Bifidobacterium adolescentis for relieving obesity and reducing visceral adipose tissues and application of bifidobacterium adolescentis

Abstract

The invention discloses bifidobacterium adolescentis for relieving obesity and reducing visceral adipose tissues and application of the bifidobacterium adolescentis, and belongs to the technical field of microorganisms. The bifidobacterium adolescentis CCFM1521 provided by the invention can obviously reduce the weight gain of obese mice induced by high fat diet, and reduce the liver weight gain and epididymis fat weight gain of obese mice induced by low and high fat diet; the disorder, caused by high-fat diet, of mouse lipid metabolism factors, namely high-density lipoprotein cholesterol and low-density lipoprotein cholesterol is recovered; the abnormal Bifidobacterium abundance in the intestinal flora of the mouse caused by high fat can be recovered, and the pathological injury, lipid accumulation and inflammation accumulation of the liver tissue of an obese individual caused by high fat diet can be obviously improved; the content of triglyceride and total cholesterol in serum of obese individuals caused by high-fat diet is improved, and the compound can be used for preparing medical products or health-care products for regulating intestinal flora, and has wide application value.

Description

Technical Field

[0001] This invention relates to a strain of Bifidobacterium adolescentis that alleviates obesity and reduces visceral fat tissue, and its applications, belonging to the field of microbial technology. Background Technology

[0002] Obesity is a chronic metabolic disease caused by excessive accumulation or abnormal distribution of fat due to various factors. Obesity is not merely weight gain, but a metabolic disorder. It significantly increases the risk of type 2 diabetes, fatty liver disease, cardiovascular disease, and certain cancers (such as breast cancer and colorectal cancer).

[0003] In obesity-related metabolic disorders, excessive accumulation of visceral fat is considered a key factor in the occurrence and development of the disease. Compared to subcutaneous fat, visceral fat is distributed deep within the abdominal cavity around organs, exhibiting higher metabolic activity and a stronger impact on endocrine function. Visceral fat cells readily produce excessive free fatty acids (FFA) and inflammatory factors (such as TNF-α and IL-6), accompanied by imbalances in adipokines secretion, such as leptin resistance and decreased adiponectin, leading to decreased insulin sensitivity and a systemic chronic inflammatory state. These abnormal signals significantly promote hepatic lipid deposition through the gut-liver-fat axis, leading to non-alcoholic fatty liver disease (NAFLD) and becoming a core driver of obesity-related complications such as diabetes and cardiovascular disease. Therefore, reducing visceral fat accumulation and improving its metabolic activity are crucial entry points for preventing and controlling obesity-related metabolic diseases.

[0004] Currently, the main interventions for obesity and excessive visceral fat include lifestyle modifications (such as dietary control and exercise intervention), drug therapy, bariatric surgery, and methods such as gut microbiota regulation (such as probiotics and prebiotics). Among these, probiotics have received widespread attention in obesity prevention and treatment due to their good safety profile and ability to regulate gut homeostasis. Obesity is often accompanied by gut microbiota dysbiosis. Gut microbiota dysbiosis can further lead to intestinal dysfunction and, through the gut-liver axis, cause significant liver dysfunction. This can result in intestinal inflammation, liver lipid accumulation, and abnormal levels of tryptophan and its metabolites such as indoleacetic acid and 3-indoleformin in the intestines and feces. Human and animal studies have shown that certain probiotics and prebiotics can repair gut microbiota imbalance and alleviate obesity. However, not all probiotics can promote weight loss; Dahi yogurt, containing Lactobacillus acidophilus, has not shown an anti-obesity effect.

[0005] Therefore, most probiotic products currently on the market lack the ability to precisely target visceral fat accumulation. Many reported strains have insufficiently in-depth research on their mechanisms of action, particularly regarding their specific targeting of visceral fat, improvement of adipose tissue inflammation, and insulin resistance pathways. The weight loss effects observed in practical applications are generally mild (especially in non-severely obese individuals), and evidence for visceral fat reduction requires higher-quality clinical data. Furthermore, the colonization ability of exogenous probiotics in the body varies, and the effects are often difficult to sustain. In addition, there are currently no patented inventions using probiotics to regulate host tryptophan metabolism and increase the level of 3-indolecarboxaldehyde (3-IAA) to reduce visceral fat. Therefore, there is an urgent need to screen for new probiotics that utilize novel pathways, effectively alleviate obesity, particularly significantly reduce visceral fat accumulation, improve related metabolic indicators, and have a well-defined safety profile, to provide safer and more effective novel intervention strategies. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to address the above-mentioned problems existing in the prior art by providing Bifidobacterium adolescentis that can colonize the human gut, alleviate the increase in tryptophan abundance associated with obesity, improve obesity, and reduce visceral adipose tissue.

[0007] This invention provides a Bifidobacterium adoltescentis strain CCFM1521, classified as Bifidobacterium adoltescentis, which was deposited on August 4, 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: 66802.

[0008] The present invention provides a composition containing the aforementioned Bifidobacterium adolescentis CCFM1521.

[0009] In one embodiment, the composition is a bacterial agent containing the Bifidobacterium adolescentis CCFM1521.

[0010] In one embodiment, the viable count of *Bifidobacterium adolescentis* CCFM1521 in the bacterial agent is ≥1×10⁻⁶. 6 CFU / g.

[0011] In one embodiment, the microbial agent can be prepared using conventional methods.

[0012] In some embodiments, the preparation method of the bacterial agent includes: culturing the Bifidobacterium adolescentis CCFM1521 in a culture medium under anaerobic conditions at 35-39°C for 24-48 hours, collecting the bacterial cells, and resuspending them with a protectant to obtain the bacterial agent.

[0013] In one embodiment, the method further includes: resuspending the bacterial cells with a protectant to achieve a bacterial concentration of 1×10⁻⁶. 10 CFU / mL.

[0014] In one embodiment, the culture medium is a modified MRS medium (mMRS), which is an MRS medium containing 0.5 g / L L-cysteine ​​hydrochloride.

[0015] In one embodiment, the modified MRS culture medium is prepared as follows: 10g tryptone, 10g beef extract, 5g yeast powder, 20g glucose, 5g sodium acetate, 2g diammonium hydrogen citrate, 2g dipotassium hydrogen phosphate, 0.5g magnesium sulfate heptahydrate, 1mL Tween 80, 0.25g manganese sulfate monohydrate, and 0.5g cysteine ​​hydrochloride are diluted with water to 1000mL, the pH is adjusted to 6.5, and sterilized at 115℃ for 15-25min.

[0016] In one embodiment, the protective agent is a mixture containing 100g / L-150g / L skim milk powder, 100g / L-150g / L maltodextrin, and 140g / L-160g / L trehalose at a final concentration.

[0017] In one embodiment, the bacterial cells are washed 2 to 4 times with phosphate buffer and then a protectant is added, wherein the pH of the phosphate buffer is 6.8-7.2.

[0018] In one embodiment, the drying can be carried out using any bacterial liquid drying process, including but not limited to vacuum freeze drying.

[0019] In some embodiments, the drying process involves pre-freezing at -15 to -20°C for 8-14 hours followed by vacuum freeze-drying.

[0020] This invention provides the use of the aforementioned Bifidobacterium adolescentis CCFM1521 in the preparation of drugs for relieving obesity, regulating tryptophan metabolism disorders, reducing visceral fat, and / or alleviating lipid accumulation.

[0021] In one embodiment, the reduction of visceral fat includes, but is not limited to, reducing the fat around visceral organs such as the liver, pancreas, intestines, kidneys, and stomach.

[0022] In one embodiment, reducing visceral fat includes reducing liver fat accumulation and lowering liver fat content; reducing epididymal fat weight and reducing epididymal fat accumulation.

[0023] In one embodiment, the obesity relief includes, but is not limited to, at least one of the following functions:

[0024] (1) Restore the gut microbiota imbalance caused by obesity;

[0025] (2) Improves the increase in liver weight and epididymal fat weight caused by obesity;

[0026] (3) Improves liver tissue damage and adipose tissue damage caused by obesity;

[0027] (4) Improves elevated total cholesterol (TC) levels caused by obesity;

[0028] (5) Improves elevated triglyceride (TG) levels caused by obesity;

[0029] (6) Reduce the elevated levels of alanine aminotransferase (ALT) caused by obesity;

[0030] (7) Reduces the levels of liver inflammatory factors IL-6, IL-1β, and TNF-α in obese individuals;

[0031] (8) Reduces the levels of total cholesterol (TC) and triglycerides (TG) in the liver of obese individuals;

[0032] In one embodiment, the regulation of tryptophan metabolism disorder includes reducing the level of tryptophan in the gut environment of obese individuals and increasing the level of 3-indoleform in the gut environment of obese individuals.

[0033] In one embodiment, the liver tissue damage includes, but is not limited to, symptoms such as hepatocyte vesicular steatosis and interstitial inflammatory cell infiltration.

[0034] In one embodiment, the adipose tissue damage includes, but is not limited to, irregularly shaped and unevenly distributed fat cells.

[0035] In one embodiment, the drug uses Bifidobacterium adolescentis CCFM1521 as the sole active ingredient.

[0036] In one embodiment, the drug comprises Bifidobacterium adolescentis CCFM1521 and a pharmaceutically acceptable drug carrier or excipient.

[0037] The present invention also provides the application of the aforementioned Bifidobacterium adolescentis CCFM1521 in the preparation of health products that help control body fat and / or regulate gut microbiota.

[0038] In one embodiment, the health product contains the Bifidobacterium adolescentis CCFM1521.

[0039] In one embodiment, the health product includes, but is not limited to, fermented foods.

[0040] Beneficial effects: This invention provides a strain of Bifidobacterium adolescentis CCFM1521 that can rapidly colonize the intestines and can be used to alleviate obesity, reduce visceral adipose tissue, and regulate tryptophan metabolism disorders, specifically in the following ways:

[0041] (1) It has the function of regulating the gut-liver axis and can alleviate lipid accumulation and inflammation associated with obesity;

[0042] (2) Restore the reduction in individual fecal 3-indolecarboxaldehyde levels caused by a high-fat diet;

[0043] (3) Restore the abnormal abundance of Bifidobacterium in the individual gut microbiota caused by high fat intake;

[0044] (4) Significantly improves pathological damage and lipid accumulation in liver tissue of obese individuals caused by high-fat diet; significantly reduces abnormal increase in liver weight and epididymal fat weight induced by high-fat diet in obese individuals.

[0045] (5) Significantly improves the levels of liver lipid metabolism factors and liver inflammatory factors in obese individuals induced by a high-fat diet, and alleviates the increase in serum triglyceride and total cholesterol levels caused by a high-fat diet.

[0046] The Bifidobacterium adolescentis CCFM1521 provided by this invention can be used to prepare drugs that alleviate obesity, reduce visceral adipose tissue, regulate intestinal flora, and regulate obesity-related lipid accumulation and tryptophan metabolism disorders, and has a very broad application prospect.

[0047] Biological Preservation Instructions

[0048] Bifidobacterium adoltescentis (CCFM1521), classified as Bifidobacterium adoltescentis, was deposited on August 4, 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: 66802. Attached Figure Description

[0049] Figure 1 This is a colony characteristic of Bifidobacterium adolescentis CCFM1521.

[0050] Figure 2 The effect of Bifidobacterium adolescentis CCFM1521 on weight gain in high-fat diet-induced obese mice.

[0051] Figure 3 The effect of Bifidobacterium adolescentis CCFM1521 on liver tissue structure in high-fat diet-induced obese mice.

[0052] Figure 4 The effect of Bifidobacterium adolescentis CCFM1521 on the abundance of gut microbiota in obese mice induced by a high-fat diet; (P<0.05).

[0053] Figure 5 The effect of Bifidobacterium adolescentis CCFM1521 on liver weight and epididymal fat weight in obese mice induced by a high-fat diet; (P<0.05).

[0054] Figure 6 The effect of Bifidobacterium adolescentis CCFM1521 on total cholesterol (TC) and triglycerides (TG) in the liver of obese mice induced by a high-fat diet (P<0.05).

[0055] Figure 7 The effect of Bifidobacterium adolescentis CCFM1521 on liver inflammatory factors IL-6, IL-1β, and TNF-α in obese mice induced by a high-fat diet; (P<0.05).

[0056] Figure 8 The effect of Bifidobacterium adolescentis CCFM1521 on serum total cholesterol (TC) in obese mice induced by a high-fat diet; (P<0.05).

[0057] Figure 9 The effect of Bifidobacterium adolescentis CCFM1521 on serum triglyceride (TG) in obese mice induced by a high-fat diet; (P<0.05).

[0058] Figure 10 The effect of Bifidobacterium adolescentis CCFM1521 on tryptophan and 3-indoleformin levels in high-fat diet-induced obese mice; (P<0.05).

[0059] Figure 11 The effect of Bifidobacterium adolescentis CCFM1521 on serum alanine aminotransferase (ALT) in obese mice induced by a high-fat diet; (P<0.05). Detailed Implementation

[0060] To further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0061] Unless otherwise specified, all reagents involved in the embodiments of this invention are commercially available products and can be purchased through commercial channels.

[0062] (I) Strain Information

[0063] The Bifidobacterium adolescentis CCFM1521 described in this invention has the following biological characteristics:

[0064] (1) Bacterial characteristics: Gram-positive bacteria that do not form spores and are non-motile.

[0065] (2) Colony characteristics: After 36 hours of anaerobic culture, distinct colonies are formed, with a diameter between 0.5-2 mm. The colonies are round on the front and raised on the side, with neat edges, milky white color, opaque, moist and smooth surface, and do not produce pigments. See Appendix. Figure 1 .

[0066] (3) Growth characteristics: Under constant temperature anaerobic conditions at 37℃, the logarithmic phase is reached after about 24 hours of culture in mMRS medium.

[0067] (4) It can significantly alleviate the increase in individual weight caused by a high-fat diet;

[0068] (5) It can significantly improve pathological damage and lipid accumulation in individual liver tissue caused by a high-fat diet;

[0069] (6) It can significantly reduce the increase of liver lipid metabolism factors and liver inflammatory factors induced by high-fat diet in individual mice;

[0070] (7) It can regulate the levels of triglycerides and total cholesterol in an individual's serum, reducing them to normal levels;

[0071] (8) It can reduce the level of tryptophan in individual feces and increase the level of 3-indolecarboxaldehyde;

[0072] (9) It can significantly restore the imbalance of Bifidobacterium flora in the gut caused by a high-fat diet.

[0073] (II) Culture medium

[0074] The modified MRS medium (mMRS) is prepared as follows: 10g tryptone, 10g beef extract, 5g yeast powder, 20g glucose, 5g sodium acetate, 2g diammonium hydrogen citrate, 2g dipotassium hydrogen phosphate, 0.5g magnesium sulfate heptahydrate, 1mL Tween 80, 0.25g manganese sulfate monohydrate, and 0.5g cysteine ​​hydrochloride are diluted with water to 1000mL. The pH is adjusted to 6.5, and the medium is sterilized at 115℃ for 15-25 minutes.

[0075] Example 1: Screening and identification of Bifidobacterium adolescentis CCFM1521

[0076] (I) Isolation and screening of Bifidobacterium adolescentis

[0077] (1) Take 1g of fresh feces from a 35-year-old Mongolian woman in Wusu City, Xinjiang Uygur Autonomous Region. After serial dilution, spread it on mMRS solid medium and incubate at 37℃ for 72 hours under anaerobic conditions.

[0078] (2) Observe and record the colony morphology, and pick up colonies to streak and purify.

[0079] (3) In mMRS liquid medium, culture at 37°C for 48 hours, and Gram stain the obtained colonies to record the colony morphology.

[0080] (4) Discard the Gram-negative bacterial strains and Gram-positive cocci in the colonies and select the Gram-positive bacilli.

[0081] (5) After catalase analysis, discard catalase-positive strains and retain catalase-negative strains.

[0082] (II) Preliminary identification of Bifidobacteria: The fructose-6-phosphate phosphoketonease assay was used, and the specific steps were as follows:

[0083] (l) The lactic acid bacteria screened in step (i) are cultured in liquid mMRS culture medium for 24 h, and then 1 mL of culture is centrifuged at 8000 rpm for 2 min.

[0084] (2) Wash twice with a 0.05M KH2PO4 solution containing 0.05% (mass percentage) cysteine ​​hydrochloride at pH 6.5;

[0085] (3) Resuspend in 200 μL of the above phosphate buffer with 0.25% (w / w) Triton X-100 added;

[0086] (4) Add 50 μL of a mixture of sodium fluoride at a concentration of 6 mg / mL and sodium iodoacetate at a concentration of 10 mg / mL, and 50 μL of fructose-6-phosphate at a concentration of 80 mg / mL, and incubate at 37°C for 1 h;

[0087] (5) Add 300 μL of hydroxylamine hydrochloride at a concentration of 0.139 g / mL and pH 6.5, and let it stand at room temperature for 10 min;

[0088] (6) Add 200 μL of 15% (mass percentage) trichloroacetic acid and 4M HCl respectively;

[0089] (7) Add 200 μL of 0.1 M HCl containing 5% (mass percentage) ferric chloride. The system quickly turns red, indicating that it is F6PPK positive and is preliminarily identified as Bifidobacterium.

[0090] (III) Molecular biological identification of Bifidobacteria

[0091] (l) Single-strain genome extraction (performed according to the TIANamp Bacteria DNA kit procedure)

[0092] A. Culture the lactic acid bacteria selected in step (II) overnight, take 1 mL of bacterial suspension into a 1.5 mL centrifuge tube, centrifuge at 10,000 rpm (~11,500×g) for 1 min, and aspirate the supernatant as thoroughly as possible;

[0093] B. Add 180 μL of buffer solution (20 mM Tris, pH 8.0; 2 mM Na2-EDTA; 1.2% Triton; lysozyme with a final concentration of 20 mg / mL (lysozyme must be prepared by dissolving lysozyme powder in buffer solution, otherwise the lysozyme will be inactive)) to the bacterial pellet and treat at 37°C for at least 30 min.

[0094] C. Add 20 μL of Proteinase K solution to the tube and mix well;

[0095] D. Add 220 μL buffer GB, shake for 15 seconds, incubate at 70°C for 10 min, the solution should become clear, and briefly centrifuge to remove water droplets from the inner wall of the tube cap.

[0096] E. Add 220 μL of anhydrous ethanol and shake thoroughly for 15 seconds. At this time, flocculent precipitate may appear. Briefly centrifuge to remove water droplets from the inner wall of the tube cap.

[0097] F. Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3 (place the adsorption column in the collection tube), centrifuge at 12,000 rpm (~13,400×g) for 30 seconds, discard the waste liquid, and place the adsorption column CB3 into the collection tube;

[0098] G. Add 500 μL of buffer GD to the adsorption column CB3 (please check whether anhydrous ethanol has been added before use), centrifuge at 12,000 rpm (~13,400×g) for 30 seconds, discard the waste liquid, and put the adsorption column CB3 into the collection tube.

[0099] H. Add 600 μL of washing buffer PW to the adsorption column CB3 (please check whether anhydrous ethanol has been added before use), centrifuge at 12,000 rpm (~13,400×g) for 30 seconds, discard the waste liquid, and place the adsorption column CB3 into the collection tube; repeat the operation once.

[0100] I. Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm (~13,400 × g) for 2 min, and discard the waste liquid. Place the adsorption column CB3 at room temperature for several minutes to thoroughly dry any residual washing liquid in the adsorption material;

[0101] J. Transfer the adsorption column CB3 into a clean centrifuge tube, add 50-200 μL of elution buffer TE dropwise to the middle of the adsorption membrane, incubate at room temperature for 2-5 min, centrifuge at 12,000 rpm (~13,400×g) for 2 min, and collect the solution into the centrifuge tube.

[0102] (2) Whole genome sequencing

[0103] The extracted whole genome was sent to a professional sequencing company, where the whole genome of the bacteria was sequenced using a next-generation sequencer. The obtained sequence results were searched and compared for similarity in GeneBank using BLAST. It was identified as Bifidobacterium adolescentis, and this strain was significantly different from the previously reported Bifidobacterium adolescentis genes, thus identifying it as a new strain.

[0104] Example 2: Bifidobacterium adolescentis CCFM1521 reduces weight gain in high-fat induced obese mice

[0105] Thirty healthy male C57BL / 6J mice weighing 18-20g were used and acclimatized to the environment for one week. They were then randomly divided into six groups: low-fat control group (LFD), high-fat model control group (HFDD), Bifidobacterium adolescentis CCFM1521 intervention group (CCFM1521), Bifidobacterium adolescentis NY15 intervention group (NY15), Bifidobacterium adolescentis FXJWS23M45 intervention group (FXJWS23M45), and Bifidobacterium adolescentis HeNa1610 intervention group (HeNa1610). Each group contained eight mice.

[0106] The concentration of the bacterial suspension administered via gavage was 2.0 × 10⁻⁶. 9 CFU / mL. The grouping and treatment methods of the experimental animals are shown in Table 4. Among them, the low-fat diet was TP23302 from Nantong Trofi Company, with fat energy supply of 10%, and the high-fat diet was TP23300 from Nantong Trofi Company, with fat energy supply of 60%.

[0107] The bacterial suspension was prepared as follows: Each bacterial strain was inoculated into MRS solid medium and cultured at 37℃ for 24 h to obtain single colonies. These single colonies were then inoculated into MRS liquid medium and cultured at 37℃ for 12 h for activation. After three generations of activation, the bacterial suspension was inoculated at a rate of 2% into 1 L of MRS liquid medium, shaken to mix, and then cultured in an anaerobic incubator at 37℃ for 24 h. The suspension was centrifuged at 8000 g / min at 4℃ for 15 min, the supernatant was removed, and the suspension was washed twice with sterile physiological saline. After centrifugation under the same conditions and removal of the supernatant, the suspension was resuspended in 30% glycerol to obtain the bacterial suspension for gavage. This suspension was then stored at -80℃ for one week. Before animal experiments, the frozen bacterial suspension was removed from the freezer, centrifuged at 6000 r / min for 5 min, washed twice with sterile physiological saline, resuspended in physiological saline, shaken to mix, and the number of viable bacteria was determined using the pour plate method initially and after one week of storage. The results showed that the initial viable count was 4.5 × 10⁻⁶. 9 CFU / mL, viable bacterial count after 1 week was 3.8 × 10⁻⁶. 9 The cfu / mL concentration did not change in order of magnitude, indicating that freezing the bacterial culture would not affect the experiment and it can be used for animal experiments.

[0108] Table 1. Grouping and treatment methods of experimental animals

[0109]

[0110] The day before the end of the experiment, the mice were placed in a weighing scale, weighed precisely, and the weight of each mouse was recorded. The experimental results are as follows: Figure 2 As shown, compared to the low-fat control group (LFD), the high-fat model control group (HFD) mice experienced a significantly increased weight gain, representing 400% of the LFD gain. In the *Bifidobacterium adolescentis* CCFM1521 intervention group (CCFM1521), the intake of *Bifidobacterium adolescentis* CCFM1521 reduced the weight gain induced by the high-fat diet by 100% compared to the low-fat diet, and its ability to reduce weight gain was significantly stronger than that of *Bifidobacterium adolescentis* FXJWS23M45 (published in the paper "Mechanism Study of Bifidobacterium Biotransformation of Conjugated Linoleic Acid"), *Bifidobacterium adolescentis* HeNa1610 (published in the paper "Analysis of Antibiotic Resistance and Transfer Risk Assessment of Bifidobacterium"), and *Bifidobacterium adolescentis* NY15 (published in the paper "Analysis of Antibiotic Resistance and Transfer Risk Assessment of Bifidobacterium"). *Bifidobacterium adolescentis* FXJWS23M45, *Bifidobacterium adolescentis* HeNa1610, and *Bifidobacterium adolescentis* NY15 did not show a significant ability to reduce weight gain.

[0111] Example 3: Bifidobacterium adolescentis CCFM1521 has a good restorative effect on liver tissue damage and fat accumulation in high-fat induced obese mice.

[0112] The grouping, modeling, and treatment methods for C57BL / 6J mice were the same as in Example 2. The difference was that at the end of the experiment, the mice were fasted for 12 hours but allowed free access to water. After anesthetizing the mice with isoflurane, blood was collected from the orbital cavity, and the mice were euthanized by cervical dislocation. The collected blood was centrifuged at 3000×g and 4℃ for 10 min, and the supernatant was collected and frozen at -80℃ for later use. Liver and adipose tissue were collected and quickly rinsed in ice-cold physiological saline to remove blood, then fixed in paraformaldehyde. Small intestine was collected and immediately placed in liquid nitrogen at -80℃ for later use.

[0113] Paraffin sections were prepared from liver and adipose tissue, stained with hematoxylin and eosin (HE), and observed and photographed under a light microscope for pathological evaluation. The experimental results are as follows: Figure 3 As shown. The specific steps for HE staining are as follows:

[0114] (1) Fixation: Wash the tissue sample with physiological saline and immediately immerse it in neutral paraformaldehyde fixative (4%) for fixation. The fixation time is generally within 72 hours.

[0115] (2) Washing: Rinse with running water or soak for several hours or overnight.

[0116] (3) Dehydration: The sample was dehydrated in 70%, 80% and 90% ethanol solutions for 30 min each, then placed in 95% ethanol solution once for 20 min and 100% ethanol solution twice for 10 min each time.

[0117] (4) To make it clear: 1 / 2 pure alcohol + 1 / 2 xylene mixture for 10 min, xylene I for 10 min, xylene II for 10 min (until it becomes clear).

[0118] (5) Wax impregnation: Immerse the sample in paraffin (62℃) for 2 hours.

[0119] (6) Embedding: The largest surface is placed at the bottom layer so that the cut surface tissue surface occupies the largest area.

[0120] (7) Slicing: Use a manual slicer to slice the wax block into segments with a thickness of 5μm.

[0121] (8) Spreading and sticking slices: Turn on the water bath and keep the water temperature at 42°C so that the slices are spread flat on the water surface.

[0122] (9) Baking the slides: Place the slides along with the slide holder into a drying oven at 55°C for about 2 hours until the wax melts.

[0123] (10) Hydration: Paraffin sections were dewaxed by xylene I and II for 10 min each, then placed in 100%, 95%, 90%, 80%, and 70% alcohol solutions for 5 min each, and then placed in distilled water for 3 min.

[0124] (11) Initial staining: Place the slices in hematoxylin and stain for about 20 seconds.

[0125] (12) Washing: Rinse with running tap water for about 15 minutes. This will turn the sections blue, but be careful not to let the water flow too strong to prevent the sections from falling off.

[0126] (13) Differentiation: Place the slices in a 1% hydrochloric acid ethanol solution to decolorize for 7 seconds. The slices should turn red and the color should be light.

[0127] (14) Rinse: Rinse the slices again in running tap water for 15-20 minutes to restore their blue color.

[0128] (15) Counterstaining: Immerse in eosin staining solution and immediately remove for dehydration.

[0129] (16) Dehydration: The slices were sequentially rinsed with 95% ethanol I, 95% ethanol II, and 70% ethanol, then immersed in 80% ethanol for 50 seconds and anhydrous ethanol for 2 minutes.

[0130] (17) Transparency: Place the slice in 1 / 2 anhydrous ethanol and 1 / 2 xylene for 1 min, and then in xylene I and II for 2 min each.

[0131] (18) Mounting: After the sections are cleared with xylene, neutral resin is used as a mounting agent. The resin can be diluted with xylene to a suitable consistency.

[0132] like Figure 3 As shown, compared with a low-fat diet (LFD), a high-fat diet (HFD) can lead to vesicular steatosis of hepatocytes, and some mice have interstitial inflammatory cell infiltration. The intervention group of Bifidobacterium adolescentis CCFM1521 (CCFM1521) can significantly improve the above-mentioned lesions by gavage administration of Bifidobacterium adolescentis CCFM1521, and the effect is significantly better than Bifidobacterium adolescentis FXJWS23M45, Bifidobacterium adolescentis HeNa1610 and Bifidobacterium adolescentis NY15 groups.

[0133] Example 4: Bifidobacterium adolescentis CCFM1521 has a restorative effect on intestinal flora imbalance caused by a high-fat diet.

[0134] The grouping, modeling, and treatment methods for C57BL / 6J mice are shown in Table 4. Before the end of the experiment, fresh feces were collected from the mice, metagenomic data were extracted, and the gut microbiota structure was analyzed using a next-generation sequencer.

[0135] The results are as follows Figure 4As shown in the figure, compared to the LFD group, the relative abundance of *Bifidobacterium* gut microbiota in the feces of mice in the high-fat diet control group (HFD) was significantly reduced, by approximately 700%. However, in the *Bifidobacterium adolescentis* CCFM1521 intervention group (CCFM1521), the intake of *Bifidobacterium adolescentis* CCFM1521 significantly increased the relative abundance of this genus, with an increase 17 times that of the high-fat diet group and twice that of the low-fat diet group. This indicates that *Bifidobacterium adolescentis* CCFM1521 can not only colonize the gut but also increase the content of *Bifidobacterium* in the gut. Analysis suggests that *Bifidobacterium adolescentis* CCFM1521 can interact with other gut microbiota, thereby more effectively utilizing tryptophan and producing downstream metabolites, such as indoles or tryptophan-derived signaling molecules, thereby regulating host obesity and the metabolic environment, indirectly promoting its own growth and that of other *Bifidobacterium* species. Other strains, after entering the gut, have a weaker ability to regulate the overall tryptophan metabolic pathway of the gut microbiota, and therefore have a limited effect on increasing the overall abundance of *Bifidobacterium*.

[0136] Example 5: Bifidobacterium adolescentis CCFM1521 can inhibit the increase of liver and epididymal fat weight in high-fat induced obese mice.

[0137] The grouping, modeling, and treatment methods for C57BL / 6J mice were the same as in Example 2. The difference was that at the end of the experiment, the mice were fasted for 12 hours but allowed free water. After the mice were sacrificed, liver tissue and epididymal adipose tissue were obtained. The entire liver of the mice was weighed on an analytical balance, and the entire epididymal adipose tissue of the mice was weighed on an analytical balance. The results were recorded.

[0138] The results are as follows Figure 5 As shown, compared to the low-fat diet group (LFD), the liver weight and epididymal fat weight of high-fat diet model (HFD) mice were significantly increased, with liver weight increasing by 172% and epididymal fat weight increasing by 339%. The *Bifidobacterium adolescentis* CCFM1521 intervention group showed a 21% reduction in liver weight and epididymal fat weight compared to the HFD model mice, resulting in a liver weight increase of only 137% of that in the LFD group. Its ability to reduce liver and epididymal fat weight was significantly stronger than that of *Bifidobacterium adolescentis* FXJWS23M45, *Bifidobacterium adolescentis* HeNa1610, and *Bifidobacterium adolescentis* NY15. *Bifidobacterium adolescentis* FXJWS23M45, *Bifidobacterium adolescentis* HeNa1610, and *Bifidobacterium adolescentis* NY15 did not show a significant ability to reduce the increase in liver and epididymal fat weight in HFD mice.

[0139] Example 6: Bifidobacterium adolescentis CCFM1521 can reduce the level of liver lipid metabolism in high-fat induced obese mice.

[0140] The grouping, modeling, and treatment methods for C57BL / 6J mice were the same as in Example 2. The difference was that at the end of the experiment, the mice were fasted for 12 hours but allowed free access to water. After the mice were sacrificed, their liver tissue was obtained, and the content of total triglycerides (TG) and total glycerol cholesterol (TC) in the liver tissue was determined according to the detection method of a commercial kit.

[0141] The results are as follows Figure 6 As shown, compared to the low-fat diet group (LFD), the total triglycerides (TG) and total glycerol cholesterol (TC) in the liver of high-fat diet model (HFD) mice were significantly increased, with TC increasing by 183% and TG increasing by 228%. The *Bifidobacterium adolescentis* CCFM1521 intervention group reduced total triglycerides (TG) and total glycerol cholesterol (TC) in the liver of HFD mice by 22% and 35%, respectively, resulting in an increase in TG of only 144% and an increase in TC of only 145% of the LFD group. Its ability to reduce total triglycerides (TG) and total glycerol cholesterol (TC) in the liver of mice was significantly stronger than that of *Bifidobacterium adolescentis* FXJWS23M45, *Bifidobacterium adolescentis* HeNa1610, and *Bifidobacterium adolescentis* NY15. *Bifidobacterium adolescentis* FXJWS23M45, *Bifidobacterium adolescentis* HeNa1610, and *Bifidobacterium adolescentis* NY15 did not show a significant ability to reduce the increase in liver TC and TG.

[0142] Example 7: Bifidobacterium adolescentis CCFM1521 can enhance the accumulation of inflammatory factors in the liver of high-fat induced obese mice.

[0143] The grouping, modeling, and treatment methods for C57BL / 6J mice were the same as in Example 2. The difference was that at the end of the experiment, the mice were fasted for 12 hours but allowed free access to water. After the mice were sacrificed, their liver tissue was obtained, and the levels of interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α in the liver tissue were measured according to the detection method of the kit.

[0144] like Figure 7As shown, compared to the LFD group mice, the high-fat diet model (HFD) induced obese mice showed significant accumulation of liver inflammation. The CCFM1521 intervention group (Bifidobacterium adolescentis CCFM1521) administered by gavage reduced interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α by 18%, 23%, and 7%, respectively, compared to the model mice. CCFM1521 also showed stronger effects than Bifidobacterium adolescentis FXJWS23M45, HeNa1610, and NY15. Bifidobacterium adolescentis FXJWS23M45, HeNa1610, and NY15 did not show significant ability to reduce liver inflammation. These results are consistent with the liver pathological tissue damage detection results, suggesting that Bifidobacterium adolescentis CCFM1521 can alleviate liver inflammation and inflammatory factor accumulation in high-fat diet-induced obese mice.

[0145] Example 8: Bifidobacterium adolescentis CCFM1521 can reduce serum total cholesterol (TC) levels in high-fat induced obese mice.

[0146] The grouping, modeling, and treatment methods for C57BL / 6J mice were the same as in Example 2. The difference was that at the end of the experiment, the mice were fasted for 12 hours but allowed free access to water. After isoflurane inhalation anesthesia, blood was collected from the orbital cavity. The mice were then euthanized by cervical dislocation. The obtained blood was centrifuged at 3000×g and 4℃ for 10 min, and the supernatant was used to determine the total cholesterol (TC) content in the blood according to the kit's detection method. The experimental results are as follows: Figure 8 As shown.

[0147] like Figure 8 As shown, compared with low-fat diet mice (LFD), the serum total cholesterol (TC) level in the high-fat diet model control group (HFD) was significantly abnormal. The serum total cholesterol level in the *Bifidobacterium adolescentis* CCFM1521 intervention group was reduced by 14% compared with the HFD model mice, and its effect was stronger than that of *Bifidobacterium adolescentis* FXJWS23M45, *Bifidobacterium adolescentis* HeNa1610, and *Bifidobacterium adolescentis* NY15. *Bifidobacterium adolescentis* FXJWS23M45, *Bifidobacterium adolescentis* HeNa1610, and *Bifidobacterium adolescentis* NY15 did not show a significant ability to reduce serum TC in mice.

[0148] Example 9: Bifidobacterium adolescentis CCFM1521 can reduce serum total triglyceride (TG) levels in mice with metabolic syndrome.

[0149] The grouping, modeling, and treatment methods for C57BL / 6J mice were the same as in Example 2. The difference was that at the end of the experiment, the mice were fasted for 12 hours but allowed free access to water. After isoflurane inhalation anesthesia, blood was collected from the orbital cavity. The mice were then euthanized by cervical dislocation. The obtained blood was centrifuged at 3000×g and 4℃ for 10 min, and the supernatant was used to determine the total triglyceride (TG) content in the blood according to the kit's detection method. The experimental results are as follows: Figure 9 As shown.

[0150] Depend on Figure 9 It can be seen that, compared with the low-fat diet group (LFD) mice, the serum total triglyceride (TG) levels in the high-fat model control group (HFD) mice were significantly abnormal. The serum total triglyceride levels in the *Bifidobacterium adolescentis* CCFM1521 intervention group (CCFM1521) were reduced by 33% compared with the HFD model mice, and the effect was stronger than that of *Bifidobacterium adolescentis* FXJWS23M45, *Bifidobacterium adolescentis* HeNa1610, and *Bifidobacterium adolescentis* NY15. *Bifidobacterium adolescentis* FXJWS23M45, *Bifidobacterium adolescentis* HeNa1610, and *Bifidobacterium adolescentis* NY15 did not show a significant ability to reduce serum TG.

[0151] Example 10: Effects of Bifidobacterium adolescentis CCFM1521 on fecal tryptophan and 3-indolecarboxaldehyde in high-fat induced obese mice

[0152] The grouping, modeling, and treatment methods for C57BL / 6J mice were the same as in Example 2. The difference was that, one day before the end of the experiment, the mice were placed in metabolic cages, their feces were collected, and stored at -80℃ for later use. The obtained feces underwent grinding, crushing, and multiple extraction processes, the specific extraction process of which is as follows:

[0153] (1) Freeze-drying: The obtained fecal samples were placed in a freeze dryer and freeze-dried for 48 hours before being taken out.

[0154] (2) Weighing: Weigh the sample on a balance and record its weight.

[0155] (3) Extraction: Add 50 mg of lyophilized sample to 900 μL of MeOH / H2O (1:1). Vortex for 15 s, homogenize using a tissue homogenizer (the homogenized sample can be incubated overnight at 4°C), then centrifuge at 15000×g for 10 min. Collect the supernatant and concentrate under vacuum at 45°C (approximately 2–4 h). Resuspend in 100 μL of MeOH / H2O (1:9), centrifuge at 15000×g for 10 min, filter through a 0.2 μm filter membrane, and detect using a Thermo Fisher QE liquid chromatography-mass spectrometry system.

[0156] like Figure 10As shown, *Bifidobacterium adolescentis* CCFM1521 reduced the abundance of tryptophan (Trp) in the feces of HFD-treated mice by 44% and increased the abundance of 3-indolecarboxaldehyde (IAID) in the feces by 171%, while other *Bifidobacterium adolescentis* strains FXJWS23M45, HeNa1610, and NY15 did not have this significant effect. These results, combined with the increase in mouse body weight, hepatic lipid accumulation, and accumulation of inflammatory factors, suggest that only *Bifidobacterium adolescentis* CCFM1521 can alleviate liver inflammatory damage and inflammatory factor accumulation in high-fat diet-induced obese mice by regulating tryptophan levels.

[0157] Example 11: Bifidobacterium adolescentis CCFM1521 can reduce serum alanine aminotransferase (ALT) levels in mice with metabolic syndrome.

[0158] The grouping, modeling, and treatment methods for C57BL / 6J mice were the same as in Example 2. The difference was that at the end of the experiment, the mice were fasted for 12 hours but allowed free access to water. After isoflurane inhalation anesthesia, blood was collected from the orbital cavity. The mice were then euthanized by cervical dislocation. The obtained blood was centrifuged at 3000×g and 4℃ for 10 min, and the supernatant was used to determine the alanine aminotransferase (ALT) content in the blood according to the kit's detection method. The experimental results are as follows: Figure 11 As shown.

[0159] Depend on Figure 11 It can be seen that, compared with the low-fat diet group (LFD) mice, the high-fat model control group (HFD) mice had abnormal serum alanine aminotransferase (ALT) levels. The serum ALT levels in the Bifidobacterium adolescentis CCFM1521 intervention group were reduced by 43% compared with HFD mice, and the effect was stronger than that of Bifidobacterium adolescentis FXJWS23M45, HeNa1610, and NY15. Bifidobacterium adolescentis FXJWS23M45, HeNa1610, and NY15 did not show a significant ability to reduce serum ALT in mice.

[0160] Example 12: Comparison of the effects of Bifidobacterium adolescentis CCFM1521 and Bifidobacterium adolescentis CCFM1062

[0161] After normalizing the data, a comparison was made between the effects of Bifidobacterium adolescentis CCFM1521 of this invention and Bifidobacterium adolescentis CCFM1062 disclosed in CN110432332A. The results showed that, compared with Bifidobacterium adolescentis CCFM1062, Bifidobacterium adolescentis CCFM1521 significantly reduced liver weight and epididymal fat weight in mice induced by a high-fat diet, while CCFM1062 did not show this effect. Furthermore, compared with other data from Bifidobacterium adolescentis CCFM1062 intervention, Bifidobacterium adolescentis CCFM1521 showed a 14% increase in reducing total cholesterol (TC) levels in the liver of mice induced by a high-fat diet, a 3% increase in reducing total triglyceride (TG) levels in the liver of mice induced by a high-fat diet, and a 5% increase in reducing serum ALT levels in mice induced by a high-fat diet, demonstrating superior effects in alleviating obesity and reducing visceral adipose tissue content.

[0162] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Bifidobacterium adoltescentis CCFM1521 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 4, 2025, with accession number GDMCC No: 66802.

2. An agent containing the Bifidobacterium adolescentis CCFM1521 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The viable count of *Bifidobacterium adolescentis* CCFM1521 in the bacterial agent is ≥1×10⁻⁶. 6 CFU / g.

4. A drug containing the Bifidobacterium adolescentis CCFM1521 as described in claim 1.

5. The use of Bifidobacterium adolescentis CCFM1521 as described in claim 1 in the preparation of a medicament for relieving obesity, regulating tryptophan metabolism disorders, reducing visceral fat, and / or alleviating lipid accumulation.

6. The application according to claim 5, characterized in that, The reduction of visceral fat includes reducing liver fat accumulation and / or lowering liver fat content.

7. The application according to claim 5, characterized in that, The obesity relief function includes, but is not limited to, at least one of the following: (1) Restore the gut microbiota imbalance caused by obesity; (2) Improves the increase in liver weight and epididymal fat weight caused by obesity; (3) Improves liver tissue damage and adipose tissue damage caused by obesity; (4) Improves elevated total cholesterol levels caused by obesity; (5) Improves elevated triglyceride levels caused by obesity; (6) Reduce the elevated alanine aminotransferase levels caused by obesity; (7) Reduces the levels of liver inflammatory factors IL-6, IL-1β, and TNF-α in obese individuals; (8) Reduce the levels of total cholesterol and triglycerides in the liver of obese individuals.

8. The application according to any one of claims 5 to 7, characterized in that, The drug includes Bifidobacterium adolescentis CCFM1521, and pharmaceutically acceptable drug carriers or excipients.

9. The use of Bifidobacterium adolescentis CCFM1521 as described in claim 1 in the preparation of health products that help control body fat and / or regulate gut microbiota.

10. The application according to claim 9, characterized in that, The health products mentioned include, but are not limited to, fermented foods.

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