Application of Lactobacillus paracasei in preparations for lowering serum cholesterol levels

Abstract

The invention belongs to the technical field of microbial application, and specifically relates to an application of Lactobacillus paracasei in a preparation for reducing serum cholesterol level. The Lactobacillus paracasei has a preservation number of CGMCC No. 24626, and is applied in the preparation for reducing serum cholesterol level, which can regulate serum cholesterol level by significantly reducing liver cholesterol level, promoting bile acid excretion and regulating intestinal flora disorder; and has no toxic side effects and high safety.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial application, and particularly relates to application of Lactobacillus paracasei in a preparation for reducing serum cholesterol level. Background Art

[0002] Cholesterol is the most abundant steroid compound in the body. It is not only a component of cell biological membranes, but also a precursor of steroid hormones, bile acids and vitamin D. For most tissues, it is very important to ensure the supply of cholesterol and maintain its metabolic balance. However, when cholesterol metabolism is unbalanced and the cholesterol level in serum is too high, it may cause intravascular deposition and damage the cardiovascular and cerebrovascular vessels, forming atherosclerosis and causing cardiovascular and cerebrovascular diseases. It is generally believed that the cholesterol content in serum is the main factor inducing cardiovascular and cerebrovascular diseases. In addition, studies have shown that in the Oriental population, for every 0.6mmol / L increase in serum total cholesterol, the risk of coronary heart disease will increase by 34%. Therefore, regulating serum cholesterol levels is of great significance to contemporary human health.

[0003] Existing studies have shown that some probiotics have the effect of lowering serum cholesterol levels; in addition, some studies have reported that the intestinal flora of people with elevated serum cholesterol levels is different from that of normal people, which means that some probiotics can lower serum cholesterol levels by regulating the intestinal flora.

[0004] Therefore, it is of great significance to discover a probiotic that can lower serum cholesterol levels. Summary of the invention

[0005] In view of the above problems, the present invention aims to provide an application of Lactobacillus paracasei in the preparation of a preparation for reducing serum cholesterol levels, wherein the Lactobacillus paracasei can significantly reduce the serum cholesterol level in the body.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] The invention provides, on one hand, an application of Lactobacillus paracasei in a preparation for reducing serum cholesterol level. The preservation number of Lactobacillus paracasei is CGMCC No.24626.

[0008] The preservation information of Lactobacillus paracasei in the present invention is as follows: preservation institution: General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC); preservation address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; preservation date: April 1, 2022; preservation number: CGMCC No. 24626; classification name: Lactobacillus paracasei.

[0009] The beneficial effects of the present invention include: the Lactobacillus paracasei in the present invention is used in the preparation of a preparation for reducing serum cholesterol levels, can significantly reduce liver cholesterol levels, promote bile acid excretion and regulate intestinal flora disorders, thereby regulating serum cholesterol levels; and has no toxic side effects and high safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 is the colony morphology of Lactobacillus paracasei TY-G05;

[0011] Figure 2 is the Gram stain of Lactobacillus paracasei TY-G05;

[0012] Figure 3 The graphs are the food intake curves of mice in each group;

[0013] Figure 4 The water drinking curve of mice in each group;

[0014] Figure 5 The weight curve of mice in each group;

[0015] Figure 6 is the cholesterol level in the serum of mice in each group;

[0016] Figure 7 is the cholesterol level in the liver of mice in each group;

[0017] Figure 8 is the bile acid level in the feces of mice in each group;

[0018] Fig. 9 is the chao index of the alpha diversity of the intestinal flora of each group of mice;

[0019] Fig.10 is the Shannon index of alpha diversity of intestinal flora of mice in each group;

[0020] Fig.11 is the beta diversity of intestinal flora of mice in each group;

[0021] Fig.12 The composition of the intestinal flora at the genus level of each group of mice;

[0022] Fig.13 is the abundance of Bifidobacterium in the intestinal flora of mice in each group;

[0023] Fig.14 is the abundance of Lactobacillus in the intestinal flora of mice in each group.

[0024] In the above figures, the experimental data are expressed as mean ± standard error (mean ± SEM); "*", "**", "***", and "****" indicate that there are statistical differences between the two groups, indicating p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively. DETAILED DESCRIPTION

[0025] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0026] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0027] The embodiment of the present invention provides an application of Lactobacillus paracasei in a preparation for reducing serum cholesterol levels, and the preservation number of Lactobacillus paracasei is CGMCC No. 24626. It should be noted that the strain of Lactobacillus paracasei is deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration; the preservation address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the preservation date is April 1, 2022, and its 16S rDNA sequence is shown in SEQ ID No. 1.

[0028] It should be noted that the Lactobacillus paracasei in the present invention does not specifically refer to live Bifidobacterium lactis, but also includes its inactivated bacteria form, lysate form, fermentation liquid form or metabolite form or a mixed form of the above forms. The specific form can be selected by those skilled in the art according to specific needs.

[0029] It should also be noted that the Lactobacillus paracasei in the present invention has strong tolerance to gastric juice and bile salts, with a survival rate of 82.19% in gastric juice and a growth efficiency of 16.47% in bile salts, indicating that it can function well in the gastric environment.

[0030] It should also be noted that, in some specific embodiments, the cholesterol level of the high-fat and high-cholesterol mouse model using the Lactobacillus paracasei of the present invention was reduced by about 27.2% compared with the cholesterol level of the high-fat and high-cholesterol mouse model not using Lactobacillus paracasei, that is, the Lactobacillus paracasei of the present invention is significantly effective in lowering serum cholesterol levels.

[0031] In some specific embodiments, the above application includes the application of one or more combinations of the following: (a) application of Lactobacillus paracasei in the preparation of a preparation for lowering liver cholesterol levels; (b) application of Lactobacillus paracasei in the preparation of a preparation for promoting bile acid excretion; (c) application of Lactobacillus paracasei in the preparation of a preparation for regulating intestinal flora disorders.

[0032] It should be noted that the liver is the main regulating organ that participates in cholesterol synthesis and decomposition, and 70%-80% of the cholesterol in the human body is synthesized by the liver, so the liver cholesterol level is measured and can reflect the function of liver lipid metabolism, that is, can control the cholesterol level in the serum by controlling the liver cholesterol level. In some specific embodiments, the liver cholesterol level of the high-fat high-cholesterol mouse model of the lactobacillus paracasei of the present invention has been used, and the liver cholesterol level of the high-fat high-cholesterol mouse model of the lactobacillus paracasei has been decreased by about 32.2%. It is shown that the lactobacillus paracasei of the present invention can effectively regulate the cholesterol level in the serum by regulating the liver cholesterol level, and the effect is remarkable.

[0033] It should also be noted that bile acid is the most important conversion product of cholesterol in animal bodies and the most important form of cholesterol excretion in the body. Promoting the excretion of bile acid to the outside is one of the feasible ways to reduce human serum cholesterol levels. Generally, the metabolic pool of human bile acid contains 3g-5g bile acid, but 98%-99% of the bile acid secreted into the intestinal tract is reabsorbed each time, and only 1%-2% of the bile acid is excreted to the outside with feces. The Lactobacillus paracasei in the present invention is colonized in the intestinal tract, which can effectively reduce the recovery of bile acid in the intestinal tract and increase the excretion of bile acid in feces, thereby reducing serum cholesterol levels. In some specific embodiments, the bile acid excretion level of the high-fat and high-cholesterol mouse model using the Lactobacillus paracasei in the present invention is about 22.3% higher than the bile acid excretion level of the high-fat and high-cholesterol mouse model without using Lactobacillus paracasei. It is shown that the Lactobacillus paracasei in the present invention can significantly promote the excretion of bile acid to reduce the cholesterol level in serum.

[0034] It should also be noted that the intestinal flora of people with elevated serum cholesterol levels is different from that of normal people, and regulating the intestinal flora is also one of the ways to lower serum cholesterol. In some specific embodiments, the use of Lactobacillus paracasei in the present invention can significantly improve the richness and diversity of the intestinal flora of a high-fat and high-cholesterol mouse model, thereby regulating the disorder of the intestinal flora and achieving the purpose of lowering the cholesterol level in serum.

[0035] In some specific embodiments, in the above-mentioned application, application (c) includes: application of Lactobacillus paracasei in the preparation of increasing the abundance of Bifidobacterium and / or Lactobacillus in the intestinal flora. It should be noted that Lactobacillus paracasei in the present invention can not only regulate the richness and diversity of the flora in the intestinal tract, but also increase the abundance of Bifidobacterium and / or Lactobacillus beneficial bacteria. In some specific embodiments, the abundance of Bifidobacterium in the high-fat and high-cholesterol mouse model using Lactobacillus paracasei is about 7 times the abundance of Bifidobacterium in the high-fat and high-cholesterol mouse model without Lactobacillus paracasei; The abundance of Lactobacillus in the high-fat and high-cholesterol mouse model using Lactobacillus paracasei is about 6 times the abundance of Lactobacillus in the high-fat and high-cholesterol mouse model without Lactobacillus paracasei.

[0036] In some specific embodiments, in the above application, the preparation includes a pharmaceutical preparation. It should be noted that the preparation in the present invention is not specifically a pharmaceutical preparation, that is, the Lactobacillus paracasei in the present invention can be added with a pharmaceutical carrier to prepare a pharmaceutical preparation, and the specific pharmaceutical preparation can be selected according to the specific clinical situation.

[0037] In some specific embodiments, in the above-mentioned application, the preparation includes solid dosage form, liquid dosage form, paste dosage form or emulsion dosage form. It should be noted that the lactobacillus paracasei in the present invention can be added with solid dosage form adjuvants to prepare solid dosage forms, such as tablets and pills, and the solid dosage form adjuvants are known to the art, generally including diluents (such as starch, dextrin, sucrose or mannose, etc.), absorbents (calcium sulfate, calcium hydrogen phosphate or light magnesium oxide, etc.), binders (polyvidone, syrup or hypromellose, etc.), wetting agents (water, etc.) or disintegrants (dry starch, sodium hydroxymethyl starch or cross-linked polyvinylpyrrolidone, etc.), etc.; in some specific embodiments, the solid dosage form can be a probiotic tablet, a probiotic pill or a probiotic granule; the same, the lactobacillus paracasei in the present invention can also be added with liquid dosage form adjuvants (such as a bulking agent, a suspending agent, an emulsifier or a coloring agent, etc.) to prepare a liquid dosage form; paste dosage form adjuvants can also be added to prepare a paste dosage form; emulsion dosage form adjuvants can also be added to prepare an emulsion dosage form. The specific dosage form can be selected by those skilled in the art according to specific needs.

[0038] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0039] It should be noted that in the following examples, the Lactobacillus paracasei used has a deposit number of CGMCC No. 24626, hereinafter also referred to as Lactobacillus paracasei TY-G05 or TY-G05.

[0040] Example 1 Isolation, purification and identification of Lactobacillus paracasei TY-G05

[0041] (1) Experimental Materials

[0042] Collect homemade Qula from herders in Qinghai Province: Use a sterile spoon to take homemade Qula from herders, put it into a 15mL sterile screw-cap centrifuge tube, tighten the cap, put it in a refrigerator, and transport it back to the laboratory for immediate purification and separation of lactic acid bacteria.

[0043] (2) Isolation and purification of TY-G05

[0044] Under sterile conditions, take 5g of Qula sample and add it to 45mL of sterilized skim milk, place it in a 37℃ incubator for enrichment and culture, and after the milk is coagulated, break the milk. Under sterile conditions, take 1mL of the broken milk sample and add it to 9mL of sterile saline, vortex it to mix it, and the result is 10 -1 The sample diluent was then diluted 10-fold to 10 -7 , select 10 -5 , 10 -6 and 10 -7100 μL of the dilution solution at the dilution degree was evenly spread on the MRS plate and inverted for incubation at 37°C for 48 hours. After the incubation, the morphology of the colonies on the MRS plate was observed, and medium-sized, raised, slightly white or slightly yellow, moist, with neat edges and round shapes of colonies were selected for plate streak method to purify the strains. The streak operation was repeated until the purified strains were obtained.

[0045] (3) Morphological and structural observation

[0046] The purified strain in (2) was inoculated into 5 mL of MRS liquid medium and cultured at 37°C for 18 h; 1 mL of bacterial solution was centrifuged at 12000 r / min for 1 min, washed twice with sterile saline, and then an equal volume of sterile saline was added to resuspend the bacteria. Finally, a small amount was taken with an inoculation loop and evenly spread on a glass slide. After fixation, Gram staining, microscopic examination, and photography were performed; Gram-positive bacteria (G + ) cells were blue-purple after staining, and Gram-negative bacteria (G - ) cells are red; observe and record the bacterial colony morphology and Gram staining results;

[0047] The colony morphology of the strains Figure 1 As shown, after the strain was purified, it formed single colonies in solid culture medium, and the colonies had the same morphology, were hemispherical, slightly white, with smooth and moist surfaces, and flat edges;

[0048] Gram staining results Figure 2 As shown, purple cells were observed under a microscope after Gram staining ( Figure 2 The original image is purple, and the shape is rod-shaped, which is determined to be Gram-positive bacteria (G + ).

[0049] (4) PCR amplification of 16S rDNA sequence

[0050] 16SrDNA gene amplification was performed using a 25 μL reaction system and the following PCR amplification program: 25 μL reaction system: 1 μL template, 1 μL upstream primer (sequence as shown in SEQ ID NO.2) (10 μM), 1 μL downstream primer (sequence as shown in SEQ ID NO.3) (10 μM), 12.5 μL 2×TaqPCRMasterMix, and the mixture was made up to 25 μL with sterile ultrapure water; PCR amplification program: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1 min, for a total of 35 cycles; 72°C end extension for 10 min; after sequence amplification, the qualified PCR amplification products were sequenced by Shanghai Sangon Biotechnology Co., Ltd., and the measured sequence was shown in SEQ ID No. 1; BLAST (http: / / www.ncbi.nlm.nih.gov / BLAST) was used to search and homology analysis in GenBank, and the homology analysis results showed that TY-G05 was Lactobacillus paracasei.

[0051] Example 2 Gastrointestinal survival rate verification of TY-G05

[0052] (1) Experimental Materials

[0053] Lactobacillus paracasei TY-G05: isolated from homemade Qu La by herders in Labei Mountain, Qinghai Province, and deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number CGMCC No.24626.

[0054] (2) Survival rate of TY-G05 in pH 3.0 artificial gastric juice

[0055] Probiotics that enter the human body must have good digestive tract tolerance in order to exert their corresponding functional activities. Before entering the human intestine to exert their functions, probiotics must first pass through the stomach (gastric juice pH is around 3.0, and the residence time is 1h-3h). The highly acidic stomach environment is not conducive to the survival of probiotics. Therefore, the first condition that probiotics should have is the ability to tolerate the effects of gastric juice in the human body.

[0056] TY-G05 was inoculated in MRS liquid culture medium at an inoculum rate of 2%, and after culturing at 37°C for 18h, 10mL was taken and centrifuged at a speed of 4000r / min for 10min, the bacterial precipitate was collected, washed twice with sterile saline, and resuspended in an equal volume of sterile saline to obtain a bacterial suspension; the prepared bacterial suspension was mixed with artificial gastric juice (0.2% NaCl and 0.35% pepsin 1:10000, pH=3.0 adjusted with 1mol / L HCl, filtered and sterilized for use) at a ratio of 1:9, and after mixing, placed in a constant temperature oscillator and cultured at 37°C at a speed of 100r / min for 3h. The number of viable bacteria at 0h and 3h was determined by the pour plate method; the survival rate of the strain tolerant to pH3.0 artificial gastric juice was calculated according to formula (1).

[0057]

[0058] The experimental and calculation results show that the survival rate of TY-G05 cultured in artificial gastric juice with pH = 3.0 for 3 hours is 82.19%, which has a high gastric juice survival rate.

[0059] (3) Growth efficiency of TY-G05 in 0.3% bile salt

[0060] After the probiotics that survive the gastric juice treatment enter the intestine, they will be inhibited and poisoned by the bile salts in the small intestine. Therefore, the strain's tolerance to bile salts is also one of the important indicators for probiotic screening. The mass concentration of bile salts in the human body fluctuates in the range of 0.03%-0.3%.

[0061] TY-G05 was inoculated in MRS liquid medium at 2% inoculation volume, and after culturing at 37°C for 18 hours, the culture medium was inoculated in MRS-THIO medium containing 0.0% and 0.3% pig bile salts (0.2% sodium thioglycolate was added to MRS liquid medium) at 2% inoculation volume, mixed and placed in a constant temperature oscillator, and cultured at 37°C at a speed of 100r / min for 24 hours. The uninoculated MRS-THIO medium was used as a blank control, and the OD of the culture medium containing 0.0% and 0.3% pig bile salts was measured respectively. 600 The growth efficiency of the strain in bile salt was calculated according to formula (2).

[0062]

[0063] Experimental and calculation results show that the growth efficiency of TY-G05 in a bile salt concentration of 0.3% is 16.47%, and it has good bile salt tolerance.

[0064] Example 3 Animal Test

[0065] (1) Experimental mouse grouping and intervention

[0066] Ten SPF wild-type male C57BL / 6 mice and ten SPF male ApoE - / - Twenty mice, all 8 weeks old, were housed in a standardized laboratory at room temperature of 25±2°C, relative humidity of 50±5%, and 12h light / 12h dark. The experiment began after one week of adaptive feeding. After the adaptation period, C57BL / 6 mice served as the blank group, and ApoE - / - The mice were randomly divided into a model group and a TY-G05 group, with 10 mice in each group.

[0067] The experimental period was 42 days, during which the mice in the blank group consumed ordinary feed, and the mice in the model group and TY-G05 group consumed high-fat and high-cholesterol feed (the specific formula is shown in Table 1). In addition, all groups of mice were gavaged daily, the blank group and the model group mice were gavaged with normal saline (200 μL), and the TY-G05 group was gavaged with the above bacterial suspension (200 μL), and the gavage time was kept consistent every day.

[0068] Table 1 High-fat and high-cholesterol feed formula

[0069] Element gram kilocalories Casein 200 800 L-Photoamine 3 12 Corn starch 212 848 Maltodextrin 71 284 sucrose 113 452 Fiber counter BW200 50 0 Soybean Oil 25 225 Cocoa Butter 155 1395 Mixed Minerals S10021 10 0 Calcium bicarbonate 13 0 Calcium carbonate 5.5 0 Potassium Citrate 16.5 0 Mixed Vitamin V10001 10 40 Choline bitartrate 2 0 cholesterol 11.25 0 Blue Dye 0.05 0 Yellow Dye 0.05 0 Red Dye 0 0 Total 897.35 4056

[0070] (2) Monitoring of mouse growth performance during the experiment

[0071] The mice were recorded for 42 days during the experimental period, with their weight recorded weekly and their food intake and water intake recorded daily (their food intake, water intake and weight changes can reflect their health status to a certain extent).

[0072] The statistical chart of the changes in food intake, water intake and body weight of mice during the experiment is as follows: Figure 3 , Figure 4 and Figure 5 As shown in the figure, during the whole experiment, the food intake and water intake of the model group and TY-G05 group mice were not significantly reduced compared with the normal group; in terms of body weight, the model group and TY-G05 group mice slightly gained weight compared with the normal group. The above results show that high-fat and high-cholesterol feed did not affect the normal feeding of mice, and Lactobacillus paracasei TY-G05 had no toxic side effects.

[0073] (3) Mouse sample collection and processing

[0074] The experimental period lasted for 42 days, after which the mice were sacrificed. The mice were fasted but not watered for 16 hours the day before being sacrificed, and the feces of the mice were collected in centrifuge tubes, immediately frozen in liquid nitrogen and stored in a -80°C refrigerator. The eyeballs were removed to collect blood, the blood was allowed to stand at 4°C for 1 hour, centrifuged at 3000r / min for 15 minutes, and the upper serum was carefully collected. The mice were dissected to separate the liver and intestinal tissues, immediately frozen in liquid nitrogen and stored in a -80°C refrigerator.

[0075] (4) Determination of serum cholesterol levels in mice

[0076] The serum refrigerated in the above (3) was taken out and the cholesterol level was detected using an automatic biochemical analyzer.

[0077] Test results such as Figure 6 As shown, the serum cholesterol level of mice in the blank group was 2.01mmol / L, and that of the model group was 27.76mmol / L. The serum cholesterol level of the model group was significantly higher than that of the blank group (p<0.0001), while the serum cholesterol level of mice in the TY-G05 group was 20.20mmol / L, which was significantly lower than that of the model group (reduced by about 27.2%) (p<0.0001). The above results show that Lactobacillus paracasei TY-G05 has the effect of regulating serum cholesterol levels.

[0078] (5) Determination of cholesterol levels in mouse liver

[0079] The liver refrigerated in the above (3) was taken out, 0.5 g was cut and accurately weighed, 5 mL of a mixture of methanol and chloroform (volume ratio 2:1) was added, and the liver tissue was fully ground using a tissue homogenizer (60 Hz, homogenization time 10 s / time, interval 30 s, 5 times in a row, at 4°C) to homogenize the liver tissue; after complete grinding, the ground mixture was transferred to a graduated test tube, and the grinder was rinsed with 4.5 mL of a mixture of methanol and chloroform (volume ratio 2:1), and the washing liquid was transferred to a graduated test tube together, the homogenate mixture collected twice before and after was fixed to 10 mL, placed in a water bath at 45°C for 1 hour, and then centrifuged at 8000g, 10 min, 4°C to collect the supernatant, and the cholesterol level was detected using an automatic biochemical analyzer.

[0080] Test results such as Figure 7 As shown, the liver cholesterol level of mice in the blank group was 2.24 μmol / g, and that of the model group was 7.70 μmol / g. The liver cholesterol level of the model group was significantly higher than that of the blank group (p < 0.0001), while the liver cholesterol level of mice in the TY-G05 group was 5.22 μmol / g, which was significantly lower than that of the model group (reduced by about 32.2%) (p < 0.0001). The above results show that Lactobacillus paracasei TY-G05 has the effect of regulating liver cholesterol levels.

[0081] (6) Determination of bile acid levels in mouse feces

[0082] The feces refrigerated in the above (3) were taken out, 0.5 g was cut and accurately weighed, 5 mL of anhydrous ethanol was added, and the feces were fully ground using a tissue homogenizer (60 Hz, homogenization time 10 s / time, interval 30 s, 5 times in a row, at 4°C) to homogenize the feces; the supernatant was collected by centrifugation at 8000 g, 10 min, 4°C, and the bile acid level was detected using an automatic biochemical analyzer.

[0083] Test results such as Figure 8 As shown, the fecal bile acid level of the blank group mice was 0.95 μmol / g, and that of the model group was 1.30 μmol / g. The fecal bile acid level of the model group was significantly higher than that of the blank group (p < 0.01), while the fecal bile acid level of the TY-G05 group mice was 1.59 μmol / g, which was significantly higher than that of the model group (increased by about 22.3%) (p < 0.05). The reason for the significant increase in the model group compared with the blank group was that the model group ingested high-cholesterol feed while the blank group ingested ordinary feed, and the cholesterol intake increased, so the bile acid excretion also increased; while the TY-G05 group also ingested high-cholesterol feed and the fecal bile acid level was further increased on the basis of the model group, indicating that TY-G05 can promote the discharge of bile acid under the same condition of exogenous cholesterol. The above results show that Lactobacillus paracasei TY-G05 has the effect of promoting the discharge of bile acid.

[0084] (7) Analysis of diversity of mouse fecal flora

[0085] The intestinal tissue refrigerated in the above (3) was taken out and the contents of the cecum were separated. The total DNA of the microbial community in the cecal contents was extracted according to the instructions of the soil DNA kit. The extraction quality of DNA was detected by 1% agarose gel electrophoresis, and NanoDrop 2000 was used to determine the DNA concentration and purity. 338F (5'-ACTCCTACGGGAGGCAGCAG-3' (SEQ ID NO.4)) and 806R (5'-GGACTACHVGGGTWTCTAAT-3' (SEQ ID NO.5)) were used to amplify the V3-V4 variable region of the 16S rRNA gene by PCR. The amplification procedure was as follows: 95°C pre-denaturation for 3 min, 25 cycles (95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 45 s), then 72°C stable extension for 10 min, and finally stored at 4°C (PCR instrument: ABI 9700); PCR reaction system: 5×TransStart FastPfu buffer 4 μL, 2.5 mM dNTPs 2 μL, upstream primer (5 μM) 0.8 μL, downstream primer (5 μM) 0.8 μL, TransStart FastPfu DNA polymerase 0.4 μL, template DNA 10 ng, ddH2O supplemented to 20 μL.

[0086] The PCR products of the same sample were mixed and recovered using 2% agarose gel. The recovered products were purified using AxyPrep DNA Gel Extraction Kit and detected by 2% agarose gel electrophoresis. TMFluorometer was used to detect and quantify the recovered products; NEXTflexTM Rapid DNA-Seq Kit was used to build the library: (1) Adapter linking; (2) Magnetic beads were used to screen and remove the self-linked fragments; (3) PCR amplification was used to enrich the library template; (4) Magnetic beads were used to recover the PCR products to obtain the final library. Sequencing was performed using Illumina's NovaSeq PE250 platform, and the sequencing was commissioned to Shanghai Meiji Biopharmaceutical Technology Co., Ltd.

[0087] Alpha diversity analysis is an important part of microbial diversity analysis, and is mainly used to study the diversity of a sample's flora. Chao index is one of the alpha diversity indices, which uses the Chao1 algorithm to estimate the number of OTUs contained in the sample, reflecting the richness of the community. Fig. 9 As shown in the figure, the Chao index of mice in the blank group was 458.1, and that of the model group was 107.9. The Chao index of mice in the model group was significantly lower than that of the blank group (p < 0.0001), while the Chao index of mice in the TY-G05 group was 154.8, which was significantly higher than that of the model group (p < 0.05). The Shannon index is an alpha diversity index that reflects the homogeneity of each species in the sample and reflects the diversity of the community. The detection calculation results are shown in the figure. Fig.10 As shown, the Shannon index of the blank group mice was 3.47, and that of the model group was 2.34, which was significantly lower in the model group than in the blank group (p < 0.0001), while the Chao index of the TY-G05 group mice was 2.67, which was significantly higher than that in the model group (p < 0.05). The above results indicate that the increase in serum cholesterol levels leads to a decrease in the richness and diversity of the intestinal flora of mice, while Lactobacillus paracasei TY-G05 restores the richness and diversity of the intestinal flora to a certain extent.

[0088] Beta diversity analysis is an important component of microbial diversity analysis, and is mainly used to study the microbial diversity between different samples. This analysis quantifies the difference in species abundance distribution between samples through statistical distance, uses statistical algorithms to calculate the distance between two samples, obtains a distance matrix, and performs visual statistical analysis on it. The present invention uses the Bray-Curtis algorithm, and the calculation results are as follows: Fig.11 As shown in the figure (the data points of different shapes in the figure represent the intestinal flora samples of each group of mice, and the closer the data points are, the more similar the intestinal flora of the two samples are), the blank group and the model group are far apart and there is no overlap at all, indicating that compared with the blank group, the intestinal flora composition of the model group mice has changed to a great extent, and the TY-G05 group is also separated by a certain distance from the model group, indicating that Lactobacillus paracasei TY-G05 has a certain degree of regulatory effect on the intestinal flora of mice.

[0089] The intestinal flora composition of each group of mice was further analyzed to explore the changes in the intestinal flora composition of mice treated with TY-G05. Fig.12 As shown in the figure, compared with the blank group, the intestinal flora composition of the model group mice changed significantly at the genus level, including an increase in the abundance of Allobaculum and a decrease in the abundance of Corynebacterium, while TY-G05 effectively inhibited this upward and downward trend. Fig.13 and Fig.14 As shown in the figure, TY-G05 increased the abundance of Bifidobacterium and Lactobacillus (p < 0.05), which are both recognized as bacteria that are beneficial to health, and the abundance of these two genera in the model group was lower than that in the blank group. The above results show that Lactobacillus paracasei TY-G05 has the effect of regulating intestinal flora.

[0090] The above results indicate that the Lactobacillus paracasei TY-G05 of the present invention can simultaneously reduce the cholesterol level in the liver, promote the excretion of bile acid and regulate the disorder of intestinal flora, thereby achieving the purpose of synergistically reducing the cholesterol level in the serum of the body.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. Lactobacillus paracasei (Lactobacillus paracasei) The application of Lactobacillus paracasei in the preparation of a pharmaceutical preparation for lowering serum cholesterol levels, the preservation number of which is CGMCC No. 24626.

2. The use according to claim 1, characterized in that: The application includes one or more of the following combinations: (a) application of Lactobacillus paracasei in preparing a pharmaceutical preparation for lowering liver cholesterol levels; (b) application of Lactobacillus paracasei in preparing a pharmaceutical preparation for promoting bile acid excretion; (c) application of Lactobacillus paracasei in preparing a pharmaceutical preparation for regulating intestinal flora disorders.

3. The use according to claim 2, characterized in that: Application (c) includes: Lactobacillus paracasei in the preparation of a method for improving the content of Bifidobacterium in intestinal flora (Bifidobacterium) and / or Lactobacillus spp. (Lactobacillus) Abundant applications in pharmaceutical preparations.

4. The use according to any one of claims 1 to 3, characterized in that: The pharmaceutical preparations include solid dosage forms, liquid dosage forms, paste dosage forms or emulsion dosage forms.

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