Lactobacillus paracasei SX-G1520 and application of lactobacillus paracasei SX-G1520 in preparation of lipid-lowering, anti-inflammatory and / or antioxidant products

Abstract

The invention discloses a strain of Lactobacillus paracasei SX-G1520 and an application of the Lactobacillus paracasei SX-G1520 in preparation of a lipid-lowering, anti-inflammatory and / or antioxidant product, the preservation number of the Lactobacillus paracasei SX-G1520 is CCTCC (China Center for Type Culture Collection) No: M 2025032, and the Lactobacillus paracasei SX-G1520 is preserved in the China Center for Type Culture Collection on January 6, 2025. The Lactobacillus paracasei SX-G1520 strain provided by the invention has lipid-lowering and antioxidant functions, significantly reduces the content of intestinal neutrophils of a zebra fish inflammatory bowel disease model, shows a significant effect on relieving intestinal inflammation, is good in safety, and has good application prospects. And a new technical choice is provided for developing probiotic preparations for regulating fat metabolism, resisting oxidative stress and preventing and treating inflammatory bowel diseases.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a strain of Lactobacillus paracasei SX-G1520 and application thereof in preparing lipid-lowering, anti-inflammatory and / or anti-oxidation products. Background Art

[0002] Probiotics are a group of active microorganisms that are extremely beneficial to maintaining health. They are mainly distributed on the surface of the host's intestinal mucosa, promoting the efficient absorption of nutrients and maintaining the health of the intestinal ecology. Lacticaseibacillus paracasei ) is a member of the probiotic family, belonging to the genus Lactobacillus. It can ferment carbohydrates to produce metabolites such as organic acids. It is acid-resistant and bile-resistant and can maintain activity in the gastrointestinal environment. In recent years, the application of Lactobacillus paracasei in the fields of food, medicine, and health care has gradually attracted attention. For example, in fermented foods, it can improve the nutritional value and flavor of food. In the medical field, it can be used as a probiotic preparation to regulate intestinal flora and enhance immunity. However, the current research and application of Lactobacillus paracasei in lipid-lowering, anti-inflammatory and antioxidant aspects is still not in-depth and extensive enough, and needs further development and utilization.

[0003] Enteritis is an inflammatory lesion of the small intestine or colon caused by infection with bacteria, viruses, fungi or parasites. The intestinal barrier function is damaged, the community structure of intestinal microorganisms changes, and oxidative stress reactions are triggered. A large number of free radicals are produced to interfere with normal lipid metabolism and increase the risk of hyperlipidemia. Hyperlipidemia usually refers to elevated levels of cholesterol and / or triglycerides in serum, which may be accompanied by low levels of high-density lipoprotein (HDL). Hyperlipidemia may cause enteritis. The accumulation of excessive lipids in the blood will aggravate inflammation and oxidative stress in the body, thereby aggravating intestinal inflammation. Current treatment technologies still have certain shortcomings. For example, drug treatment may bring side effects, non-drug treatment requires long-term adherence by patients, and has limited effects on some patients. Therefore, the development of new probiotic therapies that are safe, controllable, effective, and have few side effects is of great significance for reducing oxidative stress, intestinal inflammation, and the prevention and treatment of hyperlipidemia. Summary of the invention

[0004] In order to solve the above problems existing in the prior art, the present invention provides a strain of Lactobacillus paracasei SX-G1520 and its application in preparing lipid-lowering, anti-inflammatory and / or antioxidant products.

[0005] First object of the present invention is to provide a kind of Lactobacillus paracasei ( Lacticaseibacillus paracasei )SX-G1520.

[0006] A second object of the present invention is to provide described Lactobacillus paracasei ( Lacticaseibacillus paracasei ) Application of SX-G1520 in the preparation of lipid-lowering products.

[0007] A third object of the present invention is to provide described Lactobacillus paracasei ( Lacticaseibacillus paracasei ) Application of SX-G1520 in the preparation of anti-inflammatory products.

[0008] The fourth object of the present invention is to provide described Lactobacillus paracasei ( Lacticaseibacillus paracasei )Application of SX-G1520 in the preparation of antioxidant products.

[0009] A fifth object of the present invention is to provide said Lactobacillus paracasei ( Lacticaseibacillus paracasei ) Application of SX-G1520 in the preparation of products having any two or three functions of lipid-lowering, anti-inflammatory or antioxidant.

[0010] The sixth object of the present invention is to provide described Lactobacillus paracasei ( Lacticaseibacillus paracasei ) Use of SX-G1520 in the preparation of products for preventing and / or treating intestinal inflammation.

[0011] The 7th object of the present invention is to provide described Lactobacillus paracasei ( Lacticaseibacillus paracasei ) Use of SX-G1520 in the preparation of products for preventing and / or treating hyperlipidemia.

[0012] The eighth object of the present invention is to provide a microbial agent.

[0013] In order to achieve the above object, the present invention is implemented by the following scheme: The present invention isolates a Lactobacillus paracasei strain from fresh feces of centenarians in Suixi County, Zhanjiang City, Guangdong Province. The strain has multiple functions of lipid-lowering, anti-inflammatory and antioxidant, has good adhesion ability, and can inhibit the aggregation of neutrophils in the intestine of a DSS zebrafish model.

[0014] Therefore, the present invention requests protection for the following contents: A Lactobacillus paracasei ( Lacticaseibacillus paracasei )SX-G1520, the Lactobacillus paracasei ( Lacticaseibacillus paracasei ) The deposit number of SX-G1520 is CCTCC No: M 2025032, and it was deposited in the China Center for Type Culture Collection on January 6, 2025. The deposit address is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, opposite the First Affiliated Primary School, China Center for Type Culture Collection, Postal Code: 430072, the deposit date is January 6, 2025, and the classification name is Lactobacillus paracasei ( Lacticaseibacillus paracasei ), the deposit number is CCTCC NO: M 2025032, the deposit name is Lacticaseibacillus paracasei SX-G1520.

[0015] The Lactobacillus paracasei ( Lacticaseibacillus paracasei ) Application of SX-G1520 in the preparation of lipid-lowering products. The lipid-lowering involved in the present invention refers to lowering blood lipid levels, and the Lactobacillus paracasei ( Lacticaseibacillus paracasei )SX-G1520 is used for, including but not limited to, lowering triglyceride levels in the body or blood.

[0016] The Lactobacillus paracasei ( Lacticaseibacillus paracasei )Application of SX-G1520 in the preparation of triglyceride-degrading products.

[0017] The Lactobacillus paracasei ( Lacticaseibacillus paracasei ) Application of SX-G1520 in the preparation of anti-inflammatory products.

[0018] The Lactobacillus paracasei ( Lacticaseibacillus paracasei )Application of SX-G1520 in the preparation of antioxidant products.

[0019] The Lactobacillus paracasei ( Lacticaseibacillus paracasei ) Application of SX-G1520 in the preparation of a product for removing free radicals. The Lactobacillus paracasei described in the application ( Lacticaseibacillus paracasei ) The free radicals that SX-G1520 can scavenge include but are not limited to DPPH and hydroxyl radicals.

[0020] The Lactobacillus paracasei ( Lacticaseibacillus paracasei ) Application of SX-G1520 in the preparation of products having any two or three functions of lipid-lowering, anti-inflammatory or antioxidant.

[0021] The Lactobacillus paracasei ( Lacticaseibacillus paracasei ) Use of SX-G1520 in the preparation of products for preventing and / or treating intestinal inflammation.

[0022] The Lactobacillus paracasei ( Lacticaseibacillus paracasei ) Use of SX-G1520 in the preparation of products for preventing and / or treating hyperlipidemia.

[0023] The Lactobacillus paracasei ( Lacticaseibacillus paracasei ) Use of SX-G1520 in the preparation of products for preventing and / or treating intestinal inflammation.

[0024] The Lactobacillus paracasei ( Lacticaseibacillus paracasei ) Use of SX-G1520 in the preparation of products for preventing and / or treating hyperlipidemia and intestinal inflammation.

[0025] Preferably, the Lactobacillus paracasei ( Lacticaseibacillus paracasei ) The 16S rDNA sequence of SX-G1520 is shown in SEQ ID NO: 3.

[0026] Preferably, the Lactobacillus paracasei ( Lacticaseibacillus paracasei )SX-G1520 includes the Lactobacillus paracasei ( Lacticaseibacillus paracasei ) at least one of the live bacteria, culture solution or culture supernatant of SX-G1520.

[0027] More preferably, it also includes the Lactobacillus paracasei ( Lacticaseibacillus paracasei ) Lyophilized powder of SX-G1520 bacteria.

[0028] More preferably, it also includes the Lactobacillus paracasei ( Lacticaseibacillus paracasei ) Lyophilized powder of culture supernatant of SX-G1520.

[0029] More preferably, it also includes the Lactobacillus paracasei ( Lacticaseibacillus paracasei ) Bacterial suspension of SX-G1520.

[0030] A microbial agent comprising the Lactobacillus paracasei ( Lacticaseibacillus paracasei )SX-G1520.

[0031] Preferably, the Lactobacillus paracasei ( Lacticaseibacillus paracasei ) at least one of the live bacteria, culture solution or culture supernatant of SX-G1520.

[0032] More preferably, it also includes the Lactobacillus paracasei ( Lacticaseibacillus paracasei ) Lyophilized powder of SX-G1520 bacteria.

[0033] More preferably, it also includes the Lactobacillus paracasei ( Lacticaseibacillus paracasei ) Lyophilized powder of culture supernatant of SX-G1520.

[0034] More preferably, it also includes the Lactobacillus paracasei ( Lacticaseibacillus paracasei ) Bacterial suspension of SX-G1520.

[0035] Preferably, the Lactobacillus paracasei ( Lacticaseibacillus paracasei ) SX-G1520 is 1×10 5 CFU / mL~1×10 8 CFU / mL.

[0036] More preferably, the Lactobacillus paracasei ( Lacticaseibacillus paracasei ) SX-G1520 is 1×10 6 CFU / mL.

[0037] Compared with the prior art, the present invention has the following beneficial effects: The Lactobacillus paracasei SX-G1520 strain provided by the present invention has lipid-lowering and anti-oxidation functions, significantly reduces the intestinal neutrophil content of the zebrafish inflammatory bowel disease model, shows a significant effect in alleviating intestinal inflammation, has good safety, and provides a new technical option for the development of probiotic preparations for regulating fat metabolism, resisting oxidative stress, and preventing and treating inflammatory bowel disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the colony morphology of Lactobacillus paracasei SX-G1520.

[0039] Figure 2 This is a microscopic morphological observation of Lactobacillus paracasei SX-G1520 after Gram staining, with a magnification of 10×100.

[0040] Figure 3 This is the comparison and identification result of the 16S rDNA sequence of Lactobacillus paracasei SX-G1520 at NCBI.

[0041] Figure 4 This is a statistical graph of the in vitro triglyceride degradation rate of Lactobacillus paracasei SX-G1520. The dotted line in the figure indicates that the triglyceride degradation rate is 50%.

[0042] Figure 5 This is a statistical graph of the in vitro DPPH free radical scavenging rate of Lactobacillus paracasei SX-G1520. The dotted line in the figure indicates that the DPPH free radical scavenging rate is 50%.

[0043] Figure 6 This is a statistical graph of the in vitro hydroxyl radical scavenging rate of Lactobacillus paracasei SX-G1520. The dotted line in the figure indicates that the hydroxyl radical scavenging rate is 50%.

[0044] Figure 7This is the effect of Lactobacillus paracasei SX-G1520 on the secretion level of proinflammatory cytokines in vitro. A is the statistical results of the IL-6 secretion level of PBMC in each group, and B is the statistical results of the TNF-α secretion level of PBMC in each group.

[0045] Figure 8 This is the Gram staining image of Lactobacillus paracasei SX-G1520 after adhesion to Caco2 cells, with a magnification of 10×40.

[0046] Fig. 9 The figure shows the therapeutic effect of freeze-dried powder of Lactobacillus paracasei SX-G1520 on the zebrafish IBD model. A is the fluorescence image of each group of zebrafish, and B is the statistical graph of the number of neutrophils in the intestine of each group of zebrafish.

[0047] Fig.10 The figure shows the therapeutic effect of the bacterial suspension of Lactobacillus paracasei SX-G1520 on the zebrafish IBD model. A is the fluorescence image of each group of zebrafish, and B is the statistical graph of the number of neutrophils in the intestine of each group of zebrafish. DETAILED DESCRIPTION

[0048] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0049] Example 1 Isolation, identification and preservation of Lactobacillus paracasei SX-G1520 1. Isolation of strains The stool samples of centenarians from Suixi County, Zhanjiang City, Guangdong Province were diluted in gradients and inoculated into MRS solid medium, cultured anaerobically at 37°C for 48 hours, and single colonies on the plate were streaked. The streaking culture was performed three times to obtain the purified monoclonal strain. The monoclonal strain obtained by three generations of streaking was inoculated into MRS liquid medium, cultured anaerobically at 37°C for 48 hours, and 30 v / v% glycerol was added. The bacterial liquid and 30% glycerol were mixed in equal volumes and stored in a -80°C refrigerator.

[0050] The method for streaking the strain plate is as follows: in the anaerobic workstation, after mixing the bacterial solution, immerse the disposable inoculation loop into the bottom of the bacterial solution and scrape it repeatedly three times to ensure that the bacterial solution is smeared on the inoculation loop, and use the inoculation loop to streak three areas on the MRS culture medium plate and record the label.

[0051] 2. Morphological identification of strains (1) Colony morphology observation The isolated monoclonal strain was streaked on a plate and its colony morphology was as follows: Figure 1 As shown, it is round, with neat edges, smooth surface and milky white color.

[0052] (2) Gram staining and microscopic morphological observation After the isolated monoclonal strains were subjected to Gram staining and examined under an oil immersion microscope, Gram-positive strains appeared purple and Gram-negative strains appeared red. Figure 2 As shown, the strain is purple and is a Gram-positive bacillus.

[0053] 3. Molecular biological identification of strains The isolated monoclonal strain was subjected to full-length 16S rDNA amplification by PCR, and the amplification primers used were 16SrDNA universal primers: 1492R (5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 1)) and 27F (5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 2)), and then the amplified product was subjected to electrophoresis, and the electrophoresis product was collected for sequencing, and the 16S rDNA sequence of the monoclonal strain was obtained as follows:

[0054] The 16S rDNA sequence of the monoclonal strain (SEQ ID NO: 3) was compared with the standard bacterial species sequence at NCBI for identification. Figure 3 As shown, the comparison results showed that the monoclonal strain was Lactobacillus paracasei, and was named Lactobacillus paracasei SX-G1520.

[0055] 4. Strain preservation The above-obtained Lactobacillus paracasei SX-G1520 has been deposited in the China Center for Type Culture Collection (CCTCC, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, opposite the First Affiliated Primary School, zip code: 430072), with a preservation date of January 6, 2025, and is classified and named Lactobacillus paracasei ( Lacticaseibacillus paracasei ), the deposit number is CCTCC NO: M 2025032, the deposit name is Lacticaseibacillus paracasei SX-G1520.

[0056] Example 2 In vitro functional assay of Lactobacillus paracasei SX-G1520 1. Preparation of bacterial suspension, culture supernatant and lyophilized powder of Lactobacillus paracasei SX-G1520 The Lactobacillus paracasei SX-G1520 obtained in Example 1 was resuspended in MRS medium and cultured anaerobically at 37°C until the OD600 value of the bacterial solution was 1.6 as measured by an ultraviolet spectrophotometer. The bacterial solution was then inoculated into the autoclaved MRS medium at a ratio of 2 v / v% and cultured anaerobically at 37°C for 24 h. The cultured bacterial solution was collected and centrifuged at 8000 rpm / min for 8 min, the supernatant was collected and filtered through 0.22 The method comprises the following steps: filtering the culture supernatant of Lactobacillus paracasei SX-G1520 with a microporous membrane of 0.1 μm to obtain the culture supernatant of Lactobacillus paracasei SX-G1520, and then placing the culture supernatant of Lactobacillus paracasei SX-G1520 in a freeze dryer for freeze drying for 72 hours to obtain freeze-dried powder of the culture supernatant of Lactobacillus paracasei SX-G1520; collecting the bacterial precipitate after centrifugation, resuspending it with PBS having an equal volume to the bacterial solution before centrifugation to obtain a bacterial suspension of Lactobacillus paracasei SX-G1520, and then placing the culture supernatant of Lactobacillus paracasei in a freeze dryer for freeze drying for 72 hours to obtain freeze-dried powder of Lactobacillus paracasei SX-G1520.

[0057] 2. Determination of lipid-lowering ability (1) Experimental methods 10 mg of the lyophilized powder of Lactobacillus paracasei SX-G1520 prepared in this example was added to 1 mL of MRS liquid culture medium containing 1 g / L triglyceride as the experimental group, and anaerobically cultured at 37°C, and the cultured bacterial liquid was collected at 0 h and 48 h of culture as the test samples. 10 mg of distilled water was added to 1 mL of MRS liquid culture medium containing 1 g / L triglyceride as the blank group, and cultured and sampled in the same manner.

[0058] The triglyceride (TG) test kit (Nanjing Jiancheng Biological, A110-1-1) was used to determine the triglyceride content (C, mmol / L) in the test samples. The specific steps are as follows: first, the culture solution was centrifuged at 10000 rpm / min for 10 minutes, and the supernatant after centrifugation was collected as the sample to be tested; 2.5 μL of distilled water was added to 250 μL of working solution as a blank well; 2.5 μL of calibration substance (triglyceride, 2.68 mmol / L) was added to 250 μL of working solution as a calibration well; 2.5 μL of the sample to be tested was added to 250 μL of working solution as a sample well; after the solution in each well was fully mixed, 100 μL of the solution was pipetted into each 96-well plate, and then incubated at 37°C for 10 minutes, and the OD value at a wavelength of 500 nm was read by an enzyme reader.

[0059] (2) Result determination Formula (1): C (mmol / L) = [(A 样本 -A 空白 ) / (A 标准 -A 空白 )]×C 标准 ; Formula (2): Triglyceride degradation rate % = (C0-C 48 ) / C0×100%; Among them, A 样本 , A 标准 and A 空白 These are the OD values ​​of the sample well, calibration well, and blank well in this experiment. 500 Value; C 标准 is the concentration of the calibration substance, 2.68mmol / L; C0 is the triglyceride content in the culture solution at 0h of culture; C 48 It is the triglyceride content in the culture medium after 48 hours of cultivation.

[0060] According to the OD values ​​of the culture solution at 0h and 48h, the triglyceride content (C, mmol / L) was calculated using formula (1), and the triglyceride degradation rate (%) was calculated using formula (2). A triglyceride degradation rate of more than 50% was considered to be good in the ability to reduce triglycerides.

[0061] (3) Experimental results like Figure 4 As shown, compared with the blank group, the triglyceride degradation rate in the culture solution with Lactobacillus paracasei SX-G1520 was 62.96%, which was higher than 50%, indicating that Lactobacillus paracasei SX-G1520 had a good lipid-lowering ability.

[0062] 3. Determination of antioxidant capacity (1) DPPH (1,1-Diphenyl-2-picrylhydrazyl radical) free radical scavenging rate experiment 1) Experimental methods The DPPH free radical scavenging ability kit (Nanjing Jiancheng Biology, A153-1-1) was used to detect the DPPH free radical scavenging ability of the bacterial cell freeze-dried powder and the culture supernatant freeze-dried powder of Lactobacillus paracasei SX-G1520 prepared in this example, and the specific method is as follows: 400 μL of 80 v / v% methanol solution and 600 μL of working solution were added to 2 mL sterile EP tube 1 as a blank tube; the culture supernatant freeze-dried powder or bacterial freeze-dried powder of Lactobacillus paracasei SX-G1520 obtained in this example was dissolved with sterile water to obtain a sample to be tested with a concentration of 10 mg / mL, and 400 μL of the sample to be tested and 600 μL of 80 v / v% methanol solution were added to 2 mL sterile EP tube 2 as a control tube for the sample group; 400 μL of the sample to be tested and 600 μL of working solution were added to 2 mL sterile EP tube 3 as a measuring tube for the sample group; 400 μL of 1×PBS and 600 μL of working solution were added to 2 mL sterile EP tube 4 as a measuring tube for the blank group; 400 μL of 1×PBS and 600 μL of 80 v / v% methanol solution were added to 2 mL sterile EP tube 5 as a control tube for the blank group.

[0063] Mix the solutions in the above sterile EP tubes 1 to 5, keep them at room temperature 25°C in the dark for 30 min, centrifuge them at 4000 rpm for 5 min, pipette 800 μL of the supernatant after centrifugation into a cuvette, adjust to zero with 80% methanol, and read the OD value at a wavelength of 517 nm using a visible light spectrophotometer.

[0064] 2) Result determination Formula (3): DPPH free radical scavenging rate (%) = (1-(A 测定 -A 对照 )÷A 空白 )×100%; Among them, A 对照 , A 测定 and A 空白 are the OD values ​​of the control tube, the assay tube, and the blank tube, respectively. 517 Value. According to OD 517The DPPH free radical scavenging rate of the test tube was calculated using formula (3). If the DPPH free radical scavenging rate was above 50%, it was considered that the strain had good antioxidant capacity.

[0065] 3) Experimental results like Figure 5 As shown, compared with the blank group, the DPPH radical scavenging rates of the cell freeze-dried powder and culture supernatant freeze-dried powder of Lactobacillus paracasei SX-G1520 were 65% and 64%, respectively, both higher than 50%, indicating that Lactobacillus paracasei SX-G1520 has good antioxidant capacity.

[0066] (2) Hydroxyl radical (OH - ) Clearance rate experiment 1) Experimental methods Using hydroxyl radicals (OH - ) assay kit (Nanjing Jiancheng Biological, A018-1-1) was used to detect the hydroxyl radical scavenging ability of Lactobacillus paracasei SX-G1520. The specific method is as follows: Add 400 μL of distilled water to a 5 mL sterile centrifuge tube 1 as a blank well; add 200 μL of distilled water and 200 μL of 0.03% H2O2 standard application solution to a 5 mL sterile centrifuge tube 2 as a standard well; add 200 μL of distilled water and 200 μL of substrate application solution to a 5 mL sterile centrifuge tube 3 as a control well; dissolve the culture supernatant freeze-dried powder or bacterial freeze-dried powder of Lactobacillus paracasei SX-G1520 obtained in this example with sterile water to obtain a sample to be tested with a concentration of 10 mg / mL, and add 200 μL of substrate application solution and 200 μL of the sample to be tested to a 5 mL sterile centrifuge tube 4 as a sample well.

[0067] Add 400 μL of reagent three application solution to each of the above sterile centrifuge tubes 1 to 4, mix quickly, start timing at the same time, react at 37°C for 1 minute, then immediately add 2 mL of color developer to terminate the reaction, mix well, let stand at room temperature for 20 minutes, then pipette 100 μL of the solution into a 96-well plate, and read the OD value of each well at a wavelength of 550 nm using an enzyme reader.

[0068] 2) Result determination Formula (4): Hydroxyl free radical scavenging rate (%) = (A' 对照 -A' 测定 ) / (A' 标准 -A' 空白 )×100%; Among them, A' 对照 , A' 测定 , A' 标准 and A' 空白 are the OD values ​​of the control well, sample well, standard well, and blank well in this experiment. 550 value.

[0069] According to OD 550 The hydroxyl radical scavenging rate of the sample well was calculated using formula (4). A hydroxyl radical scavenging rate of more than 50% was considered to have good antioxidant capacity.

[0070] 3) Experimental results like Figure 6 As shown in the figure, compared with the blank group, the hydroxyl radical scavenging rates of the freeze-dried powder of Lactobacillus paracasei SX-G1520 and the freeze-dried powder of the culture supernatant were 69% and 68%, respectively, both higher than 50%, indicating that they have good antioxidant capacity.

[0071] 4. Determination of anti-inflammatory ability (1) Experimental methods The Lactobacillus paracasei SX-G1520 obtained in Example 1 was resuspended in MRS medium and anaerobically cultured at 37°C until the OD600 value of the bacterial solution was 1.6 as measured by an ultraviolet spectrophotometer. The bacterial solution was then inoculated into an autoclaved MRS medium at a ratio of 2 v / v% and anaerobically cultured at 37°C for 24 h. The bacterial precipitate after centrifugation was collected and resuspended in RPMI-1640 medium (containing 10% fetal bovine serum) to obtain a SX-G1520 bacterial suspension, and the concentration of the SX-G1520 bacterial suspension was adjusted to 1×10 8 CFU / mL.

[0072] Fresh blood was collected from healthy volunteers, and peripheral blood mononuclear cells (PBMCs) were separated using human peripheral blood lymphocyte separation medium (Solarbio, P8610 / P8900). After 4 h of static culture in a carbon dioxide incubator (37°C, 5% CO2), the cells were centrifuged at 1000 rpm for 5 min, and resuspended in RPMI-1640 medium (containing 10% fetal bovine serum) to obtain PBMC suspension, and the cell density was adjusted to 2×10 6 The PBMC suspension was added into a 96-well cell culture plate at 100 μL / well.

[0073] PBMC were divided into a blank control group and an experimental group, with 3 replicates in each group. The blank control group was added with 100 μL RPMI-1640 medium, and the experimental group was added with 100 μL SX-G1520 bacteria (the final concentration of Lactobacillus paracasei SX-G1520 was 5×10 7 CFU / mL), mixed well and placed in a carbon dioxide incubator (37°C, 5% CO2) for 72 hours. After the culture, centrifuge at 4000r / min for 5 minutes to collect the cell culture supernatant.

[0074] The concentrations of proinflammatory cytokines (interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α)) in the cell culture supernatants collected above were detected using Human IL-6 ELISA KIT (Ruixin Bio, RX106126H) and Human TNF-α ELISA KIT (Ruixin Bio, RX104793H).

[0075] (2) Data analysis: All data were statistically analyzed using GraphPad Prism 8, and experimental data were expressed as mean ± SEM. t-test analysis was used, and significant differences compared with the blank control group were expressed as: *P<0.05, **P<0.01, ***P<0.001.

[0076] Experimental results: like Figure 7 As shown in A and B, compared with the blank control group, the secretion levels of IL-6 and TNF-α in the experimental group were significantly reduced, indicating that Lactobacillus paracasei SX-G1520 has anti-inflammatory ability.

[0077] 5. Adhesion ability Adhesion ability is the first step for probiotics to colonize in the intestine. In probiotic research, adhesion ability is one of the important indicators for evaluating the potential of probiotics. Probiotics can competitively inhibit the adhesion of pathogens by adhering to intestinal epithelial cells, thereby exerting a probiotic effect. The Caco-2 cell model is a human cloned colon adenocarcinoma cell, which is similar in structure and function to differentiated small intestinal epithelial cells; the intestinal mucosal environment can be simulated by the Caco-2 cell monolayer to test the ability of probiotics to adhere to intestinal epithelial cells. This example uses a strain adhesion Caco-2 cell experiment to determine the adhesion ability of Lactobacillus paracasei SX-G1520.

[0078] (1) Experimental methods On the first day of the experiment, Caco-2 cells were cultured at 4×10 4 The cells were plated at a density of 100 μg / well in a 24-well plate and cultured in DMEM complete medium containing 10 v / v% fetal bovine serum (FBS). The next day, the culture medium was replaced with DMEM medium without double antibodies and cultured until polarized state. Then, the cells were incubated with a suspension of Lactobacillus paracasei SX-G1520 (0.5 mL, 10 8 CFU / mL) for 2 h. Gram staining was then used to observe whether the bacteria adhered to the Caco-2 cells.

[0079] At the same time, the bacterial solution before and after co-culture was diluted in a gradient manner and then plated on a bacterial plate to calculate the bacterial adhesion rate. The adhesion rate calculation method is as follows: Adhesion rate (%) = (bacterial concentration after adhesion / bacterial concentration before adhesion) × 100%.

[0080] (2) Experimental results According to statistics, the bacterial concentration of Lactobacillus paracasei SX-G1520 before adhering to Caco-2 cells was 5.50×10 8 CFU / mL, the bacterial concentration after adhering to Caco-2 cells was 2.30×10 8 CFU / mL, so the adhesion rate of Paracheese rod SX-G1520 to Caco-2 cells was 41.8%.

[0081] like Figure 8 As shown, Lactobacillus paracasei SX-G1520 can be seen on the edge and surface of Caco-2 cells, indicating that Lactobacillus paracasei SX-G1520 has the ability to adhere to intestinal epithelial cells, which is consistent with the adhesion rate results.

[0082] The above results show that the Lactobacillus paracasei SX-G1520 of the present invention has excellent lipid-lowering, antioxidant and adhesion abilities.

[0083] Example 3 In vivo functional assay of Lactobacillus paracasei SX-G1520 1. Preparation of freeze-dried powder and bacterial suspension of Lactobacillus paracasei SX-G1520 The Lactobacillus paracasei SX-G1520 obtained in Example 1 was resuspended in MRS medium and cultured anaerobically at 37°C until the OD value of the bacterial solution was measured by an ultraviolet spectrophotometer. 600 The value was 1.6, and then the bacterial liquid was inoculated into the high-pressure sterilized MRS liquid culture medium at a ratio of 2v / v%, and anaerobically cultured at 37℃ for 24h; the cultured bacterial liquid was collected and centrifuged at 8000 r / min for 8 min, and the supernatant after centrifugation was collected and filtered through a 0.22 µm microporous filter membrane to obtain the culture supernatant of Lactobacillus paracasei SX-G1520, and then the culture supernatant of Lactobacillus crispatus SX-E770 was placed in a freeze dryer and freeze-dried for 72h to obtain the culture supernatant of Lactobacillus paracasei SX-G1520 lyophilized powder, which was dissolved in E3 culture water before use and adjusted to a concentration of 0.125mg / mL to obtain a lyophilized powder solution; the bacterial precipitate after centrifugation was collected, resuspended in PBS, and the bacterial liquid concentration was adjusted to 1×10 6 CFU / mL to obtain bacterial suspension.

[0084] 2. Construction of DSS-induced zebrafish (Inflammatory Bowel Disease, IBD) model and freeze-dried powder treatment (1) Construction of DSS-induced zebrafish IBD model 3dpf transgenic Tg(mpx:EGFP) zebrafish were placed in 6-well cell culture plates and randomly divided into 4 groups, namely normal group, model group, positive group and freeze-dried powder group, with 1 well in each group and 25 fish in each well.

[0085] Table 1E3 Aquaculture water formula (1L)

[0086] E3 culture water was prepared according to the formula shown in Table 1. 4 mL of E3 culture water was added to the normal group, and 4 mL of 0.5% dextran sulfate sodium salt (DSS) was added to the other three groups to construct an intestinal inflammation model, and the cells were incubated in a 28.5°C incubator for 3 days.

[0087] (2) Treatment of DSS-induced zebrafish IBD model After the model was constructed, 4 mL of E3 aquaculture water was added to the normal group and the model group, respectively; 4 mL of 50 μg / mL mesalazine solution was added to the positive group; 4 mL of 0.125 mg / mL lyophilized powder solution was added to the lyophilized powder group; and the mice were placed in a 28.5°C incubator for 2 days.

[0088] (3) Determination of neutrophil content in zebrafish After the intervention, the solution in each well was discarded, and the zebrafish in each group were washed twice with E3 culture water. The zebrafish were then placed under an inverted fluorescence microscope for observation and photography, and the number of neutrophils in the intestine was counted.

[0089] (4) Data statistics The experimental data were expressed as mean±SEM and analyzed by t-test. Compared with the normal group: ###P<0.001; compared with the model group: *P<0.05, **P<0.01, ***P<0.001.

[0090] (5) Experimental results like Fig. 9 As shown in A and B, the content of neutrophils in the intestines of zebrafish in the model group was significantly higher than that in the normal group, indicating that the zebrafish IBD model was successfully constructed; the content of neutrophils in the intestines of zebrafish in the positive group was significantly lower than that in the model group, indicating that mesalazine effectively inhibited the aggregation of neutrophils in the zebrafish intestine; the content of neutrophils in the intestines of zebrafish in the freeze-dried powder group was significantly lower than that in the model group, indicating that the culture supernatant freeze-dried powder of Lactobacillus paracasei SX-G1520 can significantly inhibit the aggregation of neutrophils in the zebrafish intestine.

[0091] 3. Construction of DSS-induced zebrafish IBD model and bacterial suspension treatment (1) Construction of DSS-induced zebrafish IBD model 3 dpf transgenic Tg(mpx:EGFP) zebrafish were placed in a 6-well cell culture plate and randomly divided into 4 groups, namely normal group, model group, positive group and bacterial suspension group, with 1 well in each group and 25 fish in each well.

[0092] E3 culture water was prepared according to the formula shown in Table 1. 4 mL of E3 culture water was added to the normal group, and 4 mL of 0.5% DSS was added to the other three groups to construct an intestinal inflammation model, and the cells were incubated in a 28.5°C incubator for 3 days.

[0093] (2) Treatment of DSS-induced zebrafish IBD model After the model was constructed, 4 mL of E3 culture water was added to the normal group and the model group, respectively; 4 mL of 50 μg / mL mesalazine solution was added to the positive group, and 4 mL of bacterial suspension was added to the bacterial liquid group, and the cells were placed in a 28.5°C incubator for 2 days.

[0094] (3) Determination of neutrophil content in zebrafish After the incubation, the solution in each well was discarded, and the zebrafish in each group were washed twice with E3 culture water. The zebrafish were then placed under an inverted fluorescence microscope for observation and photography.

[0095] (4) Data statistics The experimental data were expressed as mean±SEM and analyzed by t-test. Compared with the normal group: ###P<0.001; compared with the model group: *P<0.05, **P<0.01, ***P<0.001.

[0096] (5) Experimental results like Fig.10 As shown in A and B, the content of neutrophils in the intestine of zebrafish in the model group was significantly higher than that in the normal group, indicating that the zebrafish IBD model was successfully constructed; the content of neutrophils in the intestine of zebrafish in the positive group was significantly lower than that in the model group, indicating that mesalazine effectively inhibited the aggregation of neutrophils in the zebrafish intestine; the content of neutrophils in the intestine of zebrafish in the bacterial suspension group was significantly lower than that in the model group, indicating that the bacterial suspension of Lactobacillus paracasei SX-G1520 can significantly inhibit the aggregation of neutrophils in the zebrafish intestine.

[0097] The above results show that the Lactobacillus paracasei SX-G1520 of the present invention has the effect of alleviating intestinal inflammation.

[0098] Example 4 Safety evaluation of Lactobacillus paracasei SX-G1520 1. Experimental methods Twelve male mice (strain ICR) were randomly divided into a saline group and a SX-G1520 group, with 6 mice in each group. The mice in the saline group were intragastrically administered with 0.1 mL / 10 g of saline, and the mice in the SX-G1520 group were intragastrically administered with 0.1 mL / 10 g of SX-G1520 bacterial solution (1×10 8 CFU / mL), intragastrically for 3 consecutive days. After 3 weeks of conventional feeding, the visceral organs (heart, liver, spleen, lung and kidney) of mice in each group were collected to calculate the organ index. The calculation formula was: weight of visceral organ / mouse body weight.

[0099] Twelve female mice were treated and their organ indexes were calculated using the above method.

[0100] 2. Experimental results Table 2 Statistical results of mouse organ index

[0101] As shown in Table 2, no significant difference was found in the organ index between the physiological saline group and the SX-G1520 group for both male and female mice, indicating that the Lactobacillus paracasei SX-G1520 of the present invention has substantially no negative impact on organs and has good safety.

[0102] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A Lactobacillus paracasei ( Lacticaseibacillus paracasei )SX-G1520, characterized by, The Lactobacillus paracasei ( Lacticaseibacillus paracasei ) The deposit number of SX-G1520 is CCTCC No: M2025032, and it was deposited in the China Center for Type Culture Collection on January 6, 2025.

2. Lactobacillus paracasei according to claim 1 ( Lacticaseibacillus paracasei ) Application of SX-G1520 in the preparation of lipid-lowering products.

3. Lactobacillus paracasei according to claim 1 ( Lacticaseibacillus paracasei ) Application of SX-G1520 in the preparation of anti-inflammatory products.

4. Lactobacillus paracasei according to claim 1 ( Lacticaseibacillus paracasei )Application of SX-G1520 in the preparation of antioxidant products.

5. Lactobacillus paracasei according to claim 1 ( Lacticaseibacillus paracasei ) Application of SX-G1520 in the preparation of products having any two or three functions of lipid-lowering, anti-inflammatory or antioxidant.

6. Lactobacillus paracasei according to claim 1 ( Lacticaseibacillus paracasei ) Use of SX-G1520 in the preparation of products for preventing and / or treating intestinal inflammation.

7. The Lactobacillus paracasei of claim 1 ( Lacticaseibacillus paracasei ) Use of SX-G1520 in the preparation of products for preventing and / or treating hyperlipidemia.

8. The use according to any one of claims 2 to 7, characterized in that: The Lactobacillus paracasei ( Lacticaseibacillus paracasei ) The 16S rDNA sequence of SX-G1520 is shown in SEQ ID NO:

3.

9. The use according to any one of claims 2 to 7, characterized in that: The Lactobacillus paracasei ( Lacticaseibacillus paracasei )SX-G1520 includes the Lactobacillus paracasei ( Lacticaseibacillus paracasei ) at least one of the live bacteria, culture solution or culture supernatant of SX-G1520.

10. A microbial agent, characterized in that: Containing the Lactobacillus paracasei described in claim 1 ( Lacticaseibacillus paracasei )SX-G1520.

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