Anti-obesity Leuconostoc mesenteroides and application thereof

Abstract

The invention discloses an anti-obesity Leuconostoc mesenteroides strain and an application thereof. According to the present invention, a Leuconostoc pseudomesenteroides CU270 strain capable of reducing cholesterol content and triglyceride content is separated and identified from fermented pickled vegetables, the strain is preserved in Guangdong Microbial Culture Collection Center on November 4, 2024, and the preservation number is GDMCC NO.65413. The invention further discloses a preparation method of the Leuconostoc pseudomesenteroides CU270 strain capable of reducing cholesterol content and triglyceride content. Researches show that the CU270 strain has strong acid resistance, cholate resistance and oxidation resistance, and can significantly reduce the content of cholesterol and triglyceride. An obese animal model research finds that the strain can relieve obesity, reduce blood fat and blood sugar levels, improve blood fat rise caused by high-fat diet and relieve liver cell injury caused by high-fat diet, has no toxic or side effect, and provides a new thought for treatment of anti-obesity diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine. More specifically, it relates to a Leuconostoc pseudomesenteroides strain with anti-obesity effect and its applications. Background Art

[0002] The global prevalence of obesity has increased significantly in the past few decades and has become a global public health problem. Obesity is a complex metabolic disorder and belongs to chronic metabolic diseases, which is characterized by excessive accumulation of body fat to the extent that it has a negative impact on health. Usually, obesity often manifests as hyperlipidemia, with elevated levels of total cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C) in serum (plasma), and a decreased level of high-density lipoprotein cholesterol (HDL-C). Obesity not only affects psychology and physiology but also triggers diseases such as diabetes, respiratory diseases, osteoarticular diseases, fatty liver, hyperuricemia, gout, and venous thrombosis. These diseases not only affect the quality of life of patients but may also endanger life.

[0003] Treatment methods for obesity include dietary and lifestyle interventions, drug treatment, and bariatric surgery. However, most weight-loss drugs have adverse reactions on the gastrointestinal tract, nerves, and cardiovascular system, and surgical operations also have certain risks. Therefore, finding safer and more effective treatment methods is the focus of current research.

[0004] As a potential treatment means, probiotics have received extensive attention in recent years. Existing studies have disclosed that probiotics such as Leuconostoc mesenteroides, Lactobacillus acidophilus, Lactobacillus plantarum, Bifidobacterium lactis, Lactobacillus rhamnosus, and Bifidobacterium sp. have lipid-lowering effects. However, there has been no report on other microbial strains that can be used to relieve obesity and simultaneously have the functions of reducing cholesterol, reducing triglyceride, and inhibiting fat accumulation. Therefore, in order to relieve obesity and reduce the diseases caused by obesity, it is necessary to develop more microbial strains or products with lipid-lowering and anti-obesity effects to expand the probiotic resource library. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortage of existing microbial strain resources that can be used to relieve obesity and simultaneously have the functions of reducing cholesterol, reducing triglyceride, and inhibiting fat accumulation, and to provide a Leuconostoc pseudomesenteroides strain with anti-obesity effect and its applications.

[0006] The first object of the present invention is to provide a strain of Leuconostoc pseudomesenteroides CU270.

[0007] The second object of the present invention is to provide a culture.

[0008] The third object of the present invention is to provide a microbial inoculum.

[0009] The fourth object of the present invention is to provide the application of the CU270 strain, or the culture, or the microbial inoculum.

[0010] The fifth object of the present invention is to provide a product.

[0011] The sixth object of the present invention is to provide a method for reducing cholesterol and triglyceride levels for non-disease treatment purposes.

[0012] The above objects of the present invention are achieved by the following technical solutions:

[0013] The present invention provides a strain of Leuconostoc pseudomesenteroides CU270, which was deposited at the Guangdong Provincial Microbial Culture Collection Center on November 4, 2024, with the deposit number GDMCC NO. 65413.

[0014] The present invention isolated and identified a strain of Leuconostoc pseudomesenteroides CU270 with cholesterol and triglyceride-lowering effects from fermented pickles. This strain is a Gram-positive bacterium, aerobic, short rod-shaped, and without spores. The colony morphology is white and convex, with a smooth surface. The CU270 strain has strong acid and bile salt tolerance, with a survival rate of 82.83% after 8 hours in MRS medium at pH = 2 and a survival rate of 83.78% after 8 hours in MRS medium with a bile salt concentration of 0.3%, and can well adapt to the gastric acid environment in vivo.

[0015] Research shows that the degradation rate of cholesterol by this strain reaches 52.80% within 24 hours in vitro, and the degradation rate of triglyceride reaches 54.94%. At the same time, the CU270 strain also has antioxidant ability, with a DPPH free radical scavenging rate of 69.23% and an ABTS + free radical scavenging rate of 40.18%. And in the study of an obese animal model, it was found that the CU270 strain can relieve obesity, reduce blood lipid and blood glucose levels, can improve the blood lipid elevation caused by a high-fat diet, and relieve the liver cell damage caused by a high-fat diet, and has no any toxic and side effects, and has the biological function of reducing fat, providing a new idea for the preparation of more anti-obesity disease products.

[0016] The present invention provides a culture, and the preparation method of the culture is as follows: inoculate strain CU270 in MRS medium and culture at 30-35 °C for 24-36 h to obtain it.

[0017] The present invention provides a microbial inoculum containing strain CU270 or its bacterial solution.

[0018] Preferably, the viable bacteria count in the inoculum is not less than 1×10 8 CFU / mL.

[0019] Furthermore, the microbial inoculum also contains a protective agent and prebiotics, and the protective agent and prebiotics contained include skim milk powder, trehalose, fructooligosaccharide gelatin, and whey protein powder.

[0020] The present invention provides the application of the above-mentioned strain CU270, or culture, or microbial inoculum in the preparation of a product for relieving obesity.

[0021] The present invention provides the application of the above-mentioned strain CU270, or culture, or microbial inoculum in the preparation of a product for reducing blood lipid levels.

[0022] The present invention provides the application of the above-mentioned strain CU270, or culture, or microbial inoculum in the preparation of an antioxidant product.

[0023] The present invention provides the application of the above-mentioned strain CU270, or culture, or microbial inoculum in the preparation of a product for relieving liver injury caused by a high-fat diet.

[0024] The present invention also provides a product containing the above-mentioned strain CU270, or culture, or microbial inoculum.

[0025] In addition, the present invention also provides a method for reducing cholesterol and triglyceride contents for non-disease treatment purposes, which is to carry out treatment with strain CU270, or culture, or microbial inoculum.

[0026] The present invention has the following beneficial effects:

[0027] The present invention has obtained a strain of Leuconostoc pseudomesenteroides CU270, which has good acid resistance, bile salt resistance, and antioxidant capacity, can effectively degrade cholesterol and triglyceride contents, can relieve obesity, reduce blood lipid and blood sugar levels, can also relieve liver cell injury caused by a high-fat diet, has no any toxic and side effects, has high safety, has a good anti-obesity effect, and can be used to prepare more products for relieving obesity. Description of the Drawings

[0028] Figure 1 It is a morphological diagram of the colony.

[0029] Figure 2It is the result diagram of Gram staining.

[0030] Figure 3 It is the phylogenetic tree of CU270.

[0031] Figure 4 It is the result diagram of the acid tolerance ability of CU270.

[0032] Figure 5 It is the result diagram of the bile salt tolerance ability of CU270.

[0033] Figure 6 It is the result diagram of the antioxidant ability of CU270.

[0034] Figure 7 It is the result diagram of the ability of CU270 to degrade TG and TC in vitro.

[0035] Figure 8 It is the result diagram of the change in the body weight of mice under the intervention of CU270 (*P<0.01, ***P<0.001, ****P<0.0001 vs. the model group, n = 6 in the figure).

[0036] Figure 9 It is the result diagram of the change in oral glucose tolerance of mice under the intervention of CU270.

[0037] Figure 10 It is the result diagram of the change in blood lipids of mice under the intervention of CU270. Specific implementation manners

[0038] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0039] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0040] The medium formula and preparation method used in the examples are as follows:

[0041] The formula and conditions of the MRS solid medium are: peptone 10.0 g, beef extract powder 8.0 g, glucose 20.0 g, anhydrous sodium acetate 5.0 g, yeast extract 4.0 g, ammonium citrate tribasic 2.0 g, dipotassium hydrogen phosphate 2.0 g, magnesium sulfate 0.2 g, manganese sulfate monohydrate 0.04 g, Tween 80 1 g. Add 1 L of distilled water, mix well until the solute is completely dissolved, and then sterilize at 121 °C for 20 min under high pressure.

[0042] Preparation of MRS-cholesterol (MRS-CHOL) medium: (1) Weigh 1.5 g of 95% cholesterol and put it into a test tube. Add 2.0 g of bile salts, 1.0 g of sucrose fatty acid ester, and 1.0 ml of Tween-80. Pipette 10 mL of absolute ethanol into the test tube, stopper the test tube, and place the test tube in a constant temperature water bath at 60 °C for 8 min. Then use a vortex mixer to vortex for 1.5 min to mix evenly and obtain a crude mixture; (2) Place the obtained crude mixture in water at a temperature of 60 °C and treat it with ultrasonic waves with a power of 80 W for 25 min in hot water. Vortex for 1 min every 6 min to mix. After the solution becomes clear and transparent, filter it through a microporous membrane with a pore size of 0.45 μm to remove bacteria and other microorganisms to obtain a cholesterol micelle solution; (3) While it is hot, add the obtained cholesterol micelle solution to 100 mL of MRS liquid medium that has been sterilized at high temperature so that the initial theoretical cholesterol concentration is 0.20 mg / mL. Stir with a magnetic stirrer at a rotation speed of 800 rpm for 10 min to uniformly disperse the cholesterol micelles in the fermentation medium system. Let it stand for more than 72 h without precipitation to obtain a culture medium solution containing cholesterol micelles.

[0043] Preparation of MRS liquid medium containing triglyceride: Mix an aqueous solution of 2% polyvinyl alcohol and vegetable oil at a volume ratio of 3:1, and after ultrasonic treatment (control parameters: ultrasonic for 5 s each time, interval time 5 s, total ultrasonic for 12 min), mix evenly to prepare a vegetable oil emulsion, which is used as the source of triglyceride. Add the prepared vegetable oil emulsion to the MRS liquid medium at a ratio of 5%, stir continuously for 20 min, adjust the pH to 6.5 ± 0.2, sterilize at 115 °C for 30 min, prepare a triglyceride medium and store it in a 4 °C refrigerator for later use.

[0044] Example 1 Screening and identification of Leuconostoc pseudomesenteroides strain CU270

[0045] 1. Isolation and purification of the strain

[0046] Select a pickled vegetable sample that has been fermented for 30 days, dilute it 10-fold serially three times with normal saline with a mass concentration of 0.9%, and then spread it on an MRS solid medium. After culturing at 37 °C for 48 h, pick out single colonies suspected to be the target strain, streak and purify them on the surface of the MRS solid medium. After streak purification for 2 generations and confirmed to be a pure strain by microscopic examination, then preserve it with glycerol with a mass concentration of 30%.

[0047] 2. Strain identification

[0048] After observing the bacterial morphology and the cell morphology after Gram staining of the strain (named CU270) obtained by isolation and purification, the cell morphology of the strain is short rod-shaped, without spores, the colony morphology is white convex, the surface is smooth, and it is aerobic. AsFigure 1 As shown; the result of Gram staining showed that it was Gram-positive, as Figure 2 shown.

[0049] 16S rRNA molecular identification: Take out the strain preserved at -80 °C, inoculate it into a centrifuge tube containing 5 mL of MRS liquid medium at a ratio of 2%, culture it at 37 °C for 24 h, then perform centrifugal separation, centrifuge at 5000 rpm for 10 min, discard the supernatant, and collect the cell suspension. Extract the genome of the strain, add universal bacterial primers for PCR amplification, and send the amplified product to Sangon Biotech (Shanghai) Co., Ltd. for sequencing and identification.

[0050] The sequencing result showed that the spliced gene sequence of strain CU270 was as shown in SEQ ID NO.1. Submit the sequenced gene sequence to the database for comparative analysis. The result was as Figure 3 shown, indicating that strain CU270 had a high homology with Leuconostoc pseudomesenteroides. Based on the above results of strain morphological characteristics and molecular biological identification, the taxonomic status of strain CU270 was identified as Leuconostoc pseudomesenteroides, and the strain was named strain CU270. It was deposited in the Guangdong Provincial Microbial Culture Collection Center on November 4, 2024, with the deposit number GDMCC NO.65413, and the deposit address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou.

[0051] Example 2 Evaluation of the probiotic function of Leuconostoc pseudomesenteroides CU270

[0052] 1. Evaluation of the acid tolerance ability of the strain:

[0053] Centrifuge the strain activated for two generations at 4 °C and 5000 r / min for 10 min, then discard the supernatant, collect the cells and wash them twice with PBS (pH 6.8) buffer. Adjust the cell concentration to 10 8 CFU / mL (OD = 2 or more) of cell suspension with PBS solution. Inoculate the cell suspension into 5 mL of MRS liquid medium with pH 2.0, pH 3.0, and pH 4.0 at an inoculation amount of 2% respectively, and culture it statically at 37 °C. Use the inoculation in ordinary MRS liquid medium as the control group, and the inoculation in MRS liquid medium with different pH values as the experimental group. Take 100 μL of the cultured bacterial liquid at 0 h, 2 h, 4 h, 6 h, and 8 h respectively, dilute the bacterial liquid to an appropriate gradient with sterile normal saline using the ten-fold serial dilution method, spot plate on the MRS plate, and perform viable count after culturing at 37 °C for 24 h. Substitute the viable count into the following formula to calculate the survival rate of the strain at different pH concentrations.

[0054] Survival rate (%) = (logN1 / logN0) × 100%

[0055] Wherein, N1 is the number of viable bacteria cultured in the experimental group; N0 is the number of viable bacteria cultured in the control group.

[0056] The results are as Figure 4 shown, indicating that strain CU270 has good acid tolerance and can survive in environments with pH = 2, pH = 3, and pH = 4. The survival rate after 8 h in the environment with pH = 2 can also reach 82.83%.

[0057] 2. Evaluation of the bile salt tolerance of the strain:

[0058] The strain activated for two generations was centrifuged at 4°C and 5000 r / min for 10 min, and then the supernatant was discarded. The bacterial cells were collected and washed twice with PBS (pH 6.8) buffer. Adjust the bacterial cell concentration to 10 8 CFU / mL (OD = 2 or above) bacterial suspension with PBS solution. The bacterial suspension was inoculated into MRS liquid medium containing 0.1% (w / v), 0.2% (w / v), 0.3% (w / v) bile salts (porcine bile salts) at an inoculation amount of 2% respectively, and cultured statically at 37°C. Inoculation in ordinary MRS liquid medium was used as the control group, and inoculation in MRS liquid medium containing different bile salts was used as the experimental group. 100 μL of the cultured bacterial liquid was taken at 0 h, 2 h, 4 h, 6 h, and 8 h respectively, and the bacterial liquid was diluted to an appropriate gradient with sterile normal saline by the ten-fold serial dilution method, spotted on an MRS plate, and cultured at 37°C for 24 h, and then the number of viable bacteria was counted. The number of viable bacteria was substituted into the following formula to calculate the survival rate of the strain at different bile salt concentrations.

[0059] Survival rate (%) = (logN1 / logN0) × 100%

[0060] Wherein, N1 is the number of viable bacteria cultured in the experimental group; N0 is the number of viable bacteria cultured in the control group.

[0061] The results are as Figure 5 shown, indicating that strain CU270 has good bile salt tolerance and can survive in bile salt environments with concentrations of 0.1%, 0.2%, and 0.3%. The survival rate after 8 h of tolerance in the environment with a bile salt concentration of 0.3% can reach 83.78%.

[0062] 3. Evaluation of the antioxidant capacity of the strain:

[0063] (1) DPPH free radical scavenging ability

[0064] Determination of the scavenging ability of 1,1-diphenyl-2-picrylhydrazyl (DPPH): Refer to the method of Ayyanna et al. (2018). After the strain was activated, the bacterial solution was centrifuged at 8000 rpm for 8 min at 4 °C, and the cells were washed with PBS buffer to prepare a bacterial suspension with a concentration of 1×10 8 CFU / mL. Equal amounts of the test solution and an ethanol solution of 0.2 mmol / L DPPH were taken, mixed well, and reacted in the dark at room temperature for 30 min. The absorbance was measured at 517 nm. Ethanol was used instead of DPPH as the control group, and ethanol was used instead of the sample in the blank group. The DPPH radical scavenging rate was calculated according to the following formula:

[0065]

[0066] In the formula: X—the scavenging rate;

[0067] A0—the absorbance value of the blank group;

[0068] A1—the absorbance value of the sample group;

[0069] A2—the absorbance value of the control group.

[0070] The measurement results are as Figure 6 shown, indicating that strain CU270 has the ability to scavenge DPPH radicals, and its ability to scavenge DPPH radicals reaches 69.23%.

[0071] (2) ABTS + Free radical scavenging ability

[0072] Equal amounts of 7.4 mmol / L ABTS and 2.6 mmol / L potassium persulfate solution were mixed and allowed to stand overnight in the dark at room temperature. The absorbance of the reacted ABTS solution at 734 nm was adjusted to 0.7±0.02. Equal amounts of the test bacterial suspension and ABTS solution were taken and reacted in the dark for 6 min. In the blank group, ethanol was used instead of the sample bacterial suspension, and in the control group, ethanol was used instead of the ABTS solution. The absorbance was measured at 734 nm. The scavenging rate of ABTS+ radicals was calculated according to the following formula:

[0073]

[0074] In the formula: X—the scavenging rate;

[0075] A0—the absorbance value of the blank group;

[0076] A1—the absorbance value of the sample group;

[0077] A2—Absorbance value of the control group.

[0078] The measurement results are as Figure 6 shown, indicating that the ability of strain CU270 to scavenge ABTS + free radicals reaches 40.18%, showing certain antioxidant effects.

[0079] 4. Evaluation of the cholesterol-degrading ability of the strain:

[0080] Determination of cholesterol content by ammonium ferric sulfate method: (1) Ammonium ferric sulfate color reagent: Dissolve 4.463 g of ammonium ferric sulfate in 100 mL of 85% phosphoric acid to obtain the stock solution. Pipette 10 mL of this stock solution and make up to 100 mL with concentrated sulfuric acid to obtain the color reagent; (2) Transfer 2 mL of the supernatant of the culture medium after centrifugation (5000 g, 10 min) to a clean test tube, add anhydrous ethanol to 10 mL, shake well, and centrifuge at 4500 g for 10 min; (3) Take 1 mL of the supernatant to a new clean test tube, then add 2 mL of ammonium ferric sulfate color reagent, mix well, and measure the absorbance at 560 nm after cooling.

[0081] Inoculate the prepared MRS-CHOL culture medium with the test strain at an inoculation amount of 2% and culture for 24 h. Use the MRS-CHOL culture medium without inoculation as a control to measure the total cholesterol content. The lower the cholesterol content detected in the supernatant, the greater the cholesterol reduction efficiency and the stronger the cholesterol-lowering ability of the strain.

[0082] Cholesterol reduction rate % = (A - B) / A × 100%

[0083] Where: A is the cholesterol content in the MRS-CHOL culture medium without inoculated lactic acid bacteria; B is the supernatant after fermentation of the test strain.

[0084] The results are as Figure 7 shown, indicating that strain CU270 has the effect of reducing cholesterol, and its ability to scavenge and degrade cholesterol reaches 52.80%.

[0085] 5. Evaluation of the triglyceride-degrading ability of the strain:

[0086] Triglyceride content (TG) determination test (single reagent GPO-PAP method): Take 1 mL each of the cultured bacterial solution and the triglyceride medium without inoculated bacteria, and centrifuge at 4000 r / min for 10 min. Take the supernatant and operate according to the instructions of the triglyceride (TG) test kit (Nanjing Jiancheng Bio- reagent Company A110-1-1). The reagents added to each well and their dosages are shown in Table 1; after shaking the well plate to mix evenly, incubate at 37 °C for 10 minutes, and measure the absorbance value of each well with an enzyme- linked immunosorbent assay (ELISA) reader at a wavelength of 500 nm, and calculate the TG content and degradation rate according to the following formula.

[0087] Table 1 Reagents added to each well

[0088] Sample addition (μL) Blank well Standard well Sample well Distilled water 2.5 Calibrator 2.5 Sample 2.5 Working solution 250 250 250

[0089] TG content (mmol / L) = (sample well - blank well A) / (standard well A - blank well A) * C standard

[0090] TG degradation rate (%) = (total TG content - residual TG content) / total TG content * 100%

[0091] In the formula: the total TG content is the triglyceride (TG) content in the triglyceride medium without inoculated bacteria; the residual TG content is the triglyceride (TG) content in the triglyceride medium after culturing the inoculated bacteria for 24 h.

[0092] The measurement results are as Figure 7 shown, indicating that strain CU270 has the effect of reducing triglycerides, and its ability to remove and degrade triglycerides reaches 54.94%.

[0093] Example 3 Application of strain CU270 in alleviating obesity

[0094] 1. Experimental animals

[0095] Select 24 SPF-grade male C57BL / 6 mice (purchased from the Animal Experiment Center of Southern Medical University), 4 weeks old, with a body weight of 17.5 ± 1.4 g, and adaptively feed them with ordinary feed in the animal house (room temperature 22 - 25 °C, humidity: 40 - 60%, lighting 10 ± 0.5 h) for one week (free diet and water).

[0096] 2. Experimental method

[0097] After adaptive feeding, the mice were randomly divided into 3 groups: a control group (Control), a model group (Model), and a CU270 group, with 8 mice in each group. The control group was given a control diet (D12450J), and the other groups were given a high-fat diet (PD6001) for feeding (both diets were purchased from Changzhou Mouse One Mouse Two Biotechnology Co., Ltd. The composition differences between D12450J and PD6001 are shown in Table 2). Water and bedding were changed twice a week, and the high-fat diet was changed daily to prevent the smell caused by fat oxidation from affecting the mice's food intake.

[0098] Table 2 Composition differences between D12450J and PD6001

[0099]

[0100] Mice in the control group and the model group were intragastrically administered 0.2 mL of sterile normal saline every day for 12 weeks. Mice in the CU270 group were intragastrically administered 0.2 mL of Leuconostoc pseudomesenteroides CU270 bacterial solution at a concentration of 1×10 10 CFU / mL for 12 weeks. During the 12 weeks of intragastric administration of the mice, the food intake and body weight changes of the mice were recorded weekly, and the epididymal and inguinal fat tissues of the mice in each group were taken out for weighing to calculate the fat coefficient. The fat coefficient reflects the degree of obesity of the human body or animals. The larger the value, the higher the degree of obesity of the individual. The specific calculation formula is:

[0101] Fat coefficient (%) = (inguinal fat weight + epididymal fat weight) / mouse body weight × 100%

[0102] 3. Test results

[0103] The statistical results of the weekly changes in the body weight of the mice in each group are shown in Table 3 and Figure 8 as shown. The weight gain and fat coefficient results of the mice in each group after 12 weeks are shown in Table 4. It can be seen that as time goes by, the body weight of the mice in the model group increases the most, significantly higher than that of the other groups, and its fat coefficient is also higher, indicating that the mice are the most obese, which is basically consistent with the body weight increment of the mice during the test period; while the weight increment of the CU270 intervention group is significantly different from that of the model group. Although the CU270 intervention group was also fed a high-fat diet, the fat coefficient of the mice in the CU270 intervention group was significantly lower than that of the model group. It can be seen that the CU270 strain has the effect of alleviating obesity.

[0104] Table 3 Weekly changes in the body weight of mice

[0105]

[0106]

[0107] Table 4 Weight gain and fat coefficient of each group of mice after 12 weeks

[0108] Blank group Model group CU270 Body weight gain / g 9.04±0.76 14.32±0.05 9.47±1.33 Fat coefficient % 1.87±0.31 4.91±1.32 3.58±0.69

[0109] Example 4: Effect of CU270 Strain on Oral Glucose Tolerance Level in Obese Mice

[0110] After 12 weeks of gavage in each group of mice in Example 3, an oral glucose tolerance test was performed on each group of mice 1 day before sacrifice. After fasting for 12 h without water deprivation, about 1 mm - 2 mm of the end of the mouse's tail was cut off with scissors, and the mouse's tail was gently squeezed along the tail vein. The first drop of blood was discarded, and the second drop of blood was used to measure the fasting blood glucose with a blood glucose meter and blood glucose test strips. The measured value was taken as the blood glucose value at 0 min. Each mouse was gavaged with a 30% glucose solution at a dose of 2 g / kg based on body weight, and the blood glucose of the mice was measured at 15 min, 30 min, 60 min, 90 min, and 120 min after gavage.

[0111] The measurement results are as Figure 9 shown. It shows that the blood glucose of the mice in the model group increased the fastest and decreased slowly after gavage with glucose, and the blood glucose of the mice in the model group was still significantly higher than that of the normal group and the CU270 group after 90 min. Under a high-fat diet, the CU270 group could reduce blood glucose and played a certain protective role in the blood glucose regulation ability of mice.

[0112] Example 5: Effect of CU270 Strain on Blood Lipid Level in Obese Mice

[0113] After 12 weeks of gavage in each group of mice in Example 3, whole blood of the mice was obtained by eye socket blood sampling, left standing at 20 °C for 2 h, centrifuged at 4 °C, 3000 r / min for 10 min, and the upper serum was taken to detect the concentrations of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) in the mouse serum. And a four-item blood lipid kit (Nanjing Jiancheng Bioengineering Institute) was used for determination.

[0114] The results in Table 5 and Figure 10 shown show that when mice are fed a high-fat diet for a long time, the accumulation of fatty acids in the body will increase significantly, which will further promote the increase in the levels of triglyceride (TG) and total cholesterol (TC) in the serum. If mice are in an obese state for a long time, the metabolic activity of lipase in the body will accelerate, which will lead to a decrease in the level of high-density lipoprotein cholesterol (HDL-C) and an increase in the level of low-density lipoprotein cholesterol (LDL-C). Judging from the data in the table, compared with the normal control group, the contents of TG, TC, and LDL-C in the model group mice were significantly increased, while the content of HDL-C was significantly decreased. The CU270 intervention group improved this situation and could relieve the significant increase in the contents of TG, TC, and LDL-C caused by the accumulation of body fat, indicating that the CU270 strain can significantly improve the blood lipid elevation caused by a high-fat diet.

[0115] Table 5 Detection results of four blood lipid indicators

[0116]

[0117] Example 6 Effect of CU270 strain on liver injury in obese mice

[0118] Take the serum of mice in each group as in Example 4, and use a kit (Nanjing Jiancheng Bioengineering Institute) to detect the concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the mouse serum. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are key metabolic enzymes in liver cells. When the liver is damaged, these two enzymes will be released from liver cells into the blood, resulting in a significant increase in the concentrations of ALT and AST in the serum. Therefore, the changes in the levels of ALT and AST in the serum can be used as important biomarkers for evaluating whether the liver is damaged.

[0119] The measurement results are shown in Table 6. From the data in the table, compared with the normal control group, the contents of ALT and AST in the model group mice were significantly increased, indicating that obese mice would have liver injury. The CU270 intervention group improved this situation and could reduce the levels of ALT and AST in the serum, indicating that the CU270 strain could significantly improve the liver cell damage caused by high-fat diet.

[0120] Table 6 Concentrations of ALT and AST

[0121]

[0122]

[0123] In summary, a Leuconostoc pseudomesenteroides CU270 strain with the functions of reducing cholesterol and triglyceride was isolated and identified from fermented pickles in the present invention. Within 24 h in vitro, the cholesterol degradation rate of this strain reached 52.80%, and the triglyceride degradation rate reached 54.94%. Moreover, it had strong acid and bile salt tolerance capabilities. The survival rate after 8 h in the MRS medium with pH = 2 was 82.83%, and the survival rate after 8 h in the MRS medium with a bile salt concentration of 0.3% was 83.78%. At the same time, the CU270 strain also had antioxidant ability, with the DPPH free radical scavenging rate reaching 69.23% and the ABTS+ free radical scavenging rate reaching 40.18%. In the study of obese animal models, it was found that the CU270 strain could relieve obesity, reduce blood lipid and blood glucose levels, improve the elevated blood lipid caused by high-fat diet, and relieve the liver cell damage caused by high-fat diet, and had no any toxic and side effects, with the biological function of reducing fat, providing new ideas for the preparation of more anti-obesity disease products.

[0124] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A strain of Leuconostoc pseudomesenteroides CU270, characterized in that, The strain was deposited at the Guangdong Microbial Culture Collection Center on November 4, 2024, with the deposit number GDMCC NO. 65413.

2. A culture, characterized in that, The preparation method of the culture is as follows: inoculate the CU270 strain described in claim 1 into MRS medium and culture it at 30-35 °C for 24-36 h to obtain the culture.

3. A microbial inoculant containing the strain or its bacterial liquid described in claim 1.

4. The microbial inoculant according to claim 3, wherein The viable count of the microbial agent is not less than 1×10 8 CFU / mL.

5. Use of the CU270 strain described in claim 1, or the culture described in claim 2, or the microbial inoculant described in claim 3 in the preparation of a product for relieving obesity.

6. Use of the CU270 strain described in claim 1, or the culture described in claim 2, or the microbial inoculant described in claim 3 in the preparation of a product for reducing blood lipid levels.

7. Use of the CU270 strain described in claim 1, or the culture described in claim 2, or the microbial inoculant described in claim 3 in the preparation of an antioxidant product.

8. Use of the CU270 strain described in claim 1, or the culture described in claim 2, or the microbial inoculant described in claim 3 in the preparation of a product for relieving liver injury caused by a high-fat diet.

9. A product, characterized in that, Containing the CU270 strain described in claim 1, or the culture described in claim 2, or the microbial inoculant described in claim 3.

10. A method for reducing cholesterol and triglyceride levels for non-disease treatment purposes, which is carried out by treating with the CU270 strain described in claim 1, or the culture described in claim 2, or the microbial inoculant described in claim 3.

Images