A probiotic composition and its use in the preparation of a product for weight loss and improvement of metabolic syndrome

Abstract

The application discloses a probiotic composition and application thereof in preparation of a product with weight loss and metabolic syndrome improvement, wherein the probiotic composition comprises Bifidobacterium animalis subsp. lactis CEGF005, Bifidobacterium animalis subsp. lactis CEGF006, Bifidobacterium longum subsp. longum CEGF007 and Ligilactobacillus salivarius CEGF008. Compared with single probiotic, the weight loss effect of the combined bacteria is 2.3-4.3 times that of each single bacterium, and the blood glucose lowering effect is 1.96 times the average level of single bacterium, and the combined bacteria have good weight loss and metabolic syndrome improvement effects and high safety.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine, functional food or microbial preparations, and more specifically, relates to a probiotic composition and its application in the preparation of drugs that can reduce weight, improve glucose tolerance and / or improve dyslipidemia. Background Technology

[0002] Metabolic syndrome (MetS) is a group of clinical syndromes characterized by obesity and insulin resistance, often accompanied by type 2 diabetes, hyperlipidemia, and metabolic-associated fatty liver disease (MASLD). It has become one of the leading challenges threatening public health worldwide. According to the International Diabetes Federation (IDF), approximately one-quarter of adults worldwide meet the diagnostic criteria for metabolic syndrome, and its prevalence continues to rise with the obesity epidemic.

[0003] Currently, clinical interventions for metabolic syndrome mainly rely on lifestyle interventions (such as diet control and exercise) and drug treatments targeting single components (such as hypoglycemic agents and lipid-lowering agents). However, lifestyle interventions require extremely high patient compliance and have limited long-term effects. Furthermore, because this syndrome involves metabolic disorders of multiple systems and multiple targets, single-target drugs (such as statins, metformin, orlistat, etc.) are often difficult to achieve comprehensive and effective interventions, and long-term use is often accompanied by adverse reactions. Therefore, there is an urgent clinical need to develop safe, effective, and multi-target comprehensive intervention strategies. Summary of the Invention

[0004] To address the aforementioned deficiencies or improvement needs of existing technologies, this invention provides a probiotic composition and its application in the preparation of drugs that combine weight loss and improvement of metabolic syndrome. The aim is to discover four new bacterial strains (Bifidobacterium animalis subsp. lactis CEGF005, Bifidobacterium animalis subsp. lactis CEGF006, Bifidobacterium longum subsp. longum CEGF007, and Ligilactobacillus salivarius CEGF008). The combined bacteria obtained by combining these strains exhibit multiple effects, including good weight loss and lipid reduction, prevention or improvement of hyperlipidemia, prevention or improvement of type 2 diabetes, and prevention or improvement of metabolic-related fatty liver disease. Furthermore, this combined bacteria composition has high safety, thereby solving the technical problem that existing drug interventions or treatments have limited efficacy and side effects.

[0005] To achieve the above objectives, according to a first aspect of the present invention, a probiotic composition is provided, comprising Bifidobacterium animalis subsp. lactis CEGF005, Bifidobacterium animalis subsp. lactis CEGF006, Bifidobacterium longum subsp. longum CEGF007 and Ligilactobacillus salivarius CEGF008;

[0006] The accession number of Bifidobacterium animalis subsp. lactis CEGF005 is CCTCC NO:M 20241079, and the accession date is May 27, 2024.

[0007] The accession number of Bifidobacterium animalis subsp. lactis CEGF006 is CCTCC NO:M 20241080, and the accession date is May 27, 2024.

[0008] The accession number of Bifidobacterium longum subsp. longum CEGF007 is CCTCC NO: M20241133, and the accession date is June 3, 2024;

[0009] The accession number of Ligilactobacillus salivarius CEGF008 is CCTCC NO: M20241111, and the accession date is May 30, 2024.

[0010] Preferably, in the probiotic composition, the 16S rDNA sequences of Bifidobacterium animalis subsp. lactis CEGF005, Bifidobacterium animalis subsp. lactis CEGF006, Bifidobacterium longum subsp. longum CEGF007, and Ligilactobacillus salivarius CEGF008 are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; the total viable count of these four probiotics in the probiotic composition is ≥1×10⁻⁶. 9 CFU / mL or ≥1×10 9CFU / g.

[0011] Preferably, the probiotic composition comprises Bifidobacterium animalis subsp. lactis CEGF005, Bifidobacterium animalis subsp. lactis CEGF006, Bifidobacterium longum subsp. longum CEGF007, and Ligilactobacillus salivarius CEGF008 in an addition ratio of 1~2:1~2:1~2:1~2.

[0012] According to a second aspect of the present invention, there is an application of the probiotic composition as described in the present invention in the preparation of a drug that combines weight loss and improvement of metabolic syndrome, wherein the metabolic syndrome is characterized by disordered glucose and lipid metabolism as one of its core clinical manifestations.

[0013] Preferably, in the application, the metabolic syndrome includes at least one of hyperlipidemia, type 2 diabetes, and metabolic-associated fatty liver disease.

[0014] Preferably, the application in which the probiotic composition is used to prepare a medicament for improving or treating hyperlipidemia.

[0015] Preferably, in the application, the hyperlipidemia is mainly manifested as hypercholesterolemia.

[0016] Preferably, the application in which the probiotic composition is used to prepare a medicament for improving or treating metabolic-related fatty liver disease.

[0017] Preferably, the application in which the probiotic composition is used to prepare a medicament for improving or treating type 2 diabetes.

[0018] Preferably, in the application, the type 2 diabetes is obesity-related type 2 diabetes.

[0019] Preferably, in the application, the ratio of Bifidobacterium animalis subsp. lactis CEGF005, Bifidobacterium animalis subsp. lactis CEGF006, Bifidobacterium longum subsp. longum CEGF007, and Ligilactobacillus salivarius CEGF008 in the drug that combines weight loss and improvement of metabolic syndrome is 1~2:1~2:1~2:1~2.

[0020] In summary, compared with the prior art, the probiotic composition provided by the present invention can achieve the following beneficial effects through the above-described technical solutions:

[0021] The probiotic composition provided by this invention includes *Bifidobacterium animalis subsp. lactis* CEGF005, *Bifidobacterium animalis subsp. lactis* CEGF006, *Bifidobacterium longum subsp. longum* CEGF007, and *Ligilactobacillus salivarius* CEGF008. Compared with single probiotics of the same viable count, mice treated with the combined strain of this invention for 28 days experienced a weight loss of 5.6 g, significantly lower than the model group (P<0.0001), and significantly superior to each individual probiotic group (P<0.05). The weight loss effect of this combined strain was 2.3–4.3 times that of each individual probiotic group (2.8 times the average weight loss of the individual probiotic groups), and its hypoglycemic effect was 1.96 times the average level of the individual probiotic groups. Compared with existing probiotic compositions, the combined strain of this invention has better effects on weight loss and improvement of metabolic syndrome, and can be used to prepare drugs that combine weight loss and improvement of metabolic syndrome. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the experimental procedure for the abundance of probiotics in vivo.

[0023] Figure 2 This is a schematic diagram of the experimental process for a probiotic intervention model mouse.

[0024] Figure 3 This is the result of H&E staining of the white adipose tissue adjacent to the gonads of mice in Example 3. Figure 3 In the diagram, (A) is the control group, (B) is the model group, (C) is the drug group, and (D) is the combined bacterial group.

[0025] Figure 4 This is the statistical result of the weight of the white adipose tissue adjacent to the gonads on one side of the mouse in Example 3. Figure 4 The bar chart, from left to right, represents the control group, model group, drug group, and combined bacterial group.

[0026] Figure 5 This refers to the serum levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL), and high-density lipoprotein cholesterol (HDL) in mice as described in Example 3. Figure 5 In the table, A represents triglyceride content; B represents total cholesterol content; C represents high-density lipoprotein cholesterol content; and D represents low-density lipoprotein cholesterol content. Figure 5The bar chart, from left to right, represents the control group, model group, drug group, and combined bacterial group.

[0027] Figure 6 The results are the serum AST and ALT levels in mice from Example 3. Figure 6 In the table, A represents serum AST level, and B represents serum ALT level.

[0028] Figure 7 This refers to the weight gain of mice in each group in Example 4. Figure 7 From left to right, the groups are: control group, model group, drug group, combined bacteria group A, combined bacteria group of the present invention, combined bacteria group B, and combined bacteria group C.

[0029] Figure 8 This refers to the area under the glucose tolerance curve for each group of mice in Example 4. Figure 8 From left to right, the groups are: control group, model group, drug group, combined bacteria group A, combined bacteria group of the present invention, combined bacteria group B, and combined bacteria group C.

[0030] Figure 9 This is the result of H&E staining of the white adipose tissue adjacent to the gonads of mice in Example 4.

[0031] Figure 10 This is the result of H&E staining of mouse liver tissue in Example 4. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0033] This invention isolated, purified, and identified two new strains of Bifidobacterium animalis subspecies, one new strain of Bifidobacterium longum subspecies, and one new strain of Lactobacillus salivarius from the intestines of healthy humans. The combined strains obtained by combining these four new strains were demonstrated in in vivo and in vitro experiments to have multiple effects, including weight loss, lipid reduction, and improvement of metabolic syndrome, such as hyperlipidemia, metabolic-associated fatty liver disease, and type 2 diabetes. Compared to individual strains, the weight loss effect of this combined strain was 2.3 to 4.3 times that of the individual strains, and its blood sugar lowering effect was 1.96 times the average level of individual strains.

[0034] Based on this, the present invention provides a probiotic, which is classified and named Bifidobacterium animalissubsp. lactis CEGF005, with accession number CCTCC NO: M 20241079 and accession date May 27, 2024.

[0035] This invention provides a probiotic, classified as Bifidobacterium animalis subsp.lactis CEGF006, with accession number CCTCC NO: M 20241080 and accession date of May 27, 2024.

[0036] This invention provides a probiotic, classified and named Bifidobacterium longum subsp. longum CEGF007, with accession number CCTCC NO: M 20241133 and accession date June 3, 2024.

[0037] This invention provides a probiotic, classified as Ligilactobacillus salivariusCEGF008, with accession number CCTCC NO: M 20241111 and accession date of May 30, 2024.

[0038] The present invention also provides a probiotic composition comprising at least two of the following: Bifidobacterium animalis subsp. lactis CEGF005, Bifidobacterium animalis subsp. lactis CEGF006, Bifidobacterium longum subsp. longum CEGF007, and Ligilactobacillus salivarius CEGF008;

[0039] The accession number of Bifidobacterium animalis subsp. lactis CEGF005 is CCTCC NO:M 20241079, and the accession date is May 27, 2024.

[0040] The accession number of Bifidobacterium animalis subsp. lactis CEGF006 is CCTCC NO:M 20241080, and the accession date is May 27, 2024.

[0041] The accession number of Bifidobacterium longum subsp. longum CEGF007 is CCTCC NO: M20241133, and the accession date is June 3, 2024;

[0042] The accession number of Ligilactobacillus salivarius CEGF008 is CCTCC NO: M20241111, and the accession date is May 30, 2024.

[0043] Preferably, the probiotic composition comprises *Bifidobacterium animalis subsp. lactis* CEGF005, *Bifidobacterium animalis subsp. lactis* CEGF006, *Bifidobacterium longum subsp. longum* CEGF007, and *Ligilactobacillus salivarius* CEGF008; the 16S rDNA sequences of *Bifidobacterium animalis subsp. lactis* CEGF005, *Bifidobacterium animalis subsp. lactis* CEGF006, *Bifidobacterium longum subsp. longum* CEGF007, and *Ligilactobacillus salivarius* CEGF008 are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; the total viable count of these four probiotics in the probiotic composition is ≥1×10⁻⁶. 9 CFU / mL or ≥1×10 9 CFU / g, and more preferably also includes prebiotics.

[0044] In some embodiments, the ratio of Bifidobacterium animalis subsp. lactis CEGF005, Bifidobacterium animalis subsp. lactis CEGF006, Bifidobacterium longum subsp. longum CEGF007, and Ligilactobacillus salivarius CEGF008 in the probiotic composition is 1~2:1~2:1~2:1~2.

[0045] In addition, the present invention also provides the application of the probiotic composition as described in the present invention in the preparation of a drug that can both reduce weight and prevent or improve metabolic syndrome, wherein metabolic syndrome is characterized by disordered glucose and lipid metabolism as one of its core clinical manifestations.

[0046] The metabolic syndrome includes at least one of hyperlipidemia, type 2 diabetes, and metabolic-related fatty liver disease.

[0047] In some embodiments, the probiotic composition includes *Bifidobacterium animalis subsp. lactis* CEGF005, *Bifidobacterium animalis subsp. lactis* CEGF006, *Bifidobacterium longum subsp. longum* CEGF007, and *Ligilactobacillus salivarius* CEGF008, and is used to prepare a medicament for the prevention, improvement, or treatment of hyperlipidemia. Preferably, the hyperlipidemia is primarily characterized by hypercholesterolemia.

[0048] In some embodiments, the probiotic composition includes *Bifidobacterium animalis subsp. lactis* CEGF005, *Bifidobacterium animalis subsp. lactis* CEGF006, *Bifidobacterium longum subsp. longum* CEGF007, and *Ligilactobacillus salivarius* CEGF008, and is used to prepare a medicament for the prevention, improvement, or treatment of metabolic-related fatty liver disease.

[0049] In some embodiments, the probiotic composition includes *Bifidobacterium animalis subsp. lactis* CEGF005, *Bifidobacterium animalis subsp. lactis* CEGF006, *Bifidobacterium longum subsp. longum* CEGF007, and *Ligilactobacillus salivarius* CEGF008, and is used as a medicine for the prevention, improvement, or treatment of type 2 diabetes. The type 2 diabetes is obesity-related type 2 diabetes.

[0050] In some embodiments, the ratio of Bifidobacterium animalis subsp. lactis CEGF005, Bifidobacterium animalis subsp. lactis CEGF006, Bifidobacterium longum subsp. longum CEGF007, and Ligilactobacillus salivarius CEGF008 in the drug that combines weight loss and prevention or improvement of metabolic syndrome is 1~2:1~2:1~2:1~2.

[0051] The following are examples.

[0052] Example 1: Isolation and Identification of Strains

[0053] Multiple strains were isolated from the human intestine and their morphological, physiological and biochemical, and molecular biological characteristics were identified in the laboratory.

[0054] 1.1 Isolation and Identification of Strains

[0055] Healthy individuals who have not used antibiotics, probiotics, or immunosuppressants within the past 3 months, and who have no history of gastrointestinal or metabolic diseases (such as obesity or diabetes), were selected. Stool samples were collected, and 1 gram of stool was weighed and mixed in 9 ml of sterile PBS buffer, vortexed, to prepare 10... -1 The diluent is then serially diluted 10-fold (e.g., 10... -2 10 -3 10 -4 10 -5 Take 50 μl of diluted sample and spread it on selective medium. Incubate it upside down in a 37°C constant temperature incubator under anaerobic or facultative anaerobic conditions for 48-72 h. Observe and select morphologically typical and isolated single colonies for further culture on solid medium. Repeat streak until pure strains are obtained and numbered CEGF005, CEGF006, CEGF007, and CEGF008. In this example, Bifidobacterium animalis selective medium is WCM 100; Bifidobacterium longum selective medium is MRSC agar (MRS medium with 0.05% cysteine ​​added and pH adjusted to 5.5); Lactobacillus salivarius selective medium is MRS medium (with 0.05% cysteine ​​added).

[0056] Observe the colony morphology: color, shape, size, edge, surface smoothness, etc. Among them, the colony morphology of CEGF005 is similar to that of the animal bifidobacterium model strain (ATCC 27536); the colony morphology of CEGF006 is similar to that of animal bifidobacterium model strain (ATCC 27536); the colony morphology of CEGF007 is similar to that of Bifidobacterium longum model strain (ATCC15707); and the colony morphology of CEGF008 is similar to that of Lactobacillus salivarius model strain (ATCC 11741).

[0057] 1.2 Molecular biological identification

[0058] Genomic DNA was extracted from the isolated strain strictly following the steps of the bacterial DNA extraction kit. Using this DNA as a template, PCR amplification was performed using a pair of classic universal primers for the bacterial 16S rRNA gene (27F, 1492R). The PCR reaction system consisted of 25 μL of 2 × EcoTaq PCR Super-Mix, 2 μL each of forward and reverse primers, 2 μL of genomic DNA, and ddH2O to a final volume of 50 μL. The PCR program was as follows: 95 ℃ for 10 min; 95 ℃ for 30 s, 55 ℃ for 30 s, 72 ℃ for 90 s, for a total of 30 cycles; and a final extension at 72 ℃ for 10 min. The PCR products were directly sequenced by Qingke Biotechnology Co., Ltd., and the 16S rDNA gene of the strain was sequenced. BLAST alignment was performed in databases such as NCBI (National Center for Biotechnology Information), and the strain with the highest homology was identified in GenBank for species identification.

[0059] Currently, based on 16S rRNA nucleotide sequence similarity, the recommended thresholds for classifying isolates at the species level are 95% and 98.65%, respectively, to determine whether a bacterial isolate belongs to an existing genus / new genus or an existing species / new species. That is, it is generally considered that if two bacterial isolates have a 16S rRNA gene sequence similarity of <98.65%, they are considered to belong to different species, and if the similarity is <95%, they are considered to belong to different genera.

[0060] In this embodiment, the 16S rDNA gene sequences obtained from the above four isolated bacteria were compared using BLAST on NCBI. The results showed that:

[0061] The 16S rDNA sequence corresponding to strain CEGF005 is shown in SEQ ID NO:1. It has the highest nucleotide similarity (97%) with the 16S rRNA of *Bifidobacterium animalis* subsp. *lactobacterium* in the database. This value is greater than the recommended threshold at the genus level (95%), indicating that strain CEGF005 belongs to the genus *Bifidobacterium*. However, this value is lower than the recommended threshold at the species level (98.65%), indicating that it does not belong to the same species as the *Bifidobacterium animalis* strain in the database.

[0062] The 16S rDNA sequence of strain CEGF006, as shown in SEQ ID NO:2, exhibits 98.83–99% nucleotide similarity to the 16S rRNA sequence of *Bifidobacterium animalis*, with the highest similarity (99%) to *Bifidobacterium animalis* subsp. *lactis*. This value exceeds the recommended threshold for species identification, indicating that strain CEGF006 belongs to *Bifidobacterium animalis* subsp. *lactis*. Furthermore, the colony morphology of strain CEGF006 is similar to but different from the type strain of *Bifidobacterium animalis*, thus identifying it as a novel strain of *Bifidobacterium animalis* subsp. *lactis*.

[0063] The 16S rDNA sequence corresponding to strain CEGF007 is shown in SEQ ID NO:3. It has the highest nucleotide similarity (98%) to the 16S rRNA sequence of Bifidobacterium longum. This value is greater than the recommended threshold at the genus level (95%), indicating that strain CEGF007 belongs to the genus Bifidobacterium. However, this value is lower than the recommended threshold at the species level (98.65%), indicating that it does not belong to the same species as the Bifidobacterium longum strains in the database.

[0064] The 16S rDNA sequence corresponding to strain CEGF008 is shown in SEQ ID NO:4. It has the highest nucleotide similarity (98%) to the 16S rRNA sequence of Lactobacillus salivarius. This value is greater than the recommended threshold at the genus level (95%), indicating that strain CEGF008 belongs to the genus Lactobacillus. However, this value is lower than the recommended threshold at the species level (98.65%), indicating that it does not belong to the same species as the Lactobacillus strains in the database.

[0065] The above identification results indicate that the strains isolated and purified in this embodiment include two new strains of Bifidobacterium animalis subsp. lactis (named Bifidobacterium animalis subsp. lactis CEGF005 and Bifidobacterium animalis subsp. lactis CEGF006, respectively), one new strain of Bifidobacterium longum subsp. longum (named Bifidobacterium longum subsp. longum CEGF007), and one new strain of Lactobacillus salivarius (named Ligilactobacillus salivarius CEGF008).

[0066] 1.3 Genomic Data Sequencing and Analysis

[0067] The extracted bacterial genomic DNA was transported to Shenzhen Kangmei BGI Genomics Co., Ltd. via dry ice for sequencing. The company handled basic data control, genome assembly, and annotation. Kangmei BGI used the DNBSEQ sequencing platform to sequence the bacterial genome. The raw data underwent quality control using methods such as Trimmomatic, removing low-quality sequences and further removing host contamination, RNA, and repetitive sequences. The genome was then assembled using Spades and Megahit tools, and functional annotation was performed using Prokka.

[0068] After annotation, the safety of the strains was evaluated using databases. Antibiotic resistance and tolerance were analyzed using the CARD database (https: / / card.mcmaster.ca / ), virulence factors were analyzed using the VFDB resistance virulence gene database (http: / / www.mgc.ac.cn / VFs / ), and pathways, metabolic enzymes, and gene families were analyzed using the KEGG database (https: / / www.kegg.jp / ) and MetaCyc database (https: / / metacyc.org / ).

[0069] 1.4 Safety evaluation of the strain

[0070] According to internationally recognized lists of probiotics, *Bifidobacterium animalis* and *Bifidobacterium longum* are typical probiotic species in the genus *Bifidobacterium*, while *Lactobacillus salivarius* is a species in the genus *Lactobacillus* that has been definitively identified as having probiotic properties. To verify the safety of the four newly isolated strains, the sequenced genomes of all functional strains were compared with the Virulence Factor Database (VFDB). Few virulence genes were found in the VFDB database, and no obvious virulence genes were found in the screened functional strains, indicating high safety for humans. In other words, through rigorous bioinformatics analysis and database comparison, these strains have been ruled out as pathogenic at the gene sequencing level and can be considered safe for subsequent functional studies and drug development.

[0071] Regarding antibiotic resistance prediction, the genomes were compared with the CARD database. These strains generally showed very little resistance to antibiotics; even when resistance was observed, it was only against rifampin, mupirocin, and tetracycline, while they were sensitive to other commonly used antibiotics. This demonstrates that these strains are relatively safe for use as probiotics because they do not carry a wide range of antibiotic resistance genes.

[0072] The two newly isolated Bifidobacterium animalis subsp. lactis strains were deposited on May 27, 2024, at the China Center for Type Culture Collection (Wuhan University Collection Center), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. The accession number CEGF005 is CCTCC NO: M 20241079, deposited on May 27, 2024, and its classification name is *Bifidobacterium animalis subsp. lactis* CEGF005; the accession number CEGF006 is CCTCC NO: M 20241080, deposited on May 27, 2024, and its classification name is *Bifidobacterium animalis subsp. lactis* CEGF006.

[0073] The newly isolated strain of *Bifidobacterium longum* subsp. longum (accession number CEGF007) was deposited on June 3, 2024, at the China Center for Type Culture Collection (Wuhan University Collection Center), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. Its accession number is CCTCC NO: M 20241133, the deposit date is June 3, 2024, and its classification name is *Bifidobacterium longum* subsp. longum CEGF007.

[0074] The newly isolated *Ligilactobacillus salivarius* strain (accession number CEGF008) was deposited on May 30, 2024, at the China Center for Type Culture Collection (Wuhan University Collection Center), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. Its accession number is CCTCC NO: M 20241111, and the deposit date is May 30, 2024. Its classification name is *Ligilactobacillus salivarius* CEGF008.

[0075] Example 2: Co-colonization of Combined Bacteria

[0076] The strains used in the experiment were the four preserved strains in Example 1: CCTCC NO: M 20241079, CCTCC NO: M20241080, CCTCC NO: M 20241133 and CCTCC NO: M 20241111.

[0077] Strain culture: *Bifidobacterium animalis* was cultured on MRS liquid medium (with 0.5 g / L cysteine ​​hydrochloride) under the following conditions: 37°C, anaerobic incubator (85% N2, 10% CO2, 5% H2) for 24-48 hours. Activation passages: Two consecutive subcultures were performed, and the logarithmic growth phase culture (OD) was collected. 600 =0.8~1.0).

[0078] Bifidobacterium longum was cultured on MRS liquid medium (with 0.5 g / L cysteine ​​hydrochloride) under the following conditions: 37°C, anaerobic incubator, for 24–48 hours. Activation passages: two consecutive subcultures were performed, and the logarithmic growth phase culture (OD200) was collected. 600 =0.8~1.0).

[0079] Lactobacillus saliva-associated with MRS liquid medium was used. Culture conditions: 37℃, anaerobic or microaerobic culture, for 24-48 hours. Activation passages: two consecutive subcultures, collecting the logarithmic growth phase (OD) culture. 600 =0.8~1.0).

[0080] Experimental materials: see Table 1.

[0081] Primers used in the experiment: see Table 2.

[0082] Table 1 Experimental Materials

[0083]

[0084] Table 2 Primers used in the experiment

[0085]

[0086] Experimental methods:

[0087] (1) In vitro co-culture

[0088] MRS medium: Weigh 54g of MRS medium, add distilled water to a final volume of 1L, stir well, and add 0.1% resazurin as an anaerobic indicator. Purge with nitrogen and heat to remove oxygen. After deoxygenation, add 0.5g / L cysteine ​​hydrochloride, dispense into anaerobic bottles, and autoclave at 121℃ for 20min. All strains inoculated with the above culture were then anaerobically cultured at 37℃ (using an anaerobic incubator).

[0089] After culturing the above four strains individually for 48 hours, they were combined in a 1:1:1:1 ratio and inoculated into the same 5 mL vial at a 1% inoculum. The mixture was then cultured at 37°C under anaerobic conditions, and the OD value was measured after 12 hours. The measured OD value (600 nm) was 2.2.

[0090] (2) In vivo colonization

[0091] The experimental procedure is as follows Figure 1 As shown, the details are as follows:

[0092] ① Antibiotic treatment: Antibiotic cocktail (ABX): ampicilin 0.2 g / L; vancomycin 0.2 g / L; neomycin 0.4 g / L; metronidazole 0.2 g / L; aspartame 3.75 g / L, prepared with distilled water and filtered for sterilization. Seven mice that had successfully developed the model were treated with ABX for 3 days before gavage to eliminate intestinal native bacteria.

[0093] ② Strains preparation: After screening, the strains were activated twice by subculture and cultured to the stationary phase. The cells were collected by centrifugation at 7000 rpm / min for 5 min, washed and resuspended with PBS, and the OD value was adjusted to 1.0. The strains were then mixed in equal proportions and concentrated 20-fold to ensure that the number of each strain administered by gavage was approximately 10. 9 about.

[0094] ③ Gavage procedure: Remove the food from the mice 4 hours before gavage, and administer 150µL of 10% NaHCO3 (filtered and sterilized) to neutralize the stomach acid (remove the water before neutralizing the stomach acid). 15 minutes later, administer 200µL of mixed probiotics. Observe the mice's condition 2 hours later and restore food and water. Administer probiotics once every 3 days, for a total of 3 times.

[0095] ④ Sample collection: Before the start of gavage after antibiotic treatment, collect control stool; after the start of gavage, start collecting experimental stool, collect stool for 7 consecutive days (100-300mg) and place it in a 1.5mL centrifuge tube, weigh it and freeze it at -80°C.

[0096] Fecal genomic DNA extraction: The procedure was performed according to the instructions for the fecal genomic DNA extraction kit (Kangwei Century, China). The steps are as follows:

[0097] ① Take out the frozen fecal sample, add 1 mL of Buffer SW, vortex for 6 min to fully dissolve the sample, centrifuge at 13000×g for 1 min, and discard the supernatant.

[0098] ② Add 1 mL of Buffer SL to the tube, vortex for 6 min to mix the sample, and incubate in a 65°C water bath for 20 min, vortexing for 20 seconds every 4 min during this period.

[0099] ③ Add 40µL of RNase A solution with a concentration of 10mg / mL, vortex for 20 seconds, and let stand at room temperature for 8 minutes. Centrifuge at 13500×g for 3 minutes, and transfer 700µL of supernatant to a new centrifuge tube.

[0100] ④ Add 700µL Buffer GL to the centrifuge tube, invert and mix several times, place on ice for 5 minutes, and centrifuge at 13000×g for 5 minutes.

[0101] ⑤ Add the supernatant to the adsorption column already placed in the collection tube, and centrifuge at 13500×g for 3-5 minutes. If there is still liquid that has not been completely centrifuged, extend the centrifugation time. After centrifugation, discard the waste liquid in the collection tube and put the adsorption column back in.

[0102] ⑥ Add 500 µL of Buffer GW1 to the adsorption column, centrifuge at 12500 pm for 1 min, discard the waste liquid, and put the adsorption column back into the tube. Repeat once more.

[0103] ⑦ Add 500µL Buffer GW2 to the adsorption column, centrifuge at 12500rpm for 1min, discard the waste liquid, and put the adsorption column back into the tube.

[0104] ⑧ Without adding any liquid, centrifuge at 12500 rpm for 2 min, discard the waste liquid, and place the adsorption column at room temperature for 5 min to completely remove residual ethanol.

[0105] ⑨ Place the adsorption column in a clean centrifuge tube, add 150µL of double-distilled water dropwise to the middle part, let it stand at room temperature for 5 minutes, centrifuge at 12500rpm for 5 minutes, and then store at -20℃.

[0106] Bacterial genome extraction: Performed according to the instructions of the universal column-based genomic DNA extraction kit (Kangwei Century, China). The specific operating steps are as follows:

[0107] ① After culturing bacteria for 24 hours, take 2 mL of the culture into a centrifuge tube, centrifuge at 12500 rpm for 1 min, and aspirate the supernatant as thoroughly as possible.

[0108] ② Prepare Enzymatic Lysis Buffer: 20 mM Tris, pH 8.0; 2 mM Na2-EDTA; 1.2% Triton X-100; Lysozyme (lysozyme) to a final concentration of 20 mg / mL. Add 180 µL of Enzymatic Lysis Buffer to the tube to resuspend the bacterial cells. Incubate at 37°C for 30 min using a constant temperature metal bath.

[0109] ③ After incubation, add 20µL Proteinase K and vortex on a vortex mixer for 15 seconds to mix thoroughly.

[0110] ④ Add 200µL BufferGL directly, vortex to mix, and incubate at 56℃ for 30min.

[0111] ⑤ Add 40 µL of RNase A solution with a concentration of 10 mg / mL, vortex for 20 seconds, and let stand at room temperature for 8 minutes. Then add 200 µL of anhydrous ethanol and vortex to mix.

[0112] ⑥ Add all the obtained solution to the adsorption column that has been loaded into the collection tube, centrifuge at 12500 rpm for 1 min, discard the waste liquid, and put the adsorption column back into the tube.

[0113] ⑦ Add 500 µL of Buffer GW1 to the adsorption column, centrifuge at 12500 pm for 1 min, discard the waste liquid, and put the adsorption column back into the tube.

[0114] ⑧ Add 500µL of Buffer GW2 to the adsorption column, centrifuge at 12500rpm for 1min, discard the waste liquid, and put the adsorption column back into the tube. Repeat once more.

[0115] ⑨ Without adding any liquid, centrifuge at 12500 rpm for 2 min, discard the waste liquid, and place the adsorption column at room temperature for 5 min to completely remove residual ethanol.

[0116] ⑩ Place the adsorption column in a clean centrifuge tube, add 100µL of double-distilled water dropwise to the middle part, let it stand at room temperature for 5 minutes, centrifuge at 12500rpm for 5 minutes, and then store at -20℃.

[0117] PCR detection of bacterial colonization in vivo: Based on the genome sequencing results, specific primers were designed for each strain using NCBI. The primer list is shown in Table 2, and the PCR reaction system and PCR reaction conditions are shown in Tables 3 and 4. Using fecal genomic DNA from different time points as templates, the presence of bacteria in feces was detected by PCR.

[0118] Table 3 PCR reaction system

[0119]

[0120] Table 4 PCR conditions

[0121]

[0122] The colonization of different strains in mice was detected by PCR, and the results are shown in Table 5.

[0123] Table 5. Colonization status of the combined bacteria

[0124]

[0125] The colonization results are shown in Table 5. PCR-agarose gel electrophoresis was used to detect fecal samples from mice 1-5 days after oral gavage. The results showed that clear target bands appeared at the expected molecular weight positions in the CEGF005 and CEGF007 groups on day 4, and in the CEGF006 and CEGF008 groups on day 3, indicating that the four target strains CEGF005, CEGF006, CEGF007, and CEGF008 could successfully colonize the mouse intestines.

[0126] Example 3: Comparison of single bacteria and combined bacteria

[0127] (1) Preparation of single bacterial cultures: The four strains screened in Example 1 (Bifidobacterium animalis subsp. lactis CEGF005, Bifidobacterium animalis subsp. lactis CEGF006, Bifidobacterium longum subsp. longum CEGF007 and Ligilactobacillus salivarius CEGF008) were activated and inoculated into the culture medium at a 1% inoculum. They were cultured in an anaerobic environment at 37℃ for 24 h to obtain the probiotic culture solutions. The cultures were centrifuged at 4,000 rpm for 15 minutes to collect the bacterial pellets. The pellets were washed twice with sterile PBS buffer (pH 7.2). Based on the viable count, the pellets were resuspended in sterile PBS to obtain a viable count of 1×10⁻⁶. 9 Single bacterial cultures with a concentration of CFU / mL are abbreviated as CEGF005, CEGF006, CEGF007, and CEGF008.

[0128] (2) Preparation of combined bacterial suspension: The probiotic culture was centrifuged at 4,000 rpm for 15 minutes, and the bacterial pellet was collected and washed twice with sterile PBS buffer (pH 7.2). The mixture of *Bifidobacterium animalis* subsp. *lactis* CEGF005, *Bifidobacterium animalis* subsp. *lactis* CEGF006, *Bifidobacterium longum* subsp. *longum* CEGF007, and *Ligilactobacillus salivarius* CEGF008 was prepared according to the addition ratio of 1:1:1:1. Based on the viable count, the mixture was resuspended in sterile PBS to obtain the combined bacterial suspension (total viable count 1 × 10⁻⁶). 9 (CFU / mL).

[0129] This embodiment uses a high-fat diet to induce an animal model of obesity combined with metabolic syndrome. The most common metabolic syndromes include hyperlipidemia, type 2 diabetes, and metabolic-associated fatty liver disease (MASLD). In this embodiment, normal mice fed a standard diet serve as the control group, while a high-fat diet is used to induce the establishment of a mouse model of obesity combined with metabolic syndrome. The experimental procedure is as follows: Figure 2 As shown, the details are as follows:

[0130] ① Experimental animals: 6-week-old male C57BL / 6J mice (SPF grade), with a uniform weight (18~22 g), were acclimatized to a normal maintenance diet (fat energy ratio ≤10%) for 1 week before the experiment began.

[0131] ②Source: Hubei Provincial Center for Disease Control and Prevention.

[0132] ③ Husbandry conditions: Mice were housed in the SPF-grade animal laboratory of Hubei Provincial Drug Safety Evaluation Center, with a constant temperature of 25±2℃ and a relative humidity of 50±5%. The light and dark cycle was 12 h. Mice in each group had free access to food and water. All experimental procedures complied with experimental animal ethics.

[0133] ④ Model building methods:

[0134] After the adaptation feeding period, the mice were randomly divided into two groups:

[0135] Normal control group (NC group): After acclimatization to normal feed for 1 week, the diet was fasted for 1 night and then given normal maintenance feed (fat energy ratio ≤10%).

[0136] High-fat diet group (HFD group): Six-week-old male C57BL / 6J mice (SPF grade), with as uniform a weight as possible, were acclimatized to a normal diet for one week, then fasted for one night and switched to a 60kcal% high-fat diet (HFD). The 60kcal% high-fat diet was selected from Changzhou Mouse No. 1 & No. 2 D12492. Mice were weighed weekly at fixed times, always before feeding, to avoid the immediate impact of the high-fat diet on weight. A significant increase in weight (P < 0.001) exceeding the average weight of mice fed a normal diet (ND) by more than 20% was considered a successful model establishment. Normal mice fed a normal diet served as the control group. Mice with large weight differences were culled, and the mice were regrouped according to the absence of differences in average weight between groups (model group, single-bacterial group, combined-bacterial group, and orlistat group), with 12 mice in each group.

[0137] ⑤ Dosing regimen:

[0138] Administration route: once every 3 days, via oral gavage;

[0139] Dosage volume: 200 μL / animal;

[0140] Drug administration cycle: 4 weeks of intervention (starting from the end of the modeling process);

[0141] Solvent control: The control group and the model group were administered an equal volume of sterile PBS by gavage. The specific details of the gavage administration for each group are as follows:

[0142] Normal control group: Normal feed + equal volume of sterile PBS buffer was administered by gavage;

[0143] Model group: High-fat diet + equal volume of sterile PBS buffer administered by gavage;

[0144] Orlistat group: high-fat diet + Orlistat (20 mg / kg);

[0145] Combined microbial group: high-fat diet + combined microbial group (combined microbial group consists of CEGF005+CEGF006+CEGF007+CEGF008, with a total viable count of 1×10⁻⁶). 9 (CFU / mL)

[0146] Single-strain CEGF005 group: high-fat diet + Bifidobacterium animalis subsp. lactis CEGF005 (live count 1×10⁻⁶) 9 (CFU / mL)

[0147] Single-strain CEGF006 group: high-fat diet + Bifidobacterium animalis subsp. lactis CEGF006 (live count 1×10⁻⁶)9 (CFU / mL)

[0148] Single-strain CEGF007 group: high-fat diet + Bifidobacterium longum subsp. longum CEGF007 (live count 1×10⁻⁶) 9 (CFU / mL)

[0149] Single-strain CEGF008 group: high-fat diet + Ligilactobacillus salivarius CEGF008 (live count 1×10⁻⁶) 9 (CFU / mL).

[0150] Before the experiment, feces were collected from each mouse and weighed to determine the starting weight. Except for the control group, all other groups were continuously fed a high-fat diet and administered medication via gavage. The control and model groups were administered an equal volume of sterile PBS buffer via gavage. The treatment protocols for each group are as follows: Figure 2 As shown in the diagram. Gavage procedure: 4 hours before gavage, mice were deprived of food and given 150µL of 10% NaHCO3 (filtered and sterilized) to neutralize stomach acid (water was removed before neutralization). 15 minutes later, the combined bacterial group was given 200µL of the combined bacterial solution, the single bacterial group was given 200µL of the single bacterial solution, the drug group was given 200µL of the drug solution, and the model and control groups were given an equal volume of sterile PBS buffer. Two hours later, the mice were observed to have resumed eating and drinking. Gavage was performed at the same time every day, once every 3 days. During the feeding period, the diet was monitored, and mouse weight and food intake were measured weekly. After gavage, mouse feces were collected to detect the colonization of the administered bacterial strains. The results showed that the four strains of the combined bacterial strains of this invention were detectable in the mice. The weight changes of each group after 28 days of intervention are shown in Table 6, and the weight gain of each group is shown in Table 7.

[0151] ⑥ Sample collection and processing

[0152] After the last administration, mice were fasted for 12 hours (but not water) and then subjected to an oral glucose tolerance test (OGTT). Serum and tissue samples were collected within 24 hours after the OGTT.

[0153] Oral glucose tolerance test (OGTT): All mice were fasted for 12 hours after the last gavage, and fasting blood glucose levels (0 min) were measured. After administering glucose at 2 g / kg according to the mice's body weight, blood was collected by tail clipping. Blood glucose levels were measured at 30 min, 60 min and 120 min after gavage using blood glucose test strips (Sinocare, China). The area under the blood glucose curve (AUC) was calculated using the trapezoidal method as follows. The blood glucose levels and AUC results for each group are shown in Table 8.

[0154] ;

[0155] Among them: G i T represents the blood glucose concentration (mmol / L or mg / dL) at the i-th time point (0, 30, 60, 120 min); i Let be the time (in minutes) at the i-th time point; n is the total number of time points.

[0156] Table 6. Changes in body weight of mice in each group.

[0157]

[0158] Table 6 shows the statistical analysis performed using SPSS 26.0. Data are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups, and Tukey's HSD test was used for post-hoc tests. In the table, day 0 of the control group is the starting point after 8 weeks of normal maintenance diet feeding following one week of acclimatization; day 0 of the other groups is the starting point after 8 weeks of high-fat diet (HFD) induction following one week of acclimatization.

[0159] During the experiment (0-28 days), the body weight of mice in each probiotic group showed significantly different changes, as shown in Table 7.

[0160] Table 7 Changes in body weight of mice in each group

[0161]

[0162] Control group: The weight fluctuated slightly between 28.5 and 30 g, and the cumulative weight gain in 28 days was 1.5 g, which is consistent with the physiological characteristics of adult mice whose weight tends to stabilize after being fed a normal diet.

[0163] Model group: On day 0, there was a highly significant statistical difference between the model group (34.6±1.9 g) and the control group (28.5±1.6 g) (P<0.0001), indicating that the high-fat diet-induced obesity model was successfully established. During the experiment, the model group's weight continued to increase, from (34.6±1.9) g to (38.2±2.9) g, with a cumulative increase of 3.6 g (10.4%), and an accelerated increase (+1.4 g) was observed from days 21 to 28, further confirming that the high-fat diet successfully induced the obesity phenotype.

[0164] In the drug group, the weight initially decreased and then stabilized, from (34.8±1.6) g to (32.8±2.4) g (-2.0 g) on ​​day 14, followed by a slight rebound and then stabilization, with a cumulative decrease of 0.8 g (2.3%). This was significantly different from the model group (P<0.005), indicating that the positive control drug effectively inhibited the weight gain induced by a high-fat diet and verified the effectiveness of the positive control drug.

[0165] Combined bacteria group: The body weight of mice after 28 days of combined bacteria intervention (32.6±1.9 g) was significantly lower than that of the model group (P<0.0001), with a weight loss of 5.6 g. This indicates that the combined bacteria of the present invention reversed the weight gain induced by high-fat diet and had a significant weight loss effect. The body weight of the combined bacteria group was further reduced compared with the drug group, suggesting that the weight loss effect of the combined bacteria is slightly better than that of the positive control drug and has the potential to replace the existing drug Orlistat.

[0166] Single-bacterial groups: Compared with the model group, the weight loss of mice in the four single-bacterial groups after 28 days of intervention ranged from 1.3 to 2.4 g. Although the weight of the single-bacterial groups decreased to some extent, in stark contrast, the combined-bacterial group (32.6 ± 1.9 g) after the same number of live bacteria intervention for the same number of days showed a weight loss of 5.6 g, which was significantly lower than that of the model group (P<0.0001) and significantly better than each single-bacterial group (P<0.05). The weight loss effect of the combined bacteria was 2.3 to 4.3 times that of the single-bacterial groups (2.8 times the average weight loss of the single-bacterial groups).

[0167] The above results indicate that the combination of the four single strains does not result in a simple additive effect, but rather a synergistic weight loss effect, with the combined strains showing a significantly better weight loss effect than the single strain intervention.

[0168] Table 8. Changes in blood glucose levels and AUC in mice of each group 0-120

[0169]

[0170] The data in Table 8 are expressed as mean ± standard deviation. There were no significant differences in baseline blood glucose levels among the groups. After oral glucose loading (0–120 min), blood glucose levels in all groups of mice showed a trend of first increasing and then decreasing, but there were significant differences between the groups.

[0171] Control group: Blood glucose reached its peak at 30 min (7.5±1.5 mmol / L), then dropped rapidly, and returned to the baseline level at 60 min (6.6±1.8 mmol / L) and 120 min (6.5±1.3 mmol / L), showing a typical normal glucose tolerance curve.

[0172] In the model group, blood glucose levels rose sharply to a peak of 26.6 ± 3.2 mmol / L at 30 min, significantly higher than that in the control group (P < 0.0001). At 60 min, blood glucose remained at a high level (17.7 ± 4.0 mmol / L), and although it decreased slightly at 120 min (13.8 ± 3.6 mmol / L), it was still significantly higher than that in the control group (P < 0.0001), indicating that the model group mice exhibited impaired glucose tolerance and insulin resistance.

[0173] In the drug group, blood glucose peaked at 30 min (18.5±3.8 mmol / L), significantly lower than that in the model group. It rapidly decreased to (12.2±4.2 mmol / L) at 60 min and further decreased to (7.5±1.9 mmol / L) at 120 min, approaching the control group level, indicating that the positive control drug can effectively promote a rapid decrease in postprandial blood glucose.

[0174] In the combined bacterial group, blood glucose also peaked at 30 min (20.0±4.2 mmol / L), with the peak value falling between that of the drug group and the model group. It decreased to (14.5±3.6 mmol / L) at 60 min and to (8.5±2.6 mmol / L) at 120 min. The overall blood glucose lowering trend was consistent with the drug group, but blood glucose levels at each time point were slightly higher than those in the drug group, suggesting that the combined bacterial group's blood glucose lowering effect was milder than that of the drug. However, there were no statistically significant differences between the two groups at any time point (P>0.05), indicating that the blood glucose lowering effect of the combined bacterial group was comparable to that of existing drugs.

[0175] Single-strain groups: The blood glucose trends of the four single-strain groups (CEGF005~008) were between those of the model group and the drug group, with peak values ​​of 23.5~25.0 mmol / L at 30 min. The CEGF006 group performed best, with a blood glucose level of (9.2±2.6) mmol / L at 120 min, significantly lower than the model group (P = 0.019). CEGF005 (12.5 mmol / L), CEGF007 (11.0 mmol / L), and CEGF008 (11.8 mmol / L) showed no statistically significant differences compared to the model group. Pairwise comparisons among the four single-strain groups showed no significant differences (P > 0.05), indicating that the effects of the probiotic interventions alone were statistically comparable.

[0176] Compared with the model group, only the CEGF006 group showed a significant difference in blood glucose at 120 min among the four single-bacterial groups (P = 0.019), while the other three groups showed no significant difference (P > 0.05), indicating that the individual intervention of each single bacterium had limited effect. However, with the same number of live bacteria and the same number of days of intervention, the combined bacteria group of this invention had a peak blood glucose level of (20.0 ± 4.2) mmol / L at 30 min, the lowest among all probiotic groups; it dropped to (8.5 ± 2.6) mmol / L at 120 min, which was not only significantly lower than the model group, but also lower than all single-bacterial groups (including the optimal single-bacterial group CEGF006 at 9.2 mmol / L), indicating that the combined bacteria intervention produced a better blood glucose lowering effect than single bacteria.

[0177] Compared to the model group, at 120 min, the combined bacterial group lowered blood glucose by 5.3 mmol / L, while the average blood glucose lowering effect of the four single bacterial groups was only 2.7 mmol / L. The blood glucose lowering effect of the combined bacteria was 1.96 times the average level of the single bacteria. Furthermore, the combined bacteria also outperformed the optimal single bacterial group, CEGF006. These results indicate that the combination of the four single bacteria did not produce a simple additive effect, but rather a synergistic blood glucose lowering effect, which was significantly better than single-strain intervention.

[0178] The trapezoidal method was used to calculate the AUC of OGTT in each group of mice using the mean. 0-120 The results showed:

[0179] Compared with the control group, the AUC in the model group was significantly increased by about 164% (2092.5 vs 792.0, P<0.001), indicating that the high-fat diet induced extremely significant glucose intolerance and insulin resistance, demonstrating the successful construction of the metabolic syndrome model.

[0180] Compared with the model group, the AUC of the drug group decreased to 1401.0 (P<0.01), indicating that orlistat effectively improved the impaired glucose tolerance of the model mice.

[0181] Compared with the model group, the AUC of the combined bacteria group significantly decreased to 1584.0 (P<0.01), indicating that the combined bacteria can significantly improve glucose tolerance, suggesting that it may exert a protective effect by improving insulin resistance. Furthermore, there was no statistically significant difference in AUC between the combined bacteria group and the drug group (1401.0) (P>0.05), suggesting that the two groups have similar effects in improving glucose tolerance. These results indicate that the combined bacteria have good potential for prevention or improvement in blocking the progression from lipid metabolism disorders to type 2 diabetes.

[0182] Obesity is not merely an increase in weight, but a disease state caused by dysfunction of adipose tissue, leading to systemic metabolic disorders throughout the body. Lipid metabolism disorders are one of the most common complications of obesity and a key bridge connecting obesity with metabolic diseases such as hyperlipidemia, diabetes, and metabolic-associated fatty liver disease (MASLD).

[0183] After the experiment, six mice were randomly selected from each group, and feces were collected from each mouse individually. The final weight and body length of each mouse were measured. The animals were fasted overnight, then euthanized. Blood, perigonial adipose tissue, liver tissue, and intestinal tissue were quickly collected, and the adipose tissue was weighed simultaneously. Tissues used for lysis were placed in centrifuge tubes and flash-frozen in liquid nitrogen, then immediately stored at -80°C before further processing. Tissues used for histological analysis were fixed overnight in 4% paraformaldehyde at room temperature for embedding and other treatments. Blood samples were centrifuged at 4°C, 2500 rpm / min for 15 min, and serum was collected for subsequent analysis. All samples were stored at -80°C before use. Serum-related markers were detected:

[0184] ① Serum triglyceride (TG) assay: According to the instructions of the serum triglyceride kit (Nanjing Jiancheng Biotechnology, China), set up standard wells, blank wells and sample wells. Add 2.5µL of standard, distilled water and serum sample to each well, then add 250µL of assay working solution to all wells, mix well and incubate at 37℃ for 10 minutes. Use an ELISA reader to measure the absorbance value of each well at a wavelength of 510nm. Calculate the serum triglyceride content according to the instructions.

[0185] ② Serum total cholesterol (TC) measurement: same as serum triglyceride measurement method.

[0186] ③ Serum High-Density Lipoprotein (HDL) Measurement: According to the instructions of the serum high-density lipoprotein kit (Nanjing Jiancheng Biotechnology, China), set up standard wells, blank wells, and sample wells. Add 2.5 µL of standard, distilled water, and serum sample to each well. Then add 180 µL of solution R1 to all wells, mix well, and incubate at 37°C for 5 minutes. Use an ELISA reader to measure the absorbance value A1 of each well at a wavelength of 546 nm. Then add 60 µL of solution R2 to all wells, mix well, and incubate at 37°C for 5 minutes. Use an ELISA reader to measure the absorbance value A2 of each well at a wavelength of 546 nm. Calculate the serum high-density lipoprotein content according to the instructions.

[0187] ④ Serum low-density lipoprotein (LDL) measurement: Same as serum high-density lipoprotein measurement method.

[0188] ⑤ Serum Alanine aminotransferase (ALT) assay: Set up test wells and control wells according to the instructions of the serum ALT kit (one test well and one control well for each sample). Add 20 µL of preheated matrix solution (37°C) to all wells. Add 5 µL of the test sample to each test well. Mix well and incubate at 37°C for 30 min. Then add 20 µL of colorimetric solution to all wells and 5 µL of the test sample to each control well. Mix well and incubate at 37°C for 20 min. Remove the plate and add 200 µL of stop solution to all wells. Mix well and let stand at room temperature for 15 min. Measure the absorbance of each well at a wavelength of 510 nm. Calculate the serum ALT content according to the instructions.

[0189] ⑥ Serum aspartate aminotransferase (AST) measurement: Same as serum alanine aminotransferase (ALT) measurement method.

[0190] ⑦ Sectioning: Tissue was fixed overnight in 4% paraformaldehyde at room temperature and then embedded in paraffin. Tissue sections were cut to 5 μm thickness according to standard protocol and stained with hematoxylin and eosin (H&E). The resulting sections were observed and photographed under a microscope. Throughout the image acquisition process, images were acquired using a Nikon digital imaging system under the same acquisition settings, recording cell staining at 200x magnification from three fields of view per section. Data were analyzed and processed using Image-Pro Plus 6.0.

[0191] To further verify the weight-loss effect of the combined bacteria, this embodiment also statistically analyzed the weight of the paragonial white adipose tissue obtained from dissection of mice and stained it with H&E. The staining results are as follows: Figure 3 As shown, Figure 3 (A) is the control group, (B) is the model group, (C) is the drug group, and (D) is the combined bacterial group; the weight of unilateral paragonal white adipose tissue is statistically analyzed as follows: Figure 4 As shown, Figure 4 The bar chart, from left to right, represents the control group, model group, drug group, and combined bacterial group.

[0192] from Figure 3 As can be seen, the adipocytes in the normal control group mice were tightly packed and small in size; while the adipocytes in the model group mice were significantly enlarged, irregularly arranged, and had blurred cell boundaries, further confirming the successful establishment of the obesity model. After intervention with the combined bacteria and the drug Orlistat, the adipocyte volume was significantly reduced, indicating that both the combined bacteria and Orlistat intervention could improve adipocyte hypertrophy and inhibit adipose tissue proliferation and hypertrophy. Furthermore, the combined bacteria group showed a significantly greater number of cells observed in the same field of view.

[0193] Figure 4 The results showed that, compared with the control group, the weight of unilateral paragonal white adipose tissue in the model group mice was significantly increased (P < 0.001), indicating that a high-fat diet induced significant epididymal fat accumulation, confirming the successful establishment of the obesity model. Compared with the model group, the weight of unilateral paragonal white adipose tissue in both the combined bacterial strain group and the positive control drug group was significantly decreased (P < 0.05), and there was no significant difference between the two intervention groups, suggesting that the combined bacterial strain of the present invention has a fat-reducing effect comparable to Orlistat, that is, the combined bacterial strain can effectively inhibit the excessive proliferation of adipose tissue induced by a high-fat diet, and the fat-reducing effect reaches the same level as the positive control drug. The above results indicate that the combined bacterial strain of the present invention has a significant weight-reduction and lipid-lowering effect, has the potential to replace the existing drug Orlistat, and can be used in the preparation of weight-reduction and lipid-lowering drugs.

[0194] The levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL), and high-density lipoprotein cholesterol (HDL) in mouse serum were measured, and the results are as follows: Figure 5 As shown, Figure 5 In the table, A represents serum triglyceride (TG) levels; B represents serum total cholesterol (TC) levels; C represents serum high-density lipoprotein cholesterol (HDL) levels; and D represents serum low-density lipoprotein cholesterol (LDL) levels. Figure 5 The bar chart, from left to right, represents the control group, model group, drug group, and combined bacterial group.

[0195] Depend on Figure 5 The results showed that the serum total cholesterol and low-density lipoprotein cholesterol levels in the model group mice were significantly higher than those in the control group, exhibiting typical biochemical characteristics of hypercholesterolemia. This indicates that the high-fat diet successfully induced hypercholesterolemia in the mice, meaning the model mice were accompanied by hypercholesterolemia. Compared to the control group, the triglyceride content in the liver of the model group mice was significantly increased (0.035 mmol / g vs. 0.018 mmol / g), indicating abnormal lipid deposition in the liver. Abnormal triglyceride deposition in the liver is an important manifestation of lipid metabolism disorder.

[0196] In the therapeutic intervention experiment, compared with the model group, the serum total cholesterol and low-density lipoprotein cholesterol levels of mice in the combined bacterial group were significantly reduced, and the liver triglyceride content (0.02 mmol / g) was also significantly decreased. The results indicate that this combined bacterial intervention effectively improved abnormal lipid deposition in the liver while significantly reducing serum total cholesterol (TC) levels, suggesting that it can improve hypercholesterolemia by regulating liver lipid metabolism through multiple targets, demonstrating good potential in intervening in lipid metabolism-related diseases.

[0197] In summary, compared with the normal control group, the model group mice showed a significant increase in body weight (P < 0.01), and HE staining of the epididymal adipose tissue revealed significantly enlarged adipocytes (P < 0.01), with loose and irregular arrangement and blurred cell boundaries, indicating the successful establishment of the obesity model. Regarding metabolic indicators, in the glucose tolerance test, the model group mice had a blood glucose level exceeding 11.1 mmol / L 30 minutes after glucose loading (P < 0.001 vs. control group), while serum total cholesterol and low-density lipoprotein cholesterol levels were significantly elevated (P < 0.001), indicating that the model mice simultaneously exhibited glucose intolerance (consistent with type 2 diabetes) and hypercholesterolemia. These results confirm that this model mouse model conforms to the characteristics of obesity-related type 2 diabetes complicated with hypercholesterolemia and can be used to evaluate the intervention effect of probiotics.

[0198] While orlistat effectively reduces body weight, total cholesterol, and LDL cholesterol, it carries a potential risk of liver and kidney damage. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are the most routine and essential indicators for assessing hepatocellular damage. When hepatocellular cells are damaged, these two enzymes are released into the bloodstream, leading to elevated serum levels. To assess the safety impact of combined bacterial intervention on liver function in mice, this example measured serum ALT and AST levels. The results are as follows: Figure 6 As shown. Figure 6 In the table, A represents the serum AST level, and B represents the serum ALT level. Figure 6 The bar chart, from left to right, represents the control group, model group, drug group, and combined bacterial group.

[0199] Depend on Figure 6 It was found that, compared with the control group, the serum AST and ALT levels of mice in the model group were significantly increased, suggesting that a high-fat diet induced hepatocellular damage, with ALT being more sensitive to liver injury. After intervention with orlistat, the serum ALT levels of mice further increased, suggesting that this drug may exacerbate liver damage. However, after intervention with the combined bacteria of this invention, the serum AST and ALT levels of mice were significantly reduced (P < 0.05 vs. model group), indicating that the combined bacteria have a significant effect in alleviating hepatocellular damage, suggesting that it may exert a protective effect against metabolic-associated fatty liver disease (MASLD) by improving hepatic lipid metabolism disorders.

[0200] Example 4 Comparison of different bacterial combinations

[0201] The strains used in the experiment were the probiotics (cb9y2, cb41m2, cb9y5, cb26y3, cb2y4, cb23y1, b15m2, cb40y4) in patent CN116286470A and the combined strains in Example 3.

[0202] Preparation of combined bacteria: The strains listed in Table 9 were transferred and activated twice, then cultured to the stationary phase. The cells were collected by centrifugation at 7000 rpm / min for 5 min, washed with PBS, and resuspended. The OD values ​​were adjusted to 1.0. The bacteria were then mixed in equal proportions and concentrated 20-fold to ensure that the number of viable bacteria for each combined bacteria via gavage was 1 × 10⁻⁶. 9 The CFU / mL values ​​are respectively designated as combined bacteria A, combined bacteria B, combined bacteria C, and the combined bacteria of this invention.

[0203] Experimental animals: 6-week-old male C57BL / 6J mice (SPF grade), with a uniform weight (18~22 g), were acclimatized to a normal maintenance diet (fat energy ratio ≤10%) for 1 week before the experiment began.

[0204] Source: Hubei Provincial Center for Disease Control and Prevention.

[0205] Husbandry conditions: Mice were housed in the SPF-grade animal laboratory of the Hubei Provincial Drug Safety Evaluation Center, under a constant temperature of 25±2℃ and a relative humidity of 50±5%. A 12-hour light-dark cycle was used. Mice in each group had free access to food and water. All experimental procedures complied with laboratory animal ethics. The experimental procedure is as follows: Figure 2 As shown, the details are as follows:

[0206] Normal mice fed a standard diet served as the control group, while a high-fat diet was used to induce an obesity model. Six-week-old male C57BL / 6J mice (SPF grade), with as uniform a weight as possible, were acclimatized to a standard irradiated diet for one week, then fasted for one night before being switched to a 60kcal% high-fat diet (HFD) to establish the obesity model. Mouse weight was measured weekly, and a weight exceeding the average weight of ND-fed mice by 20% was considered a successful model establishment indicator. The established model mice were then regrouped (model group, drug group, and four combined bacterial groups) with 6-12 mice per group, based on the absence of significant differences in mean values ​​between groups. Feces were collected from each mouse before the experiment, and the weight was used as the starting point for weight. The treatment protocols for each group are as follows: Figure 2 As shown, different solutions were administered to different groups via gavage.

[0207] Gavage procedure: 4 hours before gavage, remove the mice from the food supply and administer 150µL of 10% NaHCO3 (filtered and sterilized) to neutralize stomach acid (remove water before neutralizing stomach acid). 15 minutes later, each probiotic combination group is administered 200µL of mixed probiotics (live bacteria count 1×10⁻⁶) according to Table 9. 9 The mice were administered 200 µL of the drug (Orlistat) solution via gavage, while the model and control groups were administered an equal volume of sterile PBS buffer via gavage. Two hours later, the mice were observed to allow them to resume eating and drinking. Gavage was repeated at the same time every day for three days. Diet was monitored during the feeding period. After gavage, fecal samples were collected to assess the colonization of the administered bacterial strains. Results showed that all four strains from the combined bacteria were viable in the mice.

[0208] Table 9. List of probiotic formulations and corresponding strains for gavage.

[0209]

[0210] In Table 9, “cb9y2” refers to “Bifidobacterium animalis cb9y2 strain”; “cb2y4” refers to “Bifidobacterium animalis cb2y4 strain”; “cb41m2” refers to “Lactobacillus plantarum cb41m2 strain”; “cb9y5” refers to “Bifidobacterium longum cb9y5 strain”; “b15m2” refers to “Bifidobacterium animalis b15m2 strain”; “cb26y3” refers to “Bifidobacterium longum cb26y3 strain”; “cb23y1” refers to “Bifidobacterium longum cb23y1 strain”; “cb40y4” refers to “Bifidobacterium longum cb40y4 strain”; “CEGF005” refers to “Bifidobacterium animalis subsp. lactis CEGF005”; and “CEGF006” refers to “Bifidobacterium animalis subsp. lactis”. "CEGF006"; "CEGF007" is "Bifidobacterium longum subsp.longum CEGF007"; "CEGF008" is "Ligilactobacillus salivariusCEGF008".

[0211] Weigh the mice weekly before each feeding, and record the weight gain of each group after the experiment. Figure 7 As shown, Figure 7 From left to right, the groups are: control group, model group, drug group, combined bacteria group A, combined bacteria group of the present invention, combined bacteria group B, and combined bacteria group C.

[0212] Oral glucose tolerance test (OGTT): After the last gavage, all mice in all groups were fasted for 12 hours, and fasting blood glucose levels (0 min) were measured. Glucose was administered at 1 g / kg based on mouse body weight, and blood was collected by tail clipping. Blood glucose levels were measured at 30 min, 75 min, and 120 min after gavage using blood glucose test strips (Sinocare, China). Blood glucose curves were plotted, and the area under the OGTT curve (AUC) was calculated using the trapezoidal method. The areas under the OGTT curves for different bacterial combinations are shown below. Figure 8 As shown, Figure 8 From left to right, the groups are: control group, model group, drug group, combined bacteria group A, combined bacteria group of the present invention, combined bacteria group B, and combined bacteria group C.

[0213] After the experiment, six mice were randomly selected from each group, and feces were collected from each mouse individually. The weight and body length of the mice at the experimental endpoint were measured. The animals were fasted overnight, then euthanized. Blood, perigonial adipose tissue, liver tissue, and intestinal tissue were quickly collected, and the adipose tissue was weighed simultaneously. Tissues used for lysis were placed in centrifuge tubes and flash-frozen in liquid nitrogen, and immediately stored at -80°C before further processing. Tissues used for histological analysis were fixed overnight in 4% paraformaldehyde at room temperature for embedding and other treatments. Blood samples were centrifuged at 4°C, 2500 rpm / min for 15 min, and serum was collected for subsequent analysis. All samples were stored at -80°C before use.

[0214] Sectioning: Tissue was fixed overnight in 4% paraformaldehyde at room temperature and then embedded in paraffin. Sections were cut to 5 μm thickness according to standard protocol and stained with hematoxylin and eosin (H&E). The resulting sections were observed and photographed under a microscope. Throughout the image acquisition process, images were acquired using a Nikon digital imaging system under the same acquisition settings, recording cell staining at 200x magnification from three fields of view per section. Results are as follows: Figure 9 and Figure 10 As shown, Figure 9 and Figure 10 The groups are, in order, control group, model group, drug group, combined bacteria group A, combined bacteria group of the present invention, combined bacteria group B, and combined bacteria group C.

[0215] Compared with the control group, the model group mice showed a significant increase in body weight and markedly enlarged adipocytes (P < 0.01), with loose and irregular arrangement and blurred cell boundaries, indicating a successful establishment of the obesity model. Regarding metabolic indicators, compared with the normal control group, the model group mice showed a sharp increase in blood glucose levels to 25 mmol / L 30 minutes after glucose loading (far exceeding the diagnostic threshold of 11.1 mmol / L for impaired glucose tolerance). Simultaneously, serum total cholesterol (TC) and low-density lipoprotein (LDL) levels reached 6.7 mmol / L and 2.0 mmol / L, respectively, both showing highly significant increases (P < 0.001). These results confirm that the model mice also exhibit impaired glucose tolerance and hypercholesterolemia.

[0216] Figure 7 The results showed that, compared with the control group, the model group mice had a significantly increased body weight, indicating that the high-fat diet successfully induced an obesity phenotype in the mice. Compared with the model group, the body weight of all four probiotic combination intervention groups was significantly reduced (P < 0.05). Among them, the combined probiotic group of this invention showed the largest reduction in body weight, which was significantly lower than the other three combined probiotic groups (P < 0.05), demonstrating the best weight loss effect.

[0217] Figure 8The results showed that, compared with the control group, the blood glucose level and area under the OGTT curve of the model group mice were significantly increased, indicating that the high-fat diet successfully induced glucose intolerance and insulin resistance in mice, specifically manifested as impaired glucose tolerance and decreased insulin sensitivity.

[0218] Compared to the model group, different probiotic combinations exhibited different intervention effects. The area under the OGTT curve (AUC) of combination C was not statistically different from that of the model group (P > 0.05), while the AUCs of combination A and B were significantly lower than those of the model group (P < 0.05). In contrast, the AUC of the OGTT curve of the present invention's combination group was significantly lower than that of the model group (P < 0.01). This result indicates that the present invention's combination probiotics have the most significant effect in improving glucose tolerance disorders, superior to other tested combinations. Specifically:

[0219] Although the combined bacteria A and B had some improvement effects, their effects were not as good as those of the combined bacteria of this invention; combined bacteria C did not show a significant effect in improving glucose tolerance; the combined bacteria of this invention can significantly reduce the area under the glucose tolerance curve, suggesting that it has better potential in improving insulin resistance and restoring blood glucose regulation ability.

[0220] Figure 9 The results showed that, compared with the control group, the adipocytes of the model group mice were significantly enlarged, with an average area of ​​approximately 1.2 × 10⁻⁶. 4 µm 2 (P < 0.01), the cells were loosely and irregularly arranged with blurred cell boundaries, indicating that the obesity model was successfully constructed. Compared with the model group, the area of ​​adipocytes did not change significantly after intervention with combined bacteria C (existing bacteria) (P > 0.05), while the area of ​​adipocytes decreased significantly after intervention with combined bacteria A, combined bacteria B (existing bacteria), and the combined bacteria provided by this invention (P < 0.05). Among them, the intervention effect of the combined bacteria of this invention was the most significant, with the area of ​​adipocytes approaching the level of the control group, indicating that its effect on improving adipocyte hypertrophy is superior to that of existing bacterial combinations.

[0221] Obesity triggers systemic lipid metabolism disorders, which in turn have a significant negative impact on the liver. Excess calories are converted into fat and abnormally accumulate in hepatocytes, leading to metabolic-associated fatty liver disease (MASLD). To observe the pathological morphological changes and degree of steatosis in the liver tissues of mice in each group, this example performed H&E staining on the dissected liver tissues. The H&E staining results are as follows: Figure 10 As shown.

[0222] Under a microscope, the cytoplasm of normal hepatocytes is homogeneous. During pathological section preparation, steps such as dehydration and embedding are required. During this process, fats (oils) are dissolved by organic solvents such as alcohol and xylene. Areas where fat was originally present become vacuoles; that is, the original locations of these vacuoles were where triglycerides (i.e., fat) had accumulated within the hepatocytes. Figure 10 The results showed that the hepatocytes of the control group mice had normal morphology, tightly arranged hepatic cords, and no obvious fat vacuoles. The livers of the model group mice showed obvious diffuse macrovesicular fat vacuoles with large area and large number of vacuoles. The proportion of fat vacuoles in the liver tissue of the model group was significantly higher than that of the control group (P < 0.001), and the TG content in the liver of the model group was significantly higher than that of the control group (P < 0.05), indicating that significant lipid deposition occurred in the hepatocytes of the model mice.

[0223] Different combinations of probiotics showed differentiated effects after intervention: Group B still showed significant macrovesicular fat vacuoles, with no significant improvement compared to the model group; Groups A and C showed a small number of microfat vacuoles, with a reduced degree of fatty degeneration compared to the model group; while the combined bacteria of this invention showed intact hepatocyte structure, no fat vacuoles, and the mildest degree of fatty degeneration. Compared to the model group, the combined bacteria of this invention significantly reduced the proportion of fat vacuoles in the liver tissue of mice (P < 0.001) and significantly reduced liver TG content (P < 0.05); compared to the model group, the combined bacteria A, B, and C significantly reduced the proportion of fat vacuoles in the liver tissue of mice (P < 0.05). These results indicate that the combined bacteria of this invention are most effective in improving abnormal lipid deposition in the liver (i.e., the core pathological feature of metabolic-associated fatty liver disease).

[0224] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A probiotic composition, characterized in that, Its composition consists of Bifidobacterium animalis subsp. lactis CEGF005, Bifidobacterium animalis subsp. lactis CEGF006, Bifidobacterium longum subsp. longis CEGF007 and Lactobacillus salivati ​​CEGF008; The Latin scientific name of the Bifidobacterium animalis subsp. lactis CEGF005 is Bifidobacterium animalis subsp. lactis CEGF005, the accession number is CCTCC NO: M 20241079, and the accession date is May 27, 2024. The Latin scientific name of the Bifidobacterium animalis subsp. lactis CEGF006 is Bifidobacterium animalis subsp. lactis CEGF006, the accession number is CCTCC NO: M 20241080, and the accession date is May 27, 2024. The Latin scientific name of the Bifidobacterium longum subsp. longum CEGF007 is Bifidobacterium longum subsp. longum CEGF007, the accession number is CCTCC NO: M 20241133, and the accession date is June 3, 2024. The Latin scientific name of the Lactobacillus salivarius CEGF008 is Ligilactobacillus salivarius CEGF008, with accession number CCTCC NO: M 20241111 and accession date of May 30, 2024.

2. The probiotic composition according to claim 1, characterized in that, The 16S rDNA sequences of *Bifidobacterium animalis subsp. lactis* CEGF005, *Bifidobacterium animalis subsp. lactis* CEGF006, *Bifidobacterium longum* subsp. longum CEGF007, and *Ligilactobacillus salivarius* CEGF008 are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively; the total viable count of these four probiotics in the probiotic composition is ≥1×10⁻⁶. 9 CFU / mL or ≥1×10 9 CFU / g.

3. The probiotic composition according to claim 2, characterized in that, The Bifidobacterium animalis subsp. lactis CEGF005, Bifidobacterium animalis subsp. lactis CEGF006, Bifidobacterium longum subsp. longum CEGF007, and Ligilactobacillus salivarius CEGF008 were combined in an addition ratio of 1~2:1~2:1~2:1~2.

4. The application of a probiotic composition according to any one of claims 1 to 3, characterized in that, The probiotic composition is used in the preparation of a drug that combines weight loss and improvement of glucose intolerance.

5. The application of a probiotic composition according to any one of claims 1 to 3, characterized in that, The probiotic composition is used in the preparation of drugs that combine weight loss and improvement of dyslipidemia.

6. The use of a probiotic composition according to any one of claims 1 to 3, characterized in that, The probiotic composition is used in the preparation of drugs that can reduce weight, improve glucose tolerance, and improve dyslipidemia.

Images