Bifidobacterium adolescentis, synbiotic preparation, preparation method and application thereof

Abstract

This application discloses a strain of *Bifidobacterium adolescentis*, a synbiotic preparation, its preparation method, and its application. The *Bifidobacterium adolescentis* strain described in this application is *Bifidobacterium adolescentis* OH202, with accession number GDMCC No. 66531. This newly developed *Bifidobacterium adolescentis* OH202 is resistant to gastrointestinal stresses such as gastric acid and bile salts, exhibiting excellent intestinal colonization ability. This strain can specifically recognize and efficiently utilize active ingredients from traditional Chinese medicines such as Astragalus polysaccharides and Dendrobium polysaccharides as carbon sources; the combination of these two can form a synbiotic system with significant synergistic effects. This strain and its preparation can inhibit weight gain induced by a high-fat diet, improve abnormal lipid metabolism, and block endotoxin entry into the bloodstream by upregulating the expression of intestinal tight junction proteins and repairing the intestinal mucosal barrier, thereby reducing systemic inflammation levels. This provides a new technical solution for the biocontrol of obesity, hyperlipidemia, and metabolic syndrome.

Description

Technical Field

[0001] This application relates to the field of microbial technology, and in particular to a strain of Bifidobacterium adolescentis, a synbiotic preparation, its preparation method, and its application. Background Technology

[0002] Metabolic diseases are a major challenge threatening global public health, with obesity and its complications (such as type 2 diabetes, non-alcoholic fatty liver disease, and cardiovascular disease) showing a continuous upward trend. Epidemiological surveys show that excessive intake of high-fat diets (HFD) is one of the main causes of obesity. Obesity is not only characterized by excessive accumulation of adipose tissue, but is also considered a systemic chronic low-grade inflammatory state. Gut microbiota imbalance plays a key role in this process. High-fat diets lead to changes in gut microbiota structure, an increase in the proportion of Gram-negative bacteria, and damage to the intestinal mucosal barrier, resulting in bacterial lipopolysaccharide (LPS) entering the bloodstream and causing metabolic endotoxemia, which in turn induces systemic chronic inflammation and insulin resistance. Therefore, regulating the balance of gut microbiota to block the "enteritis-obesity" axis has become an important strategy for the prevention and treatment of metabolic diseases.

[0003] Bifidobacterium adolescentis is one of the core dominant bacterial species in the gut microbiota of healthy adults, belonging to the phylum Actinobacteria. This species possesses excellent glucose metabolism capabilities, fermenting carbohydrates to produce short-chain fatty acids such as acetic acid and lactic acid, playing a crucial role in maintaining the acidic environment of the gut, inhibiting the growth of pathogenic bacteria, and regulating host immune function. Although Bifidobacterium adolescentis has great potential in the field of gut health, the probiotic efficacy varies significantly among different strains, exhibiting strong strain specificity. Currently available or reported Bifidobacterium adolescentis strains generally suffer from slow growth rates, poor tolerance to intestinal environmental stress induced by high-fat diets, and weak colonization abilities. In particular, most strains lack significant specific improvement effects on systemic inflammation and fat accumulation caused by high-fat diets, or require extremely high ingestion levels of live bacteria to produce weak therapeutic effects.

[0004] Furthermore, existing obesity treatments, such as orlistat or GLP-1 receptor agonists, while effective, often come with side effects like gastrointestinal discomfort and nausea, and long-term use is costly. Current probiotic preparations often struggle to overcome the technical bottlenecks of *Bifidobacterium adolescentis*, a strictly anaerobic bacterium, exhibiting slow growth and low biomass during fermentation and significant activity loss during freeze-drying. Therefore, developing new strains of *Bifidobacterium adolescentis* and corresponding preparation processes is of significant clinical value and market potential for developing safe and effective products for the prevention and treatment of metabolic diseases. Summary of the Invention

[0005] The purpose of this application is to provide a novel Bifidobacterium adolescentis strain, synbiotic preparations and probiotic preparations made from the Bifidobacterium adolescentis strain, as well as the preparation methods and the application of the Bifidobacterium adolescentis strain, synbiotic preparations and probiotic preparations in metabolic diseases.

[0006] One aspect of this application discloses a strain of Bifidobacterium adolescentis, namely Bifidobacterium adolescentis OH202, with accession number GDMCC No. 66531.

[0007] It should be noted that the novel *Bifidobacterium adolescentis* strain mentioned in this application exhibits excellent tolerance to gastrointestinal stressors such as gastric acid, bile salts, and trypsin. Experimental results show that after 1 hour of incubation in simulated gastric juice at pH 2.0 and in an environment with 0.3% bile salts, its survival rates reached approximately 8.2% and 12.5%, respectively, higher than those of ordinary *Bifidobacterium adolescentis*, demonstrating corresponding intestinal colonization potential. Furthermore, this strain exhibits a unique carbon source utilization preference, specifically recognizing and efficiently utilizing active ingredients from traditional Chinese medicines such as astragalus polysaccharides and dendrobium polysaccharides. In a culture medium using astragalus polysaccharides as the sole carbon source, its proliferation efficiency is more than 1.9 times that using the common prebiotic inulin. Furthermore, it can effectively regulate metabolic disorders induced by a high-fat diet. In animal experiments, OH202 intake reduced weight gain in obese mice by approximately 21.9%, decreased serum total cholesterol (TC) and triglyceride (TG) levels by 20.2% and 18.5%, respectively, and significantly downregulated serum tumor necrosis factor-α (TNF-α) and lipopolysaccharide (LPS) levels by 42.5% and 48.0%, respectively. Mechanistic studies show that OH202 can reshape the gut microbiota, upregulate the expression of intestinal tight junction proteins ZO-1 and Occludin, and repair the intestinal mucosal barrier, thereby blocking endotoxins from entering the bloodstream and exerting a systemic anti-inflammatory and metabolic-improving effect. The Bifidobacterium adolescentis OH202 of this application can effectively treat or prevent obesity and hyperlipidemia, and help improve symptoms related to metabolic syndrome, providing a new direction for the biocontrol of metabolic diseases.

[0008] Another aspect of this application discloses a synbiotic preparation comprising the Bifidobacterium adolescentis of this application and a specific prebiotic, wherein the specific prebiotic is selected from at least one of Astragalus polysaccharide and Dendrobium polysaccharide.

[0009] Preferably, the synbiotic preparation of this application is a freeze-dried bacterial powder or a microecological capsule.

[0010] It should be noted that the key to the synbiotic formulation of this application lies in the presence of *Bifidobacterium adolescentis* OH202 and specific prebiotics that have a synergistic effect with it. Other components can be added according to requirements or dosage form. It is understood that *Bifidobacterium adolescentis* OH202, as a probiotic adapted to the intestinal microenvironment, can be used directly as the active ingredient in the formulation of this application; alternatively, the combination of *Bifidobacterium adolescentis* OH202 and specific prebiotics can be used as the active ingredient in the formulation of this application, for example, it can be a mixed powder containing bacteria and polysaccharides, or a functional food for regulating the intestinal microecology prepared using it.

[0011] Another aspect of this application discloses a microecological preparation containing the Bifidobacterium adolescentis of this application or the synbiotic preparation of this application.

[0012] Preferably, the dosage form of the microecological preparation of this application is freeze-dried bacterial powder, capsule, tablet, granule or oral liquid.

[0013] Preferably, in the microecological preparation of this application, each unit of dosage form contains ≥1×10⁻⁶ live bacteria of Bifidobacterium adolescentis OH202. 9 CFU.

[0014] Another aspect of this application discloses a method for preparing a probiotic preparation, comprising the following steps:

[0015] (1) High-density fermentation: Bifidobacterium adolescentis of this application was inoculated into liquid culture medium and cultured at 37°C under anaerobic conditions until the late logarithmic growth phase. After the culture was completed, the bacterial sludge was collected by centrifugation and washed with sterile buffer.

[0016] (2) Encapsulation of bacterial cells: Add freeze-drying protectant to the bacterial sludge obtained in step (1) and mix evenly to prepare a bacterial suspension;

[0017] (3) Vacuum freeze drying: The bacterial suspension is placed in a freeze dryer and pre-frozen at -45℃ for 3-5 hours. Then, it is sublimated and dried under a vacuum of 10-15 Pa. During the desorption drying stage, the temperature is gradually raised to 25℃. The total cycle is 20-24 hours to obtain freeze-dried bacterial powder.

[0018] (4) Compound preparation: The freeze-dried bacterial powder obtained in step (3) is mixed with a specific prebiotic to obtain a microecological preparation in the form of freeze-dried bacterial powder; wherein, the specific prebiotic is selected from at least one of Astragalus polysaccharide and Dendrobium polysaccharide.

[0019] Preferably, in step (2), the mass ratio of freeze-drying protectant to bacterial sludge is 1:1.

[0020] Preferably, the freeze-drying protectant includes skim milk, trehalose, and monosodium glutamate.

[0021] Preferably, the aqueous solution of the freeze-drying protectant contains 12% (w / v) skim milk, 8% (w / v) trehalose, and 2% (w / v) monosodium glutamate.

[0022] Preferably, in step (1), the liquid culture medium is an MRS liquid culture medium containing 0.05% (w / v) L-cysteine ​​hydrochloride.

[0023] Preferably, the viable bacteria survival rate of the freeze-dried bacterial powder obtained in step (3) after being stored at -20°C for 6 months is ≥20%.

[0024] Another aspect of this application discloses the use of the present application's Bifidobacterium adolescentis, the present application's synbiotic preparation, or the present application's probiotic preparation in the preparation of products for the prevention or treatment of metabolic diseases, wherein the use is selected from any one of the following:

[0025] (1) Use in the preparation of products for the treatment or prevention of obesity and hyperlipidemia;

[0026] (2) Use in the preparation of products that help improve metabolic syndrome and its complications, including non-alcoholic fatty liver, insulin resistance and systemic inflammation;

[0027] (3) Use in the preparation of products that regulate the balance of intestinal microecology and repair the intestinal mucosal barrier;

[0028] (4) Use in the preparation of products that reduce serum total cholesterol, triglycerides and inflammatory factor levels.

[0029] Another aspect of this application discloses a product for treating or preventing metabolic diseases, the product containing the Bifidobacterium adolescentis, the synbiotic preparation, or the probiotic preparation of this application; wherein, metabolic diseases include obesity, hyperlipidemia, metabolic syndrome caused by lipid metabolism abnormalities, and non-alcoholic fatty liver disease.

[0030] Another aspect of this application discloses a product for regulating the balance of intestinal microecology or repairing the intestinal mucosal barrier, the product containing the Bifidobacterium adolescentis of this application, the synbiotic preparation of this application, or the microecological preparation of this application; the product can upregulate the expression of intestinal tight junction proteins ZO-1 and Occludin.

[0031] Another aspect of this application discloses a product that helps lower blood lipids or reduce inflammation, the product containing the Bifidobacterium adolescentis, the synbiotic preparation, or the probiotic preparation of this application.

[0032] It should be noted that the *Bifidobacterium adolescentis* OH202 of this application also possesses some common characteristics of existing *Bifidobacterium adolescentis* strains. Therefore, referring to existing *Bifidobacterium adolescentis* strains, the *Bifidobacterium adolescentis* OH202 of this application can also be used to prepare intestinal health care health products or functional foods. Furthermore, health products or functional foods containing the *Bifidobacterium adolescentis* OH202 of this application can tolerate the gastrointestinal environment, inhibit the growth of harmful bacteria, and have effects such as treating or preventing obesity, assisting in improving dyslipidemia, and regulating the balance of intestinal microecology.

[0033] The beneficial effects of this application are as follows:

[0034] The newly developed Bifidobacterium adolescentis OH202 strain exhibits strong colonization ability and effectively tolerates human gastrointestinal stress. It forms a highly efficient synergistic effect with specific prebiotics such as Astragalus polysaccharides and Dendrobium polysaccharides, demonstrating significantly higher proliferation efficiency than ordinary prebiotic combinations. Functionally, this strain significantly inhibits high-fat diet-induced weight gain, improves abnormal lipid metabolism, and blocks endotoxin entry into the bloodstream by enhancing the expression of intestinal tight junction proteins and repairing the intestinal barrier, thereby significantly reducing systemic inflammation levels. This provides a novel intervention strategy along the "bacteria-gut-metabolism" axis for the prevention and treatment of obesity and metabolic syndrome. The freeze-dried bacterial powder prepared based on this strain and specific preservatives effectively overcomes the deficiency of Bifidobacterium spp. in the freeze-drying process. Even after freeze-drying and long-term storage, it maintains a high viable bacterial survival rate (approximately 23.3%), superior to the control strain, improving the processing stability of existing Bifidobacterium preparations and demonstrating significant industrial application value. Attached Figure Description

[0035] Figure 1 The colony morphology of Bifidobacterium adolescentis OH202 in Example 1 of this application on a modified MRS selective plate;

[0036] Figure 2 This is a comparison of the growth curves of Bifidobacterium adolescentis OH202 and the control strain ATCC 15703 in Example 1 of this application;

[0037] Figure 3 This is a comparison chart of the screening results of the effects of different carbon sources on the proliferation of Bifidobacterium adolescentis OH202 in Example 3 of this application;

[0038] Figure 4 This is a comparison chart of the survival rates of Bifidobacterium adolescentis OH202 and the control strain in Example 4 of this application after freeze-drying and long-term storage;

[0039] Figure 5 This is a graph showing the trend of body weight change in each group of mice during the 10-week intervention period in Example 5 of this application;

[0040] Figure 6This is a graph showing the regulatory effects of Bifidobacterium adolescentis OH202 on serum biochemical indicators (TC, TG) and inflammatory factors (TNF-α, LPS) in obese mice in Example 5 of this application.

[0041] Figure 7 This is a graph showing the detection results of the relative expression levels of tight junction proteins (ZO-1, Occludin) mRNA in the colon tissue of recipient mice after fecal microbiota transplantation (FMT) in Example 6 of this application.

[0042] The Bifidobacterium adolescentis strain OH202 of this application was deposited on June 16, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No. 66531. Detailed Implementation

[0043] Bifidobacterium adolescentis is one of the core dominant bacterial species in the gut microbiota of healthy adults and is widely used in the field of gut health. However, existing research reports on Bifidobacterium adolescentis generally suffer from slow growth rates, poor tolerance to intestinal environmental stress induced by high-fat diets, and weak colonization ability. Therefore, the inventors of this application conducted research on the feces of healthy adults to obtain a new Bifidobacterium adolescentis strain with superior performance.

[0044] Based on the above research and understanding, this application has developed a new strain of Bifidobacterium adolescentis, namely Bifidobacterium adolescentis OH202, with the accession number GDMCC No. 66531.

[0045] This application describes the newly developed Bifidobacterium adolescentis OH202, isolated from the feces of healthy adults. It exhibits excellent tolerance to the gastrointestinal environment, resisting stress from gastric acid, bile salts, and trypsin, and possesses good intestinal colonization potential. Studies have shown that this strain can be specifically recognized and efficiently utilized by certain active ingredients from traditional Chinese medicines such as Astragalus polysaccharides and Dendrobium polysaccharides. The combination of these two can form a synergistic system with significant synergistic effects.

[0046] In terms of functional applications, Bifidobacterium adolescentis OH202 can effectively inhibit weight gain induced by a high-fat diet, significantly reduce serum total cholesterol (TC) and triglyceride (TG) levels, and substantially reduce the concentrations of inflammatory factors such as tumor necrosis factor-α (TNF-α) and lipopolysaccharide (LPS). Further mechanistic studies have confirmed that this strain can exert systemic anti-inflammatory and metabolic regulatory effects by remodeling the intestinal microecological structure, upregulating the expression of intestinal tight junction proteins (ZO-1, Occludin), and repairing the intestinal mucosal barrier, thereby blocking endotoxin translocation.

[0047] This application also provides a process for preparing freeze-dried bacterial powder containing this strain. By employing a specific composite freeze-drying protectant (skim milk, trehalose, and monosodium glutamate) and optimized freeze-drying parameters, the technical challenge of Bifidobacterium's intolerance to freeze-drying has been overcome, significantly improving the survival rate of the strain during processing and its long-term storage stability. This strain and its preparations can be used to regulate the intestinal microecological balance, prevent or treat obesity, hyperlipidemia, and metabolic syndrome, providing a new preferred strain and technical solution for the biocontrol of metabolic diseases.

[0048] The present application will now be described in detail through specific experiments and accompanying drawings.

[0049] Unless otherwise specified, the experimental methods used in the following tests are standard methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following tests are commercially available.

[0050] Example

[0051] The culture media and reagents involved are as follows:

[0052] Modified MRS liquid culture medium: 10.0g peptone, 10.0g beef extract, 5.0g yeast powder, 20.0g glucose, 5.0g sodium acetate, 2.0g diammonium citrate, 1.0mL Tween-80, 0.4g dipotassium hydrogen phosphate, 0.58g magnesium sulfate, 0.25g manganese sulfate, 0.5g L-cysteine ​​hydrochloride, add distilled water to a final volume of 1L, adjust pH to 6.5, and autoclave at 121℃ for 15min.

[0053] Modified MRS solid medium: The formula is the same as that of modified MRS liquid medium, with an additional 15.0 g / L of agar powder.

[0054] Modified MRS selective agar plates: After sterilizing the above modified MRS solid medium, cool it to about 50°C and aseptically add mupirocin lithium salt to a final concentration of 50 mg / L.

[0055] Experiment 1: Isolation and identification of Bifidobacterium adolescentis OH202

[0056] I. Isolation and Purification of Strains

[0057] 1. Sample source and initial screening

[0058] Bifidobacterium OH202 was isolated from fecal samples of healthy adult volunteers recruited by Dalian Medical University (aged 25-35 years, BMI 18.5-23.9). Participants were required to have not used antibiotics, probiotics, or immunosuppressants within the past 3 months, and had no history of gastrointestinal or metabolic diseases (such as obesity or diabetes). Samples were immediately placed in anaerobic sampling tubes after collection and transported to the laboratory for processing within 2 hours.

[0059] 2. Separation steps

[0060] (1) Pretreatment: Take an appropriate amount of fresh fecal sample and add it to sterile physiological saline containing L-cysteine ​​hydrochloride, homogenize it, and obtain the initial bacterial suspension;

[0061] (2) Gradual dilution and selective culture: The initial bacterial suspension was subjected to conventional gradient dilution. The bacterial suspension of appropriate dilution was spread on a modified MRS selective agar plate (containing mupirocin lithium salt), placed in an anaerobic workstation, and cultured at 37°C until obvious single colonies grew.

[0062] (3) Purification and preservation: Select single colonies that conform to the typical morphological characteristics of Bifidobacterium, purify them by streaking multiple times, inoculate them into a culture medium containing a protectant, and preserve them under ultra-low temperature conditions.

[0063] II. Identification of Strains

[0064] 1. Morphological identification

[0065] The purified bacterial strain was inoculated onto a modified MRS solid plate and anaerobic incubated at 37°C for 48 hours. Colony morphology was then observed (e.g., ...). Figure 1 As shown): The colonies are round, milky white, smooth, and have neat, opaque edges; a single colony smear was Gram-stained and examined under a 1000× oil immersion microscope. The strain was Gram-positive, with no spores observed. The bacteria were mostly rod-shaped, with some cells showing Y-shaped or V-shaped branching structures at the ends. They were scattered and lacked flagella, consistent with the typical characteristics of Bifidobacterium.

[0066] 2. Growth characteristic determination

[0067] To evaluate the growth performance of the novel strain OH202, the model strain of *Bifidobacterium adolescentis*, ATCC15703, was used as a control group for comparative analysis under the same conditions. *Bifidobacterium adolescentis* OH202 and the control strain ATCC15703 were inoculated into modified MRS liquid medium at a 2% (v / v) inoculum and cultured anaerobically at 37℃. Samples were taken at 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 h of culture, and the OD of the bacterial culture was measured using a UV spectrophotometer. 600 The value (reflecting the total biomass of the bacteria) was used, and the viable count (CFU / mL) was determined by plate counting to plot the growth curve.

[0068] Growth curve (see) Figure 2 Analysis showed that the growth performance of *Bifidobacterium adolescentis* OH202 was superior to that of the control strain ATCC15703. After entering the logarithmic growth phase (4-12 h), OH202 exhibited a faster proliferation rate. The data at 12 h were compared in detail (e.g.,...). Figure 2 As shown in the figure, OH2O2 has reached its growth peak at this time, and its OD 600 The value can reach around 1.65, and the viable bacteria count is approximately 3.6 × 10⁻⁶. 9 CFU / mL; in contrast, the OD of the control strain 600 The value was only around 1.25, and the viable bacteria count was approximately 2.1 × 10⁻⁶. 9 CFU / mL. The biomass of OH202 was approximately 32% higher than that of the control strain. Furthermore, during the 12-24 h stationary period, the viable cell density of OH202 remained well maintained, without showing a significant decline trend. These results suggest that OH202 has high-density fermentation potential, which is beneficial for shortening the production cycle.

[0069] 3. Molecular biological identification

[0070] (1) DNA extraction: Take 5 mL of bacterial culture in the logarithmic growth phase, centrifuge at 12000 rpm for 2 min to collect the bacterial cells, and wash twice with sterile PBS. Total DNA was extracted using a bacterial genomic DNA extraction kit (Hunan Aike Rui Biotechnology Co., Ltd., catalog number AG21007), and the DNA quality was detected by 1% agarose gel electrophoresis;

[0071] (2) PCR amplification: Using the extracted DNA as a template, PCR amplification was performed using universal primers for the bacterial 16S rRNA gene; the upstream primer was 27F (sequence: 5'-AGAGTTTGATCCTGGCTCAG-3') (SeqID No. 1), and the downstream primer was 1492R (sequence: 5'-GGTTACCTTGTTACGACTT-3') (SeqID No. 2); the PCR reaction system was 50 μL, and the reaction conditions were: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 90 s, for a total of 30 cycles; and 72℃ final extension for 10 min.

[0072]

[0073] Experiment 2: Environmental tolerance test of Bifidobacterium adolescentis OH202

[0074] This embodiment uses a simulated gastric acid and high-concentration bile salt environment, and comprehensively evaluates the survival stability of Bifidobacterium adolescentis OH202 in the gastrointestinal environment by simulating the trypsin digestion process.

[0075] 1. Determination of acid resistance characteristics of Bifidobacterium adolescentis OH202

[0076] Adjust the pH of sterile physiological saline to 2.0 and 2.5 with hydrochloric acid, add 0.3% (w / v) pepsin, filter to sterilize, and preheat to 37°C to prepare artificial gastric fluid. Centrifuge the activated Bifidobacterium adolescentis OH202 and control strain ATCC 15703, discard the supernatant, and wash the cells twice with sterile PBS buffer to remove residual metabolites. Resuspend the cells in the artificial gastric fluid and incubate at 37°C for 1 hour. Collect the bacterial suspension at 0h and 1h, serially dilute, and spread on modified MRS agar plates. After 48h of anaerobic incubation, count the viable cells and calculate the survival rate using the following formula: Viability = (Number of viable cells at 1h / Number of viable cells at 0h) × 100%.

[0077] 2. Detection of bile salt tolerance in Bifidobacterium adolescentis OH202

[0078] The pH of the modified MRS liquid medium was adjusted to 8.0, and 0.1% and 0.3% (w / v) sodium taurocholate were added, respectively. The mixture was then autoclaved for 15 min to simulate the bile salt environment of the small intestine. *Bifidobacterium adolescentis* OH202 and the control strain ATCC15703 were inoculated at a 2% inoculum into bile-containing medium (experimental group) and bile-free ordinary modified MRS medium (blank group), respectively, and incubated anaerobically at 37℃ for 1 h. The viable bacterial count was determined using the plate count method after 1 h, and the survival rate was calculated using the following formula, with the bile-free group as the baseline: Relative survival rate = (Number of viable bacteria in experimental group / Number of viable bacteria in blank group) × 100%.

[0079] 3. Test for trypsin resistance of Bifidobacterium adolescentis OH202

[0080] Adjust the pH of sterile physiological saline to 8.0, add 0.1% (w / v) trypsin to prepare artificial intestinal fluid. Inoculate the bacterial cells into the artificial intestinal fluid at a ratio of 1:10 (v / v) and incubate at 37°C for 2 hours. Measure the number of viable bacteria before and after incubation and calculate the survival rate.

[0081] The results of the tolerance tests of the two strains in a simulated digestive tract environment are shown in Table 1.

[0082] Table 1. Gastrointestinal tolerance of Bifidobacterium adolescentis OH202

[0083] Experimental content Experimental conditions Survival rate of control strain (ATCC 15703) OH2O2 survival rate Acid resistance test pH 2.5, 1h 8.2% ± 1.5% 31.5% ± 3.8% Acid resistance test pH 2.0, 1h <0.1% 8.2% ± 0.6% Bile salt tolerance test 0.1% bile salts, 1 hour 65.4% ± 4.2% 88.6% ± 3.5% Bile salt tolerance test 0.3% bile salts, 1 hour 0.8% ± 0.2% 12.5% ± 1.2% Intestinal fluid resistance test 0.1% trypsin, 2h 87.5% ± 4.4% 95.0% ± 3.5%

[0084] Table 1 shows that *Bifidobacterium adolescentis* OH202 exhibits better tolerance to the extreme gastrointestinal environment than the control strain. Regarding acid tolerance, after treatment at pH 2.5, the survival rate of the control strain was 8.2%, while that of OH202 was 31.5%; after treatment at pH 2.0, the survival rate of the control strain was less than 0.1%, while that of OH202 was 8.2%. Comparing the two sets of data, OH202 demonstrated corresponding tolerance characteristics under the established acid gradient, providing a basis for surviving the acidic environment of the stomach.

[0085] In the bile salt tolerance test, after treatment with 0.1% bile salt concentration, the survival rate of the control strain was 65.4%, while that of OH202 was 88.6%; after treatment with 0.3% bile salt concentration, the survival rate of the control strain was 0.8%, while that of OH202 was 12.5%; in the trypsin tolerance test, the survival rate of OH202 was 95.0%. These results indicate that OH202 exhibits tolerance to bile salts and digestive enzymes in a simulated small intestinal environment.

[0086] In summary, in the in vitro evaluation system encompassing simulated gastric acid, bile salts, and trypsin, the survival rates of OH202 strain were all higher than those of the control strain. This in vitro tolerance data provides experimental support for assessing the survival and colonization potential of the strain in the intestinal environment.

[0087] Experiment 3: Prebiotic utilization characteristics of Bifidobacterium adolescentis OH202

[0088] Example 2 confirms that *Bifidobacterium adolescentis* OH202 possesses excellent tolerance to the gastrointestinal environment. However, the gut microbiota is a highly competitive and complex environment. For exogenous probiotics to rapidly establish a growth advantage and exert a long-term metabolic regulatory effect after colonization, they often require specific carbon source substrates as "proliferation factors." Given the high specificity of carbon source utilization by different strains, this experiment aims to construct a carbon source screening library containing more than 20 active ingredients from traditional Chinese medicines that are both food and medicine, and to screen for specific synergistic components that can significantly promote the proliferation of OH202, in order to construct a compound microecological preparation with synergistic effects.

[0089] 1. Screening Method

[0090] (1) Preparation of basal culture medium

[0091] Prepare a modified MRS basic liquid medium without glucose (except for the carbon source, the other components are the same as the standard modified MRS medium), adjust the pH to 6.5, and autoclave for 15 min before use.

[0092] (2) Construction of carbon source screening library

[0093] Based on the preliminary screening results from the laboratory and the catalog of medicinal and edible homologous substances, 22 kinds of Chinese herbal extracts were selected as carbon sources for testing, including: Astragalus polysaccharides, Dendrobium polysaccharides, Bletilla striata polysaccharides, Gastrodia elata polysaccharides, Schisandra chinensis polysaccharides, Ligustrum lucidum polysaccharides, Panax notoginseng polysaccharides, Dipsacus asperoides polysaccharides, Arctium lappa polysaccharides, Morinda officinalis polysaccharides, Lentinus edodes polysaccharides, tea polysaccharides, total saponins of Panax quinquefolius, Carthamus tinctorius polysaccharides, Taraxacum mongolicum polysaccharides, etc. (all of which were prepared in the laboratory or purchased from standard product companies, with a purity ≥80%). Inulin (a commercially available prebiotic) was used as a positive control, and a basal culture medium without any carbon source was used as a blank control.

[0094] (3) Cultivation and detection

[0095] Each carbon source was sterilized by filtration through a 0.22 μm microporous membrane and then added to the basal culture medium at a final concentration of 1.0% (w / v). A 2% (v / v) culture of *Bifidobacterium adolescentis* OH202 activated to the logarithmic growth phase was inoculated and incubated statically at 37℃ in an anaerobic workstation for 24 h. After incubation, the culture was thoroughly mixed, and the absorbance (OD) of the bacterial culture at 600 nm was measured using a microplate reader. 600 To evaluate the proliferative capacity of the strain under different carbon source substrates.

[0096] 2. Results Analysis

[0097] The filtering results are as follows Figure 3 As shown in the figure, experimental data revealed that *Bifidobacterium adolescentis* OH202 exhibited specific selective utilization characteristics for different carbon sources. Among the more than 20 traditional Chinese medicine components tested, the biomass of OH202 cultures was relatively high when using Astragalus polysaccharide and Dendrobium polysaccharide as carbon sources: after 24 hours of culture, the OD600 value of the Astragalus polysaccharide group was 1.68±0.05, and that of the Dendrobium polysaccharide group was 1.55±0.04, indicating that the strain could effectively utilize these two types of polysaccharides for proliferation. The OD600 value of the prebiotic inulin group, used as a positive control, was 0.85±0.03. Furthermore, the growth-promoting effects of other components in the screening library (such as Gastrodia elata polysaccharide, Bletilla striata polysaccharide, Lentinus edodes polysaccharide, etc.) were weaker than those of Astragalus polysaccharide and Dendrobium polysaccharide. These data indicate that both Astragalus polysaccharide and Dendrobium polysaccharide can serve as specific carbon sources for promoting the proliferation of *Bifidobacterium adolescentis* OH202, providing fundamental data support for the subsequent construction of a complex synbiotic system.

[0098] Experiment 4: Preparation and stability test of freeze-dried Bifidobacterium adolescentis OH202 bacterial powder

[0099] In practical applications of probiotic preparations, the "extremely anaerobic and lyophilization-intolerant" characteristics of Bifidobacterium often make it difficult for conventional preservatives to maintain the cell membrane integrity of Bifidobacterium adolescentis during the freeze-drying process, resulting in a sharp drop in viable bacterial count. This embodiment first prepared a freeze-dried bacterial powder formulation of Bifidobacterium adolescentis OH202, and then evaluated its industrial application value by verifying the processing stability of OH202 under optimized processes.

[0100] 1. Preparation process of freeze-dried bacterial powder

[0101] (1) High-density fermentation:

[0102] Bifidobacterium adolescentis OH202 and control strain ATCC 15703 were inoculated into modified MRS liquid medium (containing 0.05% L-cysteine ​​hydrochloride) and cultured at 37°C in an anaerobic fermenter until the late logarithmic growth phase, at which point the bacterial biomass reached its peak.

[0103] (2) Collection and cleaning of bacterial cells

[0104] Immediately after fermentation, the fermentation broth was centrifuged at 4°C for 15 minutes. The supernatant was discarded, and the precipitated bacterial sludge was collected. The sludge was washed once with pre-cooled sterile PBS buffer, and then centrifuged again to remove any residual culture medium.

[0105] (3) Protective agent encapsulation

[0106] A compound freeze-drying protectant was prepared with the following formula: 12% (w / v) skim milk + 8% (w / v) trehalose + 2% (w / v) monosodium glutamate (as an antioxidant), using distilled water as the solvent. The mixture was sterilized at 115°C for 15 minutes. Under aseptic conditions, the bacterial sludge and protectant were mixed at a 1:1 mass ratio and stirred at low speed until homogeneous, forming a uniform bacterial suspension.

[0107] (4) Vacuum freeze drying

[0108] The temperature-controlled freeze-drying process was adopted. The bacterial suspension was placed in a freeze dryer and pre-frozen at -45℃ for 4 hours. Then, sublimation and desorption drying were performed under a vacuum of 10-15 Pa. During the desorption period, the temperature was gradually raised to 25℃. The total cycle was 24 hours, and a loose freeze-dried bacterial powder was obtained.

[0109] 2. Long-term storage stability test

[0110] The prepared OH202 and control strain lyophilized powders were stored at -20℃ for a long period to simulate shelf life. Samples were taken immediately after lyophilization (0 months) and after 6 months of storage for serial dilution and plate counting to determine the change in viable count. The survival rate was calculated according to the following formula: Survival rate = viable count at 6 months / viable count at 0 months × 100%.

[0111] 3. Results Analysis

[0112] The stability test results are shown in Table 2. Figure 4 As shown.

[0113] Table 2. Comparison of freeze-drying stability between Bifidobacterium adolescentis OH202 and control strains.

[0114] Detection strains Immediate viable count of freeze-dried bacteria (CFU / g) viable bacteria count (CFU / g) after 6 months of storage Survival rate (%) ATCC 15703 <![CDATA[1.5×10 10 ]]> <![CDATA[8.2×10 8 ]]> 5.5% ± 0.6% OH202 <![CDATA[1.8×10 10 ]]> <![CDATA[4.2×10 9 ]]> 23.3% ± 1.5%

[0115] Experimental data showed significant differences in the viability maintenance ability of the two strains under freeze-drying and long-term storage conditions. The control strain ATCC 15703 showed a significant decrease in viable cell count after freeze-drying and 6 months of storage at -20℃, with a survival rate of only 5.5%, indicating that this strain has relatively weak tolerance to environmental stress and suffers significant viability loss during long-term storage.

[0116] In contrast, OH202 exhibited better stability. Under the same preparation process and storage conditions, the survival rate of OH202 remained at 23.3%. This result suggests that OH202 has a stronger ability to withstand the physical damage of freeze-drying and long-term low-temperature storage environments, and can more effectively maintain the integrity of the bacterial cell membrane and physiological activity. This stability characteristic exhibited by OH202 is beneficial to ensuring the effective number of viable bacteria under different application forms, laying a good material foundation for the subsequent development and application of this strain.

[0117] Experiment 5: Animal experiment on the effects of Bifidobacterium adolescentis OH202 on obesity symptoms induced by a high-fat diet.

[0118] In this embodiment, the model used was the C57BL / 6J obese mouse model induced by a high-fat diet (HFD). This model was used to evaluate the effects of Bifidobacterium adolescentis OH202 on host body weight, lipid metabolism, and systemic inflammation.

[0119] 1. Experimental grouping and treatment

[0120] Thirty 6-week-old male C57BL / 6J mice were selected and, after one week of acclimatization, were randomly divided into 3 groups (n=10 per group):

[0121] (1) HFD model group (Model): fed a high-fat diet with 60% fat as the energy source throughout the entire process, and gavaged with 0.2 mL of sterile physiological saline every day;

[0122] (2) HFD+ control group: fed a high-fat diet, and administered 0.2 mL of a solution containing 1×10⁻⁶ bacteria by gavage daily. 9 CFU ATCC 15703 bacterial suspension;

[0123] (3) HFD+OH202 group (OH202): fed a high-fat diet, and administered 0.2 mL of 1×10⁻⁶ ozoglycine daily. 9 CFU OH202 bacterial suspension.

[0124] A separate control group (ND) without high-fat induction was established as a healthy baseline. The experimental intervention lasted for 10 weeks. During this period, the mice's body weight and food intake were recorded weekly.

[0125] 2. Detection Indicators and Methods

[0126] (1) Determination of relevant metabolic indicators: After the experiment, the mice were fasted for 12 hours, and blood was collected by enucleation. The blood was allowed to stand at room temperature for 2 hours and then centrifuged to separate the upper serum layer. The levels of total cholesterol (TC) and triglycerides (TG) were measured using a kit. The serum sample was mixed with the working solution in a certain proportion and incubated at 37°C for 10 minutes. The absorbance value was measured at a wavelength of 510 nm using an ELISA reader, and the concentration was calculated according to the standard curve.

[0127] (2) Measurement of inflammatory factors: The concentrations of tumor necrosis factor-α (TNF-α) and lipopolysaccharide (LPS) in serum were measured using an ELISA kit. The diluted serum sample was added to a microplate coated with antibody, incubated, washed, and then enzyme-labeled antibody and substrate chromogenic solution were added sequentially. After the reaction was terminated, the absorbance value was measured at 450 nm using an ELISA reader.

[0128] 3. Results Analysis

[0129] (1) Effect on weight control

[0130] After 10 weeks of intervention, the trends in body weight change in each group of mice are as follows: Figure 5 As shown.

[0131] Compared with the normal diet group (ND), the body weight of mice in the HFD model group increased with prolonged feeding time, with a net weight gain of 20.5±1.8g at the end of the experiment, indicating that the obesity model was successfully established. After inoculation with the control strain ATCC 15703, the net weight gain of mice was 17.2±1.5g, which was about 16.1% lower than that of the model group. The net weight gain of the OH202 group was 16.0±1.3g, which was about 21.9% lower than that of the model group. Statistical analysis showed that the difference in body weight between the OH202 group and the model group was statistically significant (P<0.05), indicating that this strain can inhibit excessive weight gain induced by a high-fat diet to some extent.

[0132] (2) Regulatory effect on blood lipids and inflammation

[0133] Serum biochemical test results (such as) Figure 6As shown in the figure, OH202 effectively improves lipid metabolism abnormalities. Compared with the model group, the serum TC level in the OH202 group decreased by approximately 20.2%, and the TG level decreased by approximately 18.5%. Importantly, OH202 showed a good advantage in improving systemic inflammation. HFD-induced mice had significantly elevated serum TNF-α and LPS levels, indicating a state of chronic low-grade inflammation. After OH202 intervention, TNF-α levels decreased by 42.5% and LPS levels decreased by 48.0% compared with the model group; in contrast, the TNF-α reduction in the control strain group was approximately 25%. This result indicates that OH202 is significantly superior to the control strain in improving systemic inflammatory markers, suggesting that it may exert a stronger anti-inflammatory and metabolic-improving effect than the common strain by improving and enhancing intestinal barrier function and reducing endotoxin (LPS) entry into the bloodstream, thereby blocking the "enteritis-inflammation" axis.

[0134] Experiment 6: Validation of the intestinal microecological remodeling mechanism of Bifidobacterium adolescentis OH202 based on fecal microbiota transplantation (FMT)

[0135] To further investigate whether its mechanism of action depends on the remodeling of the gut microbiota, this study designed a fecal microbiota transplantation (FMT) experiment. By transplanting fecal microbiota from donor mice treated with OH202 into recipient mice with impaired gut microbiota, the effect on the intestinal barrier function of the recipient mice was observed to verify the ability of OH202 to restore a healthy gut microbiota.

[0136] 1. Preparation of donor fecal microbial solution

[0137] Mice from the HFD+OH202 group in Example 5 were selected as "positive donors," and mice from the HFD model group were selected as "negative donors." Fresh fecal samples were collected at week 10 of the intervention, and sterile PBS buffer was added under anaerobic conditions to homogenize the samples thoroughly. Food residue was removed by low-speed centrifugation, and the supernatant was collected as the FMT donor suspension.

[0138] 2. Establishment and transplantation of recipient models

[0139] Twenty 6-week-old male C57BL / 6J mice were randomly divided into two groups (n=10 per group): the FMT-HFD group (receiving feces from the HFD group) and the FMT-OH202 group (receiving feces from the OH202 group).

[0140] (1) Antibiotic pretreatment (microbial damage): In order to disrupt the original intestinal microbiota of the recipient mice, a broad-spectrum antibiotic cocktail (ABX) (containing ampicillin 1 g / L, neomycin 1 g / L, metronidazole 1 g / L, and vancomycin 0.5 g / L) was added to the drinking water of all mice for 5 consecutive days. Feces were collected aseptically and cultured anaerobically. The microbial damage model was considered to have been successfully constructed after confirming that no colony growth was observed.

[0141] (2) Microbial transplantation and feeding: After antibiotic treatment, both groups of mice were fed a high-fat diet (HFD). At the same time, 200 μL of the corresponding donor microbial suspension was administered by gavage daily. The intervention period was 4 weeks.

[0142] 3. Detection Indicators and Methods

[0143] (1) Weight monitoring: Weekly weight changes of recipient mice were recorded.

[0144] (2) Detection of intestinal barrier function: After the experiment, mouse colon tissue was collected. The relative mRNA expression levels of tight junction proteins ZO-1 and Occludin in the tissue were detected by real-time quantitative PCR (RT-qPCR).

[0145] 4. Results Analysis

[0146] (1) Transmission effect of metabolic phenotype

[0147] After four weeks of transplantation intervention, the two groups of recipient mice exhibited different trends in weight gain. Mice in the FMT-HFD group, after receiving gut microbiota from obese mice, showed faster weight gain. However, mice in the FMT-OH202 group, after receiving OH2-regulated donor gut microbiota and being fed a high-fat diet, experienced some degree of inhibition in weight gain, with a net weight gain at the end of the experiment being lower than that of the FMT-HFD group (approximately 13.5% lower). This indicates that *Bifidobacterium adolescentis* OH202 can improve the gut microbiota structure of donor mice and transfer this beneficial metabolic regulatory characteristic to recipients through fecal microbiota transplantation.

[0148] (2) Mechanisms of intestinal barrier repair

[0149] RT-qPCR test results (e.g.) Figure 7 As shown in the figure, alterations in the gut microbiota affect the host's intestinal barrier status. Compared to the FMT-HFD group, the mRNA expression levels of tight junction proteins ZO-1 and Occludin in the colon tissue of mice in the FMT-OH202 group were upregulated (approximately 2.1-fold and 1.8-fold, respectively). This result suggests that OH202 may help construct a microecological environment conducive to maintaining the integrity of the mucosal barrier, thereby exerting a systemic probiotic effect by enhancing the function of the intestinal physical barrier and reducing the penetration of harmful substances.

[0150] The above description, in conjunction with specific embodiments, provides a detailed explanation of this application and should not be construed as limiting the implementation of this application to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the basic inventive concept of this application.

Claims

1. A strain of Bifidobacterium adolescentis, characterized by: The Bifidobacterium adolescentis mentioned is Bifidobacterium adolescentis OH202, with accession number GDMCC No. 66531.

2. A synbiotic preparation, characterized in that: It comprises the Bifidobacterium adolescentis as described in claim 1 and a specific prebiotic, wherein the specific prebiotic is selected from at least one of Astragalus polysaccharide and Dendrobium polysaccharide.

3. A microecological preparation, characterized in that: Contains the Bifidobacterium adolescentis as described in claim 1 or the synbiotic preparation as described in claim 2.

4. The microecological preparation according to claim 3, characterized in that: The dosage form of the microecological preparation is freeze-dried bacterial powder, capsules, tablets, granules or oral liquid; Preferably, in the microecological preparation, each unit of dosage form contains ≥1×10⁻⁶ live bacteria of Bifidobacterium adolescentis OH202. 9 CFU.

5. The method for preparing the microecological preparation according to claim 3 or 4, characterized in that: Includes the following steps, (1) High-density fermentation: Bifidobacterium adolescentis as described in claim 1 is inoculated into liquid culture medium and cultured at 37°C under anaerobic conditions until the late logarithmic growth phase. After the culture is completed, the bacterial sludge is collected by centrifugation and washed with sterile buffer. (2) Encapsulation of bacterial cells: Add freeze-drying protectant to the bacterial sludge obtained in step (1) and mix evenly to prepare a bacterial suspension; (3) Vacuum freeze drying: The bacterial suspension is placed in a freeze dryer and pre-frozen at -45℃ for 3-5 hours. Then, it is sublimated and dried under a vacuum of 10-15 Pa. During the desorption drying stage, the temperature is gradually raised to 25℃. The total cycle is 20-24 hours to obtain freeze-dried bacterial powder. (4) Compound preparation: The freeze-dried bacterial powder obtained in step (3) is mixed with a specific prebiotic to obtain a microecological preparation in the form of freeze-dried bacterial powder; wherein the specific prebiotic is selected from at least one of Astragalus polysaccharide and Dendrobium polysaccharide.

6. The preparation method according to claim 5, characterized in that: In step (2), the mass ratio of the freeze-drying protectant to the bacterial sludge is 1:1; Preferably, the freeze-drying protectant includes skim milk, trehalose, and monosodium glutamate; Preferably, the aqueous solution of the freeze-drying protectant contains 12% (w / v) skim milk, 8% (w / v) trehalose, and 2% (w / v) monosodium glutamate. Preferably, in step (1), the liquid culture medium is an MRS liquid culture medium containing 0.05% (w / v) L-cysteine ​​hydrochloride; Preferably, the viable bacteria survival rate of the freeze-dried bacterial powder obtained in step (3) after being stored at -20°C for 6 months is ≥20%.

7. The use of the Bifidobacterium adolescentis according to claim 1, the synbiotic preparation according to claim 2, or the probiotic preparation according to claim 3 or 4 in the preparation of products for the prevention or treatment of metabolic diseases, characterized in that: The intended use is selected from any of the following: (1) Use in the preparation of products for the treatment or prevention of obesity and hyperlipidemia; (2) Use in the preparation of products that help improve metabolic syndrome and its complications, including non-alcoholic fatty liver, insulin resistance and systemic inflammation; (3) Use in the preparation of products that regulate the balance of intestinal microecology and repair the intestinal mucosal barrier; (4) Use in the preparation of products that reduce serum total cholesterol, triglycerides and inflammatory factor levels.

8. A product for treating or preventing metabolic diseases, characterized in that: The product contains Bifidobacterium adolescentis as described in claim 1, synbiotic preparation as described in claim 2, or microecological preparation as described in claim 3 or 4; the metabolic diseases include obesity, hyperlipidemia, and metabolic syndrome and non-alcoholic fatty liver disease caused by abnormal lipid metabolism.

9. A product for regulating the intestinal microecological balance or repairing the intestinal mucosal barrier, characterized in that: The product contains the Bifidobacterium adolescentis as described in claim 1, the synbiotic preparation as described in claim 2, or the probiotic preparation as described in claim 3 or 4; the product can upregulate the expression of intestinal tight junction proteins ZO-1 and Occludin.

10. A product that helps lower blood lipids or reduce inflammation, characterized in that: The product contains Bifidobacterium adolescentis as described in claim 1, synbiotic preparation as described in claim 2, or microecological preparation as described in claim 3 or 4.

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