae extract Nucleovive on target organs such as liver and skeletal muscle may be more significant in situ in tissues, but cannot be fully reflected by peripheral blood samples.

[0179] Comparative Example 1 Comparative Example 1-1 was set up with reference to Example 1. The difference from Example 1 is that the Saccharomyces cerevisiae was replaced with Saccharomyces cerevisiae strain (CICC: 30225), and then the RNA content and total nucleotide content were detected.

[0180] RNA content detection method: perchloric acid lysis + ultraviolet spectrophotometry, the operation steps are as follows: Take a certain amount of yeast slurry (about 1 mL) into a 10 mL graduated centrifuge tube, add a certain amount of distilled water and stir well. After equilibration, place the tubes symmetrically in a centrifuge and centrifuge at 2500 rpm for 10 minutes. Discard the supernatant, dilute the precipitated white yeast with water to the 10 mL mark, and mechanically stir well. Take 2 mL of the bacterial solution and 2 mL of 1N perchloric acid into a 10 mL graduated test tube and shake well. Hydrolyze in a 70℃ water bath for 20 minutes, shaking constantly during hydrolysis. Transfer the remaining 8 mL of bacterial solution to a pre-weighed petri dish with distilled water and place it in a far-infrared oven at 120℃ until constant weight. Remove and cool to room temperature in a desiccator, then weigh (W). After hydrolysis is complete, centrifuge at 2500 rpm for 10 minutes. Accurately pipette 0.2 mL of the supernatant into a 10 mL graduated test tube (depending on the concentration), dilute with distilled water to the 10 mL mark, and shake well. Using 0.01N perchloric acid as a blank control, the absorbance A at a wavelength of 260nm was measured in a 1cm quartz cuvette.

[0181] Result calculation:

[0182] In the formula: 32—1mg of nucleic acid is equivalent to 32OD value.

[0183] W—Weight of the remaining 8 mL of bacterial culture after drying to constant weight, in mg.

[0184] Total nucleotide content: Detected by liquid chromatography, the detection method is in accordance with GB / T20886.2.

[0185] Comparative Examples 1-2 were set up with reference to Example 1. The difference from Example 1 was that the Saccharomyces cerevisiae was replaced with Candida albicans strain (CICC: 31188), and then the RNA content and total nucleotide content were detected.

[0186] The experimental results are as follows: Example 1: The yeast syrup had an RNA content of 20.3% and a total nucleotide content of 35.2% in the finished product. It had a weak yeast odor and a meaty aroma.

[0187] Comparative Example 1-1: Using a common brewer's yeast strain: the yeast syrup RNA content was 8.2%, the total nucleotide content of the finished product was 12.2%, and it had a distinct yeast odor.

[0188] Comparative Examples 1-2: Using a high-nucleic acid Candida strain: the yeast syrup RNA content was 13.2%, the total nucleotide content of the finished product was 21.6%, and it had a pungent yeast odor.

[0189] Comparative Example 2 Comparative Example 2-1 was set up with reference to Example 1, except that it was only continuously fermented (without batch fermentation).

[0190] Comparative Example 2-2 was set up with reference to Example 1, except that only batch fermentation was performed (continuous fermentation was not carried out).

[0191] Example 1: The yeast extract contained 20.3% RNA, the finished product contained 35.2% total nucleotides, and 4.3% glutathione.

[0192] Comparative Example 2-1: The RNA content of yeast slurry was 21.2%, the total nucleotide content of the finished product was 34.2%, and the glutathione content was 0.2%.

[0193] Comparative Example 2-2: The yeast extract contained 9.2% RNA, the finished product contained 13.2% total nucleotides, and 3.2% glutathione.

[0194] Comparative Example 3 Comparative Example 3-1 was set up with reference to Example 1. The difference from Example 1 is that the enzyme hydrolysate was not retained and all of it was separated.

[0195] Example 1: Yeast β-glucan content 4.3%.

[0196] Comparative Example 3-1: Yeast β-glucan content 1.4%.

[0197] 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. A strain of *Saccharomyces cerevisiae*, characterized in that, The strain has the accession number CGMCC No. 38790.

2. A high-nucleotide Saccharomyces cerevisiae extract, characterized in that, The extract was prepared from the Saccharomyces cerevisiae strain described in claim 1.

3. A method for preparing the high-nucleotide Saccharomyces cerevisiae extract of claim 2, characterized in that, Includes the following steps: (a) Cultivate the brewing yeast strain according to claim 1, and ferment it to obtain yeast slurry; (b) Inactivate the yeast slurry; (c) Add nuclease to enzymatically hydrolyze the inactivated yeast slurry, so that the yeast nucleic acid is degraded into nucleotides; (d) The enzymatic hydrolysate is separated, washed, concentrated and dried to obtain the high nucleotide Saccharomyces cerevisiae extract.

4. The method according to claim 3, characterized in that, The fermentation described in step (a) includes: first continuous fermentation, then batch fermentation.

5. The method according to claim 4, characterized in that, The batch fermentation includes the step of adding an amino acid precursor solution to the yeast slurry obtained from continuous fermentation.

6. The method according to claim 5, characterized in that, The amino acid precursor solution is selected from one or more aqueous solutions of L-glutamic acid, L-cysteine, and glycine.

7. The method according to claim 4, characterized in that, The conditions for continuous fermentation include: a ventilation volume of 6000-12000 m³ / h. 3 / h, temperature 20-50℃, pH 3-7; The conditions for the batch fermentation included: an aeration rate of 5000-10000 m³ / h for the first hour. 3 / h, the second hour is 8000-15000 m 3 / h; pH 4.0-6.0; temperature 27-33℃; carbon source flow rate 500-900 kg / h; amino acid precursor solution flow rate 15-182 L / h; total fermentation time 4-10 hours.

8. The method according to claim 3, characterized in that, The inactivation described in step (b) includes: heating the yeast slurry to 55-65°C, then heating it to 85-95°C, and then cooling it to 50-60°C; The nuclease mentioned in step (c) is 5'-phosphodiesterase, and the amount added is 0.3%-1.0% of the yeast slurry mass; The enzymatic hydrolysis conditions described in step (c) are: temperature 58-62℃, pH 4.8-5.5, and time 8-15 hours; The separation and washing described in step (d) includes: retaining 5%-15% of the enzymatic hydrolysate without separation; the ratio of enzymatic hydrolysate to washing water during separation is 1:0.3-1:0.8; and washing the separated heavy phase 1-3 times. The concentration described in step (d) employs triple-effect evaporation, controlling the first-effect temperature at 75-85℃, the second-effect temperature at 65-75℃, and the third-effect temperature at 50-69℃, to concentrate the product to a solids content of 25%-45%. The drying process described in step (d) is spray drying, with an inlet air temperature of 170-185℃ and an outlet air temperature of 70-90℃.

9. The use of the high-nucleotide Saccharomyces cerevisiae extract of claim 2 or the high-nucleotide Saccharomyces cerevisiae extract prepared by any one of claims 3-8 in the preparation of functional feeds, feed additives or drugs for improving the health of aging dogs.

10. A functional feed, feed additive, or drug for improving the health of aging dogs, characterized in that, The extract comprises the high-nucleotide Saccharomyces cerevisiae extract according to claim 2 or the high-nucleotide Saccharomyces cerevisiae extract prepared by any one of claims 3-9.

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