A strain of *Lactobacillus plantarum* with weight-loss function and its uses

Abstract

This invention relates to the field of microbial technology, specifically to a *Lactobacillus plantarum* strain with weight-loss function and its uses. The *Lactobacillus plantarum* strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 25683. This *Lactobacillus plantarum* strain can produce high levels of lactic acid and short-chain fatty acids, inhibit various pathogenic bacteria, and suppress the growth of the obese strain *Enterobacter cloacae*. It adheres to intestinal epithelial cells, lowers cholesterol, inhibits the differentiation of 3T3-L1 preadipocytes, and reduces body weight, total weight gain, and total food utilization in obese rats and mice, demonstrating excellent weight-loss function.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a plant lactobacillus strain with weight-loss function, its preparation method, and its uses. Background Technology

[0002] Obesity is primarily caused by a disruption in the body's energy metabolism balance, leading to energy intake exceeding energy expenditure and excessive accumulation of body fat, resulting in metabolic diseases. Obesity is influenced by multiple factors, including genetics, environment, metabolism, and physiology, and is closely related to diseases such as hyperlipidemia, hypertension, diabetes, insulin resistance, hyperuricemia, non-alcoholic fatty liver disease, and coronary atherosclerotic heart disease. Obesity is a global public health problem. Statistics show that one-third of the world's population is overweight, and the adult obesity rate has increased by 50% in the past 20 years, while childhood and adolescent obesity has doubled. According to the latest data from the "Report on Nutrition and Chronic Diseases of Chinese Residents (2020)," more than half of the adults in China are currently overweight or obese, with an overweight rate of 34.3% and an obesity rate of 16.4% among adult residents (≥18 years old). Over the past 30 years, overweight and obesity rates have been rising across all age groups, with an average increase of approximately 2.5 times.

[0003] Probiotics can help with weight loss by regulating gut microbiota, improving intestinal inflammation, lowering cholesterol levels, and regulating hormone levels. Probiotic weight loss boasts advantages such as zero side effects, zero rebound, no water loss, no skin damage, no dieting, no exercise, and ease of use, ushering in a new era of weight loss.

[0004] A strain of *Lactobacillus plantarum* isolated from Korean kimchi has been reported in related technologies. It has a weight-loss effect, but the effect is poor and cannot meet the needs of the industry.

[0005] Therefore, a strain of *Lactobacillus plantarum* with excellent weight-loss function is needed. Summary of the Invention

[0006] This invention provides a *Lactiplantibacillus plantarum* strain with weight-loss function and its uses. The strain can produce high levels of lactic acid and short-chain fatty acids; inhibit various pathogenic bacteria; adhere to intestinal epithelial cells; inhibit the growth of the obese strain *Enterobacter cloacae*; inhibit the differentiation of 3T3-L1 preadipocytes, thereby effectively inhibiting fat deposition; and reduce cholesterol levels.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] On the one hand, the present invention provides a strain of Lactiplantibacillus plantarum, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 25683.

[0009] Optionally, the *Lactobacillus plantarum* strain contains the 16S rRNA gene represented by SEQ ID NO:1.

[0010] Optionally, the *Lactobacillus plantarum* strain is isolated from healthy human breast milk.

[0011] On the other hand, the present invention provides the use of the *Lactobacillus plantarum* strain described above for the production of lactic acid and short-chain fatty acids.

[0012] Preferably, the short-chain fatty acid is selected from one or more of formic acid, acetic acid, propionic acid, butyric acid, and isobutyric acid.

[0013] On the other hand, the present invention provides the use of the *Lactobacillus plantarum* strain described above in the preparation of compositions for inhibiting the obese strain *Enterobacter cloacae*.

[0014] On the other hand, the present invention provides the use of the *Lactobacillus plantarum* strain described above in the preparation of compositions for inhibiting the differentiation of 3T3-L1 precursor adipocytes, thereby effectively inhibiting fat deposition.

[0015] On the other hand, the present invention provides the use as described above in the preparation of pharmaceutical compositions for weight loss or reducing high blood lipids and high cholesterol caused by obesity.

[0016] Preferably, the weight loss function is to reduce cholesterol levels or inhibit the differentiation of 3T3-L1 precursor adipocytes, thereby inhibiting fat production.

[0017] Preferably, the weight loss function is selected from one or more of the following: reducing body weight, total weight gain and total food utilization rate in obese model rats; reducing body fat weight and fat / body ratio in obese model rats; increasing serum high-density lipoprotein cholesterol content; increasing intestinal short-chain fatty acid content; and reducing the expression level of serum cellular inflammatory factor TNF-α.

[0018] Preferably, the weight loss function is selected from one or more of the following: reducing cholesterol content, inhibiting the differentiation of 3T3-L1 precursor adipocytes, reducing the body weight of obese model mice, reducing the total weight gain and total food utilization rate of obese model mice.

[0019] The beneficial effects of the *Lactobacillus plantarum* strain and its products described in this invention are as follows:

[0020] 1. The *Lactobacillus plantarum* HOM2217 of the present invention can produce high levels of lactic acid and short-chain fatty acids; can inhibit a variety of pathogenic bacteria; and can adhere to intestinal epithelial cells.

[0021] 2. The plant lactobacillus HOM2217 of the present invention inhibits the growth of the obese strain Enterobacter cloacae and can inhibit the differentiation of 3T3-L1 precursor adipocytes, thereby effectively inhibiting fat deposition.

[0022] 3. The *Lactobacillus plantarum* HOM2217 of this invention can lower cholesterol levels.

[0023] 4. The *Lactobacillus plantarum* HOM2217 strain of this invention can significantly reduce the body weight, total weight gain, and total food utilization rate of obese rats, significantly reduce body fat weight and fat / body fat ratio, increase serum high-density lipoprotein cholesterol levels, effectively increase the content of short-chain fatty acids in the intestine, and reduce the expression level of the cellular inflammatory factor TNF-α in serum. It can also significantly reduce the body weight, total weight gain, and total food utilization rate of obese mice. Therefore, the *Lactobacillus plantarum* HOM2217 strain has excellent weight-loss function. Attached Figure Description

[0024] Figure 1 A biological evolutionary tree of *Lactobacillus plantarum* HOM2217 according to the present invention is shown.

[0025] Figure 2 The diagram shows the RAPD clustering analysis of Lactobacillus plantarum HOM2217 constructed based on the UPGMA method.

[0026] Figure 3 The results of growth inhibition of Enterobacter cloacae ATCC13047 by *Lactobacillus plantarum* HOM2217 according to the present invention are shown. Note: *** indicates p < 0.001 compared with the HOM2217 group.

[0027] Figure 4 The in vitro cholesterol degradation rate of *Lactobacillus plantarum* HOM2217 according to the present invention is shown. Note: *** indicates p < 0.001 compared with the HOM2217 group, ** indicates p < 0.01 compared with the HOM2217 group.

[0028] Figure 5 The effect of *Lactobacillus plantarum* HOM2217 according to the present invention on the viability of 3T3-L1 preadipocytes is shown.

[0029] Figure 6 Oil Red staining results of 3T3-L1 preadipocyte differentiation by *Lactobacillus plantarum* HOM2217 according to the present invention are shown.

[0030] Figure 7 The effect of *Lactobacillus plantarum* HOM2217 according to the present invention on lipid content after differentiation of 3T3-L1 preadipocytes is shown.

[0031] Figure 8 The effect of *Lactobacillus plantarum* HOM2217 according to the present invention on triglyceride (TG) content after differentiation of 3T3-L1 preadipocytes is shown.

[0032] Figure 9 The effect of *Lactobacillus plantarum* HOM2217 according to the present invention on the body weight of an obese rat model is shown. Note: # * indicates a significant difference compared to the negative control group (p<0.05); * indicates a significant difference compared to the model control group (p<0.05).

[0033] Figure 10 The effects of *Lactobacillus plantarum* HOM2217 according to the present invention on total body weight gain and total food utilization in obese rats are shown. Note: ## ** indicates a highly significant difference compared to the negative control group (p<0.01); ** indicates a highly significant difference compared to the model control group (p<0.01).

[0034] Figure 11 The effects of *Lactobacillus plantarum* HOM2217 according to the present invention on body fat weight and fat / body ratio in obese rat models are shown. Note: ## * indicates a highly significant difference compared to the negative control group (p<0.01); * indicates a significant difference compared to the model control group (p<0.05); ** indicates a significant difference compared to the model control group (p<0.01).

[0035] Figure 12 The effects of *Lactobacillus plantarum* HOM2217 according to the present invention on total food intake and total calorie intake in obese rat model rats are shown. Note: # * indicates a significant difference compared to the negative control group (p<0.05); * indicates a significant difference compared to the model control group (p<0.05).

[0036] Figure 13 The effects of *Lactobacillus plantarum* HOM2217 according to the present invention on serum total cholesterol (TC), serum triglycerides (TG), serum high-density lipoprotein cholesterol (HDL-C), and serum low-density lipoprotein cholesterol (LDL-C) in obese model rats are shown. Note: ##* indicates a highly significant difference compared to the negative control group (p<0.01); * indicates a significant difference compared to the model control group (p<0.05); ** indicates a highly significant difference compared to the model control group (p<0.01).

[0037] Figure 14 The effects of *Lactobacillus plantarum* HOM2217 according to the present invention on serum cytokines TNF-α and IL-6 in obese rats are shown. Note: # This indicates a highly significant difference compared to the negative control group (p<0.05); ## ** indicates a highly significant difference compared to the negative control group (p<0.01); ** indicates a highly significant difference compared to the model control group (p<0.01).

[0038] Figure 15 The effects of *Lactobacillus plantarum* HOM2217 according to the present invention on the levels of lactic acid and short-chain fatty acids (SCFA) in the cecum of obese rat model rats are shown. Note: ## * indicates a highly significant difference compared to the negative control group (p<0.01); * indicates a significant difference compared to the model control group (p<0.05); ** indicates a highly significant difference compared to the model control group (p<0.01).

[0039] Figure 16 The effect of *Lactobacillus plantarum* HOM2217 according to the present invention on the body weight of obese model mice is shown. Note: ## ** indicates a significant difference compared to the negative control group (p<0.01); ** indicates a significant difference compared to the model control group (p<0.01).

[0040] Figure 17 The effects of *Lactobacillus plantarum* HOM2217 according to the present invention on total body weight gain and total food utilization in obese mice are shown. Note: # This indicates a highly significant difference compared to the negative control group (p<0.05); ## ** indicates a highly significant difference compared to the negative control group (p<0.01); ** indicates a highly significant difference compared to the model control group (p<0.01).

[0041] Figure 18 The effects of *Lactobacillus plantarum* HOM2217 according to the present invention on food intake and caloric intake in obese model mice are shown. Note: # * indicates a significant difference compared to the negative control group (p<0.05); * indicates a significant difference compared to the model control group (p<0.05).

[0042] Microbial Preservation Instructions

[0043] Biological material: strain HOM2217, taxonomically named Lactiplantibacillus plantarum, also known as Lactobacillus plantarum, was deposited on September 9, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 25683; the address of the collection center is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing.

[0044] Identification

[0045] The Lactobacillus plantarum strain HOM2217 of this invention, also known as Lactobacillus plantarum strain HOM2217, was submitted to the Institute of Microbiology, Chinese Academy of Sciences for identification in May 2023.

[0046] The identification conclusions are as follows: Under the conditions of this laboratory, based on a comprehensive analysis of experimental data including cell morphology, physiological and biochemical characteristics, 16S rRNA gene sequence, and pheS gene sequence of the submitted bacterial strain, and referring to relevant research papers in *Bergey's Manual of Systematic Bacteriology* and the *International Journal of Systematic and Evolutionary Microbiology*, the identification result of the submitted bacterial strain (strain number: HOM2217) is: *Lactiplantibacillus plantarum*. Synonym: *Lactobacillus plantarum*.

[0047] The cell morphology of this strain is rod-shaped; its physiological and biochemical characteristics are Gram-positive, catalase-negative (-), and oxidase-negative (-); the 16S rRNA gene sequence is shown in SEQ ID NO:1, and the pheS gene sequence is shown in SEQ ID NO:9. Detailed Implementation

[0048] This invention discloses the strains, characteristics, and applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0049] To further illustrate the technical means and effects adopted by the present invention to achieve its intended purpose, the technical solution of the present invention will be further described below in conjunction with specific embodiments, but this is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention that do not depart from the spirit and scope of the present invention should be covered within the protection scope of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are implemented according to conventional methods and conditions in the art.

[0050] To make the technical problem to be solved by the present invention, the technical solution adopted, and the advantages clearer, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0051] It should be noted that, unless otherwise specified, the experimental methods used in this invention are all conventional methods.

[0052] Unless otherwise specified, all reagents and materials used in this invention are prepared using conventional methods or obtained commercially.

[0053] Lactiplantibacillus plantarum is a Gram-positive, non-motile, non-spore-forming bacterium. Its cells are rod-shaped, typically 0.9–1.2 μm × 3.0–8.0 μm, and can be single, paired, or in short chains. It is widely distributed in many foods, including fermented products such as dairy products, meat, fish, and vegetables, silage, and mucous membranes of animals and humans, such as the oral cavity, gastrointestinal tract, and vagina. This species is listed in the EU's Quality of Practice (QPS) recommended biological agents list and in the International Dairy Federation's (IDF) "List of Microorganisms with a History of Safe Use in Food." It has also received GRAS (Generally Recognized As Safe) designation from the US Food and Drug Administration. In 2010, the National Health and Family Planning Commission of China also approved Lactiplantibacillus plantarum as an edible microbial species.

[0054] This invention develops a strain with weight-loss function, which can reduce cholesterol levels, inhibit the differentiation of 3T3-L1 precursor adipocytes, significantly reduce body weight, total weight gain and total food utilization rate in obese rat models, significantly reduce body fat weight and fat / body fat ratio in obese rat models, increase serum high-density lipoprotein cholesterol levels, effectively increase the content of intestinal short-chain fatty acids, and reduce the expression of the inflammatory cytokine TNF-α in serum. It can also significantly reduce body weight in obese mouse models and reduce total weight gain and total food utilization rate in obese rat models. This strain can produce high levels of lactic acid and short-chain fatty acids, has good production performance, and can be developed into weight-loss related foods, health foods, and pharmaceuticals.

[0055] In this invention, "Lactiplantibacillus plantarum" and "Lactobacillus plantarum" can be used interchangeably.

[0056] The *Lactobacillus plantarum* strain HOM2217 used in this invention was isolated from healthy human breast milk, and the specific isolation method is as described in Example 1.

[0057] Example 1: Isolation and Identification of Lactobacillus plantarum HOM2217

[0058] (1) Preparation of artificial gastrointestinal fluid

[0059] Artificial gastric fluid: Take 16.4 mL of dilute hydrochloric acid (1 mol / L), add 800 mL of water, adjust the pH to 3.0, add 10 g of pepsin, shake well, add water to 1000 mL, centrifuge at 5000 rpm for 5 min, take the supernatant, filter it through a 0.22 μm filter membrane for sterilization, and store at -20℃ for later use.

[0060] Artificial intestinal fluid: Take 6.8g of potassium dihydrogen phosphate, add 500mL of water, and adjust the pH to 6.8 with 0.4% (0.1mol / L) sodium hydroxide solution. Separately, take 10g of pancreatic enzyme and 3g of porcine bile salt, add an appropriate amount of water to dissolve them, mix the two solutions, add water to 1000mL, centrifuge at 5000rpm for 5min, take the supernatant, filter it through a 0.22μm filter membrane for sterilization, and store at -20℃ for later use.

[0061] (2) Preparation of isolation culture medium

[0062] MRS liquid culture medium: Prepare MRS broth culture medium (product number: CM1163, OXOID, UK) according to the instructions, sterilize at 121℃ for 15 min, and store at 2-8℃ protected from light for one week.

[0063] MRS-Cys solid medium containing bromocresol purple: Add 0.04 g of bromocresol purple to each liter of MRS solid medium (catalog number: CM1175, OXOID, UK) and mix thoroughly. Sterilize at 121°C for 15 min. In a laminar flow hood, pour approximately 15 mL into each petri dish and allow to solidify before use.

[0064] (3) Isolation and screening of Lactobacillus plantarum strain HOM2217

[0065] Isolated breast milk was collected using sterile aerobic tubes and stored at low temperature under aerobic conditions. The experiment began on the day of sampling. Approximately 1g of breast milk sample was added to an aerobic tube containing 9mL of MRS liquid culture medium and incubated aerobically at 37°C for 24 hours. After centrifugation at 8000rpm for 10 minutes, the supernatant was discarded, and 10mL of artificial gastric fluid was added. After mixing, the sample was incubated aerobically at 37°C for 3 hours, centrifuged at 8000rpm for 10 minutes, and the supernatant was discarded. After adding 10mL of artificial intestinal fluid, the sample was incubated aerobicly at 37°C for 3 hours. The sample was then diluted 10-fold using the 10-fold dilution method to a final concentration of 10. -6 From the original solution to 10 -6 Take 100 μL of each sample and spread it onto an MRS solid culture medium plate containing bromocresol purple. Incubate at 37°C with aerobic conditions for 48 h.

[0066] Select single colonies with yellowing edges, streak them, and purify them 3-4 times until the colonies are uniform. Simultaneously, perform Gram staining and microscopic examination of colony morphology. Transfer the single colonies to liquid culture medium for pure culture, preserve the culture with glycerol, and store at -80°C.

[0067] (4) Morphological observation of Lactobacillus plantarum strain HOM2217

[0068] *Lactobacillus plantarum* HOM2217 cultured aerobically at 37°C for 24 h on MRS agar medium showed colonies with a diameter of 3 mm, raised, round, moist, smooth, dense, and white color, occasionally light yellow or dark yellow. Under a light microscope, it was observed to be a straight, round-terminated bacterium, approximately 0.9–1.2 μm × 3–8 μm in size, occurring singly, in pairs, or in short chains, Gram-positive, and not forming spores. Two other strains with the same morphology were also screened and named *Lactobacillus plantarum* S1-6 and S2-13.

[0069] (5) Identification of Lactobacillus plantarum strain HOM2217

[0070] 16S rRNA gene identification: DNA was extracted from the three preserved strains HOM2217, S1-6, and S2-13, and amplified using universal primers 27F (AGAGTTTGATCMTGGCTCAG) and 1492R (GGTTACCTTGTTACGACTT). PCR amplification and agarose gel electrophoresis were then performed, followed by gel extraction, sequencing, and 16S rDNA sequencing of the isolated strains. Based on the 16S rDNA sequences, alignment was performed in the NCBI database using BLAST, and a phylogenetic tree was constructed using Mega 7.0 software. The results are shown below. Figure 1 .like Figure 1As shown, all three strains were identified as *Lactiplantibacillus plantarum*, and were named *Lactiplantibacillus plantarum* HOM2217, S1-6, and S2-13, respectively. The sequence of the 16S rRNA gene of *Lactiplantibacillus plantarum* HOM2217 is shown in SEQ ID NO:1.

[0071]

[0072] In addition, *Lactobacillus plantarum* strains S1-6 and S2-13, isolated simultaneously with the *Lactobacillus plantarum* strain HOM2217 of the present invention, were used as control strains in the present invention, and their 16S rRNA gene sequences are shown in SEQ ID NO: 10 and 11, respectively. By comparing the 16S rRNA gene sequences of the *Lactobacillus plantarum* strain HOM2217 of the present invention and the *Lactobacillus plantarum* strains S1-6 and S2-13 used as control strains in the present invention, it can be seen that strains S1-6 and S2-13 share 1431 bases with the *HOM2217* strain of the present invention, with a sequence similarity of 99.38%.

[0073] (6) Identification of strains by random amplified polymorphic DNA (RAPD): DNA was extracted from the preserved strains. Using the strain DNA as a template, PCR was performed using five primers (OPA-02, OPA-18, OPL-07, OPL-16, and OPM-05), as shown in Table 1. PCR amplified polymorphic DNA fragments, which exhibited different DNA differences after gel electrophoresis. These differences were analyzed using cluster analysis software. The results are shown in Table 1. Figure 2 .like Figure 2 As shown, Lactobacillus plantarum HOM2217 is unique and differs from other Lactobacillus plantarum strains.

[0074] Table 1

[0075] Primers sequence SEQ ID NO OPA-02 5'-TGC CGA GCT G-3' 2 OPA-18 5'-AGG TGA CCG T-3' 3 OPL-07 5'-AGG CGG GAA C-3' 4 OPL-16 5'-AGG TTG CAG G-3' 5 OPM-05 5'-GGG AAC GTG T-3' 6 27F 5'-AGTTTGATCMTGGCTCAG-3' 7 1492R 5'-GGTTACCTTGTTACGACTT-3' 8

[0076] Example 2: Test on the ability to inhibit common pathogenic bacteria

[0077] (1) Activation of indicator bacteria

[0078] The indicator strains Escherichia coli ATCC8739, Staphylococcus aureus ATCC6538, Salmonella typhimurium ATCC14028, Pseudomonas aeruginosa ATCC9027, Listeria monocytogenes ATCC19111, and Clostridium difficile ATCC9689 were all purchased from the China Industrial Microbial Culture Collection Center. Indicator bacteria (Escherichia coli ATCC8739; Staphylococcus aureus ATCC6538; Salmonella typhimurium ATCC14028; Pseudomonas aeruginosa ATCC9027; Listeria monocytogenes ATCC19111) were inoculated into TSB medium at 1% of the total culture medium volume and cultured aerobically at 37°C and 180 rpm for 24 h. Clostridium difficile ATCC9689 was inoculated into BHI medium at 1% of the total culture medium volume and cultured anaerobically at 37°C for 24 h.

[0079] (2) Activation of Lactobacillus plantarum

[0080] The *Lactobacillus plantarum* HOM2217, S1-6, and S2-13 strains, cryopreserved at -80℃ as prepared in Example 1, were inoculated into sterilized MRS-Cys liquid medium as described in Example 1 at an inoculum of 1% of the total culture medium volume, and then anaerobically cultured at 37℃ for 24 hours. After two activations, the fermentation broth of the strains was obtained. The broth was then centrifuged at 8000 rpm for 10 min, and the supernatant was used for an antibacterial test.

[0081] (3) Plate preparation

[0082] Heat sterilized tryptic soy agar (TSA) medium until completely melted, pour it into a petri dish, place it on a horizontal platform to form a uniform agar layer, and allow it to solidify. Add indicator bacteria to the TSA medium, shake well, pour it into a pre-prepared TSA blank agar plate, and allow it to solidify.

[0083] (4) Antibacterial test

[0084] Using sterile forceps, gently place the Oxford cups on the agar plate, maintaining a certain distance between them. Add 0.2 mL of the supernatant of the lactic acid bacteria fermentation broth to be tested to each cup. Place the cups in a 4°C refrigerator for 24 hours to allow diffusion, then incubate at 37°C for at least 18 hours, observing the appearance of inhibition zones. After the inhibition zones form, measure them with a ruler. The liquid culture medium (MRS) from Example 1 was used as a negative control, and *Lactobacillus plantarum* S1-6 and S2-13 were used as positive control strains. Each sample was tested in triplicate. The inhibition results are shown in Table 2.

[0085] Table 2. Inhibitory effect of Lactobacillus plantarum HOM2217 on pathogenic bacteria.

[0086]

[0087] Note: "–" indicates no antibacterial activity; "+" indicates 11-16 mm; "++" indicates 17-22 mm; "+++" indicates ≥23 mm.

[0088] As shown in Table 2, compared with the positive control strains S1-6 and S2-13, *Lactobacillus plantarum* HOM2217 showed strong inhibitory effects on all six pathogenic bacteria, including *Escherichia coli*, *Salmonella*, *Pseudomonas aeruginosa*, *Listeria monocytogenes*, and *Clostridium difficile*, indicating that this strain has a good ability to inhibit pathogenic bacteria.

[0089] Example 3: Test on the ability to inhibit obesity bacteria

[0090] (1) Activation of strains

[0091] The indicator bacterium *Enterobacter cloacae* ATCC13047 and *Lactobacillus plantarum* HOM2217, S1-6, and S2-13 prepared in Example 1 were inoculated into sterilized MRS liquid medium as described in Example 1 at an inoculum concentration of 1% of the total culture medium. The medium was incubated aerobically at 37°C for 24 hours, and activated twice to obtain the fermentation broth. The bacterial cells were then collected by centrifugation at 8000 rpm for 10 min. The cells were washed three times with sterile physiological saline, and the bacterial count was adjusted to 1 × 10⁻⁶. 8 CFU / mL, for later use.

[0092] (4) Co-culture growth antagonism test

[0093] Cultured *Enterobacter cloacae* ATCC13047 and *Lactobacillus plantarum* HOM2217, S1-6, and S2-13 suspensions were inoculated into sterilized MRS liquid medium as described in Example 1 at an inoculation rate of 1% of the total culture medium. The culture was vortexed for 20 seconds. A culture inoculated only with *Enterobacter cloacae* ATCC13047 served as a positive control. The cultures were incubated aerobically at 37°C for 18 hours, and viable cell counts were performed on MRS plates. Each treatment was performed in triplicate. Data were analyzed using GraphPad Prism 9 software for one-way ANOVA and Dunnett's t-test (pairwise comparison of means between multiple experimental groups and a control group). The results are as follows: Figure 3 As shown.

[0094] Mounting evidence suggests that the gut microbiota plays a crucial role in obesity, insulin resistance, and related liver diseases. Recently, Enterobacter cloacae, discovered in obese mice, has been identified as a pathogenic bacterium in the gut and a direct culprit in obesity.

[0095] Depend on Figure 3 It can be seen that, compared with the positive control strains S1-6 and S2-13, the co-culture of *Lactobacillus plantarum* HOM2217 with *Enterobacter cloacae* for 18 hours can significantly (p<0.01) inhibit its growth, indicating its potential value in weight loss.

[0096] Example 4 Gastrointestinal transit capacity test

[0097] (1) Activation of the strain

[0098] The *Lactobacillus plantarum* HOM2217, S1-6, and S2-13 prepared in Example 1 were inoculated into the sterilized MRS liquid culture medium described in Example 1 at an inoculation amount of 1% of the total culture medium. The cultures were then incubated at 37°C with aerobic culture for 24 hours and activated twice to obtain the fermentation broth of the strains.

[0099] (2) Preparation of artificial gastric juice

[0100] Take 16.4 mL of dilute hydrochloric acid, add about 800 mL of water and 10 g of pepsin, shake well, adjust the pH to 3.0, add water to 1000 mL, filter through a 0.2 μm microporous membrane and set aside for later use.

[0101] (3) Preparation of artificial intestinal fluid

[0102] Dissolve 6.8g of potassium dihydrogen phosphate in 500mL of water, adjust the pH to 6.8, add 10g of trypsin and 3g of porcine bile salt, dissolve them, mix the two solutions, add water to 1000mL, filter under sterile conditions using a 0.22µm sterile filter membrane, and set aside for later use.

[0103] (4) Evaluation of the survival ability of the strain in the simulated gastrointestinal tract

[0104] Take 1 mL of the activated bacterial culture of the test strain and add the bacterial cells to 9 mL of artificial gastric fluid (pH 3.0). After mixing, count the viable cells and incubate at 37°C for 3 hours. After incubating in artificial gastric fluid for 3 hours, transfer all the bacterial cells to an equal volume of artificial intestinal fluid (pH 6.8), mix well, and incubate at 37°C. Perform plate viable cell counts on MRS medium at 3 hours and 24 hours, and calculate the survival rate using the following formula:

[0105] Gastric fluid 3-hour survival rate (%) = [logCFUN1 / logCFUN0] × 100%

[0106] Intestinal fluid 3-hour survival rate (%) = [logCFUN2 / logCFUN0] × 100%

[0107] 24-hour survival rate of intestinal fluid (%) = [logCFUN3 / logCFUN0] × 100%

[0108] N0 = viable count of *Lactobacillus plantarum* before treatment; N1 = viable count of *Lactobacillus plantarum* after 3 hours of gastric fluid treatment; N2 = viable count of *Lactobacillus plantarum* after 3 hours of intestinal fluid treatment; N3 = viable count of *Lactobacillus plantarum* after 24 hours of intestinal fluid treatment. The results are shown in Table 3.

[0109] Table 3 Survival rate of *Lactobacillus plantarum* HOM2217 in simulated gastrointestinal fluid.

[0110]

[0111] Table 3 shows that after 3 hours of treatment with simulated gastric fluid, the survival rate of *Lactobacillus plantarum* HOM2217 reached 100%. After further treatment with simulated intestinal fluid for 24 hours, the survival rate remained above 99%, significantly (p<0.01) superior to the intestinal fluid tolerance of the other two *Lactobacillus plantarum* strains. This indicates that *Lactobacillus plantarum* HOM2217 has a high survival rate in the intestine, laying the foundation for its colonization and effective function in the gut.

[0112] Example 5: Intestinal Epithelial Cell Adhesion Test

[0113] (1) Preparation of cell culture medium

[0114] Complete culture medium: high-glucose DMEM medium supplemented with 10% inactivated fetal bovine serum (FBS) and 1% (v / v) antibiotics (100 U / mL penicillin, 100 μg / mL streptomycin), and stored at 4°C after mixing.

[0115] Incomplete culture medium: high-glucose DMEM medium, mixed with 10% inactivated fetal bovine serum (FBS) and stored at 4°C.

[0116] (2) Cell recovery and culture

[0117] Human colorectal adenocarcinoma cells (Caco-2) were purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. Caco-2 cells were resuspended in fresh culture medium and evenly dispersed in culture flasks. They were cultured at 37°C under a gaseous atmosphere of 5% CO2 and 95% air, with the culture medium changed every 48 hours during recovery. When the cells reached good growth (80% confluence), they were digested with trypsin-EDTA solution at 37°C. After digestion, the cell concentration was adjusted to 1 × 10⁻⁶ cells / mL. 5 Cells were seeded at a density of 1 / mL in 24-well plates and cultured until the cells reached 80% confluence.

[0118] (3) Activation of Lactobacillus plantarum

[0119] The *Lactobacillus plantarum* HOM2217, S1-6, and S2-13 strains prepared in Example 1 and frozen at -80℃ were inoculated into the sterilized MRS liquid culture medium described in Example 1 at an inoculation amount of 1% of the total culture medium. The cultures were then incubated at 37℃ under aerobic conditions for 24 hours and activated twice to obtain the fermentation broth of the strains.

[0120] (4) Adhesion test

[0121] Collect bacterial cells grown in the appropriate culture medium by centrifugation at 8000 rpm for 10 min. Wash the bacterial cells three times with DPBS, resuspend the cells in incomplete culture medium, and adjust the cell concentration to 10. 7 cfu / mL. 1 mL of the above bacterial suspension was added to a 24-well plate containing a monolayer of Caco-2 cells and incubated at 37°C for 2 h in a 5% CO2 incubator. After incubation, the cells were washed three times with sterile Duchenne phosphate-buffered saline (DPBS). Caco-2 cells were digested with trypsin-EDTA solution at 37°C. The cell count and the number of viable cells before and after adhesion were recorded. Each sample was performed in triplicate. The results are shown in Table 4.

[0122] Table 4 Adhesion ability of Lactobacillus plantarum HOM2217 to Caco-2 cells

[0123]

[0124] Note: Different capital letters in the same column indicate highly significant differences (p<0.01).

[0125] Adhesion index = Number of bacteria after adhesion / Number of cells per plate

[0126] Adhesion rate = Number of bacteria after adhesion / Number of bacteria before adhesion

[0127] The results in Table 4 show that the adhesion rate of Lactobacillus plantarum HOM2217 to human colon cancer cells Caco-2 was 4.32%, and the adhesion index was 4.91. Compared with the other two Lactobacillus plantarum strains S1-6 and S2-13, it can significantly (p<0.01) improve the adhesion ability of small intestinal epithelial cells.

[0128] Example 6: Production Capacity Test of Lactic Acid and Short-Chain Fatty Acids

[0129] The *Lactobacillus plantarum* HOM2217, S1-6, and S2-13 strains, cryopreserved at -80℃ as prepared in Example 1, were inoculated into sterilized MRS liquid medium as described in Example 1 at an inoculum of 1% of the total culture medium. The cultures were then incubated aerobically at 37℃ for 24 hours, and activated twice to obtain the fermentation broth. The broth was then centrifuged at 8000 rpm for 10 min, and the supernatant was analyzed by gas chromatography to determine the content of lactic acid and short-chain fatty acids. The MRS liquid medium described in Example 1 served as a negative control. Each sample was performed in triplicate. Data were analyzed using SPSS 25.0 software via one-way ANOVA and Duncan's multiple comparisons. The results are shown in Table 5.

[0130] Table 5. Production capacity of lactic acid and short-chain fatty acids from Lactobacillus plantarum HOM2217

[0131]

[0132] Note: Different capital letters in the same column indicate highly significant differences (p<0.01).

[0133] As shown in Table 5, compared with the other two Lactobacillus plantarum strains S1-6 and S2-13, Lactobacillus plantarum HOM2217 can significantly (p<0.01) improve the production capacity of lactic acid and short-chain fatty acids (formic acid, acetic acid, propionic acid, butyric acid and isobutyric acid).

[0134] Example 7 Antibiotic susceptibility test

[0135] The drug susceptibility test was conducted in accordance with ISO 10932-2010 Milk and Dairy Products, applicable to the determination of the minimum inhibitory concentration (MIC) of antibiotics for Bifidobacterium and non-enterococcal lactic acid bacteria (LAB).

[0136] (1) Culture medium preparation

[0137] MRS liquid culture medium: Same as in Example 1

[0138] LSM liquid medium: Weigh 21.06g of Iso-Sensitest medium (catalog number: CM0473B, OXOID, UK) and 5.2g of MRS Broth, add water to 0.5L, adjust the pH to 6.85±0.1, sterilize at 121℃ for 15min, the pH should be 6.7±0.1, and store at 2-8℃ protected from light for one week.

[0139] Activation and proliferation of Lactobacillus plantarum

[0140] The *Lactobacillus plantarum* HOM2217 prepared in Example 1 was inoculated into sterilized liquid culture medium (MRS) as described in Example 1 at an inoculum of 1% of the total culture medium volume. The culture was then incubated aerobically at 28°C for 24 hours, and activated twice to obtain the fermentation broth. The activated *Lactobacillus plantarum* HOM2217 was then multiplied on MRS agar medium and incubated aerobically at 28°C for 48 hours.

[0141] (2) Preparation of antibiotic microdilution plates

[0142] Weigh 0.0512 g of antibiotics and add 10 mL of solvent. Dissolve chloramphenicol and erythromycin in ethanol (no filtration required). Dissolve ampicillin in phosphate buffer (pH 8.0, 0.1 mol / L). Dissolve other antibiotics in water. After shaking to dissolve, filter through a 0.22 μm filter membrane and aliquot into EP tubes to a concentration of 5120 μg / mL (5.12 mg / mL). Store at -20°C for later use. Dilute the antibiotic stock solution with water (ampicillin with phosphate buffer) to the appropriate concentration range. Add 50 μL of the diluent to the wells of a microdilution plate.

[0143] (3) Preparation of bacterial suspension

[0144] Pick a single colony from an agar plate. Resuspend the obtained colony in a sterile culture tube containing 2 mL to 5 mL of sterile saline. Then, resuspend the obtained colony in pre-reconstituted LSM liquid medium. Resuspend the colony until the turbidity of the solution reaches McFarland standard 1 or the optical density at 625 nm is 0.16–0.2 using a spectrophotometer. The suspension is approximately equivalent to 3 × 10⁻⁶ cells / mL. 8 CFU / mL. Dilute the bacterial suspension with the recommended culture medium, diluting it 500-fold with MRS liquid medium, as the antibiotic solution will dilute the medium by two times. Dispense the diluted bacterial suspension within 30 minutes of preparation. When using a cryoplate, thaw the frozen antibiotic solution immediately under aerobic conditions before use. Dispense 50 μL of the diluted suspension into each well of the microdilution plate (approximately 3 × 10⁻⁶). 4CFU / well). Incubate plates at 37°C under aerobic conditions for 48 hours. When using an aerobic container, cover each plate with a lid to create a homogeneous environment within the container. Perform each experiment in triplicate, with both positive and negative control groups included. Positive control wells do not contain antibiotics but contain the test strain and culture medium containing the solvent for dissolving the highest concentration of antibiotic. Negative control wells do not contain the test strain or antibiotics but contain culture medium.

[0145] (4) Read the MIC results

[0146] After 48 hours of incubation, visually read the MIC. After incubation, check the negative control wells for visible bacterial growth. If contamination is found, reject all data produced for that strain. Note: If no growth is observed in the positive control wells, it indicates that the tested strain is sensitive to the solvent used to dissolve the antibiotic. In this case, reading the MIC for that specific antibiotic is meaningless. If the negative and positive control checks are normal, visually determine bacterial growth for each antibiotic by comparing it with the positive control. It is best to place the microdilution plate on top of a stand with a magnifying glass and a desk lamp providing indirect light for easy reading. Bacterial growth is easily detected under a magnifying glass as a deposit at the bottom of the well. Discard any series of wells where discontinuous growth is observed (e.g., growth at 16 μg / mL and 64 μg / mL, but no growth at 32 μg / mL). The endpoint is defined as the lowest antibiotic concentration at which no growth is visually observed. This concentration is the MIC for that antibiotic for that specific strain. Perform three replicates for each sample, with *Lactobacillus plantarum* ATCC14917 (the type strain, the MIC quality control specified strain) as the positive control strain. The antibiotic susceptibility results of *Lactobacillus plantarum* HOM2217 are shown in Table 6. Antibiotic resistance standards for microorganisms used in food were established by the European Food Safety Authority (EFSA) in 2012.

[0147] Table 6. Antibiotic susceptibility results of *Lactobacillus plantarum* HOM2217

[0148]

[0149] As shown in Table 6, *Lactobacillus plantarum* HOM2217 is consistent with the control strain ATCC14917, and is sensitive to erythromycin, chloramphenicol, tetracycline, ampicillin, clindamycin, and gentamicin. Therefore, the *Lactobacillus plantarum* HOM2217 strain of the present invention is relatively safe.

[0150] Example 8: In vitro cholesterol degradation test

[0151] MRS high cholesterol medium: Bile salts (final mass concentration of 3 mg / mL) and cholesterol (final mass concentration of 120 μg / mL) were added to the MRS liquid medium described in Example 1.

[0152] Lactobacillus plantarum HOM2217, S1-6, and S2-13 prepared in Example 1 were inoculated into sterilized MRS liquid medium as described in Example 1 at an inoculum of 1% of the total culture medium. The culture was incubated aerobically at 37°C for 24 hours. After two generations of activation, the culture was inoculated into MRS high-cholesterol liquid medium and anaerobically cultured at 37°C for 24 hours. The mixture was then centrifuged at 4°C and 8000 rpm for 10 minutes, and the supernatant was collected. Cholesterol was determined by gas chromatography according to the national standard GB 5009.128-2016, "Determination of Cholesterol in Food". Each sample was tested in triplicate, and the cholesterol degradation rate was calculated using the following formula.

[0153] Each treatment was performed in triplicate, and the data were analyzed using t-tests with GraphPad Prism 9 software.

[0154]

[0155] In the formula: M1 and M2 are the cholesterol masses (μg) in the supernatant before and after fermentation, respectively.

[0156] See results Figure 4 .like Figure 4 As shown, in a culture medium containing bile salts, the cholesterol degradation rate of Lactobacillus plantarum HOM2217 was 50.6%, which was significantly higher than that of the other two Lactobacillus plantarum strains S1-6 and S2-13 (p<0.01).

[0157] Example 9: In vitro inhibition of lipogenesis test

[0158] (1) Preparation of cell culture medium

[0159] Complete medium 1: High glucose DMEM medium, with 10% inactivated fetal bovine serum (NBS) and 1% (v / v) antibiotics (100 U / mL penicillin, 100 μg / mL streptomycin), mixed and stored at 4°C.

[0160] Complete medium 2: High sugar DMEM medium, mixed with 10% inactivated fetal bovine (FBS) and stored at 4°C.

[0161] Cell differentiation medium 1: 3-Isobutyl-1-methylxanthine (IBMX) was added to complete medium 2 to achieve a final concentration of 0.5 mmol / L, dexamethasone (DEX) to achieve a final concentration of 1 μmol / L, and insulin to achieve a final concentration of 10 μg / mL.

[0162] Cell differentiation medium 2: Add insulin to complete medium 2 to achieve a final concentration of 10 μg / mL, and store at 4℃.

[0163] (2) Preparation of test bacterial samples

[0164] The *Lactobacillus plantarum* HOM2217, S1-6, and S2-13 prepared in Example 1 were inoculated into the sterilized MRS liquid medium described in Example 1 at an inoculation amount of 1% of the total culture medium. The culture was incubated at 37°C with aerobic conditions for 24 hours. After two generations of continuous activation, the bacterial cells were collected by centrifugation at 8000 r / min for 10 min, washed twice with PBS, and resuspended in PBS solution (0.1 mol / L, pH 7.2–7.4) to form a bacterial suspension.

[0165] Heat-killed strain (HKS): The bacterial suspension is placed in a water bath and treated at 70°C for 30 minutes to obtain heat-killed strains, which are then stored at -80°C for later use.

[0166] (3) Culture of 3T3-L1 preadipocytes

[0167] 3T3-L1 preadipocytes (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were seeded in complete culture medium 1 at a ratio of 1:3 and passaged. Cells from passages 3 to 10 were selected for experiments.

[0168] (4) Cell viability detection

[0169] The cell number was adjusted to 2 × 10⁻² using complete culture medium 2. 5 Cells / mL were cultured at a concentration of 0.1 mL per well in a 96-well plate and cultured for 2 days. After culturing, the cell culture medium was removed from each well, and 0.1 mL of different concentrations of the test cells (adjusted with complete culture medium 2) was added to each well. The plates were incubated at 37°C with 5% CO2 for 24 hours. Cell viability was then assessed using the MTS cell proliferation and cytotoxicity assay kit from Prometheus. The simplified procedure was to add 20 μL MTS to each well, incubate in the dark for 1 hour, and read the absorbance at 490 nm using a microplate reader. A blank control was provided for each treatment using cell culture medium 2 + test cells + MTS. A negative control was provided for complete culture medium 2 + cells + MTS. Results are shown below. Figure 5 As shown.

[0170] Depend on Figure 5 The results show that when the bacterial concentration is no greater than 1×10⁻⁶, 9 At a TFU / mL concentration, the viability of 3T3-L1 preadipocytes was consistently above 100%. Therefore, a concentration of 1×10⁻⁶ TFU / mL was selected for the experiment. 9 TFU / mL, 1×10 8 TFU / mL and 1×10 7 An experiment was conducted to evaluate the lipid-lowering effect of TFU / mL on cells.

[0171] (5) Differentiation of 3T3-L1 precursor adipocytes

[0172] The cultured cells were then placed in 2×10⁻⁶ complete culture medium. 5 3T3-L1 precursor adipocytes were seeded at a rate of 1 mL / mL into each well of a 24-well plate coated with 0.1% gelatin, allowing them to reach confluence and culture until 100% confluence. Day 0 was recorded 3 days after confluence. Cell differentiation medium 1 was added, and the cells were cultured for 4 days (day 4). Then, cell differentiation medium 2 was used for induction, and the cells were cultured for 2 days (day 6). After induction, the cells were cultured in complete medium 2 for 2 days (day 8). On day 8, the 3T3-L1 precursor adipocytes differentiated into round mature adipocytes with round triglyceride granules inside. The medium was changed every 2 days during this period. Bacterial samples were added on day 0, including different concentrations of bacterial samples with cell viability higher than 100%, and co-cultured with 3T3-L1 cells from day 0 to day 8 of induction.

[0173] (6) Oil Red O staining

[0174] Differentiated adipocytes were gently washed three times with an appropriate amount of PBS, fixed with 0.5 mL of 4% paraformaldehyde for 30 min, and then gently washed three times with ultrapure water. 0.5 mL of Oil Red O working solution was added to each well, and the cells were stained at 37°C in the dark for 30 min. The staining solution was discarded, and the cells were gently washed three times with ultrapure water. The culture plate was observed under an inverted microscope, and photographs were taken. After photographing, 1 mL of 100% isopropanol was added to each well to extract the Oil Red O staining solution from the lipid droplets. The plates were incubated at room temperature for 20 min, and the extracted staining solution was transferred to a clean 96-well culture plate. The absorbance was measured at 500 nm. 100% isopropanol was used as a blank, and the absorbance was measured at 500 nm. The relative lipid content was calculated as follows: Relative lipid content (%) = (OD0.05) / (OD0.05) 样品 -OD 空白 ) / (OD 阳性对照 -OD 空白 ×100%. Each treatment was performed in triplicate. Data were analyzed using GraphPad Prism 9 software for one-way ANOVA and Dunnett's t-test, i.e., pairwise comparisons of means between multiple experimental groups and one control group. Oil staining and lipid content results are as follows. Figure 6 and Figure 7 As shown.

[0175] After differentiation and maturation, 3T3-L1 cells accumulate a large number of lipid droplets within the cell. Oil Red O staining can visually reflect the amount of lipid accumulation after adipogenic differentiation. Figure 6 It was found that 90% of the cells in the model group differentiated into mature adipocytes on day 8 of induction. These cells were further enlarged, round in shape, and filled with red lipid droplets densely clustered around the nucleus, forming a "finger-like" structure, exhibiting the typical morphology of mature adipocytes. Compared with the model control group, preadipocytes treated with heat-inactivated *Lactobacillus plantarum* HOM2217 for 8 days showed a significant reduction in the number of intracellular lipid droplets at all three concentrations (low, medium, and high). Oil Red O dye quantification results of adipocytes (…) Figure 7 The results showed that heat-inactivated *Lactobacillus plantarum* HOM2217 significantly (p<0.01) reduced intracellular lipid content in a dose-dependent manner. With increasing concentrations of heat-inactivated *Lactobacillus plantarum* HOM2217, its ability to inhibit 3T3-L1 preadipocyte differentiation increased, intracellular lipid content decreased, and its lipid-reducing effect was significantly better than that of the other two *Lactobacillus plantarum* strains S1-6 and S2-13.

[0176] (6) Measurement of intracellular triglyceride (TG) content in 3T3-L1 cells

[0177] Intracellular triglyceride (TG) content was determined according to the instructions of the triglyceride (TG) assay kit produced by Nanjing Jiancheng Bioengineering Institute. The simplified procedure was as follows: add 2.5 μL of sample and 250 μL of working solution to each well, mix well, incubate at 37°C for 10 minutes, and read the absorbance at 510 nm using a microplate reader. Protein concentration was determined using a total protein quantification kit (BCA method). The simplified procedure was as follows: add 10 μL of sample and 250 μL of working solution to each well, mix well, incubate at 37°C for 10 minutes, and read the absorbance at 562 nm using a microplate reader. The triglyceride content per gram of protein was calculated using the formula.

[0178] Calculation formula:

[0179] Each treatment was performed in triplicate. Data were analyzed using GraphPad Prism 9 software for one-way ANOVA and Dunnett's t-test, i.e., pairwise comparisons of means between multiple experimental groups and one control group. Intracellular triglyceride (TG) levels in 3T3-L1 cells were as follows: Figure 8 As shown. By Figure 8It was found that the high, medium and low heat-inactivated bacterial concentrations of *Lactobacillus plantarum* HOM2217 could significantly (p<0.01) reduce the triglyceride content in cells, and there was a dose-dependent effect. As the heat-inactivated bacterial concentration of *Lactobacillus plantarum* HOM2217 increased, the triglyceride content in cells decreased, and the lipid-reducing effect was significantly better than that of the other two *Lactobacillus plantarum* strains S1-6 and S2-13.

[0180] Example 10: Preparation process of active Lactobacillus plantarum HOM2217 bacterial powder 1

[0181] (1) Culture of bacterial strains

[0182] The *Lactobacillus plantarum* HOM2217, cryopreserved at -80℃ as prepared in Example 1, was inoculated at a 1% inoculum into sterile MRS liquid medium and cultured at 37℃ for 16–24 hours. This process was repeated twice to obtain activated seed culture medium. The seed culture was then inoculated at a 3% inoculum into fermentation medium M321 and cultured aerobically at 35℃. During fermentation, sodium hydroxide solution was automatically added to maintain a constant pH of 5.0 until acid production ceased. Fermentation was terminated when sodium hydroxide addition stopped, resulting in a high-density culture of *Lactobacillus plantarum* HOM2217 with a viable count of up to 28.6 billion CFU / mL. Table 7 shows the formulation of fermentation medium M321.

[0183] Table 7 Formula for Fermentation Medium M321

[0184]

[0185] (2) Preparation of freeze-drying protectant

[0186] A protective agent containing 100 g / L peptone, 20 g / L sucrose, 10 g / L vitamin C and 1 g / L glycerol was prepared by mixing sterile water with the raw materials of the protective agent.

[0187] (3) Freeze drying

[0188] The fermentation broth of *Lactobacillus plantarum* HOM2217, after culture, was centrifuged at 6000 rpm for 10 min at 2–8 °C. The supernatant was discarded, and the bacterial sludge was collected. The sludge was washed 1–2 times with 0.9% sterile physiological saline. The washed sludge was then mixed with the aforementioned protective agent to achieve a bacterial concentration of 10-1 in the mixed broth. 11 The mycelium was freeze-dried in a freeze dryer with a CFU / mL concentration of 6.57 × 10⁻⁶ cells. The cells were pre-frozen at -55°C for 5 hours under vacuum of 0.05 mbar, then dried for 20 hours at -30°C, followed by another 15 hours at -15°C. Finally, the cells were dried for 5 hours at 35°C. After freeze-drying, the mycelium cake was pulverized using a fine grinder to obtain the freeze-dried mycelium powder.11 CFU / g.

[0189] Example 11 Preparation process of active Lactobacillus plantarum HOM2217 powder 2

[0190] (1) Culture of bacterial strains

[0191] The *Lactobacillus plantarum* HOM2217, cryopreserved at -80℃ as prepared in Example 1, was inoculated at a 3% inoculum into sterile MRS liquid medium and cultured at 37℃ for 16–24 hours. This process was repeated twice to obtain activated seed culture medium. The seed culture was then inoculated at a 3% inoculum into fermentation medium M327 and cultured aerobically at 35℃. During fermentation, sodium hydroxide solution was automatically added to maintain a constant pH of 6.0 until acid production ceased. Fermentation was terminated when sodium hydroxide addition stopped, resulting in a high-density culture of *Lactobacillus plantarum* HOM2217 with a viable count of up to 33.6 billion CFU / mL. Table 8 shows the formulation of fermentation medium M327.

[0192] Table 8. Fermentation medium M327 formulation

[0193]

[0194] (2) Preparation of freeze-drying protectants

[0195] A protective agent containing 50 g / L peptone, 60 g / L sucrose, 5 g / L vitamin C, and 2 g / L glycerol was prepared by mixing sterile water with the raw materials of the protective agent.

[0196] (3) Freeze drying

[0197] The fermentation broth of *Lactobacillus plantarum* HOM2217, after culture, was centrifuged at 6500 rpm for 15 min at 2–8 °C. The supernatant was discarded, and the bacterial sludge was collected. The sludge was washed 1–2 times with 0.9% sterile physiological saline. The washed sludge was then mixed with the aforementioned protective agent to achieve a bacterial concentration of 10-1 in the mixed broth. 11 The mycelium was freeze-dried in a freeze dryer with a CFU / mL concentration of 8 or higher. Pre-freezing was performed at -50°C for 8 hours under vacuum of 0.03 mbar. After raising the temperature to -25°C, the mycelium was dried for 15 hours, then again at -10°C for 10 hours. Finally, the temperature was raised to 32°C for a second drying period of 8 hours. After freeze-drying, the mycelium cake was pulverized using a fine grinder to obtain the freeze-dried mycelium powder. The viable cell count of the freeze-dried mycelium powder reached 8.18 × 10⁻⁶. 11 CFU / g.

[0198] Example 12: Preparation process of *Lactobacillus plantarum* HOM2217 dead bacteria powder

[0199] (1) Culture of bacterial strains

[0200] The strain cultured in Example 11 was used.

[0201] (2) Preparation of bacterial powder

[0202] High-density fermentation yielded *Lactobacillus plantarum* HOM2217 fermentation broth, which was then treated at 80℃ for 30 minutes. 20g of maltodextrin was added per liter of fermentation broth, and the mixture was dried in a spray drying tower. The dead bacteria powder, when counted under a microscope using a hemocytometer, showed a dead bacteria count of 1.23 × 10⁻⁶. 12 CFU / g.

[0203] Example 13 In vivo experiment of Lactobacillus plantarum HOM2217 in an obese animal model 1

[0204] (1) Laboratory animals and feed

[0205] Seventy healthy SPF-grade male SD rats bred by Beijing Huafukang Biotechnology Co., Ltd. were used as experimental animals.

[0206] The feed used was high-fat feed D12492 (60% fat content) and ordinary feed D12450B (10% fat content) produced by Beijing Huafukang Biotechnology Co., Ltd.

[0207] (2) Animal models and experimental treatment

[0208] After one week of acclimatization, the animals were grouped. At the end of the acclimatization period, 70 rats were randomly divided into two groups based on body weight: 10 rats were given a maintenance diet as a negative control group, and 60 rats were given a high-calorie model diet. After two weeks of feeding, the rats given the high-calorie model diet were ranked by weight gain. The obese resistant rats with the lowest weight gain were culled, and 40 obese sensitive rats were selected and randomly divided into four groups based on body weight: a model control group and three dosage groups, with 10 rats in each group. The experimental groups used *Lactobacillus plantarum* HOM2217 powder prepared in Example 10, divided into three dosage groups: a low-dose group (2.5 × 10⁻⁶) and a low-dose group (2.5 × 10⁻⁶). 9 CFU / Kg BW), medium dose group (1.25×10 10 CFU / kg BW) and high-dose group (2.5×10) 10 (CFU / kg BW). From the start of the experiment, each animal in the model control group and each dosage group was given an equal amount of high-calorie model feed daily, while the negative control group was given an equal amount of maintenance feed in the same manner. Different dosages of the test substance were administered to each dosage group, while the negative control group and model control group were given sterile water. The experimental period was 7 weeks.

[0209] (3) Detection indicators and methods

[0210] A. Apparent Indicators

[0211] During the experiment, the amount of food given and the amount of food left over for each animal in each cage were recorded weekly, and the amount of food consumed and the total calories ingested (food intake × calories per kilogram of feed) were calculated. The weight of the rats was recorded weekly, and the weight gain and feed utilization rate were calculated.

[0212] Feed utilization rate = (body weight gain / feed intake) × 100%

[0213] B. Serum four-item test

[0214] At the end of the experiment, after fasting for 12 hours, whole blood was obtained by ocular blood sampling. After standing at room temperature for 2 hours, the blood was centrifuged at 4000 r / min for 10 minutes at 4℃ to separate the serum. The serum was then used to determine the levels of total cholesterol, triglycerides, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol in each group of rats using a fully automated biochemical analyzer.

[0215] C. Cytokine assay

[0216] At the end of the experiment, after fasting for 12 hours, whole blood was obtained by ocular blood sampling. After standing at room temperature for 2 hours, the blood was centrifuged at 4000 r / min for 10 minutes at 4℃ to separate the serum. The serum was then analyzed by enzyme-linked immunosorbent assay (ELISA) according to the kit instructions to determine the levels of serum cytokines TNF-α and IL-6.

[0217] D. Determination of fat content

[0218] After blood was collected from the rats' eyeballs, they were euthanized by dislocation of the neck. Body fat (from the testes and perirenal fat pads) was collected and weighed, and the fat / body ratio was calculated.

[0219] E. Determination of lactic acid and short-chain fatty acids in cecal contents

[0220] After blood was collected from the rats' eyeballs, they were euthanized by cervical dislocation, and the contents of their cecum were collected. The contents of lactic acid and short-chain fatty acids in the cecum were detected by gas chromatography.

[0221] (4) Data processing

[0222] Statistical analysis was performed using SPSS software and pairwise comparisons of means among multiple experimental groups and a control group using the Dunnett method. Before and after the establishment of the obesity model, the weight of rats in the negative control group and the model control group was compared using an independent samples t-test.

[0223] (5) Experimental Results

[0224] The experimental results show that Figure 9-15 middle.

[0225] Depend on Figure 9The results showed that after 14 days of administration of a high-calorie model diet, the rats in the model control group and the negative control group showed a significant increase in body weight, indicating successful model establishment. Before administration of the test substance, there was no significant difference in body weight between the various dosage groups and the model control group (p>0.05). After 49 days of oral administration of the test substance, the rats in the model control group and the negative control group showed a significant increase in body weight (p<0.05). Compared with the model control group, the high-dose group of *Lactobacillus plantarum* HOM2217 showed a significant decrease in final body weight (p<0.05).

[0226] Depend on Figure 10 The results showed that after administration of a high-calorie model diet, the total weight gain and total food utilization rate of rats in the model control group were significantly increased compared with those in the negative control group (p<0.05). After oral administration of the test substance for 49 days, compared with the model control group, the high-dose group of *Lactobacillus plantarum* HOM2217 significantly (p<0.01) reduced the total weight gain and total food utilization rate of rats.

[0227] Depend on Figure 11 The results showed that after administration of a high-calorie model diet, the body fat weight and fat-to-body ratio of rats in the model control group increased significantly compared with those in the negative control group. After oral administration of the test substance for 49 days, compared with the model control group, the medium dose of *Lactobacillus plantarum* HOM2217 significantly (p<0.05) and the high dose of *Lactobacillus plantarum* HOM2217 extremely significantly (p<0.01) reduced the body fat weight and fat-to-body ratio of rats.

[0228] Depend on Figure 12 The results showed that after being fed a high-calorie diet, the total food intake of rats in the model control group decreased significantly compared with the negative control group (p<0.05); the total calorie intake of rats increased significantly (p<0.05). After oral administration of the test substance for 49 days, there were no significant differences in total food intake and total calorie intake between the various dosage groups and the model control group (p>0.05).

[0229] Depend on Figure 13 The results showed that after oral administration of the test substance to obese rats for 49 days, compared with the negative control group, the model control group showed significantly higher serum total cholesterol and serum triglycerides, lower serum high-density lipoprotein cholesterol, and higher serum low-density lipoprotein cholesterol (p<0.05). Compared with the model control group, there were no significant differences in serum total cholesterol, serum triglycerides, and serum low-density lipoprotein cholesterol among the different dosage groups (p>0.05). Medium doses of *Lactobacillus plantarum* HOM2217 significantly (p<0.05) and high doses of *Lactobacillus plantarum* HOM2217 highly significantly (p<0.01) increased serum high-density lipoprotein cholesterol levels in rats.

[0230] Depend on Figure 14 The results showed that after oral administration of the test substance to obese rats for 49 days, serum TNF-α and IL-6 levels were significantly elevated compared to the negative control group. Compared to the model control group, serum IL-6 levels in all dose groups showed a decreasing trend, but the difference was not statistically significant (p>0.05). Medium and high doses of *Lactobacillus plantarum* HOM2217 significantly and extremely significantly (p<0.01) reduced TNF-α levels. Obesity is widely recognized as a chronic low-grade inflammatory state. Inflammation plays a crucial role in the abnormal hypertrophy of adipocytes and the increase in endoplasmic reticulum pressure in adipocyte cytoplasm. Macrophages in adipose tissue are considered an important source of inflammation, secreting inflammatory factors, including tumor necrosis factor-α (TNF-α). TNF-α expression was significantly enhanced in adipocytes of obese animal models. TNF-α release from adipose tissue significantly increased during visceral fat accumulation. TNF-α expression was significantly increased in both adipose and muscle tissues in obese patients with insulin resistance (IR) and obese patients with type 2 diabetes. Therefore, it can be inferred that *Lactobacillus plantarum* HOM2217 may achieve weight loss by reducing TNF-α expression and inflammation.

[0231] Depend on Figure 15 The results showed that after oral administration of the test substance to obese rats for 49 days, the levels of lactic acid, formic acid, acetic acid, propionic acid, and butyric acid in the cecum were significantly lower in the model control group compared with the negative control group (p<0.05). Compared with the model control group, the medium dose of *Lactobacillus plantarum* HOM2217 significantly (p<0.05) increased the formic acid content in the cecum, the high dose of *Lactobacillus plantarum* HOM2217 significantly (p<0.05) increased the lactic acid content in the cecum, and highly significantly (p<0.01) increased the formic acid, acetic acid, and butyric acid content in the cecum.

[0232] In summary, administering *Lactobacillus plantarum* HOM2217... After 49 days, compared with the model control group, the test substance in *Lactobacillus plantarum* HOM2217 at the specified doses could reduce body fat weight (p<0.05) and fat / body ratio (p<0.05) and increase serum high-density lipoprotein cholesterol (p<0.05) in obese model rats. High doses of *Lactobacillus plantarum* HOM2217 could reduce body weight (p<0.05), reduce total weight gain (p<0.01) and total food utilization rate (p<0.01) in obese model rats, reduce body fat weight (p<0.01) and fat / body ratio (p<0.01), increase serum high-density lipoprotein cholesterol (p<0.01), reduce serum cytokine TNF-α (p<0.01), and increase the levels of lactic acid (p<0.05), formic acid (p<0.01), acetic acid (p<0.01), and butyric acid (p<0.01) in the cecum. There were no significant differences in total food intake and total calorie intake between the rats in each dosage group and the model control group (p>0.05). Therefore, it can be concluded that *Lactobacillus plantarum* can achieve weight loss by producing short-chain fatty acids through metabolism, lowering intestinal pH, inhibiting the reproduction of obese bacteria, and reducing TNF-α expression to reduce inflammatory response, without affecting food intake.

[0233] Example 14: In vivo experiment of Lactobacillus plantarum HOM2217 in an obese animal model 2

[0234] (1) Laboratory animals and feed

[0235] Eighty-five healthy SPF-grade C57BL / 6J (male, 4 weeks old, 15–17 g) mice bred by Beijing Vital River Laboratory Animal Technology Co., Ltd. were used as experimental animals.

[0236] The feed used was high-fat feed D12492 (fat content 60%) and ordinary feed D12450B (fat content 10%) produced by Beijing Keao Xieli Feed Co., Ltd.

[0237] (2) Animal models and experimental treatment

[0238] After one week of acclimatization, the animals were grouped. At the end of the acclimatization period, 68 mice were randomly divided into two groups based on body weight: 8 mice were given a maintenance diet as a negative control group, and 60 mice were given a high-calorie model diet. After 14 days of feeding, the mice given the high-calorie model diet were ranked by weight gain. The obese resistant mice with the lowest weight gain were culled, and 40 obese sensitive mice were selected and randomly divided into five groups based on body weight: a model control group, a positive control group (orlistat, 0.4 mg / mouse / day), and a low-dose HOM2217 live bacteria group (1 × 10⁻⁶ mg / mouse / day). 9CFU / animal / day (using *Lactobacillus plantarum* HOM2217 bacterial powder prepared in Example 11), high-dose group of HOM2217 live bacteria (5×10) 9 CFU / animal / day (using *Lactobacillus plantarum* HOM2217 bacterial powder prepared in Example 11), HOM2217 dead bacterial group (5×10) 9 TFU / animal / day (using *Lactobacillus plantarum* HOM2217 bacterial powder prepared in Example 12), 8 animals per group. From the start of the experiment, each animal in the model control group and each dosage group was given an equal amount of high-calorie model feed daily, while the negative control group was given an equal amount of maintenance feed in the same manner. Different doses of the test substance were given to each dosage group, while sterile water was given to the negative control group and the model control group. The experimental period was 11 weeks.

[0239] (3) Detection indicators and methods

[0240] During the experiment, the amount of food given and leftover food for each cage of animals was recorded weekly, and the daily food intake and calories consumed by each mouse were calculated. The weight of the mice was recorded weekly, and the weight gain and feed utilization rate were calculated.

[0241] Feed utilization rate = (body weight gain / feed intake) × 100%

[0242] (4) Data processing

[0243] Statistical analysis was performed using SPSS software and pairwise comparisons of means among multiple experimental groups and a control group using the Dunnett method. Before and after the establishment of the obesity model, the weight of rats in the negative control group and the model control group was compared using an independent samples t-test.

[0244] (5) Experimental Results

[0245] The experimental results show that Figure 16-18 middle.

[0246] Depend on Figure 16The results showed that after 2 weeks of administration of a high-calorie model diet, the weight of mice in the model control group increased significantly compared with the negative control group (p<0.01), indicating that the model was successfully established. Before administration of the test substance, there was no significant difference in weight between the various dosage groups and the model control group. After 11 weeks of oral administration of the test substance, the weight of mice in the model control group increased significantly compared with the negative control group (p<0.01). Compared with the model control group (average weight 33.9g), the low-dose group of live *Lactobacillus plantarum* HOM2217 (average weight 29.8g), the high-dose group of live *Lactobacillus plantarum* HOM2217 (average weight 29.1g), the group of dead *Lactobacillus plantarum* HOM2217 (average weight 30.4g), and the positive control group (orlistat) (average weight 29.2g) all significantly (p<0.01) reduced the final weight of mice, with weight reduction rates of 12.1%, 14.2%, 10.3%, and 13.9%, respectively. The reduction rate of body weight in the low-dose, high-dose and dead groups of live Lactobacillus plantarum HOM2217 was comparable to that in the positive control group (orlistat), with no significant difference (p>0.05).

[0247] Depend on Figure 17 The results showed that after administration of a high-calorie model diet, the total weight gain and total food utilization rate of mice were significantly higher in the model control group compared with the negative control group (p<0.01). After 11 weeks of oral administration of the test substance, compared with the model control group, the low-dose group, high-dose group, dead bacteria group, and positive control group (orlistat) of *Lactobacillus plantarum* HOM2217 all significantly (p<0.01) reduced the total weight gain of mice. Compared with the model control group, the high-dose group of *Lactobacillus plantarum* HOM2217 and the positive control group (orlistat) both significantly (p<0.05) reduced the total food utilization rate of mice. The total weight gain of the low-dose, high-dose, and dead Lactobacillus plantarum HOM2217 live bacteria groups was comparable to that of the positive control group (orlistat), with no significant difference (p>0.05). The total feed utilization rate of the high-dose Lactobacillus plantarum HOM2217 live bacteria group was comparable to that of the positive control group (orlistat), with no significant difference (p>0.05).

[0248] Depend on Figure 18 The results showed that after being fed a high-calorie diet, the total food intake of mice in the model control group was significantly lower than that in the negative control group (p<0.05). After 11 weeks of oral administration of the test substance, there were no significant differences in total food intake and total calorie intake between the experimental groups and the model control group (p>0.05).

[0249] In conclusion, after 11 weeks of administration of *Lactobacillus plantarum* HOM2217, compared with the model control group, the low-dose group, high-dose group, and dead group of *Lactobacillus plantarum* HOM2217 all significantly reduced the body weight (p<0.01) and total weight gain (p<0.01) of obese model mice, with weight loss effects comparable to the positive control group (orlistat). There were no significant differences in total food intake and total calorie intake between the various dosage groups and the model control group (p>0.05). Therefore, it can be concluded that *Lactobacillus plantarum* has a weight loss effect without affecting food intake.

[0250] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details shown and described herein.

Claims

1. A strain of *Lactiplantibacillus plantarum*, characterized in that, The *Lactobacillus plantarum* strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 25683. The *Lactobacillus plantarum* strain described herein has the effects of inhibiting the growth of the obese strain *Enterobacter cloacae*, inhibiting the differentiation of 3T3-L1 precursor adipocytes, thereby effectively inhibiting fat deposition, reducing cholesterol levels, reducing weight, total weight gain, body fat mass, and serum inflammatory factor TNF-α levels, and increasing the content of intestinal short-chain fatty acids. The *Lactobacillus plantarum* strain contains the 16S rRNA gene represented by SEQ ID NO:

1.

2. The *Lactobacillus plantarum* strain according to claim 1, characterized in that, The *Lactobacillus plantarum* strain was isolated from healthy human breast milk.

3. Use of the *Lactobacillus plantarum* strain according to claim 1 or 2 for the production of lactic acid and short-chain fatty acids.

4. The use according to claim 3, characterized in that, The short-chain fatty acid is selected from one or more of formic acid, acetic acid, propionic acid, butyric acid, and isobutyric acid.

5. Use of the *Lactobacillus plantarum* strain according to claim 1 or 2 in the preparation of a composition for inhibiting the obese strain *Enterobacter cloacae*.

6. Use of the *Lactobacillus plantarum* strain according to claim 1 or 2 in the preparation of a composition for inhibiting the differentiation of 3T3-L1 precursor adipocytes, thereby effectively inhibiting fat deposition.

7. Use of the *Lactobacillus plantarum* strain according to claim 1 or 2 in the preparation of a pharmaceutical composition for weight loss or to reduce hyperlipidemia and hypercholesterolemia caused by obesity.

8. The use according to claim 7, characterized in that, The weight loss function is to reduce cholesterol levels or inhibit the differentiation of 3T3-L1 preadipocytes, thereby inhibiting fat production.

9. The use according to claim 7, characterized in that, The weight loss function is selected from one or more of the following: reducing body weight, total weight gain and total food utilization in obese model rats; reducing body fat weight and fat / body ratio in obese model rats; increasing serum high-density lipoprotein cholesterol content; increasing intestinal short-chain fatty acid content; and reducing the expression level of serum cellular inflammatory factor TNF-α.

10. The use according to any one of claims 7 to 9, characterized in that, The weight loss function is selected from one or more of the following: reducing cholesterol levels, inhibiting the differentiation of 3T3-L1 precursor adipocytes, reducing the body weight of obese model mice, reducing the total weight gain and total food utilization rate of obese model mice.

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