Breast milk source fermentation lactobacillus mucus ELFE07 and application thereof

Abstract

The invention discloses breast milk source fermentation lactobacillus mucus ELFE07 and application thereof, and relates to the technical field of microorganisms. The lactobacillus mucus ELFE07 disclosed by the invention is preserved in the China Center for Type Culture Collection, and the preservation number of the lactobacillus mucus ELFE07 is CCTCC (China Center for Type Culture Collection) NO: M 20242947. The fermented lactobacillus mucus ELFE07 provided by the invention is obtained by screening from breast milk of healthy women, has good probiotic performance, and has the capability of inhibiting the activity of alpha-glucosidase and removing cholesterol; the composition can play a role in relieving obesity by reducing the weight of a mouse, restoring the glucose metabolism capability of the mouse and reducing the content of cholesterol and triglyceride through independent action or combined action of the composition and 2 '-fucosyllactose (2'-FL).

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly relates to a breast milk-derived fermented Lactobacillus mucosae ELFE07 and its application. Background Art

[0002] Breast milk is rich in nutrients and microorganisms, and is an important source of the intestinal microbiota during the growth and development of newborns, directly affecting the future risk of various diseases in infants. The microorganisms in breast milk are mainly composed of Lactobacillus and Bifidobacterium. Research shows that the microorganisms in breast milk can enhance the host's immune response to infection, inhibit the adhesion of pathogenic microorganisms to intestinal epithelial cells, regulate the intestinal microbiota, and improve gastrointestinal function.

[0003] Probiotics refer to live microorganisms that are beneficial to the host when ingested in sufficient amounts. Research shows that probiotics can affect the pathogenesis of metabolic diseases by regulating the intestinal microbiota, and thus participate in physiological processes such as energy metabolism and immune regulation. Research shows that Lactobacillus reuteri ( Limosilactobacillus reuteri ) DSM17938 shows significant efficacy in preventing liver metabolic disorders induced by a Western diet, demonstrating its great potential in reducing obesity; oral Blautia wexlerae ( Blautia wexlerae ) JCM 17041 can improve obesity and type 2 diabetes by remodeling the intestinal microbiota; Lactobacillus sakei subsp. sakei ( Latilactobacillus sakei ) Probio65 and Lactobacillus plantarum ( Lactiplantibacillus plantarum ) Probio-093 can effectively reduce obesity and improve adipocyte function. Summary of the Invention

[0004] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art, and to provide a breast milk-derived fermented Lactobacillus mucosae ELFE07 and its application.

[0005] The technical solution of the present invention is as follows: The first aspect of the present invention provides a breast milk-derived fermented Lactobacillus mucosae ELFE07, and the fermented Lactobacillus mucosae ( Limosilactobacillus fermentum ) ELFE07 was deposited at the China Center for Type Culture Collection on December 30, 2024, with the deposit number CCTCC NO: M 20242947. The deposit address is Wuhan University, Wuhan, China.

[0006] The 16S rDNA sequence of the fermented Lactobacillus mucosae ELFE07 is shown in SEQ ID NO. 1.

[0007] The fermented Lactobacillus mucosae ELFE07 was screened from the breast milk of healthy women.

[0008] The fermented Lactobacillus mucosae ELFE07 is a Gram-positive bacterium, facultatively anaerobic and non-spore-forming.

[0009] The fermented Lactobacillus mucosae ELFE07 of the present invention can grow on MRS agar medium. It is a Gram-positive bacterium, facultatively anaerobic and non-spore-forming. To stably preserve the excellent properties of the fermented Lactobacillus mucosae ELFE07 of the present invention, it is stored in a mixed preservation solution containing 15% glycerol and cryopreserved at -80°C, or freeze-dried and preserved with 10% skim milk.

[0010] For the identification and classification of microorganisms, 16S rDNA sequence analysis was performed on the fermented Lactobacillus mucosae ELFE07 of the present invention. The result shows that it has the highest homology (99.93%) with Lactobacillus mucosae Limosilactobacillus fermentum ) 6012, showing the closest phylogenetic relationship at the molecular systematic level with Lactobacillus mucosae. Therefore, this microorganism was identified as Lactobacillus mucosae and named Lactobacillus mucosae ELFE07.

[0011] The second aspect of the present invention provides the use of the above-mentioned Lactobacillus mucosae ELFE07 alone or in combination with 2'-FL in the preparation of products for relieving obesity.

[0012] The third aspect of the present invention provides the use of the above-mentioned Lactobacillus mucosae ELFE07 alone or in combination with 2'-FL in the preparation of products for relieving glucose metabolism disorders and lipid metabolism disorders.

[0013] The fourth aspect of the present invention provides the use of the above-mentioned Lactobacillus mucosae ELFE07 alone or in combination with 2'-FL in the preparation of products for improving glucose metabolism and insulin resistance.

[0014] 2'-FL is short for 2'-fucosyllactose, which is a major human milk oligosaccharide in breast milk. The applicant has found through research that the combined action of Lactobacillus mucosae ELFE07 and 2'-FL can play an anti-obesity effect by reducing the body weight of mice, restoring the glucose metabolism ability of mice, and decreasing the contents of cholesterol and triglycerides, thereby relieving obesity, glucose metabolism disorders, lipid metabolism disorders, and improving glucose metabolism and insulin resistance.

[0015] The dosage of the fermented Lactobacillus mucosae ELFE07 is that the number of viable bacteria of the ingested fermented Lactobacillus mucosae ELFE07 is (5 - 20) × 109 CFU / d, and the dosage of 2'-FL is 2 - 6 g / d.

[0016] The products include at least one of biological products, drugs, and health products.

[0017] The fifth aspect of the present invention provides a biological product for alleviating obesity, which comprises the above-mentioned fermented Lactobacillus mucosae ELFE07, or comprises the above-mentioned fermented Lactobacillus mucosae ELFE07 and 2'-FL.

[0018] The present invention has at least one of the following beneficial effects: 1. A strain of fermented Lactobacillus mucosae ELFE07 with good probiotic properties is screened from the breast milk of healthy women in the present invention. It has the ability to inhibit α-glucosidase activity in vitro and remove cholesterol. It can play an anti-obesity effect by reducing the body weight of mice, restoring the glucose metabolism ability of mice and reducing the triglyceride content, and can be applied to the preparation of products for alleviating obesity, alleviating sugar metabolism disorders, lipid metabolism disorders, and improving glucose metabolism and insulin resistance.

[0019] 2. The combined action of the screened fermented Lactobacillus mucosae ELFE07 and 2'-FL in the present invention can be applied to the preparation of products for alleviating obesity, alleviating sugar metabolism disorders, lipid metabolism disorders, and improving glucose metabolism and insulin resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The figures show the simulated gastric juice and simulated intestinal juice conditions of Lactobacillus mucosae ELFE01-ELFE07. Among them, A is the simulated gastric juice and B is the simulated intestinal juice.

[0021] Figure 2 The figures show the inhibition of α-glucosidase activity by Lactobacillus mucosae ELFE01-ELFE07.

[0022] Figure 3 The figures show the in vitro cholesterol removal by Lactobacillus mucosae ELFE01-ELFE07.

[0023] Figure 4 The figures show the weight and fat gain of C57BL / 6 obese mice induced by high-fat diet alleviated by Lactobacillus mucosae ELFE07 alone and in combination with 2'-FL.

[0024] Figure 5 The figures show the impaired glucose metabolism ability of C57BL / 6 obese mice induced by high-fat diet improved by Lactobacillus mucosae ELFE07 alone and in combination with 2'-FL. Among them, A is glucose tolerance and B is the area under the curve.

[0025] Figure 6 The figures show the improvement of insulin resistance in C57BL / 6 obese mice induced by high-fat diet by Lactobacillus mucosae ELFE07 alone and in combination with 2'-FL. Among them, A is the fasting blood glucose of mice, B is the fasting insulin, and C is the insulin resistance index.

[0026] Figure 7 The figure shows the situation of Lactobacillus fermentum mucosae ELFE07 alone and its co - intervention with 2'-FL in reducing triglyceride and total cholesterol in C57BL / 6 obese mice induced by high - fat diet. Detailed implementation mode

[0027] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0028] Example 1 Isolation and identification of breast milk - derived Lactobacillus fermentum mucosae ELFE07 Probiotics were isolated from the breast milk of healthy women. After cleaning the surrounding skin with sterile water and discarding the initial milk droplets, the breast milk samples were collected into sterile tubes. The breast milk was diluted and spread on MRS agar plates and anaerobically cultured at 37°C for 24 hours. Colonies with different morphologies were picked with an inoculation loop for streak purification, and 7 strains of bacteria were screened, named ELFE01 - ELFE07 respectively, and 16S rDNA sequencing was performed.

[0029] Among them, the base sequence of 16S rDNA of ELFE07 is shown as SEQ ID No:1.

[0030]

[0031] The fermented Lactobacillus mucosae ELFE07 screened in this example can grow on MRS agar medium, is a Gram-positive bacterium, facultatively anaerobic, and does not produce spores. To stably preserve the excellent properties of fermented Lactobacillus mucosae ELFE07, it is stored in a mixed preservation solution containing 15% glycerol and cryopreserved at -80 °C, or freeze-dried and preserved with 10% skim milk.

[0032] The breast milk-derived fermented Lactobacillus mucosae ELFE01 - ELFE07 in Example 1 was resuspended in 10% skim milk and inoculated into simulated gastric juice (0.3% pepsin, 7 mM KCl, 125 mM NaCl, 45 mM NaHCO 3 , pH 2.5) and simulated intestinal fluid (0.1 g / L trypsin, 0.3 g / L bile salts, 0.835 g / L KCl, 6.5 g / L NaCl) at an inoculation amount of 1%, incubated for 120 min, and viable cell counts were performed.

[0033] The results are as Figure 1 shown, where A represents simulated gastric juice and B represents simulated intestinal fluid. As Figure 1 shown in the results of A's tolerance to gastric juice, the fermented Lactobacillus mucosae ELFE01 - ELFE07 all had good tolerance to gastric juice. Figure 1 shown in the results of B's tolerance to intestinal fluid, after the fermented Lactobacillus mucosae ELFE01 - ELFE07 was treated with intestinal fluid for 2 h, although the viable cell count decreased, the viable cell count only decreased by less than one order of magnitude, indicating that it also had good tolerance to intestinal fluid.

[0034] Example 2 Experiment on the inhibition of α-glucosidase activity by fermented Lactobacillus mucosae ELFE07 The breast milk-derived fermented Lactobacillus mucosae ELFE01 - ELFE07 in Example 1 was cultured in MRS liquid medium at 37 °C for 14 h, centrifuged at 8000 rpm for 10 min, the cells were collected, washed 3 times with PBS and resuspended, and the cell suspension concentration was adjusted to 10 9 CFU / mL, sonicated for 15 min, and centrifuged at 12000 rpm for 20 min to obtain cell-free extract (CFE). 25 μL of 2.5 mM p-nitrophenyl α-D-glucopyranoside and 25 μL of CFE sample were added to a 96-well plate, incubated at 37 °C for 10 min, then 50 μL of 0.2 U / mL α-glucosidase was added, and incubated at 37 °C for 30 min. 100 μL of 0.2 M Na 2 CO 3 was added to terminate the reaction, and then incubated at 37 °C for another 15 min. By measuring OD 405。Using Acarbose ( Figure 1 Acarbose in Figure 1 ) as a control, the inhibition rate was calculated.

[0035] As Figure 2 shown in Figure 2 , the results of the α-glucosidase activity of Lactobacillus mucosae ELFE01 - ELFE07 fermentation are presented. Among them, Lactobacillus mucosae ELFE07 has a strong inhibitory effect on α-glucosidase activity, and its inhibition rate on α-glucosidase activity is 50.04%.

[0036] Example 3 Experiment on the removal of cholesterol by Lactobacillus mucosae ELFE07 in vitro The breast milk-derived Lactobacillus mucosae ELFE01 - ELFE07 from Example 1 was cultured in MRS liquid medium supplemented with 0.1 mg / mL cholesterol, 0.2% sodium thioglycolate, and 0.05% bile salts at 37 °C for 24 h, centrifuged at 8000 rpm for 5 min. To 100 μL of the supernatant, 40 μL of 1 g / L o-phthalaldehyde solution was added, and after standing for 10 min, 800 μL of a mixed acid (concentrated sulfuric acid: glacial acetic acid = 1:1) was added. After standing for another 10 min, OD 550 .

[0037] As Figure 3 shown in Figure 3 , the experimental results of the removal of cholesterol by Lactobacillus mucosae ELFE01 - ELFE07 in vitro are presented. Among them, Lactobacillus mucosae ELFE07 has a good effect on cholesterol removal, and its cholesterol removal rate is 14.70%.

[0038] In summary, compared with Lactobacillus mucosae ELFE01 - ELFE06, ELFE07 has the best inhibitory effect on α-glucosidase activity and also the best effect on cholesterol removal. Therefore, ELFE07 was selected for subsequent experiments.

[0039] Example 4 Effects of Lactobacillus mucosae ELFE07 and the co-intervention of ELFE07 and 2'-FL on the body weight, epididymal fat, and visceral fat weight of C57BL / 6 mice Male C57BL / 6 mice at 6 - 8 weeks of age were selected. After two weeks of adaptation, they were divided into 4 groups for gavage: 1) Normal control group (NC): gavaged with 0.2 mL of sterile 0.01 M PBS daily for 8 consecutive weeks; 2) Model group (HFD): given HFD (high-fat diet) daily and gavaged with 0.2 mL of sterile 0.01 M PBS for 8 consecutive weeks; 3) ELFE07 group (HFD + ELFE07): given HFD (high-fat diet) daily and gavaged with 0.2 mL of Lactobacillus mucosae ELFE07 (5×10 9CFU / mL) bacterial suspension, and intragastrically administered for 8 consecutive weeks; 4) ELFE07 + 2'-FL group (HFD + ELFE07 + 2'-FL): Given HFD (high-fat diet) every day and intragastrically administered 0.2 mL of fermented Lactobacillus mucosae ELFE07 (5×10 9 CFU / mL) bacterial suspension and 0.4 g / kg 2'-FL, and intragastrically administered for 8 consecutive weeks.

[0040] Weigh the mice every day. On the 57th day, draw blood from the mouse eyeballs and euthanize the mice. Immerse the euthanized mice in 75% ethanol for about 8 minutes, and accurately weigh the organ indices of the epididymal fat and visceral (perirenal) fat of the mice.

[0041] The results are as Figure 4 shown. Compared with the model group (HFD), the body weights and organ indices of fat of the mice in the ELFE07 group (HFD + ELFE07) and the ELFE07 + 2'-FL group (HFD + ELFE07 + 2'-FL) are significantly lower. Therefore, as Figure 4 shown, both the intervention of fermented Lactobacillus mucosae ELFE07 and the combined intervention of ELFE07 and 2'-FL can significantly reduce the body weight and organ index of fat of the mice. Among them, Figure 4 are the organ indices of the epididymal fat and visceral fat of the mice.

[0042] Example 5 Effects of the combined intervention of fermented Lactobacillus mucosae ELFE07 and ELFE07 and 2'-FL on oral glucose tolerance in C57BL / 6 mice On the 56th day of the experiment in Example 5, an oral glucose tolerance test was conducted. The mice were fasted for 12 h the previous night, and each mouse was intragastrically administered a 50% glucose solution at a dose of 2 g / kg. Before and 15 min, 30 min, 60 min, and 120 min after intragastric administration, whole blood was collected from the tail vein, the blood glucose level was measured with a standard blood glucose meter, and a glucose tolerance test curve was plotted. The glucose tolerance of the mice was analyzed by calculating the area under the curve.

[0043] The results are as Figure 5 shown. Among them, A represents glucose tolerance, and B represents the area under the curve. As Figure 5 shown by the oral glucose tolerance results, the mice in the model group showed a phenomenon of glucose metabolism disorder. In the fermented Lactobacillus mucosae ELFE07 group and the ELFE07 + 2'-FL group of the present invention, their glucose metabolism ability was significantly restored.

[0044] Example 6 Effects of the combined intervention of fermented Lactobacillus mucosae ELFE07 and ELFE07 and 2'-FL on insulin resistance in C57BL / 6 mice The serum glucose and insulin levels of the mice in Example 4 were measured using an enzymatic kit and an ELISA kit, respectively, and the insulin resistance index was calculated according to the following formula: Insulin Resistance Index (HOMA-IR) = Glucose level (mM) × Insulin level (mIU / L) / 22.5.

[0045] The results are shown as Figure 6 follows, where A represents the fasting blood glucose of the mice, B represents the fasting insulin, and C represents the insulin resistance index (HOMA-IR). As Figure 6 shown in the insulin resistance results of C, insulin resistance occurred in the model group of mice. In the group of fermented Lactobacillus mucosae ELFE07 of the present invention, its insulin resistance was alleviated to a certain extent, while in the ELFE07 + 2'-FL group, its insulin resistance was significantly alleviated. Therefore, compared with the ELFE07 group, the ELFE07 + 2'-FL group had a better effect on alleviating insulin resistance, indicating that there may be a synergistic effect between ELFE07 and 2'-FL.

[0046] Example 7 Effects of Fermented Lactobacillus mucosae ELFE07 and the Co-intervention of ELFE07 and 2'-FL on Blood Lipids in C57BL / 6 Mice The levels of total cholesterol (TC) and triglyceride (TG) in the serum of the mice in Example 4 were measured using a kit.

[0047] As Figure 7 shown, the levels of total cholesterol (TC) and triglyceride (TG) in the model group of mice were significantly higher than those in the normal control group. The levels of total cholesterol and triglyceride in the groups of fermented Lactobacillus mucosae ELFE07 and ELFE07 + 2'-FL of the present invention were significantly decreased, and even the level of triglyceride was completely restored to the normal level, indicating that fermented Lactobacillus mucosae ELFE07 and the co-action of ELFE07 and 2'-FL can reduce the levels of total cholesterol (TC) and triglyceride (TG).

[0048] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and should be covered by the protection scope of the present invention.

Claims

1. A breast milk-derived fermented Lactobacillus mucosa ELFE07, characterized in that: The fermented Lactobacillus mucilaginosus ( Limosilactobacillus fermentum )ELFE07 was deposited in the China Center for Type Culture Collection on December 30, 2024, with the deposit number CCTCC NO: M 20242947.

2. The fermented mucus Lactobacillus ELFE07 according to claim 1, characterized in that The 16S rDNA sequence of the fermentative Lactobacillus mucilaginosus ELFE07 is shown in SEQ ID NO.

1.

3. The fermented mucus lactobacillus ELFE07 according to claim 1, characterized in that The fermented mucus lactobacillus ELFE07 is screened from the breast milk of healthy women.

4. The fermented mucus Lactobacillus ELFE07 according to claim 1, characterized in that The fermented mucus lactobacillus ELFE07 is a Gram-positive bacterium, facultative anaerobic, and non-spore-forming.

5. Use of the fermented mucus lactobacillus ELFE07 according to any one of claims 1 to 4 alone or together with 2'-FL in the preparation of a product for alleviating obesity.

6. Use of the fermented mucus lactobacillus ELFE07 according to any one of claims 1 to 4 alone or together with 2'-FL in the preparation of products for alleviating sugar metabolism disorders and lipid metabolism disorders.

7. Use of the fermented Lactobacillus mucilaginosus ELFE07 according to any one of claims 1 to 4 alone or together with 2'-FL in the preparation of products for improving glucose metabolism and insulin resistance.

8. The use according to any one of claims 5 to 7, characterized in that: The dosage of the fermented mucus lactobacillus ELFE07 is (5-20)×10 9 CFU / d, and the dosage of 2'-FL is 2-6 g / d.

9. The use according to any one of claims 5 to 7, characterized in that: The product includes at least one of biological products, medicines and health products.

10. A biological product for alleviating obesity, characterized in that: The biological product comprises the fermented Lactobacillus mucilaginosus ELFE07 according to claim 1, or comprises the fermented Lactobacillus mucilaginosus ELFE07 according to claim 1 and 2'-FL.

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