Bifidobacterium adolescentis YF-01 and application thereof in fermented milk

Abstract

The application relates to the field of biological medicine, and discloses a bifidobacterium adolescentis YF-01 and application of the bifidobacterium adolescentis YF-01 in fermented milk. The bifidobacterium adolescentis YF-01 is isolated from adult feces and is preserved in the China Center for Type Culture Collection, with a preservation number of CCTCC NO: M 20251308. Experiments prove that the bifidobacterium adolescentis YF-01 has good gastrointestinal fluid tolerance, the survival rate can reach 90.1% after being treated by simulated gastric juice for 3 hours, and the survival rate can reach 72% after being treated by simulated intestinal juice for 4 hours. The self-aggregation rate of the strain is 88.83%, and the hydrophobicity is 79.41%; the strain has significant scavenging capacity for DPPH free radicals and hydroxyl free radicals, and shows good antioxidant activity and adhesion capacity, which provides a theoretical basis for the application of the strain in fermented milk. The strain and metabolites thereof can be used for preparing food, medicine or health care products with the functions of regulating intestinal flora and enhancing antioxidant capacity, and have significant application potential in the development of functional fermented milk products.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a strain of Bifidobacterium adolescentis YF-01 and its application in fermented milk. Background Technology

[0002] Probiotics have important physiological functions for the body, such as regulating the balance of intestinal flora, lowering cholesterol, anti-oxidation, anti-infection, and enhancing human immunity. They have broad application prospects in the food and medical fields.

[0003] Bifidobacteria, as dominant microorganisms in the gastrointestinal tract, play an irreplaceable role in maintaining intestinal health. In recent years, various clinical, in vivo, and in vitro studies have demonstrated that Bifidobacteria can play an important role in diseases such as inflammatory bowel disease, irritable bowel syndrome, cancer, diarrhea, and lactose intolerance, protecting the integrity of the intestinal barrier and thus maintaining a healthy and stable state of the body.

[0004] Fermented milk is rich in protein, calcium, and various nutrients. After fermentation by lactic acid bacteria, proteins are broken down into peptides and amino acids, and lactose is hydrolyzed into galactose and glucose, which are then converted into lactic acid. The resulting fine curds are easier for the gastrointestinal tract to digest and absorb, making it suitable for people with lactose intolerance. Furthermore, fermented milk performs well in regulating gut microbiota and preventing and treating diabetes, cardiovascular diseases, and liver diseases, making it a popular functional food.

[0005] Bifidobacterium adolescentis, a dominant bacterium in the healthy human gut, possesses a variety of physiological functions. It can specifically induce Th17 cells in the gut, regulate immunity without causing inflammation; supplementation with Bifidobacterium adolescentis can alleviate post-fracture sequelae, enhance intestinal barrier function, inhibit systemic inflammatory responses, and accelerate fracture healing; its metabolites and antibacterial substances can antagonize pathogenic bacteria and promote the restoration of the intestinal microecology. Furthermore, Bifidobacterium adolescentis can treat chronic diarrhea and constipation, has anti-aging effects, regulates intestinal flora, and alleviate metabolic diseases such as non-alcoholic fatty liver disease, metabolic syndrome, and type 2 diabetes. However, Bifidobacterium adolescentis has poor oxygen tolerance, is difficult to cultivate, is intolerant to high temperatures, and is easily contaminated, factors that limit its industrial development. Currently, the key technical direction is to domesticate the strain to adapt to different physicochemical environments, protect the bacterial cells to ensure a sufficient number of viable bacteria, and develop products that meet consumer tastes and nutritional needs. The application of Bifidobacterium adolescentis in fermented milk is expected to further enhance the nutritional value and functional characteristics of fermented milk, meeting consumers' demand for healthy foods. Summary of the Invention

[0006] The purpose of this invention is to provide a strain of Bifidobacterium adolescentis YF-01 and its application in fermented milk. This strain has excellent characteristics such as acid and bile salt tolerance and the ability to utilize a variety of carbohydrates. By compounding it with commercial starter cultures for the preparation of fermented milk, the viable cell count, texture, sensory quality and storage stability of fermented milk can be improved.

[0007] To achieve the above objectives, this invention provides the application of a strain of Bifidobacterium adolescentis YF-01 in the preparation of products that regulate intestinal flora, promote intestinal health, or enhance the body's antioxidant capacity. The Bifidobacterium adolescentis YF-01 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20251308, deposit date June 9, 2025, deposit address Wuhan University, Wuhan, China, and classified as Bifidobacterium adolescentis YF-01. Its 16S rDNA sequence is shown in SEQ ID NO.1.

[0008] Furthermore, the product includes fermented milk.

[0009] Furthermore, when applied, Bifidobacterium adolescentis YF-01 is combined with commercial starter culture PYS-010. When applied to fermented milk, it can shorten the fermentation time, improve the texture and flavor of fermented milk, and maintain a high number of viable bacteria and stable properties during storage.

[0010] Furthermore, when Bifidobacterium adolescentis YF-01 is combined with the commercial starter culture PYS-010, the inoculum size of Bifidobacterium adolescentis YF-01 is 1×10⁻⁶. 6 CFU / mL, the inoculum size of commercial fermentation agent PYS-010 is 0.03‰.

[0011] Furthermore, the present invention also provides a product containing live bacteria, inactivated bacteria, and / or metabolites of the aforementioned Bifidobacterium adolescentis YF-01, wherein the number of live Bifidobacterium adolescentis YF-01 in the product is not less than 1 × 10⁻⁶. 8 CFU / mL or 1×10 8 CFU / g.

[0012] Furthermore, the aforementioned products are fermented milk, dairy products, or solid beverages.

[0013] Furthermore, the above-mentioned products have the functions of regulating intestinal flora, promoting intestinal health, or enhancing the body's antioxidant capacity.

[0014] Furthermore, this invention also provides the application of secondary metabolites of Bifidobacterium adolescentis YF-01 in the preparation of products that regulate intestinal flora, promote intestinal health, or enhance the body's antioxidant capacity. The secondary metabolites include lucidenolactone, ampeloside Bs1, and lucidenic acid F. A strain of Bifidobacterium adolescentis YF-01 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20251308, deposited on June 9, 2025, at Wuhan University, Wuhan, China, and classified as Bifidobacterium adolescentis YF-01. Its 16S rDNA sequence is shown in SEQ ID NO.1.

[0015] Furthermore, during application, the viable count of Bifidobacterium adolescentis YF-01 should not be less than 1×10⁻⁶. 8 CFU / mL or 1×10 8 CFU / g.

[0016] Furthermore, the present invention also provides a product for regulating intestinal flora, promoting intestinal health, or enhancing the body's antioxidant capacity, comprising the secondary metabolites of the aforementioned Bifidobacterium adolescentis YF-01, wherein the product is a capsule, tablet, dry powder, food, or beverage.

[0017] Therefore, the Bifidobacterium adolescentis strain YF-01 described in this invention and its application in fermented milk have the following beneficial effects:

[0018] This invention provides a strain of Bifidobacterium adolescentis YF-01, whose preservation number is CCTCCNO: M 20251308. Experiments have shown that Bifidobacterium adolescentis YF-01 exhibits good tolerance to simulated gastrointestinal fluids. When combined with commercial starter cultures for fermentation, it can shorten fermentation time and improve the quality of fermented milk. During storage, the fermented milk exhibits suitable acidity, good water-holding capacity and viscosity, excellent textural properties, and a high viable count. Secondary metabolites include lucidenolactone, ampeloside Bs1, and lucidenic acid F, which can regulate bodily health. Its excellent performance during fermentation and its positive regulatory effects on bodily health make it highly promising for the preparation of food or pharmaceutical products that regulate intestinal flora, promote intestinal health, and / or enhance the body's antioxidant capacity. In addition, Bifidobacterium adolescentis YF-01 is a beneficial intestinal bacterium and is listed in the list of bacteria that can be used in food. Therefore, Bifidobacterium adolescentis YF-01 and products containing Bifidobacterium adolescentis YF-01 as their active ingredient have the advantage of food safety and are beneficial to human health with long-term use.

[0019] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0020] Figure 1 Phylogenetic tree of Bifidobacterium adolescentis YF-01;

[0021] Figure 2 Gram staining image of Bifidobacterium adolescentis YF-01;

[0022] Figure 3 In diagram A, we see the self-aggregation rate of Bifidobacterium adolescentis YF-01; in diagram B, we see the hydrophobicity of Bifidobacterium adolescentis YF-01.

[0023] Figure 4 In the diagram, A represents the DPPH scavenging rate of Bifidobacterium adolescentis YF-01, and B represents the hydroxyl radical scavenging rate of Bifidobacterium adolescentis YF-01.

[0024] Figure 5 In diagram A, we have a PCA diagram of Bifidobacterium adolescentis YF-01 before and after fermentation, and in diagram B, we have a PLS-DA diagram of Bifidobacterium adolescentis YF-01 before and after fermentation. YN represents before fermentation, and B represents after fermentation.

[0025] Figure 6 A is a metabolic volcano diagram of Bifidobacterium adolescentis YF-01 before and after fermentation (gray represents metabolites with no significant changes, red represents upregulated metabolites, and purple represents downregulated metabolites; the darker the color, the more significant the change). B is a metabolic bubble diagram of Bifidobacterium adolescentis YF-01 before and after fermentation (the size of the dots represents the number of metabolites in the pathway, and the color represents the p-value; the smaller the p-value and the redder the color, the more significant the enrichment).

[0026] Figure 7 In diagram A, we see the metabolic pathways of starch and sucrose before and after fermentation of Bifidobacterium adolescentis YF-01; in diagram B, we see the metabolic pathway of galactose; in diagram C, we see the metabolic pathway of pyruvate; and in diagram D, we see the metabolic pathway of pyruvate.

[0027] Figure 8 In the diagram, A represents the PCA metabolic diagram of Bifidobacterium adolescentis YF-01 commercial fermentation and compound fermentation group, and B represents the PLS-DA metabolic diagram of Bifidobacterium adolescentis YF-01 commercial fermentation and compound fermentation group. Among them, B represents the compound fermentation group of Bifidobacterium adolescentis and commercial fermentation agent, and S represents the commercial fermentation agent group.

[0028] Figure 9A is a metabolic volcano plot of Bifidobacterium adolescentis YF-01 Shangfa and compound groups (gray represents metabolites with no significant changes, red represents upregulated metabolites, and purple represents downregulated metabolites; the darker the color, the more significant the change). B is a metabolic bubble plot of Bifidobacterium adolescentis YF-01 Shangfa and compound groups (the size of the dots represents the number of metabolites in the pathway, and the color represents the p-value; the smaller the p-value and the redder the color, the more significant the enrichment).

[0029] Figure 10 In Figure A, pH changes during the storage of Bifidobacterium adolescentis YF-01 are shown; in Figure B, pH changes during the storage of Bifidobacterium adolescentis YF-01 are shown as titration changes in acidity.

[0030] Figure 11 Changes in the viable count of Bifidobacterium adolescentis YF-01 during storage;

[0031] Figure 12 In Figure A, the change in water-holding capacity of Bifidobacterium adolescentis YF-01 during storage is shown; in Figure B, the change in water-holding capacity and viscosity of Bifidobacterium adolescentis YF-01 during storage is shown.

[0032] Figure 13 Sensory evaluation results of Bifidobacterium adolescentis YF-01 during storage;

[0033] Figure 14 Graphs showing the main components of fermented milk containing Bifidobacterium adolescentis YF-01 before and after storage;

[0034] Figure 15 Thermographic cluster analysis of fermented milk containing Bifidobacterium adolescentis YF-01 before and after storage;

[0035] Figure 16 Metabolic pathway enrichment diagram of differential metabolites of Bifidobacterium adolescentis YF-01 fermented milk before and after storage;

[0036] Figure 17 The TIC chromatograms are for the Bifidobacterium adolescentis YF-01 compound group. A is in anion mode, and B is in cation mode. The blue chromatogram is the total ion chromatogram, and the pink chromatogram is the XIC chromatogram after dynamic background subtraction.

[0037] Information on the preservation of biological materials

[0038] Bifidobacterium adolescentis YF-01 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20251308, deposited on June 9, 2025, at Wuhan University, Wuhan, China, and classified as Bifidobacterium adolescentis YF-01. Detailed Implementation

[0039] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments. Unless otherwise defined, the technical or scientific terms used in this invention should be understood in their ordinary sense by those skilled in the art. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available raw materials.

[0040] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0041] The method for detecting the number of viable bacteria in the following examples is as follows: the national standard GB 4789.35-2016, "National Food Safety Standard for Microbiological Testing of Food - Lactic Acid Bacteria Detection", is adopted.

[0042] A strain of Bifidobacterium adolescentis YF-01 of the present invention was isolated from the feces of volunteers and deposited at the China Center for Type Culture Collection on June 9, 2025, with accession number CCTCC NO: M 20251308, deposited at Wuhan University, Wuhan, China, and classified as Bifidobacterium adolescentis YF-01.

[0043] Example 1: Isolation and identification of Bifidobacterium adolescentis YF-01

[0044] 1.1 Isolation of Bifidobacterium adolescentis YF-01.

[0045] The collected volunteer feces were spread to isolate bacterial strains. First, the samples were serially diluted, and 10 μL of the diluted sample was taken. -5 and 10 -6 200 μL of each dilution gradient sample was evenly spread onto RCM solid medium plates. After even spreading, the plates were placed in an anaerobic digester along with the anaerobic gas-generating bag and transported back to the laboratory. Anaerobic culture was carried out at 37°C for 72–96 h in an A35 anaerobic workstation (80% N2, 10% H2, 10% CO2). Single colonies of different morphology, size, and color were picked and inoculated into liquid medium, and incubated at 37°C for 24–36 h. After the strains showed good growth, Gram staining and microscopic examination were performed. The isolates were preserved, and genomic DNA was extracted for subsequent analysis.

[0046] The RCM medium consists of: 10g peptone, 10g beef extract, 3g yeast extract, 5g glucose, 1g soluble starch, 5g sodium chloride, 3g sodium acetate, 0.5g L-cysteine ​​hydrochloride, 1L distilled water, pH adjusted to approximately 6.8, 15g agar, and sterilized at 121℃ for 15 minutes.

[0047] 1.2 Physiological and biochemical identification, 16S rRNA molecular identification.

[0048] The cryopreserved test strain was inoculated into ML liquid medium and cultured at 37℃ for 24–36 h. After 2–3 passages, 30 mL of the bacterial culture at the end of the logarithmic growth phase was placed in a sterile tube and centrifuged at 8000×g for 5 min (4℃). The bacterial cells were collected, the supernatant was discarded, and the genomic DNA of the strain was extracted using the CTAB freeze-thaw method for lactic acid bacteria.

[0049] The ML liquid culture medium consists of: 10g peptone, 4g yeast extract, 8g beef extract, 20g glucose, 5g anhydrous sodium acetate, 2g triamine citrate, 2g dimethyl hydrogen phosphate, 1mL Tween-80, 0.2g magnesium sulfate heptahydrate, 0.05g manganese sulfate tetrahydrate, 1L distilled water, and 0.5g L-cysteine. The pH is adjusted to approximately 6.2, and the medium is sterilized at 21°C for 15 minutes.

[0050] The isolated strain underwent physiological and biochemical identification and 16S rRNA molecular identification. Identification was performed using the bacterial API50CH assay (bioMérieux) according to Berger's Manual of Bacterial Identification. DNA samples were extracted from the strain, and the samples were subjected to 16S rRNA molecular identification by Shanghai Meiji Biomedical Technology Co., Ltd. The molecular sequence was compared with the NCBI BLASTN database, confirming that *Bifidobacterium adolescentis* YF-01 is indeed *Bifidobacterium adolescentis* YF-01, and its 16S rRNA sequence is shown in SEQ ID NO. 1.

[0051] SEQ ID NO.1

[0052] TGTGGGGGGGGTCTACCATGCAGTCGACGGGATCCCAGGAGCTTGCTCCTGGGTGAGAGTGGCGAACGGGTGAGTAATGCGTGACCGACCTGCCCCATACACCGGAATAGCTCCTGGAAACGGGTGGTAATGCCGGATGCTCCAGTTGACCGCATGGTCCTCTGGGAAAGCTTTTGCGGTATGGGATGGGGTCGCGTCCTATCAGCTTGATGGCGGGGTAACGGCCCACCATGGCTTCGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGCGGGATGACGGCCTTCGGGTTGTAAACCGCTTTTGACTGGGAGCAAGCCCTTCGGGGTGAGTGTACCTTTCGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTATCCGGAATTATTGGGCGTAAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAAGTCCATCGCTTAACGGTGGATCCGCGCCGGGTACGGGCGGGCTTGAGTGCGGTAGGGGAGACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAACACCAATGGCGAAGGCAGGTCTCTGGGCCGTCACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGATGCTGGATGTGGGGACCATTCCACGGTCTCCGTGTCGGAGCCAACGCGTTAAGCATCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGTTCCCGACAGCCGTAGAGATACGGTCTCCCTTCGGGGCGGGTTCACAGGTGGTGCATG。

[0053] 1.3 Colony morphology characteristics.

[0054] The Bifidobacterium adolescentis YF-01 of the present invention forms opaque, milky-white colonies with a diameter of 1-2 mm, irregular edges, and rough texture on RCM solid medium.

[0055] Inside the clean bench, use an inoculation loop to pick up purified Bifidobacterium sludge and prepare a smear. After drying, perform Gram staining. Figure 2 ).

[0056] The Bifidobacterium adolescentis YF-01 of the present invention has the following morphological characteristics: the bacterial cells, when observed under a microscope, are mainly short root-like with rounded ends, and are arranged singly or in clusters.

[0057] 1.4 Optimal growth temperature and pH.

[0058] Bifidobacterium adolescentis YF-01 was activated by inoculating it at 2% (v / v) in ML liquid medium. Temperature gradients were set at 28, 32, 37, 41, and 45℃, with a pH of 6.0 for all conditions. After incubation at these temperatures for 24 hours, the OD value was measured at 600 nm. Three replicates were set for each temperature condition to determine the optimal growth temperature range. A pH gradient of ML medium was also set at 4, 5, 6, 7, 8, and 9. The strain was activated by inoculating it at 2% (v / v) in ML liquid medium. After inoculation, the ML liquid medium was incubated at 37℃ for 24 hours, and the OD value was measured at 600 nm. Three replicates were set for each pH condition to determine the optimal pH for strain growth. The results showed that the optimal growth temperature for Bifidobacterium adolescentis YF-01 was 37℃, and the optimal growth pH was 7.0. Cy5.5-NH2 and GRb1 nanoparticles were mixed at a ratio of 1:10. The mixture was sonicated at room temperature and stirred overnight in the dark. The solution was then transferred to an ultrafiltration tube with a molecular weight cutoff of 3000 Da. After centrifugation at 6000 rpm for 10 minutes, the supernatant was collected for further use.

[0059] 1.5 Carbohydrate utilization.

[0060] Bifidobacterium adolescentis YF-01 was inoculated into ML liquid medium at 2% (v / v), activated and passaged twice under optimal temperature and pH conditions, then centrifuged for 10 min, and the bacterial sludge was collected. The sludge was repeatedly washed with 0.85% sterile physiological saline and centrifuged until no residue remained. The small groove in the culture box was filled with sterile distilled water to create a humid environment. The above bacterial solution was added to sterile physiological saline, shaken well, and compared with a turbidity comparison tube. After the turbidity was consistent, the bacterial solution was inoculated into ampoules containing culture medium. Then, the inoculated culture medium was added to the test strip tube according to the API 50 CHL instructions, and the tube opening was sealed with sterile liquid lime vinegar. Anaerobic culture was carried out at the optimal temperature for 48 h. After culture, the results were observed, and the color change of the test strip was recorded by photograph, and the carbohydrate metabolism capacity of the strain was determined.

[0061] The API 50CHL carbohydrate utilization results of the strain showed that Bifidobacterium adolescentis YF-01 can utilize 18 carbohydrates, namely D-xylose, D-galactose, D-glucose, D-fructose, sorbitol, methyl-α-D-glucopyranoside, amygdalin, salicin, maltose, D-lactose, inulin, D-raffinose, starch, glycogen, D-gentiobiose, and D-thulose.

[0062] 1.6 Gastrointestinal fluid tolerance of Bifidobacterium adolescentis YF-01

[0063] Preparation method of simulated gastrointestinal fluid: After sterilizing PBS, adjust the pH value to 2.5 with 1 mol / L HCl, add 3.0 mg / ml pepsin, and filter sterilize with a 0.22 μm microporous membrane to prepare simulated gastric fluid; After sterilizing PBS, adjust the pH value to 8.0 with 0.1 mol / L NaOH, add 0.1% trypsin and 1.8% ox bile salt, and filter sterilize with a 0.22 μm microporous membrane to prepare simulated pancreatic juice.

[0064] Gastrointestinal fluid tolerance: The isolated and purified bacterial strain was activated and cultured for two generations, centrifuged and washed twice, and the bacterial cells were collected. 0.5 mL of the re-screened bacterial suspension was added to 4.5 mL of pH 2.5 simulated gastric fluid and digested at 37°C for 3 h. Viable cell counts were determined at 0 h and 3 h using the ML solid medium pour method. Then, 0.5 mL of the digested 3 h artificial bacterial gastric fluid was added to 4.5 mL of simulated intestinal fluid and incubated at 37°C. Viable cell counts were determined at 4 h and 8 h using the ML solid medium pour method. Each sample was performed in quadruplicate. Viability = [N1 / N0] × 100%. Where N0 represents the viable cell count at 0 h; N1 represents the viable cell count after simulated digestion for 3 h or 8 h.

[0065] Tolerability: The results of treatment with simulated gastric and intestinal fluids of Bifidobacterium adolescentis YF-01 are shown in Table 1 below:

[0066] Table 1. Survival status of Bifidobacterium adolescentis YF-01 in simulated gastrointestinal digestive fluids.

[0067] Bifidobacterium adolescentis YF-01 90.1 72

[0068] As shown in Table 1, Bifidobacterium adolescentis YF-01 exhibits good tolerance characteristics, with a survival rate of up to 72% after 4 hours of treatment with simulated intestinal fluid. Based on the gastrointestinal fluid tolerance effect of this embodiment, it can be concluded that Bifidobacterium adolescentis YF-01 has good tolerance to gastrointestinal fluid in the intestine and possesses probiotic properties.

[0069] 2.1 Self-aggregation ability of Bifidobacterium adolescentis YF-01.

[0070] Autoagglutination of bacterial strains refers to the phenomenon where bacteria of the same species aggregate to form multicellular clusters. Figure 3 As shown in A, the self-aggregation rate of Bifidobacterium adolescentis YF-01 was higher than that of the other three strains (P<0.05), with a self-aggregation rate of 88.83%, indicating that the tested strain has a strong adhesion ability.

[0071] 2.2 Hydrophobicity of Bifidobacterium adolescentis YF-01.

[0072] The strength of probiotic adhesion is a prerequisite for them to exert their beneficial effects; therefore, determining the initial screening indicators of probiotic adhesion is of great significance for probiotic research. Figure 3 According to the results of the study, Bifidobacterium adolescentis YF-01 had a significantly higher hydrophobicity than the other three strains (P<0.05), with a hydrophobicity of 79.41%, indicating that it had a better intestinal colonization ability.

[0073] 2.3 DPPH free radical scavenging ability of Bifidobacterium adolescentis YF-01.

[0074] DPPH radicals are very stable, synthetically produced nitrogen-centered free radicals. The DPPH method is a very common and effective method for screening and evaluating antioxidant effects. Figure 4 As shown in Figure A, the fermentation supernatant of YF-01 exhibited the highest DPPH scavenging capacity of 60.32%. The DPPH scavenging capacity of the fermentation supernatant of the same strain was significantly higher than that of the cell suspension (P < 0.05).

[0075] 2.4 Hydroxyl radical scavenging ability of Bifidobacterium adolescentis YF-01.

[0076] Hydroxyl radicals are the most reactive and oxidizing free radicals, with a strong binding ability to DNA, proteins, and lipids, and are a major factor causing oxidative damage in the body. Figure 4 The scavenging rate of hydroxyl radicals in the fermentation supernatant of different strains of strain B was significantly lower than that in the positive control VC group (P<0.05). The scavenging rate of hydroxyl radicals by all strains was above 65%, with YF-01 showing the highest scavenging rate at 67.51%. There were significant differences in the scavenging rate of hydroxyl radicals in the cell suspensions of different strains, with the scavenging rate significantly lower than that in the positive VC control group. Among them, the cell suspension of YF-01 showed the highest scavenging capacity for hydroxyl radicals at 66.13%.

[0077] 2.5 Drug susceptibility of Bifidobacterium adolescentis YF-01.

[0078] Antibiotic susceptibility testing is a key focus of probiotic research, used to identify probiotics for different applications. The resistance of YF-01 to 20 antibiotics was tested using the disk diffusion method. Antibiotic susceptibility was assessed by measuring the size of the inhibition zone, following the guidelines of the Clinical and Laboratory Standards Institute (CLSI). The results are shown in Table 2. YF-01 exhibited extremely high susceptibility to 14 antibiotics, including penicillin, ampicillin, and carbenicillin, with clear inhibition zones. It showed resistance to 6 antibiotics, including oxacillin, amikacin, and gentamicin.

[0079] Table 2. Survival status of Bifidobacterium adolescentis YF-01 in simulated gastrointestinal digestive fluids.

[0080] 1 penicillin 31.29±2.07 S 2 Oxyphenidyl 16.84±0.27 R 3 Ampicillin 30.06±2.43 S 4 Carbenicillin 34.06±3.74 S 5 Piperacillin 25.296±0.73 S 6 Cephalexin 22.84±0.82 S 7 cefazolin 26.68±1.00 S 8 Cefadroxil 23.69±1.73 S 9 Cefuroxime 32.95±1.48 S 10 Ceftazidime 24.64±0.85 S 11 Ceftriaxone 37.44±0.97 S 12 Cefoperazone 27.15±3.00 S 13 Amikacin 8.01±0.16 R 14 Gentamicin 8.12±0.54 R 15 Kanamycin 7.07±0.60 R 16 Neomycin 6.83±0.19 R 17 tetracycline 12.27±0.29 R 18 Doxycycline 17.38±0.97 S 19 Minocycline 17.19±1.46 S 20 Erythromycin 36.96±1.22 S

[0081] 2.6 Hemolytic activity of Bifidobacterium adolescentis YF-01.

[0082] The hemolysis test is used to detect whether a bacterial strain produces a toxin that causes the lysis of host red blood cells, and it is one of the evaluation indicators for the in vitro safety of bacterial strains. α-hemolytic colonies form a grass-green hemolytic ring around their colonies, also known as grass-green hemolysis; β-hemolytic colonies form a transparent hemolytic ring around their colonies, also known as complete hemolysis; γ-hemolytic colonies do not form a hemolytic ring, meaning they do not hemolyze. The hemolytic activity evaluation results of YF-01 screened in this experiment showed that after anaerobic incubation at 37℃ for 72 hours on 5 sheep blood agar plates, there was no significant change around the colonies, indicating that the hemolytic type is γ-hemolytic, meaning it does not possess hemolytic activity.

[0083] Example 3: Fermentation characteristics of Bifidobacterium adolescentis YF-01 compound commercial starter

[0084] 3.1 Preparation of fermented milk.

[0085] Bifidobacterium adolescentis YF-01 (a highly tolerant strain) was combined with the commercial starter culture PYS-010 (containing Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus). Fermented milk containing the commercial starter culture (0.03‰) served as the control group (Group S). Different inoculum amounts of Bifidobacterium adolescentis YF-01 combined with the commercial starter culture were set up as fermentation groups (Group A: Bifidobacterium adolescentis YF-01 inoculum 1×10⁻⁶). 5 Group B: CFU / mL + commercial starter culture (0.03‰), Bifidobacterium adolescentis YF-01 inoculum 1×10⁻⁶ 6 CFU / mL + commercial starter culture (0.03‰), Group C: Bifidobacterium adolescentis YF-01 inoculum 1×10 7 CFU / mL + commercial starter culture (0.03‰)), and a single-strain fermentation group of Bifidobacterium adolescentis YF-01 (Group D: Bifidobacterium adolescentis YF-01 inoculum 1×10⁻⁶). 5 (CFU / mL). For fermented milk preparation, commercially available pure milk was preheated to 60-65℃, 6.5% (v / v) white saccharifying agent was added for 15 minutes, homogenized (pressure 20 MPa), sterilized at 95℃ for 30 minutes, and rapidly cooled. The starter culture was inoculated according to the above inoculation amount, and fermentation was carried out at the optimal growth temperature (37℃). Fermentation was stopped when the pH reached 4.5±0.1. The fermented milk was then stored in a 4℃ cold storage, with samples taken and tested every 7 days during storage.

[0086] 3.2 Analysis of the fermentation performance of Bifidobacterium adolescentis YF-01 compound commercial starter culture.

[0087] As shown in Table 3, all five groups of fermented milk formed a stable tissue state after fermentation, but their textural properties differed significantly (P<0.05). Group B had the highest hardness, followed by Group A, and then Group C, indicating that the gel network structure formed by the combination of Groups A, B, and C with commercial starter cultures during fermentation was more robust and stable, resulting in better fermented milk quality. In terms of consistency, cohesion, and viscosity index, Groups B and C had higher values ​​for these parameters. Acid production during fermentation lowered the pH, causing casein particles to rearrange and affecting these parameters. Overall, the hardness, consistency, cohesion, and viscosity index of the compounded groups were significantly better than those of the single-strain group and the control group (P<0.05). Compound group B (1×10⁻⁶) 6 The texture parameters (CFU / mL) are optimal, and the gel network structure is more stable.

[0088] Table 3 Analysis of textural parameters of fermented milk

[0089] A 54.79±1.34a 437.47±2.96c 28.77±3.20b 12.73±0.18c B 56.06±0.39a 456.86±4.05b 29.60±0.18b 15.70±2.68b C 57.35±2.98a 467.08±2.06a 38.59±3.12a 20.38±4.10a D 20.68±2.00c 162.24±1.48e 9.80±2.80c 5.38±0.56e S 47.37±0.98b 421.37±5.8d 27.99±0.90b 8.62±0.21d

[0090] Note: Different uppercase letters indicate significant differences between groups (P<0.05), and different lowercase letters indicate significant differences within groups (P<0.05).

[0091] As shown in Table 4, the tasters' preference for the five groups of fermented milk, from highest to lowest, was B, S, C, A, and D. Compound group B (1×10⁻⁶) 6 The sensory score (86.63±1.11) of the CFU / mL + commercial starter culture (0.03‰) group was higher than that of the control group (84.49±1.25) and the single-strain group (72.32±0.81). Its fermented lactic acid-sweet ratio was appropriate, the texture was uniform, the consistency was delicate and silky, there was no graininess, and the flavor was superior.

[0092] The fermentation times of the five groups of fermented milk differed significantly (P<0.05). The compound group C had the shortest fermentation time, followed by groups B and S, with group D having the longest. Excessive fermentation time may lead to contamination by other microorganisms. Overall, the compound group's shorter fermentation time was significantly better than the control group (6.8 hours) and the single-strain group (24 hours). Therefore, single-strain fermented milk is not practically applicable for industrial production and should be further studied in combination with commercial starter cultures.

[0093] Table 4 Fermentation Time and Sensory Scores

[0094] A <![CDATA[80.34±0.65 e ]]> <![CDATA[6.5±0.02 c ]]> B <![CDATA[86.63±1.11 a ]]> <![CDATA[6.5+0.02 c ]]> C <![CDATA[82.49±1.20 d ]]> <![CDATA[6.0±0.01 d ]]> D <![CDATA[72.32±0.81 f ]]> <![CDATA[24.0±0.01 a ]]> S <![CDATA[84.49±1.25 c ]]> <![CDATA[6.8±0.02 b ]]>

[0095] Note: Different capital letters indicate significant differences between groups (P<0.05), and different lowercase letters indicate significant differences within groups (P<0.05).

[0096] 3.3 Analysis of metabolites before and after fermentation.

[0097] The experiment used a combination of principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) to determine the differences between metabolites before and after fermentation, such as... Figure 5 As shown in the figure, the contribution rates of the first and second principal components of PCA were 64.60% and 14.30%, respectively, with a total contribution rate of 78.90%, indicating that principal component analysis can describe the overall situation of the sample. The contribution rates of the first and second principal components of PLS-DA were 63.60% and 12.90%, respectively, with a total contribution rate of 76.50%. There was a clear separation between the two groups of samples, and the samples clustered between groups, indicating a significant difference in metabolites before and after fermentation. The volcano plot shows that... Figure 6 In the first phase (A), a total of 731 differentially regulated metabolites were generated before and after fermentation (P<0.05, VIP>1), including 211 upregulated metabolites and 21 downregulated metabolites. Acetaldehyde, L-lactic acid, and other substances were significantly upregulated after fermentation.

[0098] Acetaldehyde is an important flavor compound produced by lactic acid bacteria in fermented milk. During lactic acid bacteria fermentation, the bacteria break down lactose, amino acids, and other components in the milk to produce acetaldehyde. This substance has a unique pungent odor and a refreshing sour aroma, making it one of the key flavor markers that distinguishes fermented milk from raw milk. Studies have shown that the level of acetaldehyde is directly related to the intensity of the "typical sour milk flavor" of fermented milk. An appropriate amount of acetaldehyde can bring a fresh and lively aroma to the product, enhancing the consumer's taste experience. In addition, acetaldehyde may also have a synergistic effect with other volatile compounds in the fermented milk system, collectively forming a complex flavor profile.

[0099] L-Lactic acid is the main organic acid product of lactic acid bacteria metabolism in fermented milk. Firstly, in terms of acidity regulation, the accumulation of L-lactic acid lowers the pH of the milk system, causing casein to coagulate moderately, forming the curd structure unique to fermented milk and giving the product a smooth and delicate texture. Secondly, in terms of flavor contribution, L-lactic acid, as an organic acid, provides a mild sour taste and works synergistically with other flavor substances (such as acetaldehyde and diacetyl) to balance the sweet and sour taste of the product and enhance its richness. Furthermore, L-lactic acid has antibacterial properties, inhibiting the growth of pathogenic and putrefactive bacteria in the intestines, helping to maintain the microbial stability of fermented milk, and may also have beneficial effects on the human gut microbiota, such as promoting the colonization of probiotics and regulating the intestinal acid-base environment. From a nutritional perspective, L-lactic acid, as an isomer that can be directly utilized by the human body, can be converted into energy or participate in gluconeogenesis during metabolism, possessing certain nutritional value.

[0100] By annotating differential metabolites before and after fermentation using KEGG, 13 metabolic pathways were identified, and further enrichment analysis was performed on these pathways. Figure 6 (B) Based on p-values, the enrichment of starch and sucrose metabolism, galactose metabolism, and pyruvate metabolism was most significant, while the number of metabolites from galactose metabolism and glutathione metabolism was the highest. Figure 7 ).

[0101] Example 4: Metabolic characteristics of fermented milk formulated with Bifidobacterium adolescentis YF-01 and commercial starter

[0102] 4.1 Comparative analysis of metabolites between the compound group and the commercial group.

[0103] The experiment used a combination of principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) to determine the differences in metabolites between commercial starter cultures and compound cultures. Figure 8The contribution rates of the first and second principal components of PCA were 56.70% and 22.90%, respectively, with a total contribution rate of 79.60%, indicating that principal component analysis can describe the overall situation of the sample. The contribution rates of the first and second principal components of PLS-DA were 51.70% and 16.00%, respectively, with a total contribution rate of 67.70%. There was a clear separation between the two groups of samples, and the samples clustered between groups, indicating a significant difference in metabolites before and after fermentation. The volcano plot shows... Figure 9 In the middle A), a total of 1514 differential metabolites were generated before and after fermentation (P<0.05, VIP>1), including 14 upregulated metabolites and 11 downregulated metabolites.

[0104] Among the metabolites specific to fermented milk using compound starter cultures are lucidenolactone, ampeloside Bs1, and lucidenic acid F. The fermentation broth containing these metabolites was separated and identified using an ultra-high performance liquid chromatography (UHPLC) system with a Waters ACQUITYUPLCBEHAmide column. Retention times were as follows: Figure 17 As shown, in anion exchange mode, spirololactone and amperoside Bs1 were detected at a retention time of 8.19 min, exhibiting a co-elution peak (a single peak at the same retention time) in the TIC chromatogram. The abundance of this peak was approximately 3.0e7–3.5e7, suggesting that the two substances may have eluted simultaneously due to similar polarity or consistent retention behavior under chromatographic conditions. At a retention time of 8.34 min, ganoderic acid F was detected, corresponding to an independent peak with an abundance of approximately 3.5e7–4.0e7 in the TIC chromatogram. The peak shape was sharp, and the retention time matched the expected value of the standard, indicating that this substance can be effectively ionized and separated in anion exchange mode. In cationic mode, spirololactone and amperoside Bs1 were detected at a retention time of 8.19 minutes, with a co-elution peak abundance of approximately 6.0e7–6.5e7, which is higher than that in anionic mode. These two substances may have stronger ionization efficiency in cationic mode. At a retention time of 8.34 minutes, ganoderic acid F was detected, with a corresponding peak abundance of approximately 8.0e7–8.5e7. It is one of the high abundance peaks in the TIC spectrum in cationic mode, and its retention time is consistent with that in anionic mode, indicating that it has good detection response in both positive and negative ion modes.

[0105] Lactobacillus lactone is a terpene lactone compound with antioxidant, anti-inflammatory, and metabolic regulating functions. Its antioxidant properties can scavenge free radicals in the intestine and reduce oxidative stress damage; its anti-inflammatory effects may alleviate intestinal inflammation by inhibiting inflammatory pathways such as NF-κB; in addition, terpenoids may also regulate lipid metabolism and help improve metabolic syndrome. Upregulation of this substance may enhance the health benefits of fermented milk, making it especially suitable for people with antioxidant needs.

[0106] Amprolium Bs1 belongs to the flavonoid glycoside class of compounds and possesses antibacterial, antiviral, and angiogenic protective effects. Flavonoid glycosides can exert a prebiotic effect by inhibiting the adhesion of pathogenic bacteria in the gut and regulating the balance of gut microbiota. They may also protect the intestinal mucosal barrier through antioxidant and anti-inflammatory mechanisms. In fermented milk, they may synergistically enhance antibacterial activity with milk proteins and organic acids, especially showing potential preventive effects against bacterial diarrhea or intestinal infections.

[0107] Ganoderma lucidum acid F is a triterpenoid compound of the ganoderic acid class, possessing hepatoprotective, antitumor, and immunomodulatory functions. Triterpenoid compounds can alleviate liver damage by activating liver detoxification enzymes (such as glutathione S-transferase) and enhance the body's immunity by regulating immune cells (such as enhancing the phagocytic capacity of macrophages). In fermented milk, it may have an auxiliary conditioning effect on people with alcoholic liver damage or weakened immunity, while also endowing fermented milk with the functional characteristics of "food and medicine from the same source".

[0108] By annotating differential metabolites before and after fermentation using KEGG, a total of 61 metabolic pathways were identified. Further enrichment analysis was performed on these pathways before and after fermentation. Figure 9 (B) Based on the p-value, the enrichment of arachidonic acid metabolism, steroid biosynthesis, starch and sucrose metabolism was most significant, while nitrogen metabolism, arginine biosynthesis, and arachidonic acid metabolism had the largest number of metabolites.

[0109] Example 5: Storage characteristics of Bifidobacterium adolescentis YF-01 compound commercial starter in fermented milk

[0110] 5.1 Analysis of the storage quality of fermented milk formulated with Bifidobacterium adolescentis YF-01.

[0111] like Figure 10 As shown, during the 28-day storage period, the pH of the compound groups A, B, and C, as well as the control group S, generally showed a decreasing trend. Group C showed a significant difference from groups A, B, and S after 28 days of storage (P<0.05), likely due to the highest addition of *Bifidobacterium adolescentis*, resulting in greater acid production and a faster pH decrease. The titratable acidity of all four groups generally showed an increasing trend. After 28 days of storage, the titratable acidity of the compound groups was significantly higher than that of the control group (P<0.05), with group C showing the highest. Optimal production results were achieved by controlling the fermentation acidity between 70°T and 110°T. The titratable acidity of groups A, B, and S fell within this range, while group C showed a faster increase in titratable acidity in the later stages of storage and was higher at the end of storage. Therefore, *Bifidobacterium adolescentis* and commercial starter cultures can continue to decompose lactose and produce lactic acid at 4°C.

[0112] like Figure 11As shown, during storage at 4℃, the viable bacterial count of fermented milk first increased and then decreased, reaching its maximum at 14 days of storage, after which it showed a decreasing trend (P<0.05). This is because *Bifidobacterium adolescentis* is a strict anaerobic bacterium. At the end of storage, the viable bacterial counts of compound groups A, B, and C were significantly higher than those of the control group S (P<0.05), and the viable bacterial count of the compound groups remained at 10 even after 28 days of storage. 8 CFU / mL or higher, meeting the standards for probiotic products (>10). 6 (CFU / mL) can play a positive role in health.

[0113] like Figure 12 As shown, the water-holding capacity and viscosity of the four fermented milk groups first increased and then decreased over time. The water-holding capacity and viscosity of the compound group reached their peak after 14 days of storage, significantly higher than those of the control group (P<0.05). This indicates that the compound Bifidobacterium adolescentis YF-01 can improve the water-holding capacity and viscosity of fermented milk and stabilize them during storage, thus helping to improve the texture of the fermented milk. Furthermore, the viscosity and water-holding capacity of group B were significantly higher than those of the other groups; therefore, the inoculum size was 1×10⁻⁶. 6 Fermentation with CFU / ml Bifidobacterium adolescentis YF-01 in combination with commercial starter culture can stabilize the viscosity and water-holding capacity of fermented milk and improve its storage stability.

[0114] As shown in Table 5, the hardness, consistency, cohesiveness, and viscosity index of the four groups of fermented milk all increased first and then decreased during storage, reaching a peak at 14 days of storage, consistent with the trend of water-holding capacity and viscosity. The decrease in pH value in the early stage of storage increased the gel strength of the fermented milk, improving its textural characteristics; the later post-acidification phenomenon destroyed the gel structure, leading to a decrease in textural characteristics. Throughout the storage period, the hardness, consistency, cohesiveness, and viscosity index of groups B and C in the compound fermentation experiment were significantly higher than those of group S (P<0.05). The textural characteristic values ​​of group B fermented milk were higher than those of the other groups, indicating that an inoculum size of 1×10⁻⁶ was optimal. 6 The combination of CFU / ml YF-01 and the starter culture helps to form a stronger gel network structure, improving the stability of fermented milk.

[0115] Table 5 Texture parameters of fermented milk during storage

[0116]

[0117] like Figure 13As shown, the sensory evaluation scores of the four groups of fermented milk samples during storage indicated that group B had the highest score, followed by group S, and group C had the lowest. The highest sensory scores were achieved on day 14 of storage. Subsequently, with prolonged storage, issues such as increased acidity, whey separation, decreased viscosity, and reduced water-holding capacity led to a decline in sensory scores. Overall, all four samples initially exhibited a uniform milky white color, a moderate sweet and sour taste, and a fine texture. Group B had a significantly higher sensory score than the other groups during storage (P<0.05), and group S scored better than groups A and C, indicating that an inoculum size of 1×10⁻⁶ was optimal. 6 Fermented milk formulated with CFU / mL and commercial starter culture curdles faster, has moderate viscosity, good water-holding capacity, appropriate sweet-sour ratio, and the highest sensory score.

[0118] 5.2 Metabolic characteristics of fermented milk during storage.

[0119] Using a variety of metabolomics analysis methods, the study investigated an inoculum size of 1×10⁻⁶. 6 This study investigated the metabolic characteristics of fermented milk containing CFU / mL YF-01 and commercial starter culture before and after storage, exploring differential metabolites and their effects on fermented milk quality. Non-targeted metabolomics analysis revealed that the inoculum size was 1×10⁻⁶ before and after storage. 6 Significant differences were found in the metabolites of fermented milk fermented with YF-01 at CFU / mL compared to those fermented with commercial starter cultures. A total of 50 differential metabolites were identified, mainly involving carbohydrates and their derivatives, lipids and fatty acid derivatives, short peptides and amino acid derivatives. The differential metabolites identified in the fermented milk were enriched in 16 metabolic pathways, including fatty acid metabolism and arginine metabolism via the pentose phosphate pathway. Changes in these metabolites are closely related to the flavor, stability, and bioactivity of fermented milk, further revealing the metabolic mechanism of YF-01 in fermented milk and its role in improving product quality.

[0120] The stability of the instrument and the reliability of metabolite detection were assessed by analyzing the total ion current chromatograms of different fermented milk QC samples. The total ion current curves in positive and negative ion modes almost completely overlapped, indicating that the instrument analysis system has high stability, strong experimental repeatability, and good data accuracy, meeting the requirements for subsequent analysis.

[0121] like Figure 14 As shown, principal component analysis was performed on the metabolites of fermented milk in group B after 1 day and 28 days of storage. The total variability of the samples was 72.1%, with PC1 contributing 47.7% and PC2 contributing 24.4%. The two groups showed a separation trend and concentrated distribution within each group, indicating significant differences in the metabolites of fermented milk before and after storage.

[0122] like Figure 15 and Figure 16As shown, using the MetaboAnalyst 6.0 online website, with VIP>1 and P<0.05 as criteria, 50 differentially expressed metabolites in fermented milk before and after storage were screened. These mainly included carbohydrates and their derivatives, lipids and fatty acid derivatives, short peptides and amino acid derivatives, and other small molecule metabolites. β-lactose was highly expressed at 1 day of storage, providing energy for the microbial community and affecting the sweetness of dairy products, and possessing potential anti-tumor activity. At 28 days of storage, glycosides such as 1-hexanol arabinoside were highly expressed, acting as flavor precursors and affecting the flavor of fermented milk, and may also have antioxidant, shelf-life-extending, and texture-influencing effects. At 1 day of storage, glycerol 3-phosphate, phosphatidylglycerol, and 1-hexanol were highly expressed, participating in glycerol synthesis, possessing various application potentials, and affecting the flavor of fermented milk, respectively. At 28 days of storage, hydroxyoctanoyl-CoA, nonanoyl-CoA, and acetaldehyde were highly expressed, participating in fatty acid metabolism and affecting the flavor of fermented milk. During 28 days of storage, some short peptides and amino acid derivatives, such as γ-glutamylarginine, were highly expressed, exhibiting proteolytic properties. These peptides can regulate nutrition, exert antioxidant effects, and are also associated with several metabolic processes. Analysis of differentially expressed metabolites using MetaboAnalyst 6.0 revealed their common enrichment in 16 metabolic pathways, including fatty acid metabolism, with triglyceride biosynthesis, glycerol phosphate metabolism, and cardiolipin synthesis showing the most significant enrichment. These metabolite changes are influenced by multiple factors and enhance fermented milk quality through multi-target effects, providing a scientific basis for its development as a functional food.

[0123] The study, through non-targeted metabolomics analysis, found that vaccination with 1×10 6 Fermented milk formulated with CFU / mL Bifidobacterium adolescentis YF-01 and a commercial starter culture showed 50 significantly differentially expressed metabolites during storage, mainly involving carbohydrates, lipids, and short peptides, and enriched in 16 key metabolic pathways, including fatty acid metabolism and the pentose phosphate pathway. This formulation, by synergistically regulating these metabolic pathways, significantly improved the flavor (e.g., increased fruitiness and sweetness), stability (improved water-holding capacity and viscosity), and bioactivity (e.g., antioxidant and immunomodulatory functions) of the fermented milk, while shortening the fermentation time to 6.5 hours and maintaining a viable count >10⁻⁶ after 28 days. 8 The CFU / mL ratio provides a theoretical basis and application strategy for developing high-quality probiotic fermented milk.

[0124] Therefore, the present invention uses the above-mentioned Bifidobacterium adolescentis YF-01 and its application in fermented milk. This strain has excellent characteristics such as acid and bile salt tolerance and the ability to utilize a variety of carbohydrates. By compounding it with commercial starter culture for the preparation of fermented milk, the viable count, texture, sensory quality and storage stability of fermented milk are improved.

[0125] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Bifidobacterium adolescentis ( Bifidobacterium adolescentis The application of YF-01 in the preparation of fermented milk that regulates intestinal flora, promotes intestinal health, or enhances the body's antioxidant capacity is characterized by, The described *Bifidobacterium adolescentis* YF-01 is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20251308, deposited on June 9, 2025, at Wuhan University, Wuhan, China, and classified as follows: Bifidobacterium adolescentis Its 16S rDNA sequence is shown in SEQ ID NO.1; When preparing fermented milk that regulates intestinal flora, promotes intestinal health, or enhances the body's antioxidant capacity, Bifidobacterium adolescentis YF-01 is compounded with the commercial starter culture PYS-010. The inoculum size of Bifidobacterium adolescentis YF-01 is 1×10⁻⁶. 6 The inoculum size of the commercial starter PYS-010 was 0.03‰, and the fermentation time was 6.5±0.02h. The secondary metabolites obtained from the fermentation included spore lactone, amprolium Bs1, and ganoderic acid F.

2. A fermented milk, characterized in that: The fermented milk is obtained by compound fermentation of Bifidobacterium adolescentis YF-01 as described in claim 1 and commercial starter culture PYS-010, with an inoculum amount of Bifidobacterium adolescentis YF-01 of 1×10⁻⁶. 6 The fermentation process involved using CFU / mL of commercial starter culture PYS-010 at an inoculum size of 0.03‰ and a fermentation time of 6.5 ± 0.02 h. The resulting fermented milk product contained a viable count of Bifidobacterium adolescentis YF-01 of no less than 1 × 10⁻⁶ CFU / mL. 8 CFU / mL.

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