Pediococcus pentosaceus with cholesterol lowering function and application thereof

Abstract

The invention relates to pediococcus pentosaceus with a cholesterol lowering function and application thereof, and belongs to the technical field of microorganisms. The invention discloses pediococcus pentosaceus with a cholesterol lowering function and application of the pediococcus pentosaceus. The pediococcus pentosaceus R5 (GDMCC No.65659) is separated from special fermented food stinky acid in Guizhou. The in-vitro cholesterol removal rate of the pediococcus pentosaceus R5 can reach 59%, and the pediococcus pentosaceus R5 has the characteristics of acid / cholate resistance, no hemolysis and sensitivity to common antibiotics; an in-vivo lipid-lowering experiment is carried out on a hyperlipidemia model mouse, and the result shows that the strain R5 can remarkably improve the in-vivo dyslipidemia state of the mouse, reduce liver fat accumulation and protect the liver at the same time, and can be used for developing lipid-lowering related probiotic functional products. The strain R5 provided by the invention provides a natural and safe probiotic for preventing and / or treating hyperlipemia.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology and relates to a Pediococcus pentosaceus strain with cholesterol-lowering function and its application. Background Art

[0002] In 2002, the American Heart Association pointed out that cardiovascular disease (CVD) is the most important cause of death in developed countries and is closely related to hypercholesterolemia. The risk of heart attack in patients with hypercholesterolemia is three times higher than that of patients with normal blood lipids. The World Health Organization predicts that by 2030, cardiovascular disease will still be the main cause threatening human death and will affect approximately 23.6 million people worldwide.

[0003] Statins used clinically reduce the endogenous synthesis of cholesterol by inhibiting the activity of hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase, thereby reducing serum cholesterol levels. The clinical drug ezetimibe inhibits NPC1L1 to reduce cholesterol absorption and transport; in addition, some traditional Chinese medicines also have a certain effect on blood lipid regulation. Among the currently commonly used drugs, statins have good clinical efficacy. Statins are HMG-CoA reductase inhibitors. Such drugs competitively inhibit the rate-limiting enzyme (HMG-CoA) reductase for endogenous cholesterol synthesis, block the mevalonic acid metabolic pathway in cells, reduce intracellular cholesterol synthesis, and thus feedback-stimulate an increase in the number and activity of low-density lipoprotein (LDL) receptors on the cell membrane surface (mainly hepatocytes), increase serum cholesterol clearance, and lower the level. In addition to their lipid-regulating effects, statins can inhibit the inflammatory response of vascular endothelium, stabilize atherosclerotic plaques, and improve vascular endothelial function when applied early in patients with acute coronary syndrome. They also have effects such as delaying the degree of atherosclerosis (AS), anti-inflammation, protecting nerves, and anti-thrombosis. However, the current manufacture of statins mainly relies on chemical synthesis, and synthetic drugs may have some adverse reactions, such as general body discomfort, fever; abdominal discomfort, belching, flatulence, hepatitis, cholestasis; skeletal muscle pain, muscle fatigue, neck pain, joint swelling, etc.

[0004] The search for safe and effective functional foods, such as probiotics with human health functions, is attracting increasing attention. Currently, screening functional probiotics with cholesterol-lowering effects in the human body has become a research hotspot.

[0005] Lactic acid bacteria are a class of generally recognized as safe (GRAS) food-grade microorganisms with a long history of safe consumption.

[0006] Probiotics have been widely used in Japanese foods, beverages, medicine, etc. The research on probiotics in places such as France, Russia, and Germany has been gradually deepening. Adding probiotics to fermented milk plays a certain health care role. The research and application of probiotics for cholesterol reduction in China started relatively late. At present, there is still a blank in domestic products of lactic acid bacteria for cholesterol reduction, and not many have been truly put into application. Most of the research on lactic acid bacteria for cholesterol reduction is only for in vitro research, and there is relatively little screening of lactic acid bacteria for comprehensive evaluation of probiotic characteristics, safety evaluation, and in vivo function evaluation. Currently, there are few strains developed and applied from the lactic acid bacteria strain resources in China. Therefore, further development is needed to screen functional lactic acid bacteria that can regulate human blood lipids. Summary of the Invention

[0007] The object of the present invention is to provide a Pediococcus pentosaceus with cholesterol-lowering function and its application. The Pediococcus pentosaceus R5 of the present invention is a strain of the genus Pediococcus pentosaceus deposited under the accession number GDMCC No. 65659. It is isolated from the Guizhou characteristic fermented food "Chou Suan" and has good blood lipid-lowering effects in animals and great potential in human blood lipid-lowering. Through screening, the present invention obtains a lactic acid bacteria strain with cholesterol-lowering function. By comparing the 16S rDNA sequences, the strain is identified as Pediococcus pentosaceus and named Pediococcus pentosaceus R5. It has been experimentally proven that the cholesterol removal ability of Pediococcus pentosaceus R5 in vitro is as high as 59.27%. At the same time, it has strong acid / bile salt tolerance, is easy to pass through the stomach and colonize in the intestine. It has no hemolytic property and is sensitive to common antibiotics. In vivo experiments prove that Pediococcus pentosaceus R5 can significantly improve the in vivo lipid abnormalities caused by hyperlipidemia in mice, significantly slow down weight gain and reduce the organ index. Compared with the drug simvastatin, it can achieve lipid-lowering effects while protecting the liver. It has great application potential and value in the development of functional foods for preventing and treating hyperlipidemia and other diseases.

[0008] The object of the present invention can be achieved by the following technical solutions:

[0009] A Pediococcus pentosaceus with cholesterol-lowering function, the taxonomic name of the Pediococcus pentosaceus is Pediococcus pentosaceus, the deposit number is GDMCC No. 65659, it is deposited in the Guangdong Provincial Microbial Culture Collection Center, the deposit date is December 23, 2024, and the deposit address is the 5th floor, Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou.

[0010] Furthermore, the Pediococcus pentosaceus is Pediococcus pentosaceus R5, its 16S rDNA sequence is SEQ ID NO.1, and its complete genome sequence is SEQ ID NO.2.

[0011] Furthermore, the applications of the Pediococcus pentosaceus with cholesterol-lowering function include probiotic agents, health products or functional foods, and fat-reducing products.

[0012] Furthermore, the health products or functional foods contain Pediococcus pentosaceus or probiotic agents.

[0013] Furthermore, the probiotic agent includes probiotic liquid or probiotic freeze-dried powder.

[0014] Furthermore, the fat-reducing effect of the fat-reducing product includes reducing the accumulation of body fat and lowering the content of indicators; the indicators in the lowering of indicator content include at least one of body weight, organ index, total cholesterol, total triglyceride, and low-density lipoprotein cholesterol.

[0015] Advantages of the present invention:

[0016] The Pediococcus pentosaceus R5 of the present invention is the R5 strain of the genus Pediococcus pentosaceus deposited under GDMCC No. 65659, isolated from the Guizhou characteristic fermented food Chou Suan, and has a good lipid-lowering effect in animals and great potential in lipid-lowering in humans. The present invention has screened a lactic acid bacterium with cholesterol-lowering function, and through 16S rDNA sequence alignment, the strain is identified as Pediococcus pentosaceus and named Pediococcus pentosaceus R5. It has been experimentally proven that the cholesterol removal ability of Pediococcus pentosaceus R5 in vitro is as high as 59.27%, and at the same time, it has strong acid / bile salt tolerance, is easy to pass through the stomach and colonize in the intestine. It has no hemolytic property and is sensitive to common antibiotics. In vivo experiments have shown that Pediococcus pentosaceus R5 can significantly improve the in vivo lipid abnormality caused by hyperlipidemia in mice, significantly slow down the weight gain and reduce the organ index, and can achieve lipid-lowering effect while protecting the liver compared with simvastatin drug. It has great application potential and value in the development of functional foods for preventing and treating hyperlipidemia and the like. Description of the Drawings

[0017] For the convenience of those skilled in the art to understand, the present invention will be further described below with reference to the accompanying drawings.

[0018] Figure 1 It is the colony and Gram staining diagram of Pediococcus pentosaceus R5;

[0019] Figure 2 It is the 16S rRNA phylogenetic tree of Pediococcus pentosaceus R5;

[0020] Figure 3 It is a graph showing the experimental results of the in vitro lipid-lowering ability of Pediococcus pentosaceus R5 within 24 h. Among them, (A) is the in vitro cholesterol-lowering level;

[0021] Figure 4 It is the blood lipid levels of mice with hyperlipidemia treated with Pediococcus pentosaceus R5. Among them, (A) is the TC level in serum, (B) is the TG level in serum, (C) is the LDL-C level in serum, (D) is the HDL-C level in serum, (E) is the AST level in the liver, and (F) is the ALT level in the liver. The data are expressed as the mean ± standard deviation of three trials. There are statistically significant differences when comparing the model group with the control group: *P < 0.05, **P < 0.01;

[0022] Figure 5 It is the result of H&E staining analysis of mouse liver. Detailed implementation manners

[0023] To further elaborate on the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the following, in combination with the accompanying drawings and preferred embodiments, details the specific implementation manners, structures, features, and their effects of the present invention as follows.

[0024] Unless otherwise specified, the test methods used in the following examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0025] Example 1: Isolation and identification of Pediococcus pentosaceus

[0026] (1) Material preparation

[0027] MPS liquid medium (1 L): peptone 10 g, diammonium hydrogen phosphate 2 g, potassium dihydrogen phosphate 2 g, sodium acetate 5 g, ammonium citrate 2 g, magnesium sulfate heptahydrate 0.58 g, manganese sulfate monohydrate 0.25 g, beef extract 5 g, yeast extract 4 g, Tween-80 1 mL, glucose 20 g;

[0028] MRS solid medium: 2% technical agar powder is added to the MRS liquid medium;

[0029] CaCO 3 -MRS medium: 1% CaCO 3 ;

[0030] BSH identification medium: After melting MRS solid medium, add 0.3% sodium deoxycholate, 0.2% sodium thioglycollate, and 0.37 g / L CaCl 2 ;

[0031] MRS-CHOL liquid medium (1 L): Add 0.15 g cholesterol, 0.3 g bile salts, 0.15 g sucrose fatty acid ester, 2.0 mL Tween-80, and 5.0 - 10.0 mL absolute ethanol (for solubilization) to a beaker. Obtain a crude cholesterol solution in a 95°C water bath. Mix the crude solution thoroughly with MRS liquid medium to obtain it;

[0032] All the above media are autoclaved at 121°C for 20 min. In the formula of the same type of liquid medium, agar is not added, and the contents of the other reagents are the same.

[0033] (2) Screening and isolation of strains

[0034] Take the characteristic fermented food "Chou Suan" from Guizhou region of China and perform gradient dilution with MRS culture solution to obtain diluted bacterial solutions with concentrations of 10 0 (stock solution), 10 -3 , 10 -4 , 10 -5 respectively. Pipette 100 μL of each and spread them on CaCO 3 -MRS medium. Incubate upright for 1 h and then invert for 48 h. Select single colonies with larger calcium dissolution zones and perform continuous subculturing 4 times on MRS solid medium by the streak plate method. Pick typical colonies on the plate, perform streak purification to obtain pure colonies, preserve the strains, and extract bacterial DNA. For PCR amplification primer selection, refer to Table 1. Reaction conditions: Pre-denaturation at 95°C for 5 min; 95°C for 30 s, 56°C for 30 s, 72°C for 45 s for a total of 35 cycles, and annealing and extension at 72°C for 10 min. Perform first-generation sequencing on the PCR products, compare the sequences with the NCBI database for homology analysis, construct a phylogenetic tree for the 16S rDNA sequence of the strains, and the 16S rDNA sequence of Pediococcus pentosaceus R5 is shown as SEQ ID NO.1.

[0035] Table 1 Primer sequences

[0036] Primer sequence 27F: 5’-AGAGTTTGATCCTGGCTCAG-3’ 1492R: 5’-CTACGGCTACCTTGTTACGA-3’

[0037] Use the 16S rRNA amplification method to perform molecular identification on the purified strains. The phylogenetic tree constructed from their 16S rRNA sequences is as Figure 2 shown. Combining physiological and biochemical characteristics, the identified strain is Pediococcus pentosaceus, and it is named R5.

[0038] Based on the complete genome sequencing analysis of the second and third generation R5 strains, after comparison with the genomes of all 151 strains of Pediococcus pentosaceus in the existing database, the site chr:597597-598531 in the R5 genome was extracted, and the sequence of 934 bp was unique to the R5 strain. Its function prediction showed that it might be a site-specific DNA methyltransferase or CTP synthase. Its characteristic sequence is shown in SEQ ID NO.2.

[0039] The colonies of Pediococcus pentosaceus R5 are round or oval, with a diameter of about 0.5 to 1 μm. The colonies are milky white, hard in texture, flat or slightly convex in surface, smooth in surface, without burrs or protrusions, and have neat edges without bifurcations or roughness. Figure 1 As shown, Gram staining microscopy showed that it was Gram-positive, with cocci and cells arranged in spherical forms.

[0040] Pediococcus pentosaceus R5 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on December 23, 2024. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCCNo.65659.

[0041] Sequence SEQ ID NO.1:

[0042] CGAACGAACTTCCGTTAATTGATTATGACGTACTTGTACTGATTGAGATTTTAACACGAAGTGAGTGGCGAACGGGTGAGTAACACGTGGGTAACCTGCCCAGAAGTAGGGGATAACACCTGGAAACAGATGCTAATACCGTATAACAGAGAAAACCGCATGGTTTTCTTTTAAAAGATGGCTCTGCTATCACTTCTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCAGTGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTAAAGAAGAACGTGGGTAAGAGTAACTGTTTACCCAGTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTCTTTTAAGTCTAATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGATTACTAAGTG-TTGGAGGGTTTCCGCCCTTCAGTGC TGCAGCTAACGCATTAAGTAATCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAA;

[0043] Sequence SEQ ID NO.2:

[0044] >chr:597597-598531

[0045] ATTTTGAACGCTGGTGGTGTGGATTATAACGACATATTGTCGATTTAATAACTAAATTTGAGGGGATAACAGAGTATGGGAAATTGGTTTAACACAAATGATAATTGGTTTTTGTCAAACAGAAAGTATTTATATAATGATCTGCTTTCTGAACAGTTTGTTAAAGTGCACGATGAGCAATTAAGTAAAAACTATTATTAGTTTATTCCTTCGGACTTTGATATTGCATTAGTCGAGGCAATAGCAACTGAAAAAAGTAAATATACAAATAATAACAATCAGTATGATGAGGGTCGAACCCCAGAAAAAAAGAAAAACAAACAATTTGAAGGCTGTTTGGCAGAATTAGCAATAGCAAAATTTCTCGTGCAAATTTTTAACGAAACACCCAGTAATATTCATATTTATGATGCAGAACGAGCTGATTTTGAATACAGGGCTGGGGAAGAATATGATATTAAGGTCATTAAAAATAGCGTAGAAAAGAAATGTGAAGTTCGAAACTCATGGTCTTATAAAACTACATTTCTGATTTTTGTAGAATGTACGATATTTTGGGCACTTACACTCATGAAAGTAAAAAAACAGAAGAAATGTCGGATTTCTTTTTAGACCAATATTACAGCTAAATGAATTGAGTGATGCAATTCCTAAAAATTCAATCGAACTAGTCAAGTCTAAAAAGGTCAAATTGTATATAGTTGCGGCTTGCGATAAGCAACAGATGATATCTAAAGGTAACTATAATAAATGGATGAGTAAAGGGCAAACAAAGTACCATACAACAAAAATTAATCTTTTAAATTCAGTTGATAGTTTTGAAGATTTATACAATAATTTGTTTGAACGATAAATTAAGGAGAAAATGGTATGGAAGGTAAAAAGTTACGACCAGTTAATCAATTTTCTGATGATGTAAGAAACACGACGATTC。

[0046] Example 2: Lipid-lowering ability of the strain in vitro within 24 h - Determination of cholesterol removal ability within 24 h

[0047] Sample inoculation: Select the o-phthalaldehyde method. The strain is activated in MRS medium before being inoculated into the high-cholesterol medium. After the strain is activated in MRS medium for 24 h, a bacterial suspension with the same concentration is prepared, and then inoculated according to an inoculation amount of 2%;

[0048] Experimental method for cholesterol standard curve: Pipette 0.0, 0.1, 0.2, 0.3, 0.4, 0.5 mL of cholesterol working solution into 10-mL test tubes respectively, take 3 parallel samples respectively, volatilize the solvent completely in an oven at 75 °C, add 2 mL of chromogenic reagent, let it stand at room temperature for 10 min, then slowly add 1 mL of concentrated sulfuric acid, mix well on a vortex mixer, and within 10 - 90 min, perform colorimetry at a wavelength of 550 nm. Use the OD value as the ordinate and the cholesterol standard concentration of 100 μg / mL as the abscissa to make a standard curve;

[0049] Reference cell: Slowly add 2 mL of chromogenic reagent to 1 mL of concentrated sulfuric acid, and mix well at level 2 of the shaker for 10 s;

[0050] Sample: Take 1 mL of the culture solution, centrifuge (centrifuge at 10000 r / min for 5 min), then take 0.2 mL of the supernatant, add 2 mL of chromogenic reagent, slowly add 1 mL of concentrated sulfuric acid along the wall of the test tube, mix well on the shaker for 10 s, then let it stand at room temperature for 10 min, and perform colorimetry with a spectrophotometer at OD 550nm for colorimetry;

[0051] Cholesterol removal rate % = (A - B) / A × 100%, where A is the OD value of the supernatant of the medium without inoculated bacteria at a wavelength of 550 nm, and B is the OD value of the supernatant of the medium with inoculated bacteria at a wavelength of 550 nm.

[0052] As Figure 3 shown in (A), the in vitro cholesterol removal ability of Pediococcus pentosaceus R5 can reach 59.53% in 24 h.

[0053] Example 3: Evaluation of cholesterol-lowering in vivo of the strain

[0054] (1) Grouping of experimental animals and determination of physiological and biochemical indexes

[0055] Five-week-old male C57BL / 6J mice, weighing 17 - 18 g, were obtained from the Guangdong Provincial Medical Laboratory Animal Center. The mice were placed under controlled environmental conditions (temperature 18 - 25°C, relative humidity 55% - 70%, light / dark cycle 12 / 12 h, with free access to water and food during the experiment). The mice started to acclimatize one week before the experiment and were randomly divided into four groups. The control group (Negative Control, NC) mice were fed a Co60-irradiated maintenance diet, and the high-cholesterol group (High Cholesterol, HC) was fed a Co60-irradiated high-cholesterol and high-fat diet (HCD) with the following additives: sucrose 20%, lard 15%, cholesterol 1.2%, sodium cholate 0.2%, casein 10%, calcium bicarbonate 0.6%, stone powder 0.4%, premix 0.4%, and basal diet 52.2%. It was purchased from the Guangdong Provincial Medical Laboratory Animal Center. Among them, the Pediococcus pentosaceus R5 group (HC + R5) was fed the high-cholesterol and high-fat diet with the Pediococcus pentosaceus bacterial suspension (HCD + 1×10 9 CFU / ml / 100 g), and the simvastatin group (HC + Simvastatin, HC + Sim) was fed the high-cholesterol and high-fat diet with simvastatin drug (HCD + 0.14 g / 100 ml). The drug group and the R5 group were intragastrically administered according to the plan daily, and the control group and the high-cholesterol group were intragastrically administered an equal amount of normal saline daily.

[0056] The experiment was conducted for 8 weeks. During the experiment, the body weight was measured weekly. After 8 weeks of feeding, the mice were fasted for 12 h without water deprivation, then blood was collected from the heart and the mice were sacrificed. Immediately, the heart, liver, spleen, and kidneys were excised, rinsed, weighed, snap-frozen in liquid nitrogen, and stored at -80°C until analysis. The recorded body weight changes and organ indices are shown in Table 2 below. This experimental design was approved by the Experimental Animal Ethics Committee of Guangdong Pharmaceutical University.

[0057] Table 2 Body weight changes and organ indices

[0058]

[0059]

[0060] In Table 2, the differences between each group and the HC group in terms of body weight gain and organ index, n = 6, ap < 0.05, bp < 0.01.

[0061] (2) Determination of serum lipid levels

[0062] The blood of the experimental animals was centrifuged at 4000 r / min for 10 min at 4°C to collect the serum. According to the instructions of the commercially available kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), the levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL) cholesterol, and high-density lipoprotein (HDL) cholesterol in the serum were measured. The measurement results are as Figure 4As shown

[0063] As Figure 4 (A), (B), (C), and (D) show that under the condition of continuous intragastric administration of Pediococcus pentosaceus R5 for 8 weeks, the TC, TG, and LDL-C of high-fat mice all decreased, while HDL-C increased, indicating that Pediococcus pentosaceus R5 has a down-regulating effect on the cholesterol levels of TC, TG, and LDL-C in the serum of high-fat mice and can alleviate the increase in blood lipid levels caused by a high-fat diet.

[0064] (3) Determination of liver transaminase levels

[0065] After washing the liver tissue of the experimental animals with normal saline, blotting the surface moisture with absorbent paper, weighing it, and adding 9 times the volume according to (mass: volume = 1:9), homogenize it under ice bath conditions, and then centrifuge it at (2500 g, 4 °C) for 10 min. Measure the contents of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the supernatant according to the instructions of the commercially available kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The measurement results are as Figure 4 shown.

[0066] As Figure 4 (E) and (F) show that under the action of a high-fat diet, the liver transaminase levels of mice increased significantly. Under the condition of continuous intragastric administration of Pediococcus pentosaceus R5 for 8 weeks, the liver transaminase levels of mice decreased significantly, indicating that Pediococcus pentosaceus R5 can significantly alleviate the abnormal liver transaminase levels caused by a high-fat diet. After 8 weeks of treatment with simvastatin, the liver transaminase levels of high-fat mice increased slightly compared with the model group, indicating that simvastatin caused liver damage to mice to a certain extent.

[0067] (4) Liver and H&E staining analysis

[0068] Fixation: After sacrificing the mice, open the chest and abdomen and quickly remove the liver, and trim it to a thickness of 3 mm for the liver sample to be tested for H&E staining. Then immediately fix it with 5-10 times 4% paraformaldehyde for more than 24 h to prevent sticking to the wall;

[0069] Dehydration: 75% alcohol for 4 h → 85% alcohol for 2 h → 90% alcohol for 1.5 h → 95% alcohol for 1.5 h → 100% alcohol for 30 min - 1 h → 100% alcohol for 30 min - 1 h → alcohol-benzene mixture for 10 - 20 min → xylene for 10 - 20 min → xylene for 10 - 20 min → paraffin; Paraffin embedding - paraffin sectioning - wax trimming;

[0070] Deparaffinization: Put the paraffin section samples baked on the staining rack in the oven into xylene I and soak for 20 min. Then transfer the samples to xylene II and soak for 20 min. After the wax on the samples is dissolved, transfer the samples to absolute ethanol I and soak for 5 min, then transfer the samples to absolute ethanol II and soak for 5 min. Finally, brush the samples with absolute ethanol for 20 s, transfer them to a water basin, and rinse the alcohol on the samples thoroughly with running tap water;

[0071] Staining and dehydration: Immerse the sections in HE staining solution 1 in the staining cylinder and stain for 3 - 5 min. Then take out the sections and wash them with water in a staining cup until the sections are colorless. Next, immerse the sections in HE staining solution 2 in the staining cylinder for 3 - 5 s, quickly wash with water, and then immerse the sections in HE staining solution 3 in the staining cylinder for 3 - 5 s, quickly wash with water. Finally, immerse the sections successively in 85% ethanol, 95% ethanol, HE staining solution 4, absolute ethanol I, absolute ethanol II, absolute ethanol III, n-butanol, xylene I, and xylene II in each cylinder and soak for 3 - 5 min.

[0072] Mounting: Take out the sections, quickly dry them in the air outlet, mount them with neutral balsam, and examine them under a microscope. The test results are as Figure 5 shown.

[0073] As Figure 5 shown, compared with the normal group, the lipid deposition in the liver tissue of the HC high-fat diet group is severe, the cell nucleus is located at the edge, the cells are swollen, and lipid droplets of different sizes can be seen in the cells. Compared with the HC group, the vacuoles in the simvastatin drug group become smaller and the fatty degeneration becomes less, but there is infiltration of some inflammatory cells. The hepatocytes in the Pediococcus pentosaceus R5 group are slightly clearer, the morphology of the cell nucleus is basically regular, the number of lipid droplets is significantly less, and there is slight nuclear margination, indicating that R5 can significantly improve the fatty degeneration of the liver caused by high-fat diet.

[0074] The above is only a preferred embodiment of the present invention, and it does not impose any form of limitation on the present invention. Although the present invention has been disclosed above with a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or equivalent changes and modifications within the scope of the technical solution of the present invention by using the above-disclosed technical content. However, any indirect modification, equivalent change, and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A strain of Pediococcus pentosaceus having cholesterol-lowering function, characterized in that: The preservation number of the Pediococcus pentosaceus is GDMCC No.65659, which is preserved in the Guangdong Provincial Microbiological Culture Collection Center. The preservation date is December 23, 2024, and the preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The strain of Pediococcus pentosaceus having cholesterol-lowering function according to claim 1, characterized in that: The Pediococcus pentosaceus is Pediococcus pentosaceus R5, whose 16S rDNA sequence is SEQ ID NO.1, and whose complete genome sequence is SEQ ID NO.

2.

3. A use of Pediococcus pentosaceus having cholesterol-lowering function as claimed in any one of claims 1 to 2, characterized in that: The applications of the Pediococcus pentosaceus having cholesterol-lowering function include probiotics, health products or functional foods, and fat-reducing products.

4. The use of Pediococcus pentosaceus having cholesterol-lowering function according to claim 3, characterized in that: The health product or functional food contains Pediococcus pentosaceus or a probiotic bacterial agent.

5. The use of Pediococcus pentosaceus having cholesterol-lowering function according to claim 3 or 4, characterized in that: The probiotic agent includes probiotic liquid or probiotic freeze-dried powder.

6. The use of Pediococcus pentosaceus having cholesterol-lowering function according to claim 3, characterized in that: The fat-reducing effect of the fat-reducing product includes reducing body fat accumulation and reducing index content; the index in reducing index content includes at least one of body weight, organ index, total cholesterol, total triglycerides, and low-density lipoprotein cholesterol.

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