Pediococcus acidilactici XZFMCC101.23156, microbial preparation, antibacterial composition and application of Pediococcus acidilactici XZFMCC101.23156

Abstract

The invention provides pediococcus acidilactici XZFMCC101.23156, a microbial preparation, an antibacterial composition and application of the pediococcus acidilactici XZFMCC101.23156, and belongs to the technical field of functional microorganisms. The preservation number of the pediococcus acidilactici XZFMCC101.23156 is GDMCC No: 66860, and the pediococcus acidilactici XZFMCC101.23156 can be used as a functional microbial agent to be applied in vivo to improve diseases such as hyperlipidemia and the like, and can also achieve the effect of reducing body weight. The result of the embodiment shows that the culture supernatant of the pediococcus acidilactici XZFMCC101.23156 can effectively reduce the total cholesterol, the triglyceride, the low-density lipoprotein and the high-density lipoprotein in a rat body. The pediococcus acidilactici XZFMCC101.23156 provides a new way for the development of oral probiotic preparations and the preparation of foods, drinks, health care products and medicines with the function of degrading cholesterol.

Description

Technical Field

[0001] This invention belongs to the field of functional microbial technology, specifically relating to a strain of Pediococcus lactis XZFMCC101.23156, microbial preparations, antibacterial compositions and their applications. Background Technology

[0002] Cholesterol is a widely distributed steroid in the body. It is not only an important component of cell membranes but also a precursor to substances essential for maintaining human health, playing an indispensable physiological role. However, when the concentration of at least one of the following in blood plasma—total cholesterol, triglycerides, and low-density lipoprotein cholesterol—is too high, while the concentration of high-density lipoprotein cholesterol is too low, the incidence of cardiovascular and cerebrovascular diseases such as atherosclerosis, coronary heart disease, hypertension, and stroke will increase sharply.

[0003] Currently, while commonly used lipid-lowering drugs can rapidly regulate blood lipid levels, long-term use may cause adverse reactions such as liver and kidney damage, muscle soreness, and gastrointestinal discomfort, and some patients develop drug resistance. Therefore, there is an urgent need to develop safe, gentle, and stable natural lipid-lowering methods. Probiotics, as live microorganisms beneficial to host health, have received widespread attention for their role in regulating blood lipids. Studies have shown that probiotics participate in cholesterol metabolism through multiple mechanisms: first, by directly binding to cholesterol in the intestine through adsorption sites on the cell surface, such as polysaccharides and proteins, reducing intestinal absorption and excretion in feces; second, by producing active substances such as bile salt hydrolase (BSH) through metabolism, breaking down bound bile acids into free bile acids, promoting bile acid excretion, and thus prompting the liver to use cholesterol to synthesize new bile acids, reducing cholesterol reserves in the body; and third, by regulating the structure of the host's intestinal flora, improving the expression of lipid metabolism-related genes, and inhibiting endogenous cholesterol synthesis and intestinal absorption. However, current research on probiotics for lowering lipids still has significant limitations: on the one hand, the reported lipid-lowering probiotic strains are mostly concentrated in a few categories such as Lactobacillus and Bifidobacterium, resulting in relatively limited strain resources, and the lipid-lowering efficiency varies significantly among different strains; on the other hand, the lipid-lowering stability of existing strains is insufficient, easily affected by the intestinal environment, such as gastric acid, bile salts, and the host's dietary structure, leading to a decrease in their colonization ability and metabolic activity in the intestine, making it difficult to achieve a sustained and effective lipid-lowering effect. Furthermore, the safety evaluation of some probiotic strains is not comprehensive enough, limiting their application in functional foods or health products. Therefore, screening a novel probiotic strain with high lipid-lowering efficiency, strong intestinal tolerance, and good safety is of great significance for developing natural lipid-lowering products and meeting clinical and market demands. Summary of the Invention

[0004] To address the deficiencies in the existing technology, the present invention aims to provide a *Pediococcus lactis* strain XZFMCC101.23156, whose fermentation supernatant can significantly improve blood lipids, reduce cholesterol, triglyceride, and low-density lipoprotein levels, and increase high-density lipoprotein levels.

[0005] The objective of this invention is achieved through the following technical solution: This invention provides a strain of Pediococcus lactis ( Pediococcus acidilactici The preservation number of the lactic acid cocci XZFMCC101.23156 is GDMCC No: 66860.

[0006] This invention provides a microbial preparation comprising the above-described Pediococcus lactis XZFMCC101.23156 or its culture supernatant.

[0007] Preferably, the viability of *Pediococcus lactis* in the microbial preparation is ≥10. 8 CFU / mL.

[0008] This invention provides a method for preparing the microbial preparation described in the above technical solution, comprising: Microbial preparations were obtained by culturing Pyotrophic Lateral Species XZFMCC101.23156 in a culture medium.

[0009] Preferably, the culture temperature is 30~37℃; the culture time is 24~48 h.

[0010] The present invention provides an antibacterial composition comprising the *Pediococcus lactis* XZFMCC101.23156 described in the above technical solution and an antibiotic; the antibiotic comprises any one or more of ceftazidime, vancomycin, gentamicin, kanamycin, streptomycin, amikacin, cephalexin, and polymyxin B.

[0011] This invention provides the application of the above-described Pediococcus lactis XZFMCC101.23156, the above-described microbial preparation, the above-described microbial preparation obtained by the above-described preparation method, or the above-described antibacterial composition in the preparation of products that inhibit pathogenic bacteria; the pathogenic bacteria include any one or more of Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus.

[0012] This invention provides the application of the above-described Porphyromonas lactis XZFMCC101.23156, the microbial preparation described in the above-described technical solution, or the microbial preparation obtained by the preparation method described in the above-described technical solution in the preparation of products for improving blood lipids and / or losing weight.

[0013] Preferably, the improvement of blood lipids includes any one or more of the following: reducing cholesterol levels, reducing triglyceride levels, reducing low-density lipoprotein levels, and increasing high-density lipoprotein levels.

[0014] Preferably, the product includes one or more of the following: pharmaceuticals, health products, and food.

[0015] The beneficial effects of this invention are: This invention provides a strain of *Pediococcus lactis* XZFMCC101.23156, whose preservation number is GDMCC No: 66860. The *Pediococcus lactis* XZFMCC101.23156 provided by this invention can degrade cholesterol in vitro. This *Pediococcus lactis* XZFMCC101.23156 is acid-producing and exhibits good tolerance to acidity and bile salts. It has high self-aggregation and hydrophobicity, and can survive well in gastric and intestinal fluids. It is not sensitive to glycopeptides (vancomycin), aminoglycosides (gentamicin, kanamycin, streptomycin, amikacin), β-lactams (ceftazidime, cephalexin), and polymyxin B. The *Pediococcus lactis* XZFMCC101.23156 can be used as a functional microbial agent in vivo to improve conditions such as hyperlipidemia, and can also achieve weight loss. The results of the embodiments of this invention show that the culture supernatant of *Pediococcus lactis* XZFMCC101.23156 can effectively reduce total cholesterol, triglycerides, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol in rats. The *Pediococcus lactis* strain XZFMCC101.23156 provided by this invention offers a new approach for the development of oral probiotic preparations, cholesterol-degrading functional foods and beverages, health products, and pharmaceuticals.

[0016] Biological Preservation Information Pediococcus lactis XZFMCC101.23156, Latin scientific name: Pediococcus acidilactici It was deposited on August 20, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 66860. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 Images showing partial colony morphology and microscopic examination. Figure 2 The graph shows the cholesterol scavenging ability of the strain. Figure 3 This is a growth curve of the strain; Figure 4 Acid production curve of the strain; Figure 5 This is a graph showing the strain's pH tolerance. Figure 6 This is a graph showing the results of the strain's tolerance to bile salts; Figure 7 The results show the hydrophobicity and self-polymerization properties of the strain. Figure 8 The graph shows the results of the strain's tolerance to gastrointestinal fluids. Figure 9 Phylogenetic tree diagram of strain 156; Figure 10 The complete genome map of strain 156; Figure 11 This is a graph showing the genome annotation results of strain 156 in the COG database; Figure 12 The figure shows the genome annotation results of strain 156 in the GO database; Figure 13 The figure shows the genome annotation results of strain 156 in the KEGG database; Figure 14 The graph shows the effect of strain 156 on rat body weight. Figure 15 The graph shows the effect of strain 156 on cholesterol levels in rats. Figure 16 The graph shows the effect of strain 156 on triglyceride levels in rats. Figure 17 The graph shows the effect of strain 156 on rat low-density lipoprotein. Figure 18 The figure shows the effect of strain 156 on rat high-density lipoprotein. Detailed Implementation

[0019] This invention provides a strain of Pediococcus lactis ( Pediococcus acidilactici The preservation number of the lactic acid cocci XZFMCC101.23156 is GDMCC No: 66860.

[0020] The *Pediococcus lactis* XZFMCC101.23156 provided by this invention was isolated and screened from yogurt in Chengguan District, Lhasa City. The nucleotide sequence of the 16S rDNA of *Pediococcus lactis* XZFMCC101.23156 is shown in SEQ ID NO.1. The colonies of *Pediococcus lactis* XZFMCC101.23156 have a raised, round, smooth, dense, white surface and are relatively small; the cell diameter is between 0.8 and 1.5 mm, and they are rod-shaped or cocci, and are Gram-negative bacteria. *Pediococcus lactis* XZFMCC101.23156 can degrade cholesterol in vitro. The *Pediococcus lactis* XZFMCC101.23156 strain described herein is acid-producing and exhibits good tolerance to acidity and bile salts. It possesses high self-aggregation and hydrophobicity, allowing it to survive well in gastric and intestinal fluids. It is insensitive to glycopeptides (vancomycin), aminoglycosides (gentamicin, kanamycin, streptomycin, amikacin), β-lactams (ceftazidime, cephalexin), and polymyxin B. *Pediococcus lactis* XZFMCC101.23156 can inhibit common pathogenic bacteria such as *Escherichia coli*, *Salmonella typhimurium*, and *Staphylococcus aureus*, and has high safety, making it suitable for direct oral administration.

[0021] This invention provides a microbial preparation comprising *Pediococcus lactis* XZFMCC101.23156 or its culture supernatant as described in the above-mentioned technical solution. As an optional embodiment of this invention, the viability of *Pediococcus lactis* in the microbial preparation can be ≥10⁻⁶. 8 CFU / mL, or 10 8 ~10 9 CFU / mL.

[0022] This invention provides a method for preparing the microbial preparation described in the above-mentioned technical solution, comprising: culturing *Pediococcus lactis* XZFMCC101.23156 in a culture medium to obtain the microbial preparation. As an optional embodiment of this invention, the culture medium can be MRS broth medium; the culture temperature can be 30-37℃, or 37℃; the culture time can be 24-48 h, or 48 h. After culture, a culture solution is obtained, which can be directly used as the microbial preparation. Alternatively, the obtained culture solution can be further separated to obtain a culture supernatant and bacterial cells, which can also be used as microbial preparations respectively.

[0023] This invention provides an antibacterial composition comprising *Pediococcus lactis* XZFMCC101.23156 as described in the above-mentioned technical solution and an antibiotic; the antibiotic includes any one or more of ceftazidime, vancomycin, gentamicin, kanamycin, streptomycin, amikacin, cephalexin, and polymyxin B. This invention combines *Pediococcus lactis* XZFMCC101.23156 with an antibiotic, resulting in a synergistic antibacterial effect and significantly improved antibacterial efficiency. The results of the embodiments of this invention show that the combined use of *Pediococcus lactis* XZFMCC101.23156 with ceftazidime can significantly improve the antibacterial effect against *Escherichia coli*.

[0024] This invention provides the application of the above-described Pediococcus lactis XZFMCC101.23156, the above-described microbial preparation, the above-described microbial preparation obtained by the above-described preparation method, or the above-described antibacterial composition in the preparation of products that inhibit pathogenic bacteria; the pathogenic bacteria include any one or more of Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus.

[0025] This invention provides the application of *Pediococcus lactis* XZFMCC101.23156, the microbial preparation described in the above-mentioned technical solution, or the microbial preparation obtained by the preparation method described in the above-mentioned technical solution, in the preparation of products for improving blood lipids and / or losing weight. As an optional embodiment of this invention, improving blood lipids includes any one or more of the following: reducing cholesterol levels, reducing triglyceride levels, reducing low-density lipoprotein (LDL) levels, and increasing high-density lipoprotein (HDL) levels. The results of the embodiments of this invention show that the culture supernatant of *Pediococcus lactis* XZFMCC101.23156 can significantly reduce cholesterol, triglyceride, and LDL levels in hyperlipidemic rats. The results of the embodiments of this invention show that the culture supernatant of *Pediococcus lactis* XZFMCC101.23156 can significantly reduce weight gain caused by a high-sugar, high-fat diet, achieving a weight-loss effect, and thus can be applied to weight loss for the preparation of weight-loss products. As an optional embodiment of this invention, the product includes any one or more of the following: pharmaceuticals, health products, and food. As an optional embodiment of the present invention, the product may be a product with cholesterol-degrading ability, a product with antibacterial effect against pathogens, or a weight-loss product.

[0026] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0027] Example 1: Isolation and Purification of Strains Fermented milk from Tibet was serially diluted to 10 μL according to the national standard GB 4789.2-2022 "National Food Safety Standard - Microbiological Examination of Food - Determination of Total Colony Count". -7 10 -8 10 -9 1 mL of each sample was spread onto an MRS solid plate and incubated at 37°C for 48 h. Single colonies with different morphologies were selected and purified strains were obtained by streak plating.

[0028] Example 2: Morphological detection of strains The bacterial strains isolated in Example 1 were observed, examined under a microscope, and subjected to Gram staining experiments. Colony status, cell morphology, and staining photographs are shown below. Figure 1 The colonies are raised, round, smooth, dense, and white in color, with relatively small cell diameters ranging from 0.8 to 1.5 mm. They are rod-shaped or cocci and were identified as Gram-negative bacteria after testing. A total of 160 strains were preliminarily identified as bacteria.

[0029] Example 3: Determination of total cholesterol content in vitro by strain The bacterial cell concentration of the strain isolated in Example 2 was adjusted to 1×10⁻⁶. 8 CFU / mL bacterial suspension was inoculated at 3% (V / V) into 0.1 g / L high-cholesterol MRS broth and cultured at 37°C for 24 h. The cholesterol content in the culture medium of different strains was determined using the total cholesterol content kit from Shanghai Yuyan Biotechnology Co., Ltd.

[0030] Cholesterol degradation rate D% = [[A blank - (A assay - A control)] ÷ A blank] × 100%.

[0031] Changes in cholesterol content in the culture medium may be due to cholesterol consumption during the growth of lactic acid bacteria, cholesterol consumption by metabolic products produced by probiotics, or both. The results showed that 147 strains had a degradation rate below 50%, indicating that the strains had cholesterol-degrading capabilities, but the degradation ability varied, and most did not meet the requirements for practical production applications. Thirteen strains achieved a degradation rate of over 50%, namely XZFMCC101.23156 (abbreviated as 156), XZFMCC101.23120 (abbreviated as 120), XZFMCC101.23121 (abbreviated as 121), XZFMCC107.2347 (abbreviated as 47), XZFMCC101.23296 (abbreviated as 296), XZFMCC101.23291 (abbreviated as 291), and XZ... The degradation rates of the following PLCs are as follows: FMCC101.23166 (abbreviated as 166), XZFMCC101.23134 (abbreviated as 134), XZFMCC103.2302 (abbreviated as 02), XZFMCC107.2321 (abbreviated as 21), XZFMCC101.23161 (abbreviated as 161), XZFMCC107.2312 (abbreviated as 12), and XZFMCC101.23178 (abbreviated as 178). Figure 2 As shown in the figure, strain 156 had the highest degradation rate, reaching (66.83±2.8)%. To ensure that the biological characteristics of the strains met the requirements for actual production applications, strains with a degradation capacity of over 50% were selected for subsequent experiments.

[0032] Example 4: Growth curves and acid production curves of the strain The bacterial cell concentration of the strain in Example 3 was adjusted to 1×10⁻⁶. 8 A bacterial suspension of CFU / mL was inoculated into MRS broth at a 3% (v / v) inoculum and incubated at 37°C for 48 h. The growth curve was sampled every 2 h to assess bacterial growth over 48 h. The acid production curve was sampled every 2 h for the first 8 h, and every 4 h from 8 to 48 h to measure pH.

[0033] The growth curve and acid production curve of the strain are as follows: Figures 3-4As shown in the growth curves, strains 12, 47, 120, 121, 134, 161, 178, 291, and 269 reached the control growth phase within 12 hours, with an OD value close to 1.7. Strain 156 reached a stationary phase after 28 hours, with an OD value around 1.5, while strain O2 reached a stationary phase within about 8 hours, with an OD value around 0.7. This study indicates that within the same timeframe, a higher OD value correlates with stronger growth ability. Therefore, strains 12, 47, 120, 134, 178, 291, and 269 exhibited stronger growth abilities, followed by 121, 161, 156, and 21, while O2 showed the weakest growth ability. The acid production curves revealed that the strains continuously produced acid from 0 to 10 hours, ceasing acid production after 10 hours. This research suggests a correlation between acid production and antibacterial activity; higher acid production is more beneficial for extending product shelf life. The graph shows that strains 134, 161, and 166 have strong acid-producing abilities, followed by strains 47, 120, 121, 156, 178, 291, and 296, while strains 02 and 21 have weak acid-producing abilities. Based on the growth and acid-producing curves, strain 02 has lower growth and acid-producing abilities than the other strains, and therefore no further experiments were conducted.

[0034] Example 5: Determination of the strain's tolerance to acidity The pH of the MRS broth was adjusted to 3.0 using 1 mol / L HCl, and the bacterial concentration was adjusted to 1×10⁻⁶. 8 A bacterial suspension of CFU / mL was prepared by adding 100 μL of the suspension to MRS broth at pH 3 and incubating at 37°C for 4 h. The OD value was then measured. 600 nm value.

[0035] Under acidic conditions, the survival rate of lactic acid bacteria gradually decreases with prolonged culture time. The pH of gastric juice in the human body is generally 3.0±0.5; only lactic acid bacteria with excellent acid tolerance can successfully enter the intestines and exert their beneficial effects. Furthermore, the acid produced during the fermentation process of lactic acid bacteria lowers the pH of the fermented product; therefore, acid tolerance is also crucial for lactic acid bacteria to exert their beneficial effects in fermented products. The tolerance of different strains, such as... Figure 5 As shown, at pH 3, the survival rates of strains 120 and 121 reached over 90%, specifically (91.55±0.3)% and (91.52±2.7)%, respectively, indicating strong acid resistance. Strain 12 had the lowest survival rate (31.94±2.12)%, indicating poor acid resistance, therefore strain 12 was not used in subsequent experiments.

[0036] Example 6: Determination of the strain's tolerance to bile salts The concentration of ox bile salts added to the MRS broth was adjusted to 3.0 g / L, and the culture medium was sterilized and cooled to 30–40°C at room temperature. The activated test bacteria were inoculated into the bile-containing MRS broth at a rate of 3% (v / v), with a bile-free medium used as a control group. The cultures were incubated in an anaerobic incubator at 37°C for 3 h, and samples were taken to measure the OD of each bacterial culture. 600 Value. Calculate the survival rate using three parallel trials.

[0037] Survival rate (%) = A / B × 100%; where: A is the OD600nm value of lactic acid bacteria grown in MRS liquid medium supplemented with ox bile salts; B is the OD600nm value of lactic acid bacteria grown in untreated MRS liquid medium. 600 nm value.

[0038] The bile salt content in the small intestine fluctuates within the range of 0.03% to 0.3%. The small intestine is a crucial site for lactic acid bacteria to exert their probiotic effects. Different concentrations of bile salts can damage the cell nucleus and alter cell membrane permeability, affecting bacterial colonization and survival. Therefore, bile salt tolerance is an important indicator for evaluating whether lactic acid bacteria can exert their probiotic effects in the human gastrointestinal tract. The survival rate results for different strains are as follows... Figure 6 As shown, when the bile salt concentration was 0.3%, strain 178 had the highest survival rate (94.41±5.33)%, indicating strong bile salt tolerance; strain 47 had a weaker survival rate (41.43±0.71)%, so strain 47 was not included in subsequent experiments.

[0039] Example 7: Determination of the self-aggregation and hydrophobicity of the strain Single colonies of the strains with high bile salt test survival rates from Example 6 were collected from MRS solid plates, placed in MRS broth, and incubated statically at 37°C for 24 h. The bacterial cells were collected by centrifugation at 4000 r / min for 5 min, and washed twice with physiological saline. Two tubes of each strain were activated. One tube of strain served as a blank control with physiological saline, and the bacterial concentration after washing was adjusted to OD0.05. 600 The nm value is approximately 1. Take 10 mL of bacterial suspension and let it stand at room temperature in a test tube. Take samples of the upper bacterial suspension at 0, 1, 2, 3, 4, and 5 h to measure the OD value. 600 The nm value, denoted as At (t=1, 2, 3, 4, 5), is used to calculate the bacterial autoaggregation rate. Another tube of the test strain is used to adjust the bacterial concentration to OD0. 580 =1 nm and record it as Aj. Take 2 mL of bacterial suspension, mix it with 2 mL of chloroform, vortex for 10 min, and let it stand at room temperature for 2 h. Take 1 mL of bacterial suspension, measure the absorbance at a wavelength of 580 nm and record it as A1, and calculate the hydrophobicity.

[0040] Self-polymerization rate (%) = (A0 – At) / A0 × 100%; In the formula: At is the OD measured after standing for th. 600 nm value.

[0041] Hydrophobicity (%) = (Aj – A1) / Aj × 100%; In the formula: Aj and A1 are the values ​​obtained by measuring the aqueous phase before and after mixing with chloroform, respectively.

[0042] Strain self-aggregation refers to the ability of cells of the same bacterial strain to aggregate. This ability has a significant impact on bacterial colonization in the host, biofilm formation, and intermicrobial interactions. Strain surface hydrophobicity refers to the characteristic of the strain's cell surface having a low affinity for water but a high affinity for hydrophobic substances (such as nonpolar substances like oils and lipids). The strength of hydrophobicity can serve as an indicator of the probiotic's ability to adhere to intestinal epithelial cells. Strain adhesion to intestinal epithelial cells is a crucial prerequisite for its efficacy, and the strength of its adhesion ability is positively correlated with self-aggregation and hydrophobicity. The results of hydrophobicity and self-aggregation are as follows... Figure 7 As shown, based on the hydrophobicity results, strain 161 exhibited the highest hydrophobicity (47.26±0.36)%), demonstrating strong aggregation ability; strain 120 showed the weakest hydrophobicity (8.99±2.4)%). Regarding self-aggregation, the self-aggregation rate increased over time within 1-5 hours, with strains 291, 166, 121, and 156 showing the fastest growth. Strain 291 reached a self-aggregation rate of (63.43±0.47)% at 5 hours, while strain 134 achieved a self-aggregation rate of (13.45±0.2)%. In summary, higher self-aggregation and hydrophobicity indicate stronger colonization ability. Therefore, strains 21, 156, 161, 178, and 296, with both self-aggregation and hydrophobicity exceeding 15%, were selected for subsequent experiments.

[0043] Example 8: Determination of the survival rate of the strain in artificial gastric fluid In the real human gut environment, pepsin and trypsin hydrolyze the proteins of microorganisms, and may even inhibit or kill probiotics.

[0044] The five purified bacterial strains screened in Example 7 were cultured in MRS medium for 24 h, and the bacterial cells were collected by centrifugation at 4000 r / min for 5 min. After washing twice with sterile physiological saline, the cells were resuspended in an equal volume of sterile physiological saline to prepare a bacterial suspension. The bacterial concentration was adjusted to 10-1. 8 CFU / mL. Mix 1.0 mL of bacterial suspension with 9.0 mL of simulated gastric fluid and incubate at 37°C. Take samples at 0 h and 3 h to calculate the viable count. Then, mix 1.0 mL of bacterial suspension treated with simulated gastric fluid for 3 h with 9.0 mL of simulated intestinal fluid, incubate at 37°C for 4 h, and take samples to calculate the viable count. Calculate the survival rate of simulated gastric fluid and intestinal fluid according to the following formula.

[0045] Gastric juice survival rate (%) = ( ) × 100% In the formula: N represents the number of viable bacteria after treatment with artificial gastric juice, CFU / mL; N0 represents the number of viable bacteria without treatment with artificial simulated gastric juice, CFU / mL.

[0046] Intestinal fluid survival rate (%) = ( ) × 100% In the formula: N1 represents the number of viable bacteria after treatment with artificial simulated intestinal fluid, CFU / mL; N represents the number of viable bacteria after treatment with artificial gastric fluid, CFU / mL.

[0047] Gastrointestinal tolerance test results as follows Figure 8 As shown, the results indicated that the survival rate of all five tested bacterial strains exceeded 30% after 3 hours of treatment with simulated gastric fluid. Strain 161 had the highest survival rate, reaching (70.46±1.61)%, followed by strain 296, reaching (65.77±3.15)%. Strains 21 and 178 had survival rates below 50%, indicating poor gastric fluid tolerance. After 3 hours of treatment with simulated intestinal fluid, the survival rate of strain 296 decreased to (31.88±0.54)%, possibly due to the alkaline environment and trypsin. The survival rates of the remaining strains all exceeded 80%. The study found that strain 296 had a high survival rate in simulated gastric fluid but a significant decrease in intestinal fluid, suggesting that while its acid-resistant mechanism could cope with the gastric environment, it could not resist the inhibitory effects of trypsin and other substances in intestinal fluid. Strain 178 had a poor survival rate in gastric fluid, suggesting that the strain could not survive in gastric fluid for a long time. Considering all factors, strains 21, 156, and 161 were selected for subsequent experiments.

[0048] Example 9: Determination of drug susceptibility of bacterial strains The Kirby-Bauer disk diffusion method was used to assess the resistance phenotypes of the strains to 20 antibiotics. The bacterial suspension from Example 8 was adjusted to 10... 8 CFU / mL, 1 mL was evenly spread on an MRS agar plate, antibiotic discs were fixed in place, and incubated at 37℃ for 24 h. The antibiotic discs were then attached to the plate, and a blank antibiotic susceptibility test disc was used as a control. The plate was then incubated anaerobically at 37℃ for 48 h, and the diameter of the inhibition zone was measured. Triple replicates were performed for each antibiotic. The results are shown in Table 1.

[0049] Table 1 Results of drug sensitivity testing

[0050] Note: In the table, "-", "I", "R", and "S" represent insensitivity, moderate sensitivity, drug resistance, and sensitivity, respectively.

[0051] Three strains showed resistance to tetracyclines (a type of tetracycline) but sensitivity to minocycline. They also showed sensitivity and resistance to seven β-lactam antibiotics (ceftriaxone, piperacillin, cefazolin, cefuroxime sodium, cefoperazone, and ampicillin). Strain 156 was not sensitive to ceftazidime and cephalexin, but showed resistance to penicillin, indicating that it could be taken concurrently with ceftazidime or cephalexin.

[0052] Example 10: Determination of the antibacterial activity of the strain Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus were activated separately in LB medium (incubated at 37°C for 24 h). 100 μL of each was evenly spread onto MRS agar plates. After drying, Oxford cups were placed vertically on the agar medium, and a single bacterial culture was added to each cup at a concentration of 1 × 10⁻⁶. 8 The concentration of CFU / mL was increased to 0.1 mL, and the mixture was incubated at 37℃ for 24 h. The diameter of the inhibition zone was measured and recorded. The results are shown in Table 2.

[0053] Table 2 Determination of the antibacterial activity of the strains

[0054] Note: Different letters indicate significant differences (P<0.05).

[0055] Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus were selected as indicator bacteria, and the antibacterial activity of the strains was determined based on the diameter of the inhibition zone. The results are shown in Table 2. Strain 21 showed good antibacterial activity against Escherichia coli and Salmonella typhimurium, but moderate antibacterial activity against Staphylococcus aureus; strain 156 showed good antibacterial activity against Salmonella typhimurium and Staphylococcus aureus, but moderate antibacterial activity against Escherichia coli; strain 161 showed good antibacterial activity against Salmonella typhimurium, but moderate antibacterial activity against Escherichia coli and Staphylococcus aureus.

[0056] Example 11: Strain 156 combined with ceftazidime inhibits Escherichia coli Strain strain 156 was inoculated into MRS broth and cultured at 37°C for 24 h. After centrifugation at 4000 rpm for 5 min, the supernatant was discarded, and the bacterial sludge was retained. Physiological saline was added to adjust the bacterial suspension to 1×10⁻⁶. 8 CFU / mL, E. coli were activated in LB medium (cultured at 37°C for 24 h) and the bacterial suspension was adjusted to 1×10⁻⁶. 8 CFU / mL (suspension method as above). Spread 1 mL of *E. coli* evenly onto an MRS agar plate. After drying, place an Oxford cup vertically on the agar medium. Add 0.1 mL of strain 156 suspension to control Oxford cup 1. The suspension concentration is 1×10⁻⁶. 8CFU / mL; Control 2: 0.1 mL of 30 µg / mL ceftazidime was added to the Oxford cup; Experiment 3: 0.1 mL of bacterial suspension of strain 156 was added to the Oxford cup along with 0.1 mL of 30 µg / mL ceftazidime. The mixtures were incubated at 37℃ for 24 h. The diameter of the inhibition zone was measured in triplicate and recorded. The results are shown in Table 3.

[0057] Table 3. Inhibition of Escherichia coli by combination of strain 156 and ceftazidime

[0058] Note: Different letters indicate significant differences (P<0.05).

[0059] Ceftazidime has an inhibitory effect on *Escherichia coli*, and strain 156, while insensitive to ceftazidime, also exhibits inhibitory activity against *E. coli*. Therefore, a combined approach was chosen to inhibit *E. coli*. Results showed that the inhibition zone of both strain 156 and ceftazidime was significantly larger than that of either strain 156 or ceftazidime alone, indicating that the combination of strain 156 and ceftazidime significantly enhanced the inhibitory effect on *E. coli*. Given the prevalence of antibiotic overuse, combining bacterial agents with antibiotics to reduce antibiotic overuse may become a new research direction.

[0060] Example 12: 16S rDNA sequencing of strain 156 1. Strain 156 was sent to Shanghai Sangon Biotech for testing. Species identification was performed using bacterial 16S rDNA sequencing. The procedure included bacterial genomic DNA extraction, 16S rDNA-specific primer PCR amplification and purification, DNA sequencing, and sequence alignment. The results were then entered into the National Center for Biotechnology Information (NCBI) database for BLAST comparison. A phylogenetic tree was constructed using Mega software to determine the species. The 16S rDNA sequence of strain 156 is shown in SEQ ID NO. 1.

[0061] SEQ ID NO.1:

[0062] Strain 156 was identified as Pediococcus lactis, an edible strain, and its phylogenetic tree is shown below. Figure 9 As shown. Strain 156 and Pediococcus acidilactici The 16S rDNA sequence similarity of OM0739841 was 99.87%, and it was identified as Pediococcus lactis. The strain 156 is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 66860.

[0063] Combining the molecular and biological characteristics of the strain, strain 156 was found to have good biological properties. In terms of cholesterol degradation function, strain 156 had the highest degradation rate. Therefore, strain 156 was selected for subsequent experiments.

[0064] 2. Genome sequencing and functional gene prediction of strain 156 (1) The whole genome sequencing of strain 156 was completed by Shanghai Meiji Biotechnology Co., Ltd., based on the Illumina HiSeq platform. FastQC was used to visually evaluate the sequencing data quality of the samples, Trimmomatic was used for data processing, and SPAdes was used for assembly. Prokka was used to predict gene elements, and RepeatMasker was used to identify repetitive sequences on the genome. Functional annotation information from COG, GO, KEGG, and CAZy databases was obtained by comparative analysis of NCBI Blast+, Swissprot, and TrEMBL, KAAS, and HMMER3, respectively. Transmembrane protein prediction analysis was performed using TMHMM.

[0065] Whole-genome sequencing results showed that strain 156 consists of a single circular chromosome with a genome size of 2,103,218 bp, a GC content of 42.02%, and 2,025 protein-coding sequences identified. This chromosome contains 15 rRNAs, 57 tRNAs, and 24 sRNAs. There are 68 repetitive sequences with a total length of 20,792 bp, accounting for 1.14% of the genome. The whole-genome map of strain 156 is shown below. Figure 10 .

[0066] (2) Genome annotation results of strain 156 1) Annotation in the COG database: To infer gene sequence function, annotation is performed in the COG database. Figure 11It is known that strain 156 has 1618 genes annotated across 23 entries in the COG database, mainly focusing on general function prediction, carbohydrate transport and metabolism, transcription, and amino acid transport and metabolism. Among these, 267 genes (11.65%) are related to carbohydrate transport and metabolism. The presence of genes related to lipid transport and metabolism (I) and the corresponding biosynthesis, transport, and catabolism of secondary metabolites (Q) in the genome suggests a strong ability to break down cholesterol. Traditional Tibetan yogurt is produced using an open production method in a complex environment. The *Pediococcus lactis* 156 isolated from this environment can effectively degrade cholesterol and triglycerides using its lipid transport and metabolism capabilities, and its metabolic products also have cholesterol-degrading effects.

[0067] 2) Annotation in the GO database: To support the evolutionary and functional studies of this strain, the sequence was compared in the GO database, revealing 2195 annotated genes. See [link to GO database]. Figure 12 The results showed that in the genome of *Pediococcus lactis* 156, the genes involved in biological processes were mainly involved in metabolic processes (690 genes) and cellular processes (860 genes); in terms of molecular function, binding and activity were the main aspects (645 genes), indicating that *Pediococcus lactis* 156 has strong binding and metabolic capabilities. *Pediococcus lactis* 156 participates in fatty acid biosynthesis, containing fatty acid synthase genes, which convert lipids into fatty acids, thus possessing the ability to degrade cholesterol.

[0068] 3) KEGG database annotation results: To analyze gene function and metabolic pathways, gene annotation was performed in the KEGG database. The genome of *Pediococcus lactis* 156 contains 1240 genes, and functional annotations were obtained for 40 pathways across 6 functional categories, as shown below. Figure 13 As shown in the figure, analysis revealed numerous gene function annotations in the Pediococcus lactis 156 genome related to metabolism, environmental information processing, and genetic information processing. Genes involved in lipid metabolism pathways were consistent with the annotation results from the GO database. These included genes encoding fatty acyl-ACP thioesterase B, CBH encoding choylglycine hydrolase, and glpK encoding glycerol kinase. Fatty acyl-ACP thioesterase B, choylglycine hydrolase, and alcohol dehydrogenase are key enzymes in cholesterol metabolism. After ingestion of this probiotic, the human body utilizes the generated enzymes to metabolize cholesterol, thus lowering blood lipid levels.

[0069] Example 13 Preparation method of supernatant of XZFMCC101.23156: Inoculate strain 156 into MRS broth and incubate at 37℃ for 24 h. Inoculate into MRS broth again at (v / v) 3% and incubate at 37℃ for another 24 h. Centrifuge at 4000 rpm for 5 min, retain the supernatant and remove the bacterial sludge.

[0070] Establishing a rat model After 7 days of acclimatization, 30 rats with similar weights were randomly divided into two groups: a normal control group (6 rats) and a model group (24 rats). The normal control group was fed a normal diet, while the model group was fed a high-sugar, high-fat diet. The model was validated after 4 weeks of feeding: the weight changes of rats in each group during the modeling period were detected, and the serum triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol of rats in each group were detected after 4 weeks.

[0071] After the model was validated, the model group was divided into four groups: a model control group, a positive control group, and experimental group 1. The positive control group was administered atorvastatin calcium solution orally at a dose of 10 mL / kg (equivalent to a daily dose of 5 mg / kg) and was designated as the positive group. Experimental group 1 was administered XZFMCC101.23156 supernatant orally at a dose of 0.4 mL / animal and was designated as the sample group. The normal control group and the model control group were administered physiological saline orally at a dose of 1 mL / 100 g and were designated as the normal group and the model group, respectively. All administrations were once daily for 6 weeks. During the gavage period, the normal control group maintained a normal diet, while the other groups were fed a high-sugar, high-fat diet.

[0072] The results are shown in Tables 4-5 and Figures 14-18 As shown. Figure 14 The graph shows the effect of strain 156 on rat body weight. Figure 15 The graph shows the effect of strain 156 on cholesterol levels in rats. Figure 16 The graph shows the effect of strain 156 on triglyceride levels in rats. Figure 17 The graph shows the effect of strain 156 on the low-density lipoprotein content in rats. Figure 18 The figure shows the effect of strain 156 on the high-density lipoprotein content in rats.

[0073] Table 4. Effects of strain 156 on rat body weight

[0074] in: This indicates that p < 0.05. This indicates that p < 0.01. This means p < 0.001, and the same applies below.

[0075] Table 5. Effects of strain 156 on blood disorders in rats.

[0076] Under the experimental conditions, after 4 weeks of continuous feeding with a high-sugar, high-fat diet, the body weight of the model group (527±26.55 g) was significantly increased compared to the normal control group (451±4.42 g) after 4 weeks of modeling (P<0.01). Serum total cholesterol, triglycerides, and low-density lipoprotein cholesterol were all significantly increased, confirming the successful establishment of the model. Figures 10-14 As shown, compared with the normal group, the body weight of the model group was significantly higher than that of the normal group (P<0.001). From 1 to 4 weeks after the intervention, the body weight of rats in each group gradually increased, and from 4 to 6 weeks the positive group and the probiotic experimental group tended to stabilize. From the 4th week, the body weight of the probiotic experimental group showed a decreasing trend and approached that of the normal rat group, indicating that taking probiotics reduced the body weight of high-sugar and high-fat rats.

[0077] Compared with the model control group, after 6 weeks of intervention, the total cholesterol, triglycerides, and low-density lipoprotein cholesterol levels in the experimental group were significantly lower than those in the model group (p<0.001), decreasing by 7.6 μmol / dL, 959 mg / dL, and 19 μmol / dL, respectively; high-density lipoprotein cholesterol levels were close to those in the normal model group (p<0.001). Although the lipid-lowering effect of the experimental group was lower than that of the positive control group, it still had a significant lipid-lowering effect. Therefore, strain 156 has the effect of improving the body weight of obese rats and assisting in the regulation of blood lipid levels.

[0078] In summary, this invention screened 160 bacterial strains from fermented milk in Tibet. In vitro cholesterol content determination of the supernatant revealed that 13 strains exhibited a degradation rate exceeding 50%. Physicochemical analysis showed that strain 156 demonstrated strong tolerance (acid, bile salt, and gastrointestinal fluid tolerance), high growth viability, varying degrees of resistance or sensitivity to different antibiotics, and inhibitory effects on *Escherichia coli*, with better inhibitory effects when combined with ceftazidime. Rat experiments showed that its supernatant significantly improved the body weight of obese rats and assisted in regulating blood lipid levels. 16S rDNA sequencing, homology analysis, and phylogenetic tree construction identified it as *Pediococcus lactis*, with a genome size of 2103218 bp, a GC content of 42.02%, and 2025 protein-coding sequences identified. Gene annotation in the database revealed the presence of genes (I) related to lipid transport and metabolism, as well as the biosynthesis, transport, and catabolism of corresponding secondary metabolites (Q), indicating that strain 156 and its metabolites have a strong ability to degrade cholesterol.

[0079] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Pediococcus lactis ( Pediococcus acidilactici The preservation number of the lactic acid cocci XZFMCC101.23156 is GDMCC No: 66860.

2. A microbial preparation, characterized in that, Includes the *Pediococcus lactis* XZFMCC101.23156 of claim 1 or its culture supernatant.

3. The microbial preparation according to claim 2, characterized in that, The viability of *Pediococcus lactis* in the microbial preparation is ≥10. 8 CFU / mL.

4. A method for preparing the microbial preparation according to claim 2 or 3, characterized in that, include: Microbial preparations were obtained by culturing Pyotrophic Lateral Species XZFMCC101.23156 in a culture medium.

5. The preparation method according to claim 4, characterized in that, The culture temperature is 30~37℃; the culture time is 24~48 h.

6. An antibacterial composition, characterized in that, It includes the *Pediococcus lactis* XZFMCC101.23156 as described in claim 1 and an antibiotic; the antibiotic includes any one or more of ceftazidime, vancomycin, gentamicin, kanamycin, streptomycin, amikacin, cephalexin, and polymyxin B.

7. The use of the *Pediococcus lactis* XZFMCC101.23156 of claim 1, the microbial preparation of claim 2 or 3, the microbial preparation prepared by the preparation method of claim 4 or 5, or the antibacterial composition of claim 6 in the preparation of a product for inhibiting pathogenic bacteria; wherein the pathogenic bacteria include any one or more of *Escherichia coli*, *Salmonella typhimurium*, and *Staphylococcus aureus*.

8. The use of the microbial preparation of Pyotrophic Lateral Sclerosis XZFMCC101.23156 as described in claim 1, the microbial preparation as described in claim 2 or 3, or the microbial preparation prepared by the preparation method as described in claim 4 or 5 in the preparation of products for improving blood lipids and / or losing weight.

9. The application according to claim 8, characterized in that, The improvement of blood lipids includes any one or more of the following: lowering cholesterol levels, lowering triglyceride levels, lowering low-density lipoprotein levels, and increasing high-density lipoprotein levels.

10. The application according to claim 8, characterized in that, The products include any one or more of the following: pharmaceuticals, health products, and food.

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