A strain of Lactobacillus paragaseri SYB-PG26 and its application

Abstract

The present invention relates to the field of microorganisms, and in particular to a strain of Lactobacillus paragasseri SYB-PG26, a bacterial agent containing the same, a composition and a use thereof. The Lactobacillus paragasseri SYB-PG26 of the present invention has strong uric acid-lowering ability, cholesterol-lowering ability and anti-inflammatory effect, and also has good acid resistance and bile salt resistance, and is expected to be developed and applied to the food or pharmaceutical industry.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to a strain of Lactobacillus paragasseri SYB-PG26, a bacterial agent and a composition containing the same, and applications thereof. Background Art

[0002] Breast milk is an essential source of nutrition for infants in their early stages of growth and plays a vital role in their growth and development. The presence of probiotics in breast milk has been confirmed by a large number of studies. Probiotics colonize in the human intestine and exert their probiotic properties to regulate the balance of intestinal flora, promote the body's absorption of nutrients, relieve inflammation, reduce the incidence of metabolic diseases, and improve the body's immunity.

[0003] The National Health Commission of China has announced that there are 14 strains of probiotics that can be used in infant food in China, excluding Lactobacillus paragaseri.

[0004] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0005] Purpose of the Invention

[0006] The present invention aims to provide a Lactobacillus paragasseri SYB-PG26 having strong uric acid-lowering ability, cholesterol-lowering ability and anti-inflammatory effect, a bacterial agent and a composition containing the same, and applications thereof.

[0007] Solution

[0008] To achieve the purpose of the present invention, the present invention adopts the following technical scheme.

[0009] In the first aspect, the present invention provides a strain of Lactobacillus paragasseri SYB-PG26, which is classified and named Lactobacillus paragasseri, and was deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee on March 22, 2024; the address of the depository unit is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China; the deposit number is CGMCC No.30095.

[0010] The Lactobacillus paragaseri SYB-PG26 of the present invention is isolated from a fresh breast milk sample. In vitro experiments have shown that the strain has the following properties or effects:

[0011] 1) The Lactobacillus paragaseri SYB-PG26 of the present invention has strong acid resistance and bile salt resistance;

[0012] 2) The Lactobacillus paragaseri SYB-PG26 of the present invention has a strong uric acid-lowering ability;

[0013] Specifically, the inventors have proved through in vitro experiments that the Lactobacillus paragastris SYB-PG26 of the present invention has the ability to degrade inosine and guanosine, and inosine and guanosine are intermediate products of purine metabolism, which are ultimately metabolized to uric acid in the human body. Increased in the content of inosine and guanosine will lead to increased uric acid production. Long-term high levels of uric acid will lead to hyperuricemia. In view of this, the strain of the present invention shows the potential for treating hyperuricemia.

[0014] Furthermore, the inventors applied the strain of the present invention to a hyperuricemia animal (i.e., bee) model to observe the uric acid-lowering effect of the strain. The results showed that the Lactobacillus paragastris SYB-PG26 of the present invention can significantly reduce the hemolymph uric acid concentration of bees, thereby alleviating hyperuricemia.

[0015] 3) The Lactobacillus paragaseri SYB-PG26 of the present invention has certain anti-inflammatory effects;

[0016] In the above-mentioned hyperuricemia animal model, the inventors also observed that the Lactobacillus paragaseri SYB-PG26 of the present invention can regulate immune pathways and reduce the expression of inflammatory factors; in addition, in the LPS inflammation model, the inventors observed that at appropriate concentrations, the Lactobacillus paragaseri SYB-PG26 of the present invention can reduce the secretion of inflammatory factors IL-6 and TNF-α induced by LPS, and increase the secretion of inflammatory factor IL-10; from the above, it can be seen that the Lactobacillus paragaseri SYB-PG26 of the present invention has a certain anti-inflammatory effect;

[0017] 4) The Lactobacillus paragaseri SYB-PG26 of the present invention has a strong cholesterol-lowering effect;

[0018] 5) The Lactobacillus paragaseri SYB-PG26 of the present invention can effectively treat constipation.

[0019] In a second aspect, the present invention provides a bacterial agent, wherein the active ingredient of the bacterial agent includes the Lactobacillus paragaseri SYB-PG26 described in the first aspect.

[0020] In a specific embodiment, the bacterial agent is a solid preparation or a liquid preparation; preferably, the solid bacterial agent is a powder prepared by freeze-drying.

[0021] In a third aspect, the present invention provides a composition comprising the Lactobacillus paragaseri SYB-PG26 as described in the first aspect, or comprising the bacterial agent as described in the second aspect.

[0022] In a preferred embodiment, the composition is a pharmaceutical composition, which comprises the Lactobacillus paragaseri SYB-PG26 as described in the first aspect or the bacterial agent as described in the second aspect, and a pharmaceutically acceptable carrier.

[0023] In a feasible embodiment, the pharmaceutically acceptable carrier includes, but is not limited to, one or more of a filler, a wetting agent, a disintegrant, a binder or a lubricant.

[0024] Preferably, the pharmaceutical composition is in oral dosage form.

[0025] Further preferably, the oral dosage form is selected from the group consisting of solutions, suspensions, emulsions, powders, lozenges, pills, syrups, buccal lozenges, tablets and capsules.

[0026] In a fourth aspect, the present invention provides use of the Lactobacillus paragaseri SYB-PG26 described in the first aspect, the bacterial agent described in the second aspect, or the composition described in the third aspect in the preparation of products for the following purposes:

[0027] 1) Lowering uric acid;

[0028] 2) Preventing and / or treating hyperuricemia, gout and their complications; optionally, the complications are gouty arthritis or gouty nephropathy;

[0029] 3) Lower cholesterol;

[0030] 4) Prevention and / or treatment of hypercholesterolemia;

[0031] 5) Anti-inflammatory;

[0032] 6) Prevent and / or treat constipation.

[0033] In view of the uric acid-lowering activity, cholesterol-lowering activity, anti-inflammatory activity and the efficacy of preventing and / or treating constipation of the strain of the present invention, the strain of the present invention and its derivative products can be used to prepare products with the functions of lowering uric acid, lowering cholesterol, anti-inflammatory and preventing and / or treating constipation; and, further, they can be used alone or in combination with other products with corresponding functions; these products can be prepared as microbial preparations or medicines.

[0034] In a possible embodiment, the product is a pharmaceutical product.

[0035] Beneficial Effects

[0036] The Lactobacillus paragaseri SYB-PG26 of the present invention is isolated from a fresh breast milk sample. Compared with the prior art, the Lactobacillus paragaseri SYB-PG26 has the following advantages:

[0037] 1) It has a strong ability to degrade inosine and guanosine, and can effectively reduce the level of hemolymph uric acid, thereby alleviating and treating hyperuricemia, and can be used to prevent, alleviate and / or treat hyperuricemia and / or gout and its complications;

[0038] 2) It has strong cholesterol-lowering ability and can be used to treat hypercholesterolemia;

[0039] 3) It has certain anti-inflammatory effects;

[0040] 4) It can effectively treat constipation;

[0041] 5) It also has good acid resistance and bile salt resistance.

[0042] In view of the above advantages, the Lactobacillus paragaseri SYB-PG26 of the present invention is expected to be developed and applied in the pharmaceutical industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] One or more embodiments are exemplarily described by the pictures in the accompanying drawings, and these exemplary descriptions do not constitute limitations on the embodiments. The special word "exemplary" here means "used as an example, embodiment or illustrative". Any embodiment described as "exemplary" here is not necessarily interpreted as being superior or better than other embodiments.

[0044] Figure 1 The results of the acid resistance test of Lactobacillus paragaseri SYB-PG26 of the present invention and various control strains are shown;

[0045] Figure 2 The bile salt tolerance test results of Lactobacillus paragaseri SYB-PG26 of the present invention and various control strains are shown;

[0046] Figure 3 The results of in vitro guanosine and inosine reduction ability assays of Lactobacillus paragaseri SYB-PG26 and various control strains are shown;

[0047] Figure 4 The results of the uric acid-lowering ability test of Lactobacillus paragaseri SYB-PG26 and various control strains on a hyperuricemia bee model are shown;

[0048] Figure 5The results show that the Lactobacillus paragaseri SYB-PG26 of the present invention and each control strain reduce the gene expression levels of inflammatory pathway related factors Wnt-Rho1 (Figure A), PGRP-LC (Figure B), Atg1 (Figure C), MAPK-phl (Figure D), and Toll (Figure E) in a hyperuricemia honey bee model;

[0049] Figure 6 The results of the cholesterol-lowering ability test of Lactobacillus paragaseri SYB-PG26 of the present invention and each control strain are shown; wherein, the AD graphs respectively show the results of the test of serum TC, TG, LDL-C, and HDL-C levels of each treatment group;

[0050] Figure 7 The results of the anti-inflammatory ability test of Lactobacillus paragaseri SYB-PG26 and each control strain in the LPS inflammation model are shown; wherein, Figures AC respectively show the results of the test of the levels of IL-6, TNF-α and IL-10 secreted by cells in each treatment group;

[0051] Figure 8 The effect of the Lactobacillus paragaseri SYB-PG26 of the present invention on the small intestinal motility (i.e., ink propulsion rate) of mice is shown;

[0052] Fig. 9 The figure shows the effect of Lactobacillus paragaseri SYB-PG26 of the present invention on the feces weight (Figure A) and the number of feces particles (Figure B) of mice.

[0053] The Lactobacillus paragasseri SYB-PG26 of the present invention is classified and named as Lactobacillus paragasseri, with a preservation date of March 22, 2024; the preservation unit is the General Microbiology Center of the China Culture Collection Administration; the preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China; the preservation number is CGMCC No.30095. DETAILED DESCRIPTION

[0054] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Unless otherwise expressly stated, throughout the specification and claims, the term "including" or its variations such as "comprising" or "including" will be understood to include the stated elements or components, without excluding other elements or other components.

[0055] In addition, in order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In some embodiments, raw materials, elements, methods, means, etc. well known to those skilled in the art are not described in detail, so as to highlight the main purpose of the present invention.

[0056] In the following examples, the sources of the biological materials or reagents used are as follows:

[0057] MRS solid medium was purchased from Beijing Luqiao Technology Co., Ltd., catalog number CM188;

[0058] MRS broth medium was purchased from Beijing Luqiao Technology Co., Ltd., catalog number CM187;

[0059] Nutrient agar was purchased from Beijing Luqiao Technology Co., Ltd., catalog number CM107;

[0060] Kovacs' indigo substrate reagent was purchased from Qingdao Hibo Biotechnology;

[0061] Inosine and guanosine standard solutions were purchased from Sigma with catalog numbers I4125 and G6752, respectively;

[0062] The control strains L. plantarum 299253, L. reuteri 254476, and L. gasseri 339385 were purchased from Beina Chuanglian Biotechnology Co., Ltd., L. reuteri DSM 17938 was purchased from Bio, and L. rhamnosus LGG was purchased from Kangcuile;

[0063] L. rhamnosus B2-2 and L. rhamnosus B2-3 are two strains previously screened from breast milk by the applicant and identified as Lactobacillus rhamnosus (the applicant declares that if someone needs to use this strain to verify the relative advantages of the strain of the present invention in the future, they can come to the applicant to conduct experiments);

[0064] B1-29 and B1-30 are two strains of Lactobacillus gasseri from human milk with better effects screened by the applicant, and R2 and R3 are two strains of Lactobacillus plantarum from human milk with better effects screened by the applicant; for details, please refer to the literature: Yin Chunmei, Li Zhen, Jiang Tiemin, et al. Screening and identification of lactic acid bacteria from human breast milk and preliminary determination of their ability to lower blood pressure [J]. Food Science and Technology, 2019(8):18-22;

[0065] Dextran sulfate sodium (DSS) was from MP Company, USA, with a molecular weight of 36000-50000Da;

[0066] Al(OH)3 was from Sigma, USA;

[0067] OVA ovalbumin was from Sigma, USA;

[0068] Cytokine ELISA detection kits were purchased from Shanghai ELISA Biotechnology Co., Ltd.

[0069] Example 1: Isolation and identification of Lactobacillus paragaseri SYB-PG26

[0070] Fresh breast milk samples were selected, gradiently diluted with sterilized PBS buffer (purchased from Guangzhou Jie Te Biofiltration Co., Ltd.), and then spread on MRS solid culture medium containing 1% CaCO3, and cultured in an anaerobic incubator at 37°C for 48 hours, and single white or milky white colonies with obvious transparent circles were picked for Gram staining and microscopic examination, and rod-shaped and Gram-positive bacterial strains were selected, and strains with better growth potential were selected from a large number of strains, and enriched and cultured in an anaerobic incubator at 37°C; the screened strains were tested for bile salt resistance, and a Lactobacillus gasseri with good acid and bile salt resistance was screened, which was named SYB-PG26; the SYB-PG26 bacterial solution was mixed with sterilized 30% glycerol in a ratio of 1:1 and stored in a refrigerator at -80°C for use.

[0071] The screened strain SYB-PG26 was subjected to 16S rDNA sequencing, and its 16S rDNA sequence is shown in SEQ ID NO: 1:

[0072]

[0073] The 16S rDNA sequencing results were compared with the NCBI database by BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and combined with its colony morphology, it was identified as Lactobacillus paragaseri.

[0074] Example 2: Determination of probiotic properties of Lactobacillus paragaseri SYB-PG26

[0075] (1) Acid resistance test

[0076] Under aseptic conditions, the strains to be tested (including Lactobacillus paragaseri SYB-PG26 of the present invention and L. plantarum 299253, L. reuteri 254476, L. gasseri 339385, L. reuteri DSM 17938, and L. rhamnosus LGG as controls) were subcultured for three generations in MRS liquid medium for activation, and the bacterial solution was diluted with empty medium until the viable count was 10 8 CFU / mL; transfer the activated bacterial liquid to a sterile centrifuge tube, centrifuge at 3000rpm for 10min to collect the bacteria, wash the collected bacteria twice with liquid MRS medium and resuspend them in MRS liquid medium with a pH of 3.0, and culture them anaerobically at 37°C. Sampling was performed at 0, 1, and 3h, and each sample was diluted 10-fold with sterile PBS buffer. 100μL of the diluted samples of each dilution were spread on MRS plates, and 2 parallels were made for each dilution. The plates were cultured at 37°C for 48h and then counted. The total colony count was determined according to the national standard GB4789.2-2016 and the survival rate of the strain was calculated.

[0077] Strain survival rate (%) = N1 / N0*100;

[0078] In the above formula: N0 is the number of viable bacteria (cfu / mL) at 0h; N1 is the number of viable bacteria (cfu / mL) after the strain has been acid-resistant for 1h.

[0079] The experimental results are as follows Figure 1 As shown, Figure 1 The results showed that after surviving for 1 hour and 3 hours under the condition of pH 3.0, the survival rates of Lactobacillus paragaseri SYB-PG26 from breast milk reached 70.59% and 160.78%, respectively, which were much higher than those of the control strain.

[0080] (2) Bile salt resistance test

[0081] Under aseptic conditions, the strains to be tested (including Lactobacillus paragaseri SYB-PG26 of the present invention and L. plantarum 299253, L. reuteri 2544746, L. gasseri 339385, L. reuteri DSM 17938, and L. rhamnosus LGG as controls) were subcultured for three generations in MRS liquid medium for activation, and the bacterial solution was diluted with empty medium until the viable count was 10 8 CFU / mL; transfer the activated bacterial liquid to a sterilized centrifuge tube, centrifuge at 3000rpm for 10min to collect the bacteria, wash the collected bacteria twice with liquid MRS medium, resuspend them in MRS broth medium containing 0.3% bile salts, and culture them anaerobically at 37°C. Take samples at 0 and 1h and perform 10-fold gradient dilutions. Take 100μL of the diluted samples of each dilution and spread them on MRS solid culture medium plates. Count them after anaerobic culture at 37°C for 48h; determine the total colony count and calculate the strain survival rate according to the national standard GB4789.2-2016.

[0082] Strain survival rate (%) = N1 / N0*100;

[0083] Where: N0 is the number of viable bacteria at 0h (cfu / mL); N1 is the number of viable bacteria after the strain has been resistant to bile salt for 1h (cfu / mL).

[0084] The experimental results are as follows Figure 2 As shown, Figure 2 The results showed that after surviving for 1 hour and 3 hours under the condition of bile salt concentration of 0.3%, the survival rates of Lactobacillus paragaseri SYB-PG26 from human milk reached 81.33% and 138.67%, respectively, which were much higher than those of the control strain.

[0085] Example 2: Determination of uric acid-lowering ability of Lactobacillus paragaseri SYB-PG26

[0086] 1. In vitro uric acid-lowering ability assay

[0087] The strains to be tested (including Lactobacillus gasseri SYB-PG26 of the present invention and L. gasseri B1-29, B1-30, L. plantarum R2, L. plantarum R3, L. rhamnosus B2-2, L. rhamnosus B2-3, and L. rhamnosus LGG as controls) are inoculated into MRS broth medium after being preserved with glycerol under sterile conditions, anaerobically cultured at 37° C. for 24 hours, and activated for three generations continuously. Then, the strains are inoculated into MRS broth medium, cultured at 37° C. for 18 hours for activation. The activated strains are subcultured twice with an inoculation amount of 3%, and then the strains are cultured to a stable period and taken out for use.

[0088] Take 2mL of the strain culture medium into a sterile centrifuge tube, centrifuge at 4°C and 6000r / min for 10min, discard the supernatant, and then wash the strain three times with sterile PBS phosphate buffer; add 1mL of 2.82mmol / L inosine-guanosine solution to the strain, mix well, incubate at 37°C and anaerobic conditions with shaking (200r / min) for 1h, and then centrifuge at 4°C and 8000r / min for 10min, draw 0.9mL of the supernatant and add 0.1mL of 0.1mol / L HClO4 and mix well to terminate the reaction, filter the reaction solution through a 0.22μm filter membrane, add it to a liquid phase detection bottle for liquid phase quantitative detection, and the sample injection volume is 20μL.

[0089] The experimental results are as follows Figure 3 As shown, Figure 3 The results show that the guanosine degradation rate and inosine degradation rate of the Lactobacillus paragaseri SYB-PG26 of the present invention are 99.69% and 99.95% respectively, which are significantly higher than those of the control strain.

[0090] 2. Determination of uric acid-lowering ability in a hyperuricemia animal model (bee model)

[0091] Honey bees (Apis mellifera) were purchased from China Agricultural University. This experiment was commissioned by Suzhou Fenghua Biotechnology Co., Ltd.

[0092] The experimental method is as follows:

[0093] 1) Experimental groups: blank group, model group and intervention group, 120 bees in each group;

[0094] 2) Hyperuricemia modeling and probiotic intervention;

[0095] In the blank group, 120 bees were fed with 50% sugar water + milk powder for 12 days; the other 240 bees were used to make a hyperuric acid model - they were fed with 50% sugar water + milk powder + 1% yeast-derived RNA for 7 days. The hemolymph uric acid value of the model group increased significantly, indicating that the hyperuric acid model was successfully established. The 240 successfully modeled bees were randomly divided into a modeling group and an intervention group (120 bees in each group). The bees in the modeling group and the intervention group continued to be fed with high uric acid modeling bee food. At the same time, the intervention group was fed with probiotic samples daily, supplemented at 1010 CFU / g (probiotic powder and pollen were added to the bee feed at a ratio of 1:20, and a new diet was replaced every 24 hours to maintain the activity of the probiotics). After 5 days of probiotic nutritional intervention, uric acid evaluation was performed, and the hemolymph uric acid levels and gene expressions of intestinal tissue inflammatory pathway-related factors (including Wnt-Rho1, PGRP-LC, Atg1, MAPK-phl, and Toll) were compared among the control group, model group, and intervention group.

[0096] The results of the hemolymph uric acid level determination of each group of bees are as follows Figure 4 As shown, Figure 4 The results showed that the hyperuric acid model group was significantly higher than the blank group (P < 0.01), indicating that the hyperuric acid model of honey bees was successfully established; after the intervention of Lactobacillus paragaseri SYB-PG26, the hemolymph uric acid concentration was significantly lower than that of the model group (P < 0.001); this shows that the Lactobacillus paragaseri SYB-PG26 of the present invention can significantly reduce the hemolymph uric acid level of hyperuric acid honey bees, and therefore has a strong uric acid-lowering ability.

[0097] mRNA was extracted from the intestinal tissue of honey bees and the gene expression levels of inflammation-related factors (including Wnt-Rho1, PGRP-LC, Atg1, MAPK-phl, and Toll) were detected by RT-PCR. Figure 5 As shown in the AE figure, the results show that the mRNA levels of inflammatory pathway-related factors in the high uric acid model group were significantly increased, but after intervention with Lactobacillus paragaseri SYB-PG2, the gene expression levels of these factors were significantly decreased. Explanation: Lactobacillus paragaseri SYB-PG2 can not only significantly reduce the hemolymph uric acid concentration of bees, but also regulate the immune pathway and reduce the expression of inflammatory factors, thus having a certain anti-inflammatory effect.

[0098] Example 3: Determination of cholesterol-lowering ability of Lactobacillus paragaseri SYB-PG26

[0099] Experimental animals: 60 SPF male SD rats weighing 180±20g bred by Beijing Weitonglihua Experimental Animal Technology Co., Ltd. were selected.

[0100] Cholesterol-lowering animal model feed: add 20.0% sucrose, 15.0% lard, 1.2% cholesterol, 0.2% sodium cholate, appropriate amount of casein, calcium hydrogen phosphate, stone powder, etc. to the maintenance feed; except crude fat, the moisture, crude protein, crude fat, crude fiber, crude ash, calcium, phosphorus, calcium: phosphorus of the model feed must meet the national standards for maintenance feed.

[0101] Experimental groups:

[0102] The blank control group, model control group, positive control group (Lactobacillus paracasei + Bifidobacterium breve, 1:1 mixture), low-, medium- and high-dose groups of Lactobacillus paracasei of the present invention (the low dose was 2.5×10 8 CFU / unit; medium dose 5×10 8 CFU / unit; high dose is 1.5×10 9 CFU / each).

[0103] Experimental methods:

[0104] SD rats were randomly divided into 2 groups according to body weight, 10 rats were given maintenance feed as blank control group, and 50 rats were given model feed to make cholesterol-lowering animal model. For cholesterol-lowering animal model, after giving model feed for 1-2 weeks, the serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL-C), and high-density lipoprotein (HDL-C) levels were measured. According to TC level, the cholesterol-lowering animal model was randomly divided into 5 groups (i.e., a model control group, a positive control group, and three doses of the strain group of the present invention). After grouping, the model control group, the positive control group, and the three doses of the strain group of the present invention were compared, and it was found that there was no significant difference in the TC, TG, LDL-C, and HDL-C levels among the groups, and the model control group, the positive control group, and the three doses of the strain group of the present invention were compared with the blank control group, and it was found that the increase of TC, TG, and LDL-C was significantly different, and the model was determined to be established.

[0105] After grouping, the three doses of the strain groups of the present invention were orally administered with the test samples every day, the blank control group and the model control group were simultaneously administered with the same volume of the corresponding solvent, and the positive control group was simultaneously administered with the same dose of a 1:1 mixture of Lactobacillus paracasei and Bifidobacterium breve; the blank control group continued to be administered with maintenance feed, the model control group, the positive control group and the three doses of the strain groups of the present invention continued to be administered with model feed, and their body weights were measured regularly, and blood was collected without fasting at the end of the experiment. Serum was separated as soon as possible after blood collection, and serum TC, TG, LDL-C, and HDL-C levels were determined by enzyme-linked immunosorbent assay (ELISA).

[0106] The experimental results are as follows Figure 6 As shown in the AD diagram in the figure, these results show that: by comparing the high-dose strain group of the present invention with the model control group, it can be seen that, except for high-density lipoprotein, the differences between the two are statistically significant; indicating that the Lactobacillus paragaseri SYB-PG26 of the present invention is effective in lowering cholesterol; and there is no statistical difference between the high-dose strain group of the present invention and the positive control group, indicating that: in terms of the cholesterol-lowering effect, the Lactobacillus paragaseri SYB-PG26 of the present invention and the positive control have equivalent efficacy.

[0107] Example 4: Determination of the anti-inflammatory ability of Lactobacillus paragaseri SYB-PG26

[0108] In this example, the LPS inflammation model was used to detect the anti-inflammatory effect of Lactobacillus paragaseri SYB-PG26 of the present invention.

[0109] Experimental steps:

[0110] 1. RAW 246.7 cell culture

[0111] Mouse mononuclear macrophage RAW264.7 cells were cultured in a medium containing 10% fetal bovine serum and 100 mg / L -1 Penicillin, 100 mg L -1 The cells were cultured in high-glucose DMEM medium containing streptomycin in a constant temperature incubator at 37°C and 5% CO2, and passaged every other day.

[0112] 2. Treatment of bacterial supernatant

[0113] After the bacteria were activated for 3 generations (i.e., passed to the 3rd generation), the turbidity was adjusted to 0.5 MCF (1 MCF = 3.0 × 10 8 CFU / mL), cultured at 37°C, 180r / min for 24h; then, centrifuged at 6000r / min for 10min, and the supernatant was collected; after filtering the supernatant with a bacterial filter (0.22μm), it was diluted 10 times, 100 times, and 1000 times for later use.

[0114] 3. Construction of LPS inflammation model & establishment of experimental groups

[0115] Set up blank group, control group and experimental group, and adjust the concentration of RAW 246.7 cells to 5×10 6 cell / mL, 1.6mL per well was inoculated in a 6-well plate, 400μL cell culture medium was added to the blank group, 200μL LPS solution + 200μL cell culture medium was added to the control group, and 200μL LPS solution + 200μL bacterial supernatant was added to the experimental group; LPS concentration was 1000ng / mL (reference patent CN201810390167.1), and the concentration of bacterial supernatant was the original concentration, diluted 10 times, 100 times, and 1000 times. Cultured at 37°C and 5% CO2 for 24h, the cell supernatant was collected for the determination of inflammatory cytokines and other related indicators.

[0116] 4. Detection of inflammatory factors

[0117] The levels of cytokines IL-6, IL-10, and TNF-α were measured according to the instructions of the ELISA kit (Shanghai ELISA Biotechnology Co., Ltd.).

[0118] The experimental results are as follows Figure 7 As shown in the AC diagram.

[0119] Figure 7In the figure, Figure A shows the effect of different concentrations of the supernatant of the strain of the present invention on the LPS-induced IL-6 secretion of RAW 246.7 cells, Figure B shows the effect of different concentrations of the supernatant of the strain of the present invention on the LPS-induced TNF-α secretion of RAW 246.7 cells, and Figure C shows the effect of different concentrations of the supernatant of the strain of the present invention on the LPS-induced IL-10 secretion of RAW 246.7 cells.

[0120] As shown in Figure A, compared with the control group, the original concentration of SYB-PG26 supernatant significantly reduced the LPS-induced IL-6 secretion of RAW246.7 cells (p < 0.01), and other concentrations of supernatant had no significant effect on the secretion of IL-6.

[0121] As shown in Figure B, compared with the control group, the SYB-PG26 supernatant diluted 100 times significantly reduced the secretion of TNF-α (p < 0.05), and the SYB-PG26 supernatant diluted 1000 times extremely significantly reduced the secretion of TNF-α (p < 0.01), and there was no significant difference in TNF-α between the two and the blank group.

[0122] As shown in Figure C, compared with the control group, the original concentration of SYB-PG26 supernatant significantly increased the secretion of IL-10 (p < 0.05), and the 10-fold diluted SYB-PG26 supernatant extremely significantly increased the secretion of IL-10 (p < 0.01).

[0123] The above results show that the Lactobacillus paragaseri SYB-PG26 of the present invention can significantly inhibit the secretion of inflammatory factors IL-6 and TNF-α by the inflammatory cell model, and promote the secretion of inflammatory factor IL-10 (IL-10 can inhibit the expression of inflammatory factors such as TNF, IL-6, and IL-1 through activated macrophages, therefore, the increased secretion of IL-10 can further inhibit inflammation). Therefore, the Lactobacillus paragaseri SYB-PG26 of the present invention has a certain anti-inflammatory effect.

[0124] Example 5: Efficacy determination of Lactobacillus paragaseri SYB-PG26 for treating constipation

[0125] The experimental instruments and reagents information are as follows:

[0126] Surgical scissors, ophthalmic forceps, ruler, syringe, balance, activated carbon powder, gum arabic 20200103;

[0127] MRS solid medium was purchased from Beijing Luqiao Technology Co., Ltd., catalog number CM188;

[0128] MRS broth medium was purchased from Beijing Luqiao Technology Co., Ltd., catalog number CM187;

[0129] Loperamide hydrochloride: Xi'an Janssen Pharmaceutical Co., Ltd., product number LFJ8473.

[0130] Experimental animals: 120 SPF male Kunming mice of Weitonglihua strain weighing 18-22 g were selected.

[0131] Positive control Lactobacillus paracasei: the deposit number is GDMCC No.60960, see CN114032193A (application number CN202111331927.X, Chinese patent application with the invention name “A Lactobacillus paracasei 207-27 and its application”).

[0132] Positive control Bifidobacterium breve: the deposit number is GDMCC No.60962, see CN111944727A (application number CN202010858666.6, Chinese patent application with the invention name “A Bifidobacterium breve 207-1 and its application”).

[0133] Mouse constipation experiment, the experimental groups are shown in Table 1.

[0134] Table 1 Experimental groups

[0135]

[0136]

[0137] (1) Small intestinal motility test:

[0138] After 7 days of adaptive feeding, mice in each group were fasted but not watered for 16 hours.

[0139] The model control group, positive control group and three doses of SYB-PG26 intervention groups were gavaged with loperamide hydrochloride (2-4 mg / kg BW), and the blank control group was given distilled water.

[0140] 0.5 hours after loperamide hydrochloride was administered, the positive control group and the three doses of SYB-PG26 intervention groups were given ink containing the corresponding test samples (containing 5% activated carbon powder and 10% gum arabic), and the blank control group and the model control group were given ink by gavage. The animals were housed in single cages and fed with normal water and food.

[0141] After 25 minutes, the animals were killed immediately, the abdominal cavity was opened and the mesentery was separated, the intestinal tube from the pylorus at the upper end to the ileocecal region at the lower end was cut, and placed on a tray. The small intestine was gently pulled into a straight line, and the length of the intestinal tube was measured as the "total length of the small intestine", and the length from the pylorus to the front of the ink was the "ink advancement length". The ink advancement rate was calculated according to the following formula:

[0142]

[0143] The results are as follows Figure 8 shown.

[0144] (2) Determination of defecation time, number of stool particles and stool weight:

[0145] After 7 days of adaptive feeding, mice in each group were fasted but not watered for 16 hours.

[0146] The model control group, positive control group and three doses of SYB-PG26 intervention groups were gavaged with loperamide hydrochloride (4-7 mg / kg BW), and the blank control group was given distilled water.

[0147] 0.5 hours after loperamide hydrochloride was administered, the positive control group and the three doses of SYB-PG26 intervention groups were given ink containing the corresponding test samples (containing 5% activated carbon powder and 10% gum arabic), and the blank control group and the model control group were given ink by gavage. The animals were housed in single cages and fed with normal water and food.

[0148] Starting from the injection of ink, the time of the first black stool of each animal, the number and weight of black stools within 5 or 6 hours were recorded.

[0149] The results are as follows Fig. 9 As shown in Figures A and B.

[0150] Depend on Figure 8 The ink advancement rate test results and Fig. 9 As shown in the stool weight (Figure A) and stool particle (Figure B) measurement results, there are statistical differences between the high-dose SYB-PG26 intervention group and the model control group; this shows that Lactobacillus paragasseri SYB-PG26 is effective in treating constipation; while there is no statistical difference between the high-dose SYB-PG26 intervention group and the positive control group, which shows that the constipation treatment efficacy of Lactobacillus paragasseri SYB-PG26 and the positive control group is equivalent.

[0151] 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 it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of Lactobacillus paragaseri ( Lactobacillus paragasseri )SYB-PG26, characterized in that, It is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit date being March 22, 2024 and the deposit number being CGMCC No.30095.

2. A bacterial agent, characterized in that The active ingredient of the bacterial agent includes the Lactobacillus paragaseri as claimed in claim 1 ( Lactobacillus paragasseri )SYB-PG26.

3. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the Lactobacillus paragaseri as claimed in claim 1 ( Lactobacillus paragasseri )SYB-PG26, or comprising the bacterial agent as claimed in claim 2.

4. The pharmaceutical composition according to claim 3, characterized in that The pharmaceutical composition comprises the Lactobacillus paragaseri as claimed in claim 1 ( Lactobacillus paragasseri )SYB-PG26 or the bacterial agent as described in claim 2, and a pharmaceutically acceptable carrier.

5. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition is in oral dosage form.

6. The pharmaceutical composition according to claim 5, characterized in that The oral dosage form is selected from the group consisting of solutions, suspensions, emulsions, powders, lozenges, pills, tablets and capsules.

7. The pharmaceutical composition according to claim 5, characterized in that The oral dosage form is selected from the group consisting of: syrup and buccal lozenge.

8. The Lactobacillus paragaseri according to claim 1 ( Lactobacillus paragasseri ) Use of SYB-PG26, the bacterial agent according to claim 2 and / or the pharmaceutical composition according to claim 3 in the preparation of medicines for the following purposes: 1) Lowering uric acid; 2) Prevention and / or treatment of hyperuricemia, gout and their complications; among them, The complication is gouty arthritis or gouty nephropathy.

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