Lactiplantibacillus plantarum strain having weight-reducing function, probiotic formulation, preparation method therefor, and use thereof

Abstract

The present invention particularly relates to a Lactiplantibacillus plantarum strain having a weight-reducing function, a live bacterium formulation, a preparation method therefor, and the use thereof. The Lactiplantibacillus plantarum strain is preserved in China General Microbiological Culture Collection Center (CGMCC) of China Committee for Culture Collection of Microorganisms, the accession number thereof being CGMCC No. 25683. The Lactiplantibacillus plantarum strain and the live bacterium formulation thereof can inhibit the growth of an obesity bacterial strain Enterobacter cloacae, reduce the cholesterol, and inhibit the differentiation of 3T3-L1 preadipocytes, so as to reduce the body weight of an obesity model mouse and reduce the total weight gain and total food utilization rate of an obesity model rat, thereby achieving an excellent weight-reducing function.

Description

A plant lactobacillus strain with weight-loss function, a probiotic preparation, and its preparation method and use

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese Patent Application No. 202311294650.7 filed in China on September 28, 2023 and Chinese Patent Application No. 202311294687.X filed in China on September 28, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the technical field of microorganisms, and in particular to a Lactobacillus plantarum strain with weight-loss function, a probiotic preparation, and a preparation method and application thereof. Background Art

[0004] Obesity is primarily a metabolic disease caused by a disruption in the body's energy metabolism, resulting in energy intake exceeding energy expenditure, leading to excessive body fat accumulation. Obesity is influenced not only by genetic, environmental, metabolic, and physiological factors, but is also closely linked to conditions such as hyperlipidemia, hypertension, diabetes, insulin resistance, hyperuricemia, non-alcoholic fatty liver disease, and coronary heart disease. Obesity is a global public health issue. According to statistics, one-third of the global population is overweight. Over the past 20 years, the obesity rate among adults has increased by 50%, and obesity among children and adolescents has doubled. According to the latest data from the "2020 Report on the Nutrition and Chronic Disease Status of Chinese Residents," more than half of adults in China are overweight or obese. Among adults (aged 18 and above), the overweight rate is 34.3% and the obesity rate is 16.4%. Over the past 30 years, the rates of overweight and obesity have steadily increased across all age groups, with an average increase of approximately 2.5 times.

[0005] Probiotics can help with weight loss by regulating intestinal flora, improving intestinal inflammation, lowering cholesterol, and regulating hormone levels. Probiotic weight loss has zero side effects, no rebound, no water loss, no skin damage, no dieting or exercise required, and is simple and convenient to use, ushering weight loss into a new era.

[0006] Related art reports a strain of Lactobacillus plantarum isolated from Korean kimchi, which has a weight-loss effect, but the weight-loss effect is poor and cannot meet industry needs.

[0007] Therefore, there is a need for a plant lactobacillus strain with excellent weight-loss function and a probiotic preparation containing the same.

[0008] Summary of the Invention

[0009] The present invention provides a Lactobacillus plantarum strain with weight-loss function, a probiotic preparation containing the same, and a preparation method and use thereof. The strain and the probiotic preparation can produce high levels of lactic acid and short-chain fatty acids; can inhibit a variety of pathogenic bacteria; can adhere to intestinal epithelial cells; inhibit the growth of the obese strain Enterobacter cloacae and the differentiation of 3T3-L1 precursor adipocytes, thereby effectively inhibiting fat deposition; and can reduce cholesterol content.

[0010] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0011] In one aspect, the present invention provides a Lactobacillus plantarum strain, which is deposited in the China General Microbiology Culture Collection Center (CGMCC) with a deposit number of CGMCC No. 25683.

[0012] Preferably, the Lactobacillus plantarum strain comprises the 16S rRNA gene represented by SEQ ID NO: 1.

[0013] On the other hand, the present invention provides a method for isolating the above-mentioned Lactobacillus plantarum strain, comprising taking a suitable sample, growing it in a suitable culture medium, and then treating it with artificial gastric juice and artificial intestinal juice to obtain the strain.

[0014] Preferably, the Phytobacterium plantarum strain is isolated from healthy human breast milk.

[0015] In another aspect, the present invention provides a probiotic preparation, which is a live bacteria preparation or a dead bacteria preparation, and comprises the Lactiplantibacillus plantarum strain as described above.

[0016] Preferably, the probiotic preparation is a live bacteria preparation, and the live bacteria content is 6 to 9×10 11 CFU / g.

[0017] Preferably, the probiotic preparation is a dead bacteria preparation and contains 7 to 13×10 11 CFU / g of dead bacteria.

[0018] In another aspect, the present invention provides a method for preparing the probiotic preparation as described above, comprising the following steps:

[0019] (1) strain culture, inoculating the plant lactobacillus strain in a sterile liquid culture medium at an inoculum amount of 1 to 3% of the total amount of the culture medium, culturing for 16 to 24 hours to obtain a seed culture solution, and then inoculating the obtained seed culture solution in a fermentation medium at an inoculum amount of 1 to 3% of the total amount of the culture medium, performing fermentation culture, and obtaining a high-density culture solution of the plant lactobacillus;

[0020] (2) Drying.

[0021] Preferably, the fermentation medium used contains: 60-100 g / L glucose, 60-80 g / L yeast extract, 0.2-0.4 g / L magnesium sulfate, 0.1-0.2 g / L manganese sulfate, 2-4 g / L dipotassium hydrogen phosphate, 2-4 g / L potassium dihydrogen phosphate, 1-2 g / L Tween 80, and 0.5-2 g / L L-cysteine ​​salt.

[0022] Preferably, during the fermentation process, sodium hydroxide solution is automatically added to maintain a constant pH of 5.0-6.0 until the fermentation stops producing acid, and the fermentation is terminated when sodium hydroxide is no longer added.

[0023] In one example, the probiotic preparation is a live bacterial preparation, and the method further includes preparing a lyophilization protectant after the strain culture step and before the drying step, and freeze-drying is performed in the drying step; optionally, the lyophilization protectant used comprises: a protectant of 50-100 g / L peptone, 20-60 g / L sucrose, 5-10 g / L vitamin C and 1-3 g / L glycerol.

[0024] Preferably, the freeze-drying conditions are: pre-freezing temperature of -50 to -55°C, pre-freezing time of 5 to 8 hours, primary drying temperature of -10 to -30°C, primary drying time of 25 to 35 hours, secondary drying temperature of 32 to 35°C, and secondary drying time of 5 to 8 hours.

[0025] In another example, the probiotic preparation is a dead bacteria preparation, and the drying step is to first perform heat inactivation and then dry it in a spray drying tower to obtain the dead bacteria preparation; optionally, the heat inactivation condition is 70-100°C for 10-40 minutes.

[0026] In another aspect, the present invention provides use of the Lactobacillus plantarum strain as described above for producing lactic acid and short-chain fatty acids.

[0027] Preferably, the short-chain fatty acid is selected from one or more of formic acid, acetic acid, propionic acid, butyric acid and isobutyric acid.

[0028] In another aspect, the present invention provides use of the Lactobacillus plantarum strain as described above in the preparation of a composition for inhibiting the obesity strain Enterobacter cloacae.

[0029] In another aspect, the present invention provides use of the above-mentioned Lactobacillus plantarum strain in preparing a composition for inhibiting the differentiation of 3T3-L1 preadipocytes, thereby effectively inhibiting fat deposition.

[0030] In another aspect, the present invention provides the use of the above-mentioned composition in the preparation of a pharmaceutical composition for reducing weight or lowering hyperlipidemia and hypercholesterolemia caused by obesity.

[0031] Preferably, the weight-loss function is to reduce the cholesterol content, or inhibit the differentiation of 3T3-L1 preadipocytes to inhibit fat production.

[0032] Preferably, the weight loss function is selected from one or more of reducing the body weight, total weight gain and total food utilization rate of obese model rats, reducing the body fat weight and fat / body ratio of obese model rats, increasing the serum high-density lipoprotein cholesterol content, increasing the intestinal short-chain fatty acid content, and reducing the expression of the inflammatory factor TNF-α in the serum.

[0033] Preferably, the weight loss function is one or more selected from the group consisting of reducing cholesterol content, inhibiting differentiation of 3T3-L1 preadipocytes, reducing body weight of obese model mice, and reducing total weight gain and total food utilization rate of obese model rats.

[0034] In one embodiment, the Lactobacillus plantarum strain according to the present invention can be prepared into common food, health food or medicine.

[0035] Beneficial effects of the Lactobacillus plantarum strain and its preparation according to the present invention:

[0036] 1. The plant lactobacillus strain and its probiotic preparation of the present invention can tolerate artificial gastrointestinal fluid, can produce high amounts of lactic acid and short-chain fatty acids; can inhibit a variety of pathogenic bacteria; and can adhere to intestinal epithelial cells.

[0037] 2. The Lactobacillus plantarum strain and the probiotic preparation thereof of the present invention inhibit the growth of the obese strain Enterobacter cloacae and can inhibit the differentiation of 3T3-L1 preadipocytes, thereby achieving the effect of effectively inhibiting fat deposition.

[0038] 3. The plant lactobacillus strain and its preparation and its probiotic preparation of the present invention can reduce the cholesterol content.

[0039] 4. the plant lactobacillus strain of the present invention and its preparation and its probiotic preparation can significantly reduce the body weight of obesity model rats, reduce the total weight gain and total food utilization rate of obesity model rats, significantly reduce the body fat weight and fat / body ratio of obesity model rats, increase serum high-density lipoprotein cholesterol content, effectively increase the content of intestinal short-chain fatty acids, and reduce the expression of inflammatory factor TNF-α in serum. It can significantly reduce the body weight of obesity model mice, reduce the total weight gain and total food utilization rate of obesity model rats. Therefore, the plant lactobacillus strain of the present invention has an excellent weight-loss function.

[0040] 5. The probiotic preparation of the present invention has simple production process parameters, is easy to control, has a short cycle, ensures a high survival rate of the Lactobacillus plantarum strain, and the obtained product can be stored for a long time with stable product quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIG1 shows a biological evolutionary tree of Lactobacillus plantarum HOM2217 according to the present invention.

[0042] FIG2 shows a RAPD cluster analysis diagram of Lactobacillus plantarum HOM2217 constructed based on the UPGMA method.

[0043] Figure 3 shows the results of the growth inhibition of Enterobacter cloacae ATCC13047 by Lactobacillus plantarum HOM2217 according to the present invention. Note: *** indicates p<0.001 compared with the HOM2217 group.

[0044] Figure 4 shows the in vitro cholesterol degradation rate of Lactobacillus plantarum HOM2217 according to the present invention. Note: *** indicates p<0.001 compared with the HOM2217 group, ** indicates p<0.01 compared with the HOM2217 group.

[0045] FIG5 shows the effect of Lactobacillus plantarum HOM2217 according to the present invention on the viability of 3T3-L1 preadipocytes.

[0046] FIG6 shows the results of Oil Red staining of 3T3-L1 preadipocytes differentiated by Lactobacillus plantarum HOM2217 according to the present invention.

[0047] FIG7 shows the effect of Lactobacillus plantarum HOM2217 according to the present invention on the lipid content of 3T3-L1 preadipocytes after differentiation.

[0048] FIG8 shows the effect of Lactobacillus plantarum HOM2217 according to the present invention on the triglyceride (TG) content of 3T3-L1 preadipocytes after differentiation.

[0049] FIG9 shows the effect of Lactobacillus plantarum HOM2217 on the body weight of obese rats. #Indicates significant difference compared with the negative control group (p<0.05); * indicates significant difference compared with the model control group (p<0.05).

[0050] FIG10 shows the effect of Lactobacillus plantarum HOM2217 according to the present invention on the total weight gain and total food utilization of obese rats. ## Indicates that there is a very significant difference compared with the negative control group (p<0.01); ** indicates that there is a very significant difference compared with the model control group (p<0.01).

[0051] FIG11 shows the effect of Lactobacillus plantarum HOM2217 on body fat weight and fat / body ratio in obese rats. ## Indicates extremely significant difference compared with the negative control group (p<0.01); * indicates significant difference compared with the model control group (p<0.05); ** indicates significant difference compared with the model control group (p<0.01).

[0052] FIG12 shows the effect of Lactobacillus plantarum HOM2217 according to the present invention on the total food intake and total calorie intake of obese rats. # Indicates significant difference compared with the negative control group (p<0.05); * indicates significant difference compared with the model control group (p<0.05).

[0053] Figure 13 shows the effects of Lactobacillus plantarum HOM2217 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 rats. ## Indicates extremely significant difference compared with the negative control group (p<0.01); * indicates significant difference compared with the model control group (p<0.05); ** indicates extremely significant difference compared with the model control group (p<0.01).

[0054] FIG14 shows the effect of Lactobacillus plantarum HOM2217 according to the present invention on serum cytokines TNF-α and IL-6 in obese rats. # Indicates that there is a very significant difference compared with the negative control group (p<0.05); ## Indicates that there is a very significant difference compared with the negative control group (p<0.01); ** indicates that there is a very significant difference compared with the model control group (p<0.01).

[0055] FIG15 shows the effect of Lactobacillus plantarum HOM2217 on the content of lactic acid and short-chain fatty acids (SCFA) in the cecum of obese rats. ##Indicates extremely significant difference compared with the negative control group (p<0.01); * indicates significant difference compared with the model control group (p<0.05); ** indicates extremely significant difference compared with the model control group (p<0.01).

[0056] FIG16 shows the effect of Lactobacillus plantarum HOM2217 on the body weight of obese mice. ## Indicates significant difference compared with the negative control group (p<0.01); ** indicates significant difference compared with the model control group (p<0.01).

[0057] FIG17 shows the effect of Lactobacillus plantarum HOM2217 according to the present invention on the total weight gain and total food utilization of obese mice. # Indicates that there is a very significant difference compared with the negative control group (p<0.05); ## Indicates that there is a very significant difference compared with the negative control group (p<0.01); ** indicates that there is a very significant difference compared with the model control group (p<0.01).

[0058] FIG18 shows the effect of Lactobacillus plantarum HOM2217 on food intake and caloric intake in obese mice. # Indicates significant difference compared with the negative control group (p<0.05); * indicates significant difference compared with the model control group (p<0.05).

[0059] Microorganism Preservation Instructions

[0060] Biological materials: HOM2217 strain, classified as Lactiplantibacillus plantarum, also known as Lactobacillus plantarum, was deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms (CGMCC) on September 9, 2022, with the deposit number CGMCC No. 25683; the address of the deposit center is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing.

[0061] Identification

[0062] The Lactobacillus plantarum HOM2217 strain of the present invention, also known as the Lactobacillus plantarum HOM2217 strain, was sent to the Institute of Microbiology, Chinese Academy of Sciences for identification in May 2023.

[0063] The test and identification conclusion is as follows: Under the conditions of our laboratory, based on a comprehensive analysis of the cell morphology, physiological and biochemical characteristics, 16S rRNA gene sequence, pheS gene sequence, and other experimental data of the tested strain, and with reference to relevant research articles in the "Bergey's Manual of Systematic Bacteriology" and the International Journal of Systematic and Evolutionary Microbiology, the tested strain (strain number: HOM2217) was identified as Lactiplantibacillus plantarum. Synonyms: Lactobacillus plantarum.

[0064] The cell morphology of the strain is rod-shaped; the physiological and biochemical characteristics are: Gram-positive, catalase-negative (-), 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 DESCRIPTION

[0065] The present invention discloses strains, characteristics, and applications. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is obvious that relevant persons can modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0066] To further illustrate the technical means and effects of the present invention to achieve the intended purpose, the following technical solutions of the present invention are further described in conjunction with specific embodiments, but are not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention that does not depart from the spirit and scope of the technical solution of the present invention shall be included in the scope of protection of the present invention. Experimental methods for which specific conditions are not specified in the following implementations are carried out in accordance with conventional methods and conditions in the art.

[0067] In order to make the technical problems to be solved, the technical solutions adopted and the advantages of the present invention 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.

[0068] It should be noted that the experimental methods used in the present invention are all conventional methods unless otherwise specified.

[0069] Unless otherwise specified, the reagents and materials used in the present invention were prepared using conventional methods or obtained from commercial channels.

[0070] Lactobacillus plantarum is a Gram-positive, non-motile, non-spore-forming bacterium with straight rod-shaped cells, typically 0.9-1.2 μm x 3.0-8.0 μm, found singly, in pairs, or in short chains. It is widely distributed in many foods, including dairy, meat, fish, vegetable fermentation products, silage, and animal and human mucous membranes, such as the oral cavity, gastrointestinal tract, and vagina. This strain is included in the EU Qualification for Safety (QPS) recommended list of biological agents and on the International Dairy Federation (IDF) "List of Microorganisms with a History of Safe Use in Food." It has also received GRAS (Generally Recognized as Safe) designation from the U.S. Food and Drug Administration. In 2010, the National Health and Family Planning Commission of China also approved Lactobacillus plantarum as an edible fungus.

[0071] The present invention has developed a strain with weight-loss properties that can reduce cholesterol levels and inhibit the differentiation of 3T3-L1 preadipocytes. It significantly reduces the body weight, total weight gain, and total food utilization rate of obese rat models, as well as the body fat mass and fat-to-body ratio of obese rat models. It also increases serum high-density lipoprotein cholesterol levels, effectively increases intestinal short-chain fatty acid levels, and reduces serum expression of the inflammatory factor TNF-α. It can significantly reduce the body weight of obese mice and the total weight gain and total food utilization rate of obese rat models. This strain can produce high levels of lactic acid and short-chain fatty acids, exhibits excellent production performance, and can be developed into weight-loss-related foods, health foods, and pharmaceuticals.

[0072] In the present invention, "Lactiplantibacillus plantarum" and "Lactobacillus plantarum" are used interchangeably.

[0073] The Lactobacillus plantarum HOM2217 strain used in the present invention is isolated from healthy human breast milk, and the specific isolation method is as described in Example 1.

[0074] Example

[0075] Example 1 Isolation and identification of Lactobacillus plantarum HOM2217 strain

[0076] (1) Preparation of artificial gastrointestinal fluid

[0077] Artificial gastric juice: 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 with 0.22 μm filter membrane for sterilization, and store at -20°C for later use.

[0078] Artificial intestinal fluid: Take 6.8 g of potassium dihydrogen phosphate, add 500 mL of water, and adjust the pH to 6.8 with 0.4% (0.1 mol / L) sodium hydroxide solution. Take 10 g of pancreatic enzyme and 3 g of porcine bile salt, add appropriate amount of water to dissolve them, mix the two liquids, add water to 1000 mL, centrifuge at 5000 rpm for 5 min, take the supernatant, filter and sterilize with a 0.22 μm filter membrane, and store at -20°C for later use.

[0079] (2) Preparation of separation culture medium

[0080] MRS liquid culture medium: MRS broth culture medium (catalog number: CM1163, OXOID, UK) was prepared according to the instructions, sterilized at 121°C for 15 min, and stored at 2-8°C in the dark for one week.

[0081] MRS-Cys solid medium containing bromocresol purple: Add 0.04 g of bromocresol purple per liter of MRS solid medium (Cat. No. CM1175, OXOID, UK) and stir thoroughly. Stir at 121°C for 15 min. Pour the medium into a clean plate in a laminar flow hood, pour approximately 15 mL into each culture dish, and allow to solidify before use.

[0082] (3) Isolation and screening of Lactobacillus plantarum HOM2217 strain

[0083] Use sterile aerobic tubes to collect in vitro breast milk and store it at low temperature under aerobic environment. The experiment started on the day of sampling. Take about 1g of breast milk sample and add it to an aerobic tube containing 9mL of MRS liquid culture medium. Incubate it aerobically at 37℃ for 24h, centrifuge it at 8000rpm for 10min, discard the supernatant, add 10mL of artificial gastric juice, mix it, incubate it aerobically at 37℃ for 3h, centrifuge it at 8000rpm for 10min, discard the supernatant, add 10mL of artificial intestinal juice, mix it, incubate it aerobically at 37℃ for 3h, dilute the sample to 10 by 10-fold dilution method. -6 , from stock solution to 10 -6 100 μL of the solution was taken in sequence and spread on MRS solid culture medium plates containing bromocresol purple, and cultured aerobically at 37°C for 48 h.

[0084] Pick a single colony with yellowing edges, streak culture, and purify 3-4 times until the colony is single. Perform Gram staining and observe colony morphology under a microscope. Transfer the single colony to liquid culture medium for pure culture, preserve the seed in glycerol, and store at -80°C.

[0085] (4) Morphological observation of Lactobacillus plantarum HOM2217 strain

[0086] Lactobacillus plantarum HOM2217, cultured aerobically at 37°C for 24 hours on MRS agar, showed colonies approximately 3 mm in diameter, with raised, rounded shapes, a moist, smooth surface, and a dense, white color, occasionally showing light or dark yellow. Microscopic observation revealed round-ended, straight rods measuring approximately 0.9–1.2 μm by 3–8 μm, forming solitary, paired, or short chains. They were Gram-positive and non-spore-forming. Two other strains with the same morphology were also screened and designated Lactobacillus plantarum S1-6 and S2-13.

[0087] (5) Identification of Lactobacillus plantarum HOM2217 strain

[0088] 16S rRNA gene bacterial species identification: DNA was extracted from the three preserved strains HOM2217, S1-6 and S2-13, and DNA amplification was performed. PCR amplification and agarose gel electrophoresis were performed using universal primers 27F (SEQ ID NO: 7) and 1492R (SEQ ID NO: 8). The gel was then excised, recovered, and sequenced. The isolated strains were subjected to 16S rDNA sequencing. Based on their 16S rDNA sequences, BLAST tools were used to compare them in the NCBI database, and a biological evolutionary tree was drawn using Mega 7.0 software. The results are shown in Figure 1. As shown in Figure 1, the identification results of the three strains were all Lactiplantibacillus plantarum, and they were named Lactiplantibacillus plantarum HOM2217, S1-6 and S2-13, respectively. Among them, the sequence of the 16S rRNA gene of Lactobacillus plantarum HOM2217 is shown in SEQ ID NO: 1:

[0089] In addition, Lactobacillus plantarum strains S1-6 and S2-13, which were isolated simultaneously with the Lactobacillus plantarum strain HOM2217 of the present invention, are used as control strains in the present invention, and the sequences of their 16S rRNA genes are shown in SEQ ID NOs: 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 strain S2-13 have 1431 bases in common with the strain HOM2217 of the present invention, and the sequence similarity is 99.38%.

[0090] (6) Randomly Amplified Polymorphic DNA (RAPD) Strain Identification: DNA was extracted from the preserved strains, and PCR was performed using the strain DNA as a template using five primers, OPA-02, OPA-18, OPL-07, OPL-16, and OPM-05, as shown in Table 1. DNA fragments that exhibit polymorphism were amplified by PCR, and after gel electrophoresis, distinct DNA differences were observed. Cluster analysis software was used for analysis, and the results are shown in Figure 2. As shown in Figure 2, the Lactobacillus plantarum HOM2217 strain is distinct from other Lactobacillus plantarum strains and is unique.

[0091] Table 1

[0092] Example 2 Test on the ability to inhibit common pathogenic bacteria

[0093] (1) Activation of indicator bacteria

[0094] 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 Microorganism 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 an inoculum size of 1% of the total culture volume and cultured aerobically at 37°C, 180 rpm, for 24 hours before use. Clostridium difficile ATCC9689 was inoculated into BHI medium at an inoculum size of 1% of the total culture volume and cultured anaerobically at 37°C for 24 hours before use.

[0095] (2) Activation of Lactobacillus plantarum

[0096] Lactobacillus plantarum HOM2217, S1-6, and S2-13 strains prepared in Example 1 and stored frozen at -80°C were inoculated into sterilized MRS-Cys liquid culture medium described in Example 1 at an inoculum size of 1% of the total culture medium. The culture was then anaerobically incubated at 37°C for 24 hours. After activation twice, fermentation broths were obtained. The broths were then centrifuged at 8000 rpm for 10 minutes, and the supernatants were used for bacteriostasis testing.

[0097] (3) Plate preparation

[0098] Heat sterilized Tryptic Soy Agar (TSA) medium until completely melted, pour into a Petri dish, place on a horizontal surface to form an even agar layer, and wait for it to solidify. Add the indicator bacteria to the TSA medium, shake well, pour into a pre-prepared TSA blank agar plate, and let it stand to solidify.

[0099] (4) Antibacterial experiment

[0100] With sterile tweezers, Oxford cup is gently placed on flat board, in the centre, keep certain distance, to wherein adding respectively the lactic acid bacteria fermentation liquid supernatant of 0.2mL to be measured, place 4 ℃ of refrigerators, diffuse after 24 hours, cultivate at least 18 hours in 37 ℃ of incubators, observe the appearance of inhibition zone.After forming the inhibition zone, measure with ruler.With the liquid nutrient medium (MRS) among the embodiment 1 is negative control, plant lactobacillus S1-6 and S2-13 are as positive control bacterial strain, and each sample is done 3 parallels, and antibacterial results are shown in Table 2.

[0101] Table 2 Inhibitory effect of Lactobacillus plantarum HOM2217 strain on pathogenic bacteria Note: “–” no antibacterial activity; “+” 11-16mm; “++” 17-22mm; “+++” ≥ 23mm

[0102] As shown in Table 2, compared with the positive control strains S1-6 and S2-13, the Lactobacillus plantarum HOM2217 strain has a stronger inhibitory effect on the six pathogenic bacteria, and has a stronger inhibitory effect on Escherichia coli, Salmonella, Pseudomonas aeruginosa, Listeria monocytogenes and Clostridium difficile, indicating that the strain has a good ability to inhibit pathogenic bacteria.

[0103] Example 3 Test on the ability to inhibit obesity bacteria

[0104] (1) Strain activation

[0105] The indicator bacteria Enterobacter cloacae ATCC13047 and the Lactobacillus plantarum HOM2217, S1-6, and S2-13 strains prepared in Example 1 were inoculated into the sterilized MRS liquid culture medium described in Example 1 at an inoculum rate of 1% of the total culture medium. The culture was aerobically cultured at 37°C for 24 hours and activated twice to obtain a fermentation broth. The cells were then centrifuged at 8000 rpm for 10 minutes to collect the cells. The cells were washed three times with sterile saline and the bacterial count was adjusted to 1×10 8 CFU / mL, reserve for future use.

[0106] (4) Co-culture growth antagonism test

[0107] The cultured Enterobacter cloacae ATCC13047 and Lactobacillus plantarum HOM2217, S1-6, and S2-13 strain suspensions were inoculated into the sterilized MRS liquid medium described in Example 1 at an inoculum size of 1% of the total culture medium. The culture was vortexed for 20 seconds, and a culture inoculated with only Enterobacter cloacae ATCC13047 was used as a positive control. The cells were aerobically cultured at 37°C for 18 hours, and viable bacteria were counted on MRS medium. Three parallels were used for each treatment, and data were analyzed using GraphPad Prism 9 software for one-way analysis of variance (ANOVA) and Dunnett's t-test, i.e., pairwise comparisons of the means between multiple experimental groups and a control group. The results are shown in Figure 3.

[0108] Growing evidence suggests that the gut microbiota plays an important role in obesity, insulin resistance, and related liver diseases. Recently, Enterobacter cloacae, a bacterium found in obese mice, has been identified as a pathogenic bacteria in the gut and a direct culprit in obesity.

[0109] As shown in Figure 3, compared with the positive control strains S1-6 and S2-13, the Lactobacillus plantarum HOM2217 strain can significantly (p < 0.01) inhibit the growth of Enterobacter cloacae when co-cultured with Enterobacter cloacae for 18 hours, indicating potential value for weight loss.

[0110] Example 4 Gastrointestinal transit capacity test

[0111] (1) Activation of strains

[0112] The Lactobacillus plantarum HOM2217, S1-6, and S2-13 strains prepared in Example 1 were inoculated into the sterilized MRS liquid culture medium described in Example 1 at an inoculum amount of 1% of the total culture medium, and cultured aerobically at 37° C. for 24 hours. After activation twice, the strain fermentation broth was obtained.

[0113] (2) Preparation of artificial gastric juice

[0114] 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 with a 0.2 μm microporous membrane and set aside.

[0115] (3) Preparation of artificial intestinal fluid

[0116] Take 6.8g of potassium dihydrogen phosphate, add 500mL of water to dissolve, adjust the pH to 6.8, add 10g of trypsin and 3g of porcine bile salt, mix the two solutions after dissolution, add water to 1000mL, filter with a 0.22um sterile filter membrane under sterile environment, and set aside.

[0117] (4) Evaluation of the survival ability of strains in the simulated gastrointestinal tract

[0118] Take 1 mL of activated bacterial suspension of the strain to be tested and add the cells to 9 mL of artificial gastric fluid (pH 3.0). Mix thoroughly and count the viable cells. Place the culture in a 37°C incubator for 3 hours and then count the viable cells. After 3 hours of incubation in artificial gastric fluid, transfer all cells to an equal volume of artificial intestinal fluid (pH 6.8), mix thoroughly, and incubate at 37°C. Count the viable cells on plates using MRS medium at 3 and 24 hours. Calculate the survival rate using the following formula:

[0119] Gastric juice 3-hour survival rate (%) = [logCFUN1 / logCFUN0] × 100%

[0120] 3-hour survival rate in intestinal fluid (%) = [logCFUN2 / logCFUN0] × 100%

[0121] 24-hour survival rate in intestinal fluid (%) = [logCFUN3 / logCFUN0] × 100%

[0122] N0 = the number of viable Lactobacillus plantarum bacteria before treatment, N1 = the number of viable Lactobacillus plantarum bacteria after 3 hours of treatment with gastric juice, N2 = the number of viable Lactobacillus plantarum bacteria after 3 hours of treatment with intestinal juice, N3 = the number of viable Lactobacillus plantarum bacteria after 24 hours of treatment with intestinal juice. The results are shown in Table 3.

[0123] Table 3 Survival rate of Lactobacillus plantarum HOM2217 strain in simulated gastrointestinal fluid

[0124] Table 3 shows that after 3 hours of treatment with simulated gastric fluid, the survival rate of Lactobacillus plantarum HOM2217 reached 100%. After 24 hours of treatment with simulated intestinal fluid, its 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 intestine.

[0125] Example 5 Intestinal epithelial cell adhesion ability test

[0126] (1) Preparation of cell culture medium

[0127] 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), mixed and stored at 4°C.

[0128] Incomplete culture medium: high-glucose DMEM medium to which 10% inactivated fetal bovine serum (FBS) was added, mixed, and stored at 4°C.

[0129] (2) Cell recovery and culture

[0130] Human colorectal adenocarcinoma cells, Caco-2, were purchased from the Cell Resource Center, Institute of Basic Medicine, Chinese Academy of Medical Sciences. Caco-2 cells were resuspended in fresh culture medium and evenly dispersed in a culture flask. Culture was maintained at 37°C in an atmosphere of 5% CO₂ and 95% air. The culture medium was replaced every 48 hours during recovery. When the cells were well grown (80% confluence), they were digested with trypsin-EDTA solution at 37°C. After digestion, the cell concentration was adjusted to 1×10 5 The cells were seeded in 24-well plates and cultured until the confluence reached 80%.

[0131] (3) Activation of Lactobacillus plantarum

[0132] The Lactobacillus plantarum HOM2217, S1-6 and S2-13 strains prepared in Example 1 and stored at -80°C were inoculated into the sterilized MRS liquid culture medium described in Example 1 at an inoculum amount of 1% of the total culture medium, and cultured aerobically at 37°C for 24 hours. After activation twice, the strain fermentation broth was obtained.

[0133] (4) Adhesion test

[0134] The cells grown in the corresponding culture medium were collected by centrifugation at 8000 rpm for 10 min. The cells were washed with DPBS three times and then resuspended in incomplete culture medium and the cell concentration was adjusted to 10 7 cfu / mL. 1 mL of the above bacterial suspension was added to a 24-well plate containing Caco-2 cells grown to a monolayer and incubated in a 5% CO2 incubator at 37°C for 2 h. After incubation, the cells were washed three times with sterile Dulbecco's phosphate-buffered saline (DPBS). Caco-2 cells were digested with trypsin-EDTA solution at 37°C. The cell count and viable bacterial count before and after adhesion were counted. Each sample was performed in triplicate. The results are shown in Table 4.

[0135] Table 4 Adhesion ability of Lactobacillus plantarum HOM2217 strain to Caco-2 cells Note: The numbers with different capital letters in the same column are significantly different (p<0.01).

[0136] Adhesion index = number of bacteria after adhesion / number of cells per dish

[0137] Adhesion rate = number of bacteria after adhesion / number of bacteria before adhesion

[0138] The results in Table 4 show that the adhesion rate of Lactobacillus plantarum HOM2217 strain to human colon cancer cells Caco-2 is 4.32%, and the adhesion index is 4.91. Compared with the other two strains of Lactobacillus plantarum S1-6 and S2-13, it can significantly (p < 0.01) improve the adhesion ability of small intestinal epithelial cells.

[0139] Example 6 Lactic acid and short-chain fatty acid production capacity test

[0140] Respectively with the plant lactobacillus HOM2217, S1-6 and the S2-13 bacterial strain of-80 ℃ of freezing preservations of preparation among the embodiment 1, be accessed in the sterilized described MRS liquid nutrient medium of embodiment 1 with the inoculum size that accounts for culture medium total amount 1%, 37 ℃ left standstill aerobic culture 24 hours, activated bacterial strain fermentation liquid after twice.Then under the 8000rpm condition centrifugal 10min, get supernatant and use the content of gas chromatograph detection lactic acid and short-chain fatty acids.The described MRS liquid nutrient medium of embodiment 1 is a negative control, and each sample is done 3 parallels, and data adopt SPSS25.0 software to carry out one-way variance (ANOVA) analysis and Duncan multiple comparison, and the results are shown in Table 5.

[0141] Table 5 Lactic acid and short-chain fatty acid production capacity of Lactobacillus plantarum HOM2217 strain Note: The numbers with different capital letters in the same column are significantly different (p<0.01).

[0142] As shown in Table 5, compared with the other two strains of Lactobacillus plantarum S1-6 and S2-13, the Lactobacillus plantarum HOM2217 strain 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).

[0143] Example 7 Antibiotic Sensitivity Test

[0144] The antimicrobial susceptibility test was conducted according to the ISO10932-2010 milk and dairy products standard developed by the International Organization for Standardization, which is applicable to the determination of the minimum inhibitory concentration (MIC) of antibiotics for bifidobacteria and non-enterococcal lactic acid bacteria (LAB).

[0145] (1) Culture medium preparation

[0146] MRS liquid culture medium: same as Example 1

[0147] LSM liquid culture medium: Weigh 21.06 g of Iso-Sensitest medium (Cat. No. CM0473B, OXOID, UK), weigh 5.2 g of MRS Broth, add water to 0.5 L, adjust the pH to 6.85 ± 0.1, sterilize at 121°C for 15 min, the pH should be 6.7 ± 0.1, set aside, and store at 2-8°C in the dark for one week.

[0148] Activation and proliferation of Lactobacillus plantarum

[0149] The Lactobacillus plantarum HOM2217 strain prepared in Example 1 was inoculated into the sterilized liquid culture medium (MRS) described in Example 1 at an inoculum size of 1% of the total culture medium, and aerobic cultured at 28° C. for 24 hours. The activated strain fermentation liquid was obtained after two activations. The activated Lactobacillus plantarum HOM2217 strain was propagated on MRS agar medium and aerobic cultured at 28° C. for 48 hours.

[0150] (2) Preparation of antibiotic microdilution plates

[0151] Weigh 0.0512g of antibiotics and add 10mL of solvent. Dissolve chloramphenicol and erythromycin in ethanol (without filtration), ampicillin in phosphate buffer (pH 8.0, 0.1 mol / L), and other antibiotics in water. After shaking, filter through a 0.22μm filter and aliquot into EP tubes at a concentration of 5120μg / mL (5.12mg / mL). Store at -20°C until needed. Dilute the antibiotic stock solution with water (phosphate buffer for ampicillin) to the appropriate concentration range. Add 50μL of the dilution to the wells of a microdilution plate.

[0152] (3) Preparation of bacterial suspension

[0153] Pick up a single colony from the agar plate. Suspend the colony in a sterile culture tube filled with 2 mL to 5 mL of sterile saline. Suspend the colony in pre-reduced LSM liquid medium. Suspend the colony until the turbidity of the solution reaches McFarland standard 1 or the optical density at 625 nm is 0.16 to 0.2 using a spectrophotometer. The suspension is approximately equivalent to 3 × 10 8 CFU / mL. Dilute the bacterial suspension with the recommended culture medium. Dilute the bacterial suspension 500-fold with MRS liquid medium because the antibiotic solution will dilute the culture medium twice. Dispense the diluted bacterial suspension within 30 minutes of preparation. When using frozen plates, thaw the frozen antibiotic solution under aerobic conditions immediately before use. Dispense 50 μL of the diluted suspension into each well of the microdilution plate (approximately 3×10 4CFU / well). Incubate the plates at 37°C under static aerobic conditions for 48 hours. When using an aerobic jar, cover the plates with lids between each plate to create a uniform environment within the jar. Each experiment was repeated three times, with both positive and negative control groups. Positive control wells contained no antibiotic but the test strain and culture medium containing the solvent containing the highest concentration of antibiotic. Negative control wells contained no test strain or antibiotic but culture medium.

[0154] (4) Read the MIC result

[0155] After 48 hours of incubation, read the MIC visually. After incubation, inspect the negative control wells for visible bacterial growth. If contamination is observed, reject all data generated for that strain. Note: Lack of growth in the positive control wells indicates that the tested strain is sensitive to the solvent used to dissolve the antibiotic. In this case, reading the MIC for that particular antibiotic is meaningless. If the negative and positive controls are normal, determine bacterial growth visually for each antibiotic by comparison with the positive control. Ideally, place the microdilution plate on top of a magnifying glass rack, with a bench lamp providing indirect light for easy reading. Bacterial growth is easily detected under a magnifying glass as a pellet 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 observed visually. This concentration is the MIC for that antibiotic for that particular strain. Three replicates are performed for each sample, with Lactobacillus plantarum ATCC14917 (the model strain, designated for MIC quality control) serving as the positive control. The antibiotic susceptibility results of Lactobacillus plantarum HOM2217 are shown in Table 6. In 2012, the European Food Safety Authority (EFSA) established antibiotic resistance standards for bacteria used in food.

[0156] Table 6 Antibiotic sensitivity results of Lactobacillus plantarum HOM2217 strain

[0157] As shown in Table 6, the Lactobacillus plantarum HOM2217 strain 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.

[0158] Example 8 In vitro cholesterol degradation test

[0159] 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.

[0160] The plant lactobacillus HOM2217, S1-6 and S2-13 strains prepared in Example 1 were respectively accessed into the sterilized MRS liquid medium described in Example 1 with an inoculum amount of 1% of the total culture medium, and aerobically cultured at 37°C for 24 hours. After continuous activation for two generations, they were inoculated into MRS high cholesterol liquid medium, anaerobically cultured at 37°C for 24 hours, and then centrifuged at 4°C, 8000rpm for 10 minutes to collect the supernatant. According to the national standard GB 5009.128-2016 food safety national standard for the determination of cholesterol in food, gas chromatography was used for determination. Each sample was repeated 3 times, and the cholesterol degradation rate was calculated according to the following formula. Each treatment was repeated 3 times, and the data were analyzed by t-test using Graph Pad prism9 software.

[0161] Where: M1 and M2 are the cholesterol masses (μg) in the supernatant before and after fermentation, respectively.

[0162] The results are shown in Figure 4. As shown in Figure 4, in a medium containing bile salts, the cholesterol degradation rate of Lactobacillus plantarum HOM2217 strain was 50.6%, which was significantly higher than that of the other two Lactobacillus plantarum strains S1-6 and S2-13 (p < 0.01).

[0163] Example 9 In vitro lipogenesis inhibition test

[0164] (1) Preparation of cell culture medium

[0165] Complete medium 1: high-glucose DMEM medium, supplemented 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.

[0166] Complete medium 2: high-glucose DMEM medium to which 10% inactivated fetal bovine serum (FBS) was added and stored at 4°C.

[0167] Cell differentiation medium 1: Add 3-isobutyl 1-methylxanthine (IBMX) to complete medium 2 to a final concentration of 0.5 mmol / L, dexamethasone (DEX) to a final concentration of 1 μmol / L, and insulin to a final concentration of 10 μg / mL.

[0168] Cell differentiation medium 2: Add insulin to complete medium 2 to a final concentration of 10 μg / mL and store at 4°C until ready for use.

[0169] (2) Preparation of test bacterial samples

[0170] The plant lactobacillus HOM2217, S1-6 and S2-13 strains prepared in Example 1 were respectively accessed into the sterilized MRS liquid medium described in Example 1 with an inoculum size accounting for 1% of the total culture medium. The cells were left to stand aerobically for 24 hours at 37°C. After two generations of continuous activation, the cells were collected by centrifugation at 8000 rpm for 10 min, washed twice with PBS, and resuspended in a 0.1 mol / L PBS solution (pH 7.2-7.4) to form a bacterial suspension.

[0171] Heat-killed strain (HKS): Place the bacterial suspension in a water bath and treat at 70°C for 30 minutes to obtain heat-killed strains. Store at -80°C until ready for use.

[0172] (3) Culture of 3T3-L1 preadipocytes

[0173] 3T3-L1 preadipocytes (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were inoculated into complete medium 1 at a ratio of 1:3 for passage, and cells of passages 3 to 10 were selected for the experiment.

[0174] (4) Cell viability assay

[0175] Adjust the cell number to 2 × 10 cells using complete medium 2. 5 Cells / mL were cultured, and 0.1 mL of cell fluid was aspirated from each well and plated in a 96-well culture plate. The cells were cultured for 2 days and then used for later use. The cultured cells in the 96-well plate were taken, the cell culture medium was aspirated, and 0.1 mL of test bacteria of different concentrations adjusted with complete medium 2 was added to each well. The cells were cultured in a 37°C, 5% CO2 incubator for 24 hours. Cell viability was tested using the MTS cell proliferation and cytotoxicity assay kit produced by Promega. The brief steps were to add 20 μL of MTS to each well, incubate in an incubator in the dark for 1 hour, and read the absorbance at 490 nm using a microplate reader. Each treatment used cell culture medium 2 + test bacteria + MTS as a blank control. Complete medium 2 + cells + MTS was used as a negative control. The test results are shown in Figure 5.

[0176] From the results in Figure 5, we can see that when the bacterial concentration is not greater than 1×10 9 TFU / mL, the activity of 3T3-L1 preadipocytes was higher than 100%, so 1×10 9 TFU / mL, 1×10 8 TFU / mL and 1×107 TFU / mL was used to evaluate the cell lipid-lowering effect.

[0177] (5) 3T3-L1 preadipocyte differentiation

[0178] The cultured cells were washed with complete medium at 2 × 10 5 Cells were plated at a concentration of 1 mL / mL in a 24-well culture plate coated with 0.1% gelatin, and 3T3-L1 preadipocytes were allowed to grow and cultured until 100% confluence. After 3 days of fusion, it was recorded as day 0. Cell differentiation medium 1 was added and cultured for 4 days, which was recorded as day 4. Then, it was changed to cell differentiation medium 2 for induction and cultured for 2 days, which was recorded as day 6. After induction of differentiation, complete medium 2 was used for 2 days of culture, which was recorded as day 8. On day 8, 3T3-L1 preadipocytes differentiated into round mature adipocytes with round triglyceride particles generated inside. The culture medium was changed every 2 days during this period. Bacterial samples were added on day 0, and bacterial samples of different concentrations with cell viability higher than 100% were added and co-cultured with 3T3-L1 cells from day 0 to day 8 of induction of differentiation.

[0179] (6) Oil Red O staining

[0180] Wash the differentiated mature adipocytes 3 times with an appropriate amount of PBS, add 0.5mL 4% paraformaldehyde to fix for 30 minutes, and wash 3 times with ultrapure water; add 0.5mL Oil Red O working solution to each well, stain for 30 minutes in the dark at 37℃, discard the staining solution, and wash 3 times with ultrapure water; place the culture plate under an inverted microscope to observe the results and take pictures. After the photo is taken, add an equal amount of 1mL 100% isopropanol to each well to extract the Oil Red O stain in the lipid droplets, let it stand at room temperature for 20 minutes, aspirate the extracted staining solution and transfer it to a clean 96-well culture plate, and measure the absorbance at 500nm. Use 100% isopropanol as a blank and measure the absorbance at 500nm. The relative lipid content is calculated as follows: Relative lipid content (%) = (OD 样品 / OD 空白 ) × 100%. Each treatment was performed in triplicate, and data were analyzed using GraphPad Prism 9 software for one-way analysis of variance (ANOVA) and Dunnett's t-test (pairwise comparisons of means between multiple experimental groups and a control group). The results of oil staining and lipid content are shown in Figures 6 and 7.

[0181] After differentiation and maturation, 3T3-L1 cells accumulate a large number of lipid droplets in the cells. Oil Red O staining can intuitively reflect the amount of lipid accumulation after fat differentiation. As shown in Figure 6, 90% of the cells in the model group differentiated into mature adipocytes on the 8th day of induction. The cells were further enlarged, rounded, and covered with red lipid droplets inside the cells, densely packed around the nucleus, forming a "finger-like" structure, showing the typical morphology of mature fat. Compared with the model control group, after 8 days of induction and differentiation, the preadipocytes treated with heat-killed bacteria of Lactobacillus plantarum HOM2217 were able to significantly reduce the number of intracellular lipid droplets at low, medium, and high concentrations. The quantitative results of Oil Red O dye in adipocytes (Figure 7) showed that heat-killed bacteria of Lactobacillus plantarum HOM2217 strain could significantly (p < 0.01) reduce the intracellular lipid content, and there was a dose-dependent effect. As the concentration of heat-killed bacteria of Lactobacillus plantarum HOM2217 increased, its ability to inhibit the differentiation of 3T3-L1 preadipocytes increased, the intracellular lipid content decreased, and its fat-reducing effect was significantly better than that of the other two strains of Lactobacillus plantarum S1-6 and S2-13.

[0182] (6) Determination of triglyceride (TG) content in 3T3-L1 cells

[0183] The intracellular triglyceride content was determined according to the instructions of the triglyceride (TG) detection kit produced by Nanjing Jiancheng Bioengineering Research Institute. The brief steps are to add 2.5 μL of sample and 250 μL of working solution to each well, mix, incubate at 37°C for 10 minutes, and read the absorbance at 510 nm using a microplate reader. The protein concentration was determined using a total protein quantification kit (BCA method). The brief steps are to add 10 μL of sample and 250 μL of working solution to each well, mix, incubate at 37°C for 10 minutes, and read the absorbance at 562 nm using a microplate reader. The glyceride content per gram of protein was calculated according to the formula.

[0184] Calculation formula:

[0185] Each treatment was replicated in triplicate, and data were analyzed using GraphPad Prism 9 software using one-way analysis of variance (ANOVA) and Dunnett's t-tests (pairwise comparisons of means between multiple experimental groups and a control group). The triglyceride (TG) content in 3T3-L1 cells is shown in Figure 8. As shown in Figure 8, high, medium, and low heat-killed bacterial concentrations of Lactobacillus plantarum HOM2217 significantly (p < 0.01) reduced TG content in cells, with a dose-dependent effect. As the heat-killed bacterial concentration of Lactobacillus plantarum HOM2217 increased, TG content decreased, demonstrating a significantly better lipid-reducing effect than the other two Lactobacillus plantarum strains, S1-6 and S2-13.

[0186] Example 10 Preparation process of active bacterial powder of Lactobacillus plantarum HOM2217

[0187] (1) Culture of bacterial strains

[0188] The plant lactobacillus HOM2217 bacterial strain of-80 ℃ of freezing preservation prepared among the embodiment 1 was inoculated in aseptic MRS liquid nutrient medium with 1% inoculum size, cultivated 16~24 hours at 37 ℃, and the seed culture liquid after activation was obtained twice through passage culture like this. The seed culture liquid was inoculated in fermentation medium M321 with 3% inoculum size, and 35 ℃ of constant temperature aerobic cultivations were performed. In the fermentation process, the automatic flow addition of sodium hydroxide solution kept constant pH 5.0, causing fermentation to stop until acid production. When sodium hydroxide was no longer added, fermentation was stopped, and the plant lactobacillus HOM2217 high-density culture liquid was obtained, and the viable count could reach 28.6 billion CFU / mL. Table 7 has shown the formula of fermentation medium M321.

[0189] Table 7 Fermentation medium M321 formula

[0190] (2) Preparation of lyophilization protective agent

[0191] Sterile water was mixed with the protective agent raw materials to prepare a protective agent containing 100 g / L peptone, 20 g / L sucrose, 10 g / L vitamin C and 1 g / L glycerol.

[0192] (3) Freeze-drying

[0193] The fermented bacterial liquid of Lactobacillus plantarum HOM2217 after cultivation was centrifuged at 2-8°C and 6000 rpm for 10 min, the supernatant was discarded, the bacterial sludge was collected, and the bacterial sludge was washed 1-2 times with 0.9% sterile saline, and the washed bacterial sludge was mixed with the above protective agent to make the bacterial concentration of the mixed bacterial liquid reach 10 11 CFU / mL or more, freeze-dry in a freeze dryer, pre-freeze at -55°C for 5 hours, vacuum 0.05mbar, raise the temperature to -30°C, dry for 20 hours, then raise the temperature to -15°C and dry for 15 hours. After the temperature rises to 35°C, dry for a second time for 5 hours. After freeze-drying is completed, the cake is crushed with a fine grinder to obtain the freeze-dried powder. The number of viable bacteria in the freeze-dried powder reaches 6.57×10 11 CFU / g.

[0194] Example 11 Preparation process 2 of active bacterial powder of Lactobacillus plantarum HOM2217

[0195] (1) Culture of bacterial strains

[0196] The plant lactobacillus HOM2217 strain of-80 ℃ of cryopreservation prepared among the embodiment 1 was inoculated in sterile MRS liquid medium with an inoculum size of 3%, cultivated at 37 ℃ for 16-24 hours, and subcultured twice to obtain the seed culture liquid after activation. The seed culture liquid was inoculated in fermentation medium M327 with an inoculum size of 3%, and cultivated at 35 ℃ of constant temperature aerobic culture. During the fermentation process, sodium hydroxide solution was automatically added to maintain a constant pH of 6.0, causing fermentation to stop until acid production. Fermentation was terminated when sodium hydroxide was no longer added, obtaining a high-density culture liquid of the plant lactobacillus HOM2217 strain, with a viable count of up to 33.6 billion CFU / mL. Table 8 shows the formula of fermentation medium M327.

[0197] Table 8 Fermentation medium M327 formula

[0198] (2) Preparation of lyophilization protective agent

[0199] Sterile water was mixed with the protective agent raw materials to prepare a protective agent containing 50 g / L peptone, 60 g / L sucrose, 5 g / L vitamin C and 2 g / L glycerol.

[0200] (3) Freeze-drying

[0201] The fermented bacterial liquid of Lactobacillus plantarum HOM2217 strain after cultivation was centrifuged at 2-8°C and 6500 rpm for 15 min, the supernatant was discarded, the bacterial sludge was collected, and the bacterial sludge was washed 1-2 times with 0.9% sterile saline, and the washed bacterial sludge was mixed with the above protective agent to make the bacterial concentration of the mixed bacterial liquid reach 10 11 CFU / mL or above, freeze-dry in a freeze dryer, pre-freeze at -50°C for 8 hours, vacuum 0.03mbar, and after the temperature rises to -25°C, dry for 15 hours. Then, after the temperature rises to -10°C, dry for 10 hours. After the temperature rises to 32°C, dry for a second time for 8 hours. After freeze-drying is completed, the cake is crushed with a fine grinder to obtain the freeze-dried bacterial powder. The number of viable bacteria in the freeze-dried bacterial powder reaches 8.18×10 11 CFU / g.

[0202] Example 12 Preparation process of dead bacteria powder of Lactobacillus plantarum HOM2217 strain

[0203] (1) Culture of bacterial strains

[0204] The strain cultured in Example 11 was used.

[0205] (2) Preparation of bacterial powder

[0206] The fermentation broth of Lactobacillus plantarum HOM2217 strain was obtained by high-density fermentation, treated at 80°C for 30 minutes, and compounded with 20g of maltodextrin as an auxiliary material per liter of fermentation broth. The product was then dried in a spray dryer. The number of dead bacteria in the dead bacteria powder was 1.23×10 12 CFU / g.

[0207] Example 13 In vivo test of Lactobacillus plantarum HOM2217 strain in an obese animal model

[0208] (1) Experimental animals and feed

[0209] A total of 70 healthy SPF male SD rats bred by Beijing Huafukang Biotechnology Co., Ltd. were used as experimental animals.

[0210] The feeds used were high-fat feed D12492 (fat content 60%) and ordinary feed D12450B (fat content 10%) produced by Beijing Huafukang Biotechnology Co., Ltd.

[0211] (2) Animal models and experimental treatments

[0212] The animals were grouped after 1 week of observation and adaptation. After the adaptation period, 70 rats were randomly divided into 2 groups according to body weight, of which 10 rats were given maintenance feed as a negative control group, and 60 rats were given high-calorie model feed. After 2 weeks of feeding, the rats given high-calorie model feed were sorted according to weight gain, and the 1 / 3 obesity-resistant rats with lower weight gain were eliminated. 40 obesity-sensitive rats were screened and randomly divided into 4 groups according to body weight, namely the model control group and three dose groups, with 10 rats in each group. The experimental group used the plant lactobacillus HOM2217 powder prepared in Example 10 and was divided into three dose groups. The low-dose group (2.5×10 9 CFU / Kg BW, medium dose group (1.25×10 10 CFU / Kg BW) and the high-dose group (2.5×10 10 CFU / kg body weight). From the start of the experiment, each animal in the model control group and each dose group was given an equal amount of high-calorie model feed daily. The negative control group was similarly given an equal amount of maintenance feed. Each dose group was given different doses of the test substance, while the negative and model control groups were given sterile water. The experimental period was 7 weeks.

[0213] (3) Detection indicators and methods

[0214] A. Apparent indicators

[0215] During the experiment, the amount of food given and the remaining food in each cage were recorded weekly to calculate the food intake and total calorie intake (food intake × calories per kilogram of feed); the body weight of the rats was recorded weekly to calculate the weight gain and feed utilization rate;

[0216] Feed utilization rate = weight gain / food intake × 100%

[0217] B. Four serum tests

[0218] At the end of the experiment, after fasting for 12 hours, whole blood was obtained by eye sampling, and the blood was kept at room temperature for 2 hours. The blood was centrifuged at 4000 r / min for 10 minutes at 4°C to separate the serum. The serum was collected and the levels of total cholesterol, triglycerides, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol in the serum of each group of rats were determined using an automatic biochemical analyzer.

[0219] C. Cytokine Assay

[0220] At the end of the experiment, after fasting for 12 hours, whole blood was obtained by eye sampling, kept at room temperature for 2 hours, and centrifuged at 4000 r / min for 10 minutes at 4°C to separate the serum. The serum was collected and the levels of serum cytokines TNF-α and IL-6 were determined by enzyme-linked immunosorbent assay (ELISA) according to the kit instructions.

[0221] D. Determination of fat content

[0222] After blood was collected from the rats' eyeballs, they were killed by cervical dislocation. Body fat (testicular and perirenal fat pads) was collected and weighed, and the fat / body ratio was calculated.

[0223] E. Determination of lactate and short-chain fatty acids in cecal contents

[0224] After blood was collected from the rats' eyeballs, the rats were killed by cervical dislocation, and the cecal contents were collected. The contents of lactic acid and short-chain fatty acids in the cecal contents were detected by gas chromatography.

[0225] (4) Data processing

[0226] SPSS statistical software was used for variance analysis and Dunnett's method for pairwise comparison of means between multiple experimental groups and one control group. The independent sample t-test was used to compare the body weights of rats in the negative control group and the model control group before and after the establishment of the obesity model.

[0227] (5) Experimental results

[0228] The experimental results are shown in Figures 9-15.

[0229] As shown in Figure 9, after 14 days of administration of the high-calorie model diet, the model control group showed a significant increase in body weight compared to the negative control group, indicating that the model was successfully established. Before administration of the test substance, there was no significant difference in body weight between the model control group and the dose group (p>0.05). After 49 days of oral administration of the test substance, the model control group showed a significant increase in body weight compared to the negative control group (p<0.05). Compared to the model control group, the final body weight of rats in the high-dose Lactobacillus plantarum HOM2217 group was significantly reduced (p<0.05).

[0230] As shown in Figure 10, after administration of the high-calorie model diet, the total weight gain and total food utilization rate of the model control group increased significantly compared with the negative control group (p < 0.05). After oral administration of the test substance for 49 days, the high-dose Lactobacillus plantarum HOM2217 group significantly (p < 0.01) reduced the total weight gain and total food utilization rate of the rats compared with the model control group.

[0231] As shown in Figure 11, after administration of the high-calorie model diet, the body fat weight and fat / body ratio of the model control group increased significantly compared to the negative control group. After oral administration of the test substance for 49 days, 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 / body ratio of the rats compared to the model control group.

[0232] As shown in Figure 12, after administration of the high-calorie diet, the total food intake of the model control group was significantly lower than that of the negative control group (p<0.05); the total calorie intake of the rats was also significantly higher (p<0.05). After oral administration of the test substance for 49 days, there were no significant differences in the total food intake or total calorie intake between the rats in each dose group and the model control group (p>0.05).

[0233] As shown in the results of Figure 13, after the obese model rats were orally administered with the test substance for 49 days, the model control group compared with the negative control group, serum total cholesterol and serum triglycerides increased, serum high-density lipoprotein cholesterol decreased, and serum low-density lipoprotein cholesterol increased, with significant differences (p < 0.05). Compared with the model control group, there was no significant difference in the increase of serum total cholesterol, serum triglycerides and serum low-density lipoprotein cholesterol in each dosage group (p> 0.05). The medium dose of plant lactobacillus HOM2217 significantly (p < 0.05) and the high dose of plant lactobacillus HOM2217 extremely significantly (p < 0.01) increased the serum high-density lipoprotein cholesterol content of rats.

[0234] As shown in Figure 14, 49 days after oral administration of the test substance, serum TNF-α and IL-6 levels in obese rats were significantly elevated in the model control group 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 no significant differences were found (p>0.05). Lactobacillus plantarum HOM2217 significantly and extremely significantly (p<0.01) reduced TNF-α levels at the medium and high doses. Obesity is generally recognized as a chronic, low-grade inflammatory state. Inflammation plays a significant role in abnormal adipocyte hypertrophy and increased endoplasmic reticulum pressure in adipocytes. Macrophages in adipose tissue are considered a key source of inflammation, secreting the inflammatory cytokine tumor necrosis factor (TNF-α). TNF-α expression is significantly enhanced in adipocytes in obese animal models. Visceral fat accumulation significantly increases TNF-α release in adipose tissue. TNF-α expression is significantly increased in adipose tissue and muscle tissue in obese patients with insulin resistance (IR) and obese patients with type 2 diabetes. Therefore, it can be inferred that the Lactobacillus plantarum HOM2217 strain may achieve the purpose of weight loss by reducing the expression of TNF-α and reducing inflammation.

[0235] As shown in Figure 15, after 49 days of oral administration of the test substance to obese rats, the model control group showed a significant decrease in the levels of lactic acid, formic acid, acetic acid, propionic acid, and butyric acid in the cecum compared to the negative control group (p < 0.05). Compared to the model control group, the medium dose of Lactobacillus plantarum HOM2217 significantly (p < 0.05) increased the level of formic acid in the cecum, while the high dose of Lactobacillus plantarum HOM2217 significantly (p < 0.05) increased the level of lactic acid in the cecum and extremely significantly (p < 0.01) increased the levels of formic acid, acetic acid, and butyric acid in the cecum.

[0236] In summary, after 49 days of administration of Lactobacillus plantarum HOM2217 strain, compared with the model control group, the test substance in the medium dose of Lactobacillus plantarum HOM2217 strain can reduce the body fat weight (p<0.05) and fat / body ratio (p<0.05) of the obese rats, and increase serum high-density lipoprotein cholesterol (p<0.05); high dose of Lactobacillus plantarum HOM2217 strain can reduce the body weight of the obese rats (p<0.05), reduce the The total weight gain (p<0.01) and total food utilization rate (p<0.01) of rats were increased, body fat weight (p<0.01) and fat / body ratio (p<0.01) of obese rats were reduced, serum high-density lipoprotein cholesterol (p<0.01) was increased, serum cytokine TNF-α (p<0.01) levels were reduced, and cecaloric acid (p<0.05), formic acid (p<0.01), acetic acid (p<0.01), and butyric acid (p<0.01) levels were increased. There were no significant differences in total food intake and total caloric intake between the rats in each dose group and the model control group (p>0.05). Therefore, it can be seen that the Lactobacillus plantarum HOM2217 strain, without affecting food intake, produces short-chain fatty acids through metabolism, lowers intestinal pH, inhibits the reproduction of obese bacteria, and reduces TNF-α expression to reduce inflammatory responses, thereby achieving a weight-loss effect.

[0237] Example 14 In vivo test of Lactobacillus plantarum HOM2217 strain in an obese animal model 2

[0238] (1) Experimental animals and feed

[0239] 85 healthy SPF-grade C57BL / 6J (male, 4 weeks old, 15-17 g) mice bred by Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. were used as experimental animals.

[0240] The feeds used were high-fat feed D12492 (fat content 60%) and ordinary feed D12450B (fat content 10%) produced by Beijing Keao Xieli Feed Co., Ltd.

[0241] (2) Animal models and experimental treatments

[0242] The animals were acclimated to feeding and observed for one week before being grouped. After the acclimation period, 68 mice were randomly divided into two groups according to body weight. Eight mice were fed a maintenance diet as a negative control group, and 60 mice were fed a high-calorie model diet. After 14 days of feeding, the mice fed the high-calorie model diet were sorted by weight gain, and the 1 / 3 obesity-resistant mice with the lowest weight gain were eliminated. 40 obesity-sensitive mice were screened and randomly divided into five groups according to body weight: a model control group, a positive control group (orlistat, 0.4 mg / mouse / day), and a low-dose group of live HOM2217 (1×10 9CFU / each / day) (using the Lactobacillus plantarum HOM2217 powder prepared in Example 11), the HOM2217 live bacteria high-dose group (5×10 9 CFU / each / day) (using the Lactobacillus plantarum HOM2217 powder prepared in Example 11), HOM2217 dead bacteria group (5×10 9 TFU / animal / day) (using the Lactobacillus plantarum HOM2217 powder prepared in Example 12), with 8 animals per group. From the start of the experiment, each animal in the model control group and each dose 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. Each dose group was given different doses of the test substance, while the negative control group and the model control group were given sterile water. The experimental period was 11 weeks.

[0243] (3) Detection indicators and methods

[0244] During the experiment, the amount of food given and the amount of food left in each cage were recorded weekly, and the daily food intake and caloric intake of each mouse were calculated. The weight of the mice was recorded weekly, and the weight gain and feed utilization rate were calculated.

[0245] Feed utilization rate = weight gain / food intake × 100%

[0246] (4) Data processing

[0247] SPSS statistical software was used for variance analysis and Dunnett's method for pairwise comparison of means between multiple experimental groups and one control group. The independent sample t-test was used to compare the body weights of rats in the negative control group and the model control group before and after the establishment of the obesity model.

[0248] (5) Experimental results

[0249] The experimental results are shown in Figures 16-18.

[0250] As shown in Figure 16, after two weeks of administration of the high-calorie model diet, the model control group showed a significant increase in body weight compared to the negative control group (p < 0.01), indicating successful model establishment. Before administration of the test substance, there were no significant differences in body weight between the model control group and the dose group. After 11 weeks of oral administration of the test substance, the model control group showed a significant increase in body weight compared to the negative control group (p < 0.01). Compared with the model control group (average weight 33.9g), the low-dose live Lactobacillus plantarum HOM2217 group (average weight 29.8g), the high-dose live Lactobacillus plantarum HOM2217 group (average weight 29.1g), the killed Lactobacillus plantarum HOM2217 group (average weight 30.4g), and the positive control group (orlistat) (average weight 29.2g) all significantly (p < 0.01) reduced the final body weight of the mice, with weight reduction rates of 12.1%, 14.2%, 10.3%, and 13.9%, respectively. The weight reduction rates of the low-dose live Lactobacillus plantarum HOM2217, high-dose live Lactobacillus plantarum HOM2217, and killed Lactobacillus plantarum HOM2217 groups were comparable to those of the positive control group (orlistat), with no significant differences (p > 0.05).

[0251] As shown in the results of Figure 16, after 11 weeks of high-calorie model feed administration, the model control group showed a significant increase in weight compared to the negative control group (p < 0.01), indicating that the model was successfully established. After 11 weeks of oral administration of the test substance, the model control group showed a significant increase in weight compared to the negative control group (p < 0.05). Compared with the model control group (average weight 577g), the high dose of plantarum Lactobacillus HOM2217 (average weight 535.4g) significantly (p < 0.05) reduced the final weight of the mice, with a weight loss rate of 7.2%.

[0252] As shown in the results of Figure 17, after being given a high-calorie model feed, the total weight gain and total food utilization rate of the model control group increased compared with the negative control group, with a very significant difference (p < 0.01). After 11 weeks of oral administration of the test substance, compared with the model control group, the low-dose group of live bacteria HOM2217, the high-dose group of live bacteria, the killed bacteria group and the positive control group (orlistat) group were all able to significantly (p < 0.01) reduce the total weight gain of the mice. Compared with the model control group, the high-dose group of live bacteria HOM2217 and the positive control group (orlistat) group were able to significantly (p < 0.05) reduce the total food utilization rate of the 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).

[0253] As shown in Figure 18, after administration of a high-calorie diet, the total food intake of the model control group was significantly lower than that of the negative control group (p<0.05). After 11 weeks of oral administration of the test substance, there were no significant differences in the total food intake or total calorie intake of the mice in the test groups compared to the model control group (p>0.05).

[0254] In summary, after giving plant lactobacillus HOM2217 bacterial strain 11 weeks, compared with the model control group, plant lactobacillus HOM2217 viable bacteria low-dose group, viable bacteria high-dose group and dead bacteria group can all significantly reduce obesity model mice body weight (p < 0.01), reduce the total weight gain of obesity model mice (p < 0.01), and the weight loss effect is suitable with the positive control group (orlistat). Each dosage group mouse total food intake, total calorie intake and the model control group were compared without significant difference (p > 0.05). Therefore, it can be known that plant lactobacillus HOM2217 bacterial strain has the effect of weight loss when not affecting the food intake.

[0255] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the details shown and described herein.

Claims

1. A plant lactobacillus (Lactiplantibacillus plantarum) strain, wherein the plant lactobacillus strain is deposited in the China General Microbiological Culture Collection Center (CGMCC) with a deposit number of CGMCC No. 25683.

2. The plant lactobacillus strain according to claim 1, wherein The Lactobacillus plantarum strain comprises the 16S rRNA gene represented by SEQ ID NO:

1.

3. The plant lactobacillus strain according to claim 1, wherein The plant lactobacillus strain is isolated from healthy human breast milk. 4 . A probiotic preparation, which is a live bacteria preparation or a dead bacteria preparation, and comprises the Lactiplantibacillus plantarum strain according to claim 1 .

5. The probiotic preparation according to claim 4, wherein The probiotic preparation is a live bacteria preparation, and the live bacteria content is 6 to 9×10 11 CFU / g.

6. The probiotic preparation according to claim 4, wherein The probiotic preparation is a dead bacteria preparation and contains 7 to 13×10 11 CFU / g of dead bacteria.

7. A method for preparing a probiotic preparation according to any one of claims 4 to 6, comprising the following steps: (1) strain culture, inoculating the plant lactobacillus strain in a sterile liquid culture medium at an inoculum amount of 1 to 3% of the total amount of the culture medium, culturing for 16 to 24 hours to obtain a seed culture solution, and then inoculating the obtained seed culture solution in a fermentation medium at an inoculum amount of 1 to 3% of the total amount of the culture medium, performing fermentation culture, and obtaining a plant lactobacillus high-density culture solution; (2) Drying.

8. The method according to claim 7, wherein: The fermentation medium used contains: 60-100 g / L glucose, 60-80 g / L yeast extract, 0.2-0.4 g / L magnesium sulfate, 0.1-0.2 g / L manganese sulfate, 2-4 g / L dipotassium hydrogen phosphate, 2-4 g / L potassium dihydrogen phosphate, 1-2 g / L Tween 80, and 0.5-2 g / L L-cysteine ​​salt.

9. The method according to claim 7, wherein: During the fermentation process, sodium hydroxide solution is automatically added to maintain a constant pH of 5.0-6.0 until the fermentation stops producing acid, and the fermentation is terminated when sodium hydroxide is no longer added.

10. The method according to any one of claims 7 to 9, wherein: The probiotic preparation is a live bacteria preparation, and the method further comprises preparing a lyophilization protectant after the strain culture step and before drying, and freeze-drying is performed in the drying step; optionally, the lyophilization protectant used comprises: a protectant of 50-100 g / L peptone, 20-60 g / L sucrose, 5-10 g / L vitamin C and 1-3 g / L glycerol.

11. The method according to claim 10, wherein: The freeze-drying conditions are as follows: pre-freezing temperature is -50 to -55°C, pre-freezing time is 5 to 8 hours, primary drying temperature is -10 to -30°C, primary drying time is 25 to 35 hours, secondary drying temperature is 32 to 35°C, and secondary drying time is 5 to 8 hours.

12. The method according to claims 7 to 9, wherein: The probiotic preparation is a dead bacteria preparation, and the drying step is to first perform heat inactivation and then dry it in a spray drying tower to obtain the dead bacteria preparation; optionally, the heat inactivation condition is 70-100° C. for 10-40 minutes.

13. Use of the Lactobacillus plantarum strain according to any one of claims 1 to 3 for producing lactic acid and short-chain fatty acids.

14. The use according to claim 13, wherein The short-chain fatty acid is selected from one or more of formic acid, acetic acid, propionic acid, butyric acid and isobutyric acid.

15. Use of the Lactobacillus plantarum strain according to any one of claims 1 to 3 in the preparation of a composition for inhibiting the obesity strain Enterobacter cloacae.

16. Use of the Lactobacillus plantarum strain according to any one of claims 1 to 3 in the preparation of a composition for inhibiting the differentiation of 3T3-L1 preadipocytes, thereby effectively inhibiting fat deposition.

17. Use of the Lactobacillus plantarum strain according to any one of claims 1 to 3 in the preparation of a pharmaceutical composition for reducing weight or lowering hyperlipidemia and hypercholesterolemia caused by obesity.

18. The use according to claim 17, wherein The weight loss function is to reduce the cholesterol content, or inhibit the differentiation of 3T3-L1 preadipocytes to inhibit the formation of fat.

19. The use according to claim 17, wherein The weight loss function is selected from one or more of reducing the body weight, total weight gain and total food utilization rate of obese model rats, reducing the body fat weight and fat / body ratio of obese model rats, increasing serum high-density lipoprotein cholesterol content, increasing intestinal short-chain fatty acid content, and reducing the expression of cellular inflammatory factor TNF-α in serum.

20. The use according to any one of claims 17 to 19, wherein The weight loss function is selected from one or more of reducing cholesterol content, inhibiting differentiation of 3T3-L1 preadipocytes, reducing body weight of obese model mice, and reducing total weight gain and total food utilization of obese model rats.

Images