Lactobacillus rhamnosus YYSJ019 strain for relieving obesity and application of lactobacillus rhamnosus YYSJ019 strain

Abstract

The invention relates to a lactobacillus rhamnosus YYSJ019 strain for relieving obesity and application of the lactobacillus rhamnosus YYSJ019 strain. The lactobacillus rhamnosus YYSJ019 strain is classified and named as lactobacillus rhamnosus, and the preservation number of the lactobacillus rhamnosus YYSJ019 strain is CGMCC (China General Microbiological Culture Collection Center) No.33472. The strain has good tolerance and high surface hydrophobicity in gastric juice, and has the potential of smoothly entering the gastrointestinal tract of a human body; the bacterial strain has excellent intestinal adhesion capability, and provides a basis for continuously playing a probiotic effect; the strain can slow down weight gain of obese mice and improve fat accumulation, liver injury and inflammation in bodies of the obese mice; the blood sugar metabolism capability and the blood fat metabolism capability can also be improved, and the potential of preventing and treating hyperglycemia or hyperlipidemia is achieved; in addition, a mouse small intestine endocrine cell line (STC-1 cell line) can be stimulated to generate the glucagon-like peptide-1, and the glucagon-like peptide-1 has the potential of improving diseases caused by insufficient secretion of the glucagon-like peptide-1; the effects of reducing uric acid in vitro and reducing uric acid in vivo are achieved, and the potential of preventing and treating hyperuricemia is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of new strain development and application, and relates to a Lactobacillus rhamnosus YYSJ019 strain for alleviating obesity and an application thereof. Background Art

[0002] Obesity is a chronic metabolic disease characterized by excessive body fat accumulation, often accompanied by imbalanced energy metabolism and multiple health risks. Obesity is closely associated with metabolic syndrome, type 2 diabetes, and cardiovascular disease. While traditional interventions (such as diet, medication, and surgery) have some effectiveness, they are limited by high side effects and a high risk of relapse.

[0003] In recent years, many research results have confirmed that probiotics have become a hot strategy for improving obesity by regulating intestinal flora to promote metabolism. Obese people may have characteristic changes in intestinal flora diversity and disordered flora structure. This flora imbalance can affect host metabolism through multiple mechanisms: (1) Impaired energy metabolism regulation: reduced short-chain fatty acids produced by bacterial fermentation of dietary fiber, leading to weakened liver gluconeogenesis inhibition and increased adipose tissue lipolysis; (2) Intestinal barrier damage: downregulation of tight junction protein expression, increased endotoxin translocation, and induction of chronic low-grade inflammation; (3) Bile acid metabolism disorder: affecting the farnesoid X receptor and G protein-coupled bile acid receptor 1 signaling pathways, interfering with glucose and lipid metabolism balance; (4) Dysfunction of the brain-gut axis: affecting the secretion of metabolic regulatory peptides by enteroendocrine cells, leading to abnormal appetite regulation and excessive energy intake.

[0004] Given the close relationship between intestinal flora and obesity symptoms, microecological intervention has become a current research hotspot. Currently, there are still few types of probiotic products that can effectively improve obesity. Therefore, it is very meaningful to find and develop more probiotic intervention strategies. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the object of the present invention is to provide a rhamnosus Lactobacillus YYSJ019 strain and its application for alleviating obesity, and in particular to provide a rhamnosus Lactobacillus YYSJ019 strain and its application that can alleviate obesity, improve sugar metabolism and lipid metabolism, and improve hyperuricemia.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a rhamnosus Lactobacillus YYSJ019 strain for alleviating obesity. The rhamnosus Lactobacillus YYSJ019 strain is classified and named Lacticaseibacillus rhamnosus, with a preservation number of CGMCC No. 33472 and a preservation date of January 20, 2025.

[0008] The present invention isolates and preserves a new Lactobacillus rhamnosus strain from breast milk and names it Lacticaseibacillus rhamnosus YYSJ019. The strain has good tolerance in gastric juice and high surface hydrophobicity, and has the potential to smoothly enter the human gastrointestinal tract. The strain has excellent intestinal adhesion ability, indicating that it has a long intestinal retention time, providing a basis for continuously exerting its probiotic effects. The strain can slow down the weight gain of obese mice and improve fat accumulation, liver damage and inflammation in the obese mice. It can also improve blood sugar metabolism and blood lipid metabolism, and has the potential to prevent and treat hyperglycemia or hyperlipidemia. It can also stimulate the production of glucagon-like peptide-1 in a mouse small intestinal endocrine cell line (STC-1 cell line), and has the potential to improve diseases caused by insufficient secretion of glucagon-like peptide-1. It also has the effect of lowering uric acid in vitro and in vivo, and has the potential to prevent and treat hyperuricemia.

[0009] In addition, Lactobacillus rhamnosus is a probiotic, so the Lactobacillus rhamnosus YYSJ019 strain screened and obtained in the present invention is highly safe when used in the preparation of relevant functional products.

[0010] In a second aspect, the present invention provides a probiotic agent for alleviating obesity, wherein the strains in the agent include the Lactobacillus rhamnosus YYSJ019 strain described in the first aspect.

[0011] Preferably, the number of viable bacteria of Lactobacillus rhamnosus YYSJ019 in the probiotic agent is not less than 1×10 8 CFU / mL or 1×10 8 CFU / g, for example 1×10 8 CFU / mL, 2×10 8 CFU / mL, 5×10 8 CFU / mL, 8×10 8 CFU / mL, 1×10 9 CFU / mL, 5×10 9 CFU / mL, 1×10 10 CFU / mL, etc. Other specific point values within this numerical range can be selected and will not be described here one by one.

[0012] Preferably, the dosage form of the probiotic agent includes a solution, powder, tablet, granule or capsule. The dosage form of the agent involved in the present invention is not limited, including the most commonly used solution, powder, or further prepared capsule, tablet or granule.

[0013] Preferably, the bacterial agent may also contain excipients; the excipients include any one or a combination of at least two of a protective agent, a filler, a binder, a disintegrant, an emulsifier, a solubilizer, a solubilizing agent, an osmotic pressure regulator, a colorant, a pH regulator, an antioxidant, an antibacterial agent or a buffer.

[0014] In a third aspect, the present invention provides use of the Lactobacillus rhamnosus YYSJ019 strain according to the first aspect or the probiotic agent according to the second aspect in the preparation of a preparation for preventing or improving obesity.

[0015] In a fourth aspect, the present invention provides the use of the rhamnosus Lactobacillus YYSJ019 strain according to the first aspect or the probiotic agent according to the second aspect in the preparation of a preparation for improving fat accumulation caused by obesity, liver damage caused by obesity, or inflammation in the body caused by obesity.

[0016] In a fifth aspect, the present invention provides the use of the rhamnosus Lactobacillus YYSJ019 strain according to the first aspect or the probiotic agent according to the second aspect in the preparation of a preparation for improving the richness and diversity of intestinal flora.

[0017] In a sixth aspect, the present invention provides the use of the Lactobacillus rhamnosus YYSJ019 strain described in the first aspect or the probiotic agent described in the second aspect in the preparation of a preparation for preventing or improving sugar metabolism and / or lipid metabolism.

[0018] In a seventh aspect, the present invention provides the use of the rhamnosus Lactobacillus YYSJ019 strain described in the first aspect or the probiotic agent described in the second aspect in the preparation of a preparation for preventing or improving hyperglycemia and / or hyperlipidemia.

[0019] In an eighth aspect, the present invention provides use of the Lactobacillus rhamnosus YYSJ019 strain according to the first aspect or the probiotic agent according to the second aspect in the preparation of a glucagon-like peptide-1 release promoter.

[0020] According to the research results of the present invention, Lactobacillus rhamnosus YYSJ019 can promote the production of glucagon-like peptide-1 at the in vitro level (cellular level), that is, Lactobacillus rhamnosus YYSJ019 or its bacterial agent can be made into a preparation for exploring the physiological metabolic processes or disease mechanisms related to insufficient secretion of glucagon-like peptide-1.

[0021] In the ninth aspect, the use of the rhamnosus Lactobacillus YYSJ019 strain described in the first aspect or the probiotic agent described in the second aspect in the preparation of a uric acid degrading agent.

[0022] According to the research results of the present invention, Lactobacillus rhamnosus YYSJ019 can promote the degradation of uric acid at the in vitro level, that is, Lactobacillus rhamnosus YYSJ019 or its bacterial agent can be made into an experimental reagent for degrading uric acid in vitro.

[0023] In the tenth aspect, the use of the rhamnosus Lactobacillus YYSJ019 strain according to the first aspect or the probiotic agent according to the second aspect in the preparation of a preparation for preventing or improving hyperuricemia.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention isolates and preserves a new Lactobacillus rhamnosus strain from breast milk and names it Lacticaseibacillus rhamnosus YYSJ019. The strain has good tolerance in gastric juice and high surface hydrophobicity, and has the potential to smoothly enter the human gastrointestinal tract. The strain has excellent intestinal adhesion ability, indicating that it has a long intestinal retention time, providing a basis for continuously exerting its probiotic effects. The strain can slow down the weight gain of obese mice and improve fat accumulation, liver damage and inflammation in the obese mice. It can also improve blood sugar metabolism and blood lipid metabolism, and has the potential to prevent and treat hyperglycemia or hyperlipidemia. It can also stimulate the production of glucagon-like peptide-1 in a mouse small intestinal endocrine cell line (STC-1 cell line), and has the potential to improve diseases caused by insufficient secretion of glucagon-like peptide-1. It also has the effect of lowering uric acid in vitro and in vivo, and has the potential to prevent and treat hyperuricemia. In addition, the rhamnosus Lactobacillus YYSJ019 strain is a probiotic and is highly safe when used to prepare relevant functional products. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the colony morphology of Lactobacillus rhamnosus YYSJ019;

[0027] Figure 2 This is a microscopic image of Lactobacillus rhamnosus YYSJ019;

[0028] Figure 3 is the weight gain trend graph of mice in each group;

[0029] Figure 4 is the statistical graph of obesity index of mice in each group;

[0030] Figure 5 The weight statistics of epididymal white adipose tissue, inguinal white adipose tissue and scapular brown adipose tissue of each group of mice are shown in Figure 2.

[0031] Figure 6are H&E staining images of epididymal white adipose tissue, inguinal white adipose tissue, and scapular brown adipose tissue of mice in each group;

[0032] Figure 7 is the oral glucose tolerance curve of mice in each group;

[0033] Figure 8 This is a statistical graph of the area below the oral glucose tolerance line of each group of mice;

[0034] Figure 9 These are the appearance of liver tissue and H&E section staining images of mice in each group after the intervention;

[0035] Figure 10 The graph shows the statistical results of liver index and LPS (lipopolysaccharide), AST (aspartate aminotransferase), and ALT (alanine aminotransferase) levels in liver tissue of mice in each group;

[0036] Figure 11 This is the statistical result graph of serum lipid indexes of mice in each group;

[0037] Figure 12 is the statistical result graph of serum inflammatory factors and leptin levels of mice in each group;

[0038] Figure 13 This is the statistical result graph of the ACE index, Chao1 index and Shannon index of the intestinal flora of each group of mice;

[0039] Figure 14 This is the three-dimensional graph of the principal component PCA analysis of the intestinal flora of each group of mice;

[0040] Figure 15 This is a graph showing the relative abundance of different bacterial genera in the intestines of mice in each group;

[0041] Figure 16 is the statistical result of blood uric acid level of mice in each group;

[0042] Figure 17 This is the statistical result of serum xanthine oxidase activity of mice in each group;

[0043] Figure 18 is the statistical result graph of renal index, serum urea nitrogen and serum creatinine levels of mice in each group;

[0044] Different letters in each figure indicate significant differences among the groups (p < 0.05).

[0045] The YYSJ019 strain involved in the present invention is classified and named Lacticaseibacillus rhamnosus, the preservation time is January 20, 2025, the preservation number is CGMCC No. 33472, the preservation unit is the General Microbiology Center of the China Culture Collection Administration, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0046] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0047] The YYSJ019 strain involved in the following experiments is classified as Lacticaseibacillus rhamnosus and its deposit number is CGMCC No.33472.

[0048] The Lactobacillus rhamnosus LGG involved in the following experiments was purchased from Guangdong Provincial Microbial Culture Collection Center.

[0049] The Caco-2 cells involved in the following experiments were purchased from the Culture Collection of Wuhan University.

[0050] The C57BL / 6j male mice involved in the following experiments were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. and kept in the animal room of Hubei Provincial Center for Disease Control and Prevention. The animal experiment ethics number is: Safety Assessment Center Animal (Fu) No. 202410274.

[0051] MRS medium (g / L) used in the following experiments: 20.0 g glucose, 10.0 g yeast peptone, 10.0 g beef extract powder, 5.0 g yeast extract powder, 5.0 g anhydrous sodium acetate, 1.0 g Tween-80, 2.0 g diammonium hydrogen citrate, 2.0 g dipotassium hydrogen phosphate, 0.58 g magnesium sulfate heptahydrate, and 0.19 g manganese sulfate monohydrate. Dissolved in 1000 mL of distilled water, adjusted to pH 6.8, and autoclaved at 115°C for 20 min. Solid medium was supplemented with 1.5% agar.

[0052] Preparation of YYSJ019 bacterial suspension used in the following experiments:

[0053] The rhamnosus Lactobacillus YYSJ019 strain was inoculated into MRS liquid medium, cultured at 37°C for the second generation, and then cultured overnight to obtain rhamnosus Lactobacillus YYSJ019 bacterial liquid. The liquid was centrifuged at 5000 rpm at 4°C for 5 minutes, and the bacterial slurry was collected, resuspended with physiological saline, and adjusted to the required concentration.

[0054] Unless otherwise specified, all reagents and consumables used in the following experiments are commercially available, and the relevant experimental steps are common operations in the field and have technical meanings that can be generally understood by those skilled in the art.

[0055] Example 1

[0056] Isolation and screening of Lactobacillus rhamnosus YYSJ019:

[0057] Collected breast milk samples were aseptically diluted with sterile saline, plated on LBS agar plates, and incubated at 37°C for 72 hours. Colony morphology was visually observed, and monoclonal colonies that were milky white, round, shiny, with neat edges and a raised, moist surface were selected. These colonies were then purified by repeated streaking on MRS solid medium. After microscopic observation, initial screening and purification were performed. Purified strains were incubated in MRS medium at 37°C for 12-14 hours, centrifuged, and resuspended in sterile 30% glycerol solution and stored at -80°C.

[0058] Example 2

[0059] Morphological characteristics and 16S rDNA identification of Lactobacillus rhamnosus YYSJ019:

[0060] The colony morphology of Lactobacillus rhamnosus YYSJ019 on MRS medium and the bacteria under the microscope are shown as follows: Figure 1 and Figure 2 The YYSJ019 strain grew well on MRS solid plates, producing individual colonies that were milky white, round, raised, medium-sized, with neat edges and a glossy surface. Microscopic examination revealed rod-shaped cells arranged singly, in pairs, or in chains, consistent with the staining characteristics of Gram-positive bacteria.

[0061] The selected target strain was cultured in liquid, collected, and genomic DNA extracted. PCR amplification was performed using universal primers 27F / 1492R to generate a PCR product. The PCR product was analyzed by gel electrophoresis and then sent to Wuhan Jinkairui Bioengineering Co., Ltd. for sequencing. The identified gene sequence was submitted to the NCBI database (www.ncbi.nlm.nih.gov) for BLAST analysis and alignment. Based on molecular biological identification, the strain was identified as Lactobacillus rhamnosus, named YYSJ019, and deposited.

[0062] Example 3

[0063] Resistance of Lactobacillus rhamnosus YYSJ019 to artificial gastric juice:

[0064] Simulated artificial gastric juice: prepare 0.5% NaCl solution, adjust the pH to 3 with 1 mol / L HCl, add 0.3% pepsin, and then fully dissolve it and filter it with a 0.22 μm microporous membrane for sterilization before use.

[0065] Take 1mL of YYSJ019 bacterial solution and centrifuge it. After collecting the bacteria, add it to 1mL of prepared simulated gastric juice with pH 3 and mix it evenly. Digest it at 37℃. Take the digestion fluid at 0h and 3h to detect the number of viable bacteria and calculate the survival rate. Where, the survival rate of the strain (%) = N t / N0×100%, where N0 represents the number of viable bacteria of the strain at 0 h (CFU / mL), N t The 3-hour survival rate of the strain YYSJ019 in artificial gastric fluid at pH 3 was 100.54±1.35%, indicating that Lactobacillus rhamnosus YYSJ019 has a strong ability to tolerate the gastric fluid environment.

[0066] Example 4

[0067] Cell surface hydrophobicity of Lactobacillus rhamnosus YYSJ019:

[0068] Adjust the OD600 value of the YYSJ019 bacterial suspension to approximately 1.0, and simultaneously measure the absorbance of the bacterial suspension at 600 nm (A0). Place 12 mL of the bacterial suspension and 4 mL of xylene in a test tube, mix thoroughly, and let stand at room temperature for 10 minutes. Vortex mix again, and let stand at room temperature for 20 minutes to separate the transparent lower aqueous layer and the upper solvent layer containing the cells. Remove the aqueous phase and measure its absorbance at 600 nm (A1). Buffer solution serves as a blank control. Surface hydrophobicity is expressed as the percentage of bacterial adhesion to organic solvent. The calculation formula is: Surface hydrophobicity (%) = (1 - A1 / A0) × 100%.

[0069] The experimental results show that the surface hydrophobicity of strain YYSJ019 is 93.30±0.75 a (%), Lactobacillus rhamnosus LGG was used as the control strain to investigate its cell surface hydrophobicity ability. The results showed that the surface hydrophobicity of LGG strain was 60.44±2.06 b This indicates that the hydrophobicity of the YYSJ019 strain is significantly higher than that of LGG (different letters indicate significant differences between the two (p < 0.05)). The surface hydrophobicity of a strain can reflect its colonization in the human intestine. A higher surface hydrophobicity reflects a higher colonization rate, indicating that YYSJ019 has the potential to adhere and colonize in the gastrointestinal tract.

[0070] Example 5

[0071] Adhesion ability of Lactobacillus rhamnosus YYSJ019 to Caco-2 cells:

[0072] Caco-2 cell culture: Caco-2 cells were cultured in DMEM medium supplemented with 10% heat-inactivated (56°C, 30 min) fetal bovine serum (FBS), 1% penicillin and streptomycin in a 37°C, 90% humidity, 5% CO2 incubator.

[0073] Cell adhesion assay:

[0074] (1) Preparation of monolayer: Caco-2 cells were inoculated into DMEM medium supplemented with 20% (v / v) fetal bovine serum and transferred to 12-well cell culture plates. The amount of cells added was 10 cells / well. 5 Cells were cultured at a constant temperature of 5% CO2 and 37°C, with the medium changed every other day until a monolayer was obtained.

[0075] (2) Preparation of bacterial suspension: Adjust the bacterial suspension concentration to 10 8 CFU / mL;

[0076] (3) Co-culture: Remove the culture medium from the prepared Caco-2 cell monolayer, rinse twice with PBS buffer, remove the buffer, add 1 mL / well of the prepared bacterial suspension, mix well, and incubate at 37°C with 5% CO2 for 2 h. Remove the culture supernatant and rinse five times with sterile PBS to remove unadhered bacteria.

[0077] (4) Add 0.2 mL / well of trypsin cell digestion solution and digest for 5 min to elute the cells from the culture plate wells. The collected solution is the sample; the collected sample is serially diluted and viable bacteria are counted. Adhesion capacity is calculated using the following formula: Adhesion capacity (CFU / cell) = number of bacteria adhering to cells (CFU) / number of cells in the well (cells).

[0078] The cell adhesion experiment above used Lactobacillus rhamnosus LGG as a control strain, and each experiment was performed in parallel 3 times. Lactobacillus rhamnosus LGG is a commercial strain recognized in the industry as having good adhesion. The adhesion of the strain to Caco-2 cells was tested through adhesion experiments. The results showed that the adhesion ability of the YYSJ019 strain was 17.63±1.50 a (CFU / cell), the adhesion capacity of LGG strain was 4.19±0.17 bThe results (CFU / cell) indicate that the YYSJ019 strain exhibits remarkable adhesion, reaching significantly higher levels than the control strain LGG (different letters indicate significant differences (p < 0.05)). This excellent adhesion is a key prerequisite for intestinal colonization. Strains with strong adhesion properties have prolonged intestinal retention, providing a foundation for sustained probiotic efficacy.

[0079] Example 6

[0080] Evaluation of Lactobacillus rhamnosus YYSJ019 Stimulating Glucagon-like Peptide-1 Production:

[0081] (1) Mouse small intestinal endocrine cell line (STC-1 cell line) was inoculated into a 24-well plate and cultured until the density reached 2×10 5 / well, the cells were cultured to 80% confluence. 2+ and Mg 2+ The cells were washed twice with PBS buffer and cultured in DMEM (0.5 mL) without glucose and glutamine for 30 min.

[0082] (2) Take 0.5 mL of YYSJ019 bacterial suspension (1×10 8 CFU / mL) were added to the cell plate and incubated at 37°C for 4 hours. The supernatant was collected in a 1.5 mL centrifuge tube and centrifuged at 5900 × g at 4°C for 10 minutes to remove the cell pellet. Glucagon-like peptide-1 (GLP-1) content was measured using an ELISA kit, using the supernatant of STC-1 cells not inoculated with the bacterial suspension as a blank control.

[0083] (3) The results showed that the GLP-1 concentration in the blank control group was 23.91±0.45 b (pg / mL), and the GLP-1 concentration in the YYSJ001 group was 34.91±0.26 a (pg / mL), indicating that the YYSJ001 strain can significantly promote the release of GLP-1 from STC-1 cells (different letters indicate significant differences between the two (p < 0.05)).

[0084] Example 7

[0085] Evaluation of the uric acid-lowering ability of Lactobacillus rhamnosus YYSJ019 in vitro:

[0086] (1) Prepare uric acid solution: 0.084 g uric acid, 100 mL distilled water; pH 7.2 ± 0.2; filter through a 0.22 μm filter and set aside.

[0087] (2) The Lactobacillus rhamnosus YYSJ019 cells were washed twice with ddH2O and resuspended in 1 mL of ddH2O. The OD value (600 nm) of the bacterial solution was adjusted to 1.00 ± 0.1 using a UV spectrophotometer. Uric acid solution was added to the bacterial solution to a final uric acid concentration of 500 μmol / L. The cells were cultured at 37°C for 6 h. After 6 h, the bacterial solution was inactivated and the reaction was terminated by water bathing at 98°C for 10 min. The cells were centrifuged at 10,000 rpm for 2 min, and the remaining uric acid content in the supernatant was detected using a uric acid test kit. The uric acid degradation rate was calculated to be 15.66 ± 1.69%. This indicates that the Lactobacillus rhamnosus YYSJ019 strain has the ability to degrade uric acid in vitro.

[0088] Example 8

[0089] Improvement effect of Lactobacillus rhamnosus YYSJ019 on obese mice induced by high-fat diet:

[0090] (1) Experimental animals: Healthy male C57BL / 6j mice (5–6 weeks; 19 ± 1 g) were housed in the animal room of the Hubei Provincial Center for Disease Control and Prevention. The experimental animal use license number is SCXK (Beijing) 2024-0001. All mice were housed in a specific pathogen-free, thermostatic environment with a temperature of 22 ± 3°C, a humidity of 50 ± 10%, and a 12-h light / 12-h dark cycle. All mice had free access to food and water and received humane care.

[0091] (2) Animal grouping: After one week of adaptive feeding, 30 mice were randomly divided into three groups (10 mice in each group) according to their body weight: normal group (ND group), model group (HFD group), and probiotic group (YYSJ019 group).

[0092] (3) Animal modeling and intervention methods:

[0093] During the experiment, the normal group was fed with ordinary feed + sterile saline, the model group was fed with high-fat feed containing 60% fat + sterile saline, and the probiotic group was fed with high-fat feed containing 60% fat + probiotic solution (concentration of solution 8.0×10 9 CFU / mL, 200 μL was administered orally to each mouse every day for 9 consecutive weeks.

[0094] (4)Indicator analysis:

[0095] (4.1) Impact on weight gain and obesity rate:

[0096] The weight of each group of mice was recorded, and the obesity index of each group of mice was calculated (obesity index = (weight of mice in the model group or probiotic group - average weight of the normal group) / average weight of the normal group × 100%). Figure 3 and Figure 4 shown.

[0097] Depend on Figure 3 and Figure 4 As shown, after induction of the model using a high-fat diet, the weight of mice in the HFD group continued to increase steadily and rapidly, with weight gain exceeding that of the ND and YYSJ019 groups. Compared with the HFD group, the weight gain of mice in the YYSJ019 group was significantly less. At week 9, there was no significant difference in weight between the YYSJ019 and ND groups, indicating that YYSJ019 treatment can slow weight gain and alleviate obesity in mice. The obesity index is a commonly used criterion for determining obesity in mice, with an obesity index greater than 20% generally considered obese. The obesity index of mice in the HFD group continued to rise, reaching 23.32% at week 9, meeting the obesity standard. The obesity index of mice in the YYSJ019 treatment group showed a less pronounced upward trend and remained stable. This suggests that Lactobacillus rhamnosus YYSJ019 can effectively inhibit excessive weight gain induced by a high-fat diet and alleviate obesity in mice.

[0098] (4.2) Effects on mouse adipose tissue:

[0099] After the experiment, the mice were killed and their epididymal white adipose tissue (eWAT), inguinal white adipose tissue (iWAT) and scapular brown adipose tissue (BAT) were quickly sampled and weighed. Figure 5 As shown in the data, compared with the ND group, the epididymal white fat weight and inguinal white fat weight of the HFD group mice were significantly increased (p<0.05), indicating that a high-fat diet can induce the accumulation of lipids in adipose tissue of mice; compared with the HFD group, the YYSJ019 intervention group significantly reduced the epididymal white fat weight and inguinal white fat weight of the mice (p<0.05), indicating that the rhamnosus Lactobacillus YYSJ019 intervention can effectively reduce the proportion of white fat in mice and alleviate the accumulation of fat in mice fed a high-fat diet.

[0100] The brown fat mass of HFD-induced obese mice also increased, but its function was impaired and its activity decreased. H&E staining is a routine histopathology technique that can observe the pathological morphology of adipose tissue. Figure 6 As shown, the white and brown adipose tissue structures of mice in the ND group were normal, with no inflammatory cell infiltration. In contrast, the HFD group showed significant enlargement of both types of white adipocytes, expansion of brown adipocytes, and increased lipid droplet size. YYSJ019 reduced white adipocyte enlargement in obese mice, restored brown adipocyte morphology, and reduced lipid content.

[0101] (4.3) Effects on oral glucose tolerance in mice:

[0102] After the intervention, oral glucose tolerance test was performed on mice. The glucose dosage for glucose tolerance test was 2 g per kg body weight, and 20% glucose solution was prepared with normal saline. The mice were fasted for 16 hours, during which time they maintained normal drinking water. Before starting the glucose tolerance test, each mouse was weighed, blood was collected from the tip of the tail, and fasting blood glucose was measured with a blood glucose meter. The measured value was considered as the blood glucose value at 0 min. Glucose was gavaged and timing was started from the moment of completion. The blood glucose value of each mouse at each time point was measured at 30, 60, 90 and 120 min, and the oral glucose tolerance curve was drawn, as shown in the figure. Figure 7 As shown, calculate the area below the oral glucose tolerance line, and the statistical results are as follows Figure 8 shown.

[0103] Compared with the ND group, the HFD group showed significantly higher peak blood glucose levels and delayed recovery after glucose ingestion. However, after YYSJ019 treatment, peak blood glucose levels were reduced and recovery was more rapid. The HFD group had the largest area under the curve compared to the other groups, indicating that obese mice have a reduced ability to metabolize glucose. YYSJ019 treatment can suppress blood glucose elevation and improve glucose metabolism in obese mice.

[0104] (4.4) Effects on mouse liver:

[0105] After the intervention, liver tissues of mice in each group were collected, the fresh livers were weighed, and the liver index of each group was calculated. Part of the samples were taken for H&E section staining. The remaining tissues were pretreated and ground, and then the liver indicators of mice were measured according to the operating procedures of the Elisa kit, including LPS (lipopolysaccharide), AST (aspartate aminotransferase), and ALT (alanine aminotransferase).

[0106] The results are as follows Figure 9 As shown in the data, observation of liver appearance and H&E staining results showed that the liver status of mice in the ND group was normal, and excessive accumulation of lipid droplets in the form of macrovesicles and microvesicles was found in HFD-exposed hepatocytes, while intervention in the YYSJ019 group reduced HFD-induced lipid droplet accumulation.

[0107] like Figure 10 Observation of the liver index across the groups revealed a significant increase in the liver index in the HFD group (p < 0.05). A high-fat diet may lead to a large accumulation of triglycerides in the liver, leading to an increase in the liver index. YYSJ019 intervention significantly reduced the liver index in mice (p < 0.05). Increased AST and ALT levels in the HFD group indicate liver damage; the increased endotoxin LPS in the HFD group may also contribute to liver damage. This suggests that YYSJ019 intervention can effectively reduce inflammation in obese mice and protect the livers of these models, reducing damage.

[0108] (4.5) Effects on serum lipids in mice:

[0109] After the intervention, whole blood was collected from each group of mice and centrifuged to obtain serum. The levels of various serum indicators of each group of mice were measured, including triglyceride (TG), total cholesterol (T-CHO), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C). The TC / HDL-C ratio was also compared. This ratio has been proven to be a better indicator for assessing obesity and hyperlipidemia.

[0110] The results are as follows Figure 11 As shown in the results, compared with the ND group, the serum TG, TC, LDL-c levels and TC / HDL-C ratio of mice in the HFD group were significantly increased (p<0.05), indicating that the modeling caused an increase in the blood lipid levels of obese mice, which means that the lipid metabolism in the blood of obese mice was abnormal. The YYSJ019 intervention group significantly reduced the levels of TG, T-CHO, LDL-C and TC / HDL-C (p<0.05), and the triglyceride (TG) content of the YYSJ019 group was no different from that of the ND group. The results show that Lactobacillus rhamnosus YYSJ019 can effectively alleviate dyslipidemia in mice fed a high-fat diet, regulate lipid metabolism in the body, and improve the blood lipid levels of mice.

[0111] (4.6) Effects on serum inflammation and leptin in mice:

[0112] After the intervention, the whole blood of mice in each group was centrifuged to obtain serum. The levels of cytokines in the serum of mice were determined using ELISA kits, including TNF-α, IL-6, IL-10 and leptin (LEP). The results are shown in Figure 12 As shown in the results, compared with the ND group, the high-fat diet significantly increased the levels of pro-inflammatory factors TNF-α and IL-6 (p<0.05) and significantly decreased the level of the anti-inflammatory factor IL-10 (p<0.05). Compared with the HFD group, YYSJ019 intervention significantly reduced the levels of pro-inflammatory factors and increased the levels of anti-inflammatory factors in the serum of mice. This indicates that the YYSJ019 strain can regulate inflammation in mice fed a high-fat diet.

[0113] Leptin is an adipocytokine associated with inflammation, regulating energy expenditure, lipogenesis, and alleviating inflammation and insulin resistance. Compared with the ND group, the high-fat diet led to increased serum LEP levels in the HFD group (p<0.05), while the YYSJ019 group significantly alleviated this change. This suggests that the YYSJ019 strain can effectively regulate adipocytokine levels, thereby regulating energy expenditure and lipogenesis.

[0114] (4.7) Effects on intestinal flora of mice:

[0115] After the intervention, the mice were killed, and the cecal contents of each group were collected for microbial diversity analysis. The 16S rDNA V3-V4 hypervariable region of all bacteria in the samples was sequenced on the Mi Seq Illumina sequencing platform to determine the characteristics of the intestinal microbiome.

[0116] α-diversity reflects the species richness and diversity of the sample. The ACE index, Chao1 index and Shannon index are commonly used indicators for judging the richness and diversity of intestinal microbial colonies. Figure 13 As shown, the results showed that a high-fat diet would lead to a decrease in the richness and diversity of the body's intestinal flora, and YYSJ019 intervention could significantly increase the richness and diversity of the intestinal flora, approaching that of the healthy group.

[0117] β-diversity is used to compare the similarity of different samples in terms of species diversity. Based on the principal component PCA analysis of the three-dimensional map ( Figure 14 ) shows that beta diversity reflects differences in microbial composition. The microbial structures of the ND and HFD groups were separated, indicating that a high-fat diet (HFD) alters microbial composition. After YYSJ019 intervention, the microbial distribution in the YYSJ019 group shifted away from the HFD group, suggesting that YYSJ019 can reverse HFD-induced microbial disturbances and bring them closer to those of the healthy group.

[0118] The relative abundance of different bacterial genera in the intestines of each group of mice is shown in Figure 2. Figure 15 Results show that high-fat diet (HFD)-induced obese mice have reduced abundance of Faecalibaculum and Bifidobacterium in their guts, leading to intestinal microbial dysbiosis. It also significantly reduces the abundance of Akkermansia, a genus directly associated with obesity. Furthermore, studies have reported that the abundance of Enterobacteriaceae is generally low in healthy individuals, but may be significantly altered in patients with obese metabolic diseases. YYSJ019 treatment significantly increased the relative abundance of beneficial bacteria in the mouse gut, including Faecalibaculum, Bifidobacterium, Lactobacillus, and Akkermansia, while reducing the relative abundance of harmful bacteria, including Erysipelatoclostridium and Enterobacter. This indicates that the YYSJ019 strain can regulate the structure of intestinal flora and improve the obesity symptoms of mice caused by a high-fat diet.

[0119] Example 9

[0120] Improvement effect of Lactobacillus rhamnosus YYSJ019 on hyperuricemia mice:

[0121] (1) Experimental animals: Healthy male C57BL / 6j mice (5–6 weeks; 19 ± 1 g) were housed in the animal room of the Hubei Provincial Center for Disease Control and Prevention. The experimental animal use license number is SCXK (Beijing) 2024-0001. All mice were housed in a specific pathogen-free, thermostatic environment with a temperature of 22 ± 3°C, a humidity of 50 ± 10%, and a 12-h light / 12-h dark cycle. All mice had free access to food and water and received humane care.

[0122] (2) Animal grouping: After one week of adaptive feeding according to the above-mentioned mice, 40 mice were randomly divided into four groups (10 mice in each group): blank control group (NC group), model group (MOD group), ADC group (allopurinol group), and Lactobacillus rhamnosus YYSJ019 intervention group (YYSJ019 group).

[0123] (3) Animal modeling and intervention methods:

[0124] Except for the blank control group, the other groups were subjected to hyperuricemia modeling. The modeling method was as follows: potassium oxonate was suspended in 0.5% sodium carboxymethyl cellulose solution, adenine was suspended in normal saline, 0.2 mL adenine (50 mg / kg) was gavaged daily and 0.2 mL potassium oxonate solution (250 mg / kg) was intraperitoneally injected to induce hyperuricemia model. The blank control group mice were gavaged with the same volume of normal saline and intraperitoneally injected with the same volume of 0.5% sodium carboxymethyl cellulose solution. After 4 hours of adenine and potassium oxonate treatment, the YYSJ019 group was given 0.2 mL of bacterial solution (bacterial solution concentration was 8×10 9 CFU / mL). The ADC group was given 0.2 mL of allopurinol solution (5 mg / kg), and the NC and MOD groups were given an equal volume of normal saline. The intervention was conducted once a day for 15 consecutive days.

[0125] (4)Indicator analysis:

[0126] (4.1) Blood uric acid levels and serum xanthine oxidase activity in mice:

[0127] After the intervention, the mice in each group were killed and blood was collected from the ophthalmic vein to detect serum uric acid levels. Figure 16 As shown in the data, the blood uric acid content of mice in the MOD group was significantly higher than that in the NC group (P<0.05), indicating that the hyperuricemia model was successfully established. Compared with the MOD group, the blood uric acid content in the YYSJ019 group was significantly decreased (P<0.05), indicating that YYSJ019 intervention has the ability to reduce blood uric acid levels.

[0128] Serum xanthine oxidase activity was tested. Figure 17 As shown, xanthine oxidase (XOD) is a key metabolic enzyme in the uric acid production pathway, primarily found in the liver and blood. It catalyzes hypoxanthine into xanthine, which is then further converted to uric acid. Increased XOD activity correlates with increased uric acid production, while decreased XOD activity correlates with decreased uric acid production. XOD activity in the MOD group was significantly higher than in the NC group (P < 0.05). After YYSJ019 treatment, XOD activity was significantly decreased compared with the MOD group (P < 0.05). There was no significant difference in the effect between the YYSJ019 group and the NC or ADC groups. This suggests that the YYSJ019 strain can inhibit XOD activity, thereby lowering serum uric acid levels.

[0129] (4.2) Assessment of renal impairment:

[0130] Kidney tissue and blood were collected from each group of mice, and the renal index, serum urea nitrogen, and serum creatinine were calculated. Urea nitrogen and creatinine are indicators of kidney function. Elevated blood urea nitrogen and creatinine levels indicate renal insufficiency, potentially leading to renal failure, nephritis, acute tubular necrosis, and other pathologies.

[0131] The results are as follows Figure 18 As shown, the renal index in the MOD and ADC groups was significantly higher than that in the NC group, indicating renal enlargement and other pathological changes. The renal index in the YYSJ019 group was significantly lower (P < 0.05), with no significant difference from the NC group. Creatinine and urea nitrogen levels in the MOD and ADC groups were significantly higher than those in the NC group, while urea nitrogen and creatinine levels were significantly lower in the YYSJ019 group after intervention (P < 0.05).

[0132] The applicant declares that the present invention is illustrated by the above-described embodiments, but the present invention is not limited to the above-described embodiments. This does not mean that the present invention must rely on the above-described embodiments in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacements for raw materials in the present invention, additions of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

[0133] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0134] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A Lactobacillus rhamnosus YYSJ019 strain for alleviating obesity, characterized in that: The classification name of the rhamnosus Lactobacillus YYSJ019 strain is Lacticaseibacillus rhamnosus, the preservation number is CGMCC No.33472, and the preservation date is January 20, 2025.

2. A probiotic agent for alleviating obesity, characterized in that: The strain in the bacterial agent includes the rhamnosus Lactobacillus YYSJ019 strain according to claim 1.

3. The probiotic agent according to claim 2, characterized in that The number of viable bacteria of Lactobacillus rhamnosus YYSJ019 in the probiotic agent is not less than 1×10 8 CFU / mL or 1×10 8 CFU / g.

4. The probiotic agent according to claim 2, characterized in that The dosage form of the probiotics includes solution, powder, tablet, granule or capsule.

5. Use of the Lactobacillus rhamnosus YYSJ019 strain according to claim 1 or the probiotic agent according to any one of claims 2 to 4 in preparing a preparation for preventing or improving obesity.

6. Use of the Lactobacillus rhamnosus YYSJ019 strain according to claim 1 or the probiotic agent according to any one of claims 2 to 4 in the preparation of a preparation for improving fat accumulation caused by obesity, liver damage caused by obesity, or inflammation in the body caused by obesity.

7. Use of the Lactobacillus rhamnosus YYSJ019 strain according to claim 1 or the probiotic agent according to any one of claims 2 to 4 in the preparation of a preparation for improving the richness and diversity of intestinal flora.

8. Use of the Lactobacillus rhamnosus YYSJ019 strain according to claim 1 or the probiotic agent according to any one of claims 2 to 4 in preparing a preparation for preventing or improving sugar metabolism and / or lipid metabolism.

9. Use of the Lactobacillus rhamnosus YYSJ019 strain according to claim 1 or the probiotic agent according to any one of claims 2 to 4 in the preparation of a glucagon-like peptide-1 release promoter.

10. Use of the Lactobacillus rhamnosus YYSJ019 strain according to claim 1 or the probiotic agent according to any one of claims 2 to 4 in preparing a preparation for preventing or improving hyperuricemia.

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