Lactobacillus paracasei ELPA01 and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a lactobacillus paracasei ELPA01 strain and application thereof. The Lactobacillus paracasei ELPA01 is preserved in the China Center for Type Culture Collection on December 30, 2024, the preservation address is No. 299 on eight road, Wuchang District, Wuhan City, Hubei Province, and the preservation number is CCTCC NO: M 20242946. The Lactobacillus paracasei ELPA01 has the advantages that the Lactobacillus paracasei ELPA01 is used for preparing the Lactobacillus paracasei ELPA01, and the Lactobacillus paracasei ELPA01 is used for preparing the Lactobacillus paracasei ELPA01; according to the lactobacillus paracasei ELPA01, the lactobacillus paracasei ELPA01 with good probiotic performance is screened from breast milk of healthy women, and the lactobacillus paracasei ELPA01 has good capabilities of degrading cholesterol, adhering and generating bile salt hydrolase, and can play an anti-obesity effect by reducing the weight of mice and reducing fat accumulation.

Description

Technical Field

[0001] This application belongs to the field of microbial technology, and specifically relates to a strain of Lacticaseibacillus paracasei ELPA01 and its application. Background Art

[0002] Obesity is characterized by the excessive accumulation of adipocytes and lipids, usually due to nutritional imbalance or energy intake exceeding expenditure. Obese individuals are at higher risk of chronic diseases, including cancer, non-alcoholic fatty liver disease, circulatory and reproductive disorders, atherosclerosis, and cardiovascular diseases. Currently, chemical drugs are widely used to treat obesity due to their high efficacy, although they often have adverse reactions such as metabolic disorders. For example, orlistat is a commonly used weight loss drug that helps regulate the lipid balance of patients and exhibits anti-obesity and antioxidant properties; however, it may cause side effects, including flatulence, hepatotoxicity, and nephrotoxicity. Therefore, there is an urgent need for safe and effective alternative solutions. Summary of the Invention

[0003] The object of the present invention is to solve the deficiencies of the prior art and provide a strain of Lacticaseibacillus paracasei ELPA01 and its application, specifically adopting the following technical solutions: In the first aspect, the present invention provides a strain of Lacticaseibacillus paracasei ELPA01. The Lacticaseibacillus paracasei ELPA01 was deposited at the China Center for Type Culture Collection on December 30, 2024. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC NO: M 20242946. The 16S rDNA sequence of Lacticaseibacillus paracasei ELPA01 is shown in SEQ ID NO. 1.

[0004] SEQ ID NO. 1:

[0005] In a second aspect, the present invention provides the use of the above-mentioned Lactobacillus paracasei ELPA01 in the preparation of a product for relieving obesity and / or relieving metabolic disorders caused by obesity and / or improving liver lipid deposition caused by obesity.

[0006] As a further preferred embodiment, the above-mentioned product includes food or medicine.

[0007] In a third aspect, the present invention provides a drug for relieving metabolic disorders caused by obesity, and the drug includes the above-mentioned Lactobacillus paracasei ELPA01.

[0008] In a fourth aspect, the present invention provides a drug for improving liver lipid deposition caused by obesity, and the drug includes the above-mentioned Lactobacillus paracasei ELPA01.

[0009] In a fifth aspect, the present invention provides a drug for relieving obesity, and the drug includes the above-mentioned Lactobacillus paracasei ELPA01.

[0010] In a sixth aspect, the present invention provides a drug for relieving obesity, relieving metabolic disorders caused by obesity and improving liver lipid deposition caused by obesity, and the drug includes the above-mentioned Lactobacillus paracasei ELPA01.

[0011] As a further preferred embodiment, the drug further includes pharmaceutical excipients.

[0012] As a further preferred embodiment, the pharmaceutical excipient is at least one of water, lactose, sodium chloride, and glucose.

[0013] The beneficial effects of the present invention are as follows: The present invention discloses a strain of Lactobacillus paracasei ELPA01 with good probiotic properties screened from the breast milk of healthy women. It has good abilities of cholesterol degradation, adhesion and production of bile salt hydrolase, and can play an anti-obesity effect by reducing the body weight of mice and reducing fat accumulation. Description of the Drawings

[0014] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0015] Figure 1 The figure shows the acid tolerance result graph of Lactobacillus paracasei ELPA01; Figure 2The figure shows the cholesterol removal in vitro by Lactobacillus paracasei ELPA01; Figure 3 The figure shows the adhesion of Lactobacillus paracasei ELPA01 to Caco-2 cells; Figure 4 The figure shows the autoaggregation of Lactobacillus paracasei ELPA01; Figure 5 The figure shows the hydrophobicity of Lactobacillus paracasei ELPA01; Figure 6 The figure shows the reduction of body weight and fat gain in C57BL / 6 obese mice induced by high-fat diet by Lactobacillus paracasei ELPA01; Figure 7 The figure shows the alleviation of hepatocyte swelling, liver lipid deposition and epididymal adipocyte enlargement in C57BL / 6 obese mice induced by high-fat diet by Lactobacillus paracasei ELPA01; Detailed implementation mode Next, the technical solutions in the embodiments of the present application will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present application.

[0016] Example 1 Isolation and identification of Lactobacillus paracasei ELPA01 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 streaking to ensure no contaminants. Lactobacillus paracasei ELPA01 is a Gram-positive bacterium, facultatively anaerobic, and does not produce spores. To stably preserve the excellent properties of Lactobacillus paracasei ELPA01 of the present invention, it was stored in a mixed preservation solution containing 15% glycerol and frozen at -80°C, or freeze-dried with 10% skim milk.

[0017] 16S rDNA sequencing was performed on Lactobacillus paracasei ELPA01, and the base sequence of its 16S rDNA is shown in SEQ ID No: 1. And 16S rDNA sequence analysis was carried out, and the result was: compared with Lactobacillus paracasei ( Lacticaseibacillus paracasei) MLG3-17 has the highest homology (99.86%) and shows the closest molecular phylogenetic relationship with Lactobacillus paracasei. Therefore, this microorganism was identified as Lactobacillus paracasei and named Lactobacillus paracasei ELPA01. In this example, a control strain was also screened out and named Lactobacillus paracasei ELPA04.

[0018] The Lactobacillus paracasei derived from breast milk in Example 1 was inoculated into MRS liquid medium with pH values of 2.5, 3.5, and 6.5 at an inoculation amount of 1%, and anaerobically cultured for 3 h, followed by viable cell counting.

[0019] The results are as Figure 1 shown. Lactobacillus paracasei all has good acid tolerance. Lactobacillus paracasei ELPA01 is more tolerant to pH 3.5. When the pH is 2.5, the viable cell count decreases significantly, but the viable cell count only decreases by less than 0.5 order of magnitude, indicating that it also has good tolerance to acid with a pH of 2.5.

[0020] Example 2 Quantitative experiment on the bile salt hydrolase activity of Lactobacillus paracasei ELPA01 The Lactobacillus paracasei derived from breast milk was cultured in MRS liquid medium at 37 °C for 24 h, centrifuged at 8000 rpm for 10 min to collect the bacterial cells, washed 3 times with PBS and resuspended, and the cell 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). 100 μL of CFE was added to 1.8 mL of PBS (pH 6.0) and 100 μL of 6 mM sodium taurodeoxycholate, incubated for 30 min, and the reaction was terminated by adding 2 mL of 15% trichloroacetic acid. The supernatant was mixed with an equal volume of ninhydrin color-developing solution (containing 0.25% ninhydrin and 60% glycerol), boiled in a water bath for 15 min, and OD 570 was measured. The protein content of the sample was determined using a Brandford protein concentration assay kit, and the enzyme activity was calculated based on the amino acid concentration and protein concentration. The results are shown in Table 1: Table 1 As shown in Table 1 above, Lactobacillus paracasei ELPA01 has a relatively high bile salt hydrolase activity, and its specific enzyme activity is 65.05 ± 2.5 U / g.

[0021] Example 3 Experiment on the removal of cholesterol by Lactobacillus paracasei ELPA01 in vitro Lactobacillus paracasei ELPA01 was cultured in MRS liquid medium supplemented with 0.1 mg / mL cholesterol, 0.2% sodium thioglycollate, and 0.05% oxgall at 37 °C for 24 h, centrifuged at 8000 rpm for 5 min, 40 μL of 1 g / L o-phthalaldehyde solution was added to 100 μL of the supernatant, after standing for 10 min, 800 μL of mixed acid (concentrated sulfuric acid: glacial acetic acid = 1:1) was added, and the OD was measured after standing for 10 min. 550 。

[0022] The results are as Figure 2 shown. It can be seen from the results that Lactobacillus paracasei ELPA01 has a good cholesterol removal effect, and its cholesterol removal rate is 17.67%.

[0023] Example 4 Adhesion experiment of Lactobacillus paracasei ELPA01 to Caco-2 cells Caco-2 cells were inoculated in 6-well plates (5×10 5 cells / well), and the suspension of Lactobacillus paracasei ELPA01 (10 8 CFU / mL) was added and incubated at 37 °C for 2 h. The unadhered bacteria were washed away, and the adhered bacteria were counted on MRS agar plates. Lactobacillus rhamnosus LGG was used as a control strain.

[0024] The results are as Figure 3 shown. It can be seen from the results that Lactobacillus paracasei ELPA01 has a strong adhesion effect on Caco-2 cells, and its adhesion rate is 2.34 lg cfu / 100 cells, which is higher than that of other Lactobacillus paracasei tested.

[0025] Example 5 Autoaggregation experiment of Lactobacillus paracasei ELPA01 The concentration of Lactobacillus paracasei ELPA01 was adjusted to 10 8 CFU / mL, and the initial OD 600 was measured. Then the bacterial suspension was vortexed for 10 seconds and incubated at 37 °C for 4 h, and the OD 600 of the bacterial suspension was measured, and the autoaggregation of Lactobacillus paracasei ELPA01 was calculated.

[0026] The results are as Figure 4 shown. It can be seen from the results that the autoaggregation rate of Lactobacillus paracasei ELPA01 is 37.77%.

[0027] Example 6 α-glucosidase activity inhibition experiment Lactobacillus paracasei ELPA01 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 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). Add 25 μL of 2.5 mM p-nitrophenyl α-D-glucopyranoside and 25 μL of CFE sample into a 96-well plate, incubate at 37 °C for 10 min, then add 50 μL of 0.2 U / mL α-glucosidase, and incubate at 37 °C for 30 min. Add 100 μL of 0.2 M Na 2 CO 3 to terminate the reaction, and then incubate at 37 °C for another 15 min. By measuring OD 405 . Using acarbose ( Figure 5 Acarbose in it) as a control, the inhibition rate was calculated.

[0028] As Figure 5 showed the results of the inhibitory effect of Lactobacillus paracasei ELPA01 on α-glucosidase activity, and its inhibition rate on α-glucosidase activity was 37.66%.

[0029] Example 7 Effect of Lactobacillus paracasei ELPA01 on body weight, liver weight and epididymal fat weight of C57BL / 6 mice Male C57BL / 6 mice at 6-8 weeks old were selected. After two weeks of adaptation, they were divided into 3 groups for gavage: 1) Normal control group (NC): gavaged with 0.2 mL of sterile 0.01 M PBS every day for 8 consecutive weeks; 2) Model group (HFD): given HFD and gavaged with 0.2 mL of sterile 0.01 M PBS every day for 8 consecutive weeks; 3) ELPA01 group (HFD+ELPA01): gavaged with 0.2 mL of Lactobacillus paracasei ELPA01 (5×10 9 CFU / mL) bacterial suspension every day for 8 consecutive weeks; The body weight of the mice was weighed every day, and the mice were euthanized on the 57th day. The euthanized mice were immersed in 75% ethanol for about 8 minutes, and the organ indices of the epididymal fat and visceral (perirenal) fat of the mice were accurately weighed.

[0030] The results were as Figure 6 shown. From the results, it can be seen that Lactobacillus paracasei ELPA01 can significantly reduce the body weight and organ indices of fat in mice. Among them, Figure 6 A represents the body weight of the mice, and B represents the organ indices of the epididymal fat and visceral fat of the mice.

[0031] Example 8 Characterization of liver and epididymal fat sections of C57BL / 6 mice Cut a piece about 1 cm in size 3 The livers and fats of the mice were immersed in 10% tissue fixative for fixation and stored at 4°C. HE and Oil Red O staining were performed on liver sections, and HE staining was performed on epididymal fat sections.

[0032] The results are as Figure 7 shown in the HE staining results of the liver. It can be seen from the results that the liver cell volume of the mice in the model group increased, and severe vacuolization occurred. In the Lactobacillus paracasei ELPA01 group of the present invention, the morphology of the liver cells was significantly restored to normal ( Figure 7 A). The Oil Red O staining of the liver showed that the lipid deposition in the liver of the mice in the model group was significantly higher than that in the normal control group. In the Lactobacillus paracasei ELPA01 group of the present invention, the lipid deposition in the liver was significantly reduced ( Figure 7 B). The HE staining of the epididymal fat showed that the volume of the epididymal fat cells of the mice in the model group increased significantly. In the Lactobacillus paracasei ELPA01 group of the present invention, the volume of the fat cells decreased significantly ( Figure 7 B).

[0033] The embodiments of the present application have been described above in conjunction with the accompanying drawings. Specific examples are used in this article to elaborate on the principles and implementation manners of the present application. The description of the above embodiments is only used to help understand the core idea of the present application. However, the present application is not limited to the above specific implementation manners. The above specific implementation manners are only illustrative and not restrictive. Under the inspiration of the present application, those of ordinary skill in the art can also make many forms without departing from the purpose of the present application and the scope protected by the claims, and all belong to the protection scope of the present application.

Claims

1. A strain of Lactobacillus paracasei ELPA01, characterized in that The Lactobacillus paracasei ( Lacticaseibacillus paracasei ) ELPA01 was deposited in the China Center for Type Culture Collection on December 30, 2024. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC NO:M 20242946.

2. Lactobacillus paracasei ELPA01 according to claim 1, characterized in that The 16S rDNA sequence of the Lactobacillus paracasei ELPA01 is shown in SEQ ID NO.

1.

3. Use of the Lactobacillus paracasei ELPA01 described in any one of claims 1-2 in preparing a product for alleviating obesity and / or alleviating metabolic disorders caused by obesity and / or improving liver lipid deposition caused by obesity.

4. The use according to claim 3, characterized in that: The product includes a food or a medicine.

5. A drug for alleviating metabolic disorders caused by obesity, characterized in that: The drug comprises the Lactobacillus paracasei ELPA01 according to claim 1.

6. A drug for improving liver lipid deposition caused by obesity, characterized in that: The drug comprises the Lactobacillus paracasei ELPA01 according to claim 1.

7. A drug for alleviating obesity, characterized in that: The drug comprises the Lactobacillus paracasei ELPA01 according to claim 1.

8. A drug for alleviating obesity, alleviating metabolic disorders caused by obesity, and improving liver lipid deposition caused by obesity, characterized in that: The drug comprises the Lactobacillus paracasei ELPA01 according to claim 1.

9. The drug according to claim 8, characterized in that The medicine also includes pharmaceutical excipients.

10. The drug according to claim 9, characterized in that The pharmaceutical excipient is at least one of water, lactose, sodium chloride and glucose.

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