Pentosaccharomyces pentosaceus and application thereof

Abstract

This invention discloses a strain of *Pediococcus pentosaceus* and its applications, belonging to the field of microbial technology. The strain is *Pediococcus pentosaceus* (…). Pediococcus acidilactici L27, with accession number CCTCC NO: M 20252576, has the following outstanding characteristics: (1) strong cholesterol-lowering ability in vitro; (2) excellent tolerance to the gastrointestinal environment, maintaining a high survival rate in simulated gastric juice and bile salt-containing environments; (3) able to produce broad-spectrum antibacterial substances mainly composed of organic acids, which can inhibit a variety of foodborne pathogens and have stable antibacterial properties; (4) possessing hydrophobic cell surface and co-aggregation ability with pathogens, showing potential for intestinal colonization competition; and (5) having no hemolytic activity and good safety. The L27 strain of this invention can be used to prepare drugs, fermented dairy products, probiotic preparations or feed additives with cholesterol-lowering and / or intestinal flora regulation functions, providing a core strain resource for the development of multifunctional, efficient and stable new probiotic products.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a species of Pediococcus pentosaceus and its applications. Background Technology

[0002] Traditional fermented dairy products have always been an important source of microbial diversity and potential probiotic resources, accumulating a variety of lactic acid bacteria and other microbial communities during their natural fermentation process. In recent years, with the general increase in people's health awareness, the demand for functional foods has continued to grow, and probiotics with specific physiological functions (such as lowering cholesterol, antibacterial properties, and intestinal adhesion) have received increasing attention from consumers and the scientific community. Currently, the most common commercially available probiotics on the market are mainly represented by Lactobacillus and Bifidobacterium, and the research and application of related strains are relatively mature. However, these strains are mostly derived from conventional fermented foods or the human gut. For probiotic resources originating from specific regions and traditional food systems, the systematic exploration, functional identification, and development and application are still insufficient, especially in preserving regional characteristics and microbial genetic diversity, where there is significant room for development.

[0003] Eastern Inner Mongolia boasts a long history of nomadic herding and dairy fermentation. Its traditional dairy products (such as cheese, yogurt, and milk tofu) have developed a unique microbial ecosystem during natural fermentation, containing abundant and underdeveloped lactic acid bacteria resources. These native microorganisms, through long-term natural selection and adaptive evolution, may possess excellent acid resistance, bile salt tolerance, and low-temperature tolerance, as well as potential probiotic functions, such as lowering cholesterol, inhibiting pathogens, and regulating gut microbiota. However, current research on the systematic screening, precise functional identification, and subsequent application development of probiotics in traditional dairy products from this region is limited, and many potentially valuable strains remain unidentified and unutilized.

[0004] Therefore, isolating, screening, and identifying probiotic strains from traditional dairy products in eastern Inner Mongolia that possess high cholesterol-lowering ability, good gastrointestinal tolerance, broad-spectrum antibacterial activity, and safe use not only helps enrich my country's probiotic germplasm resource bank, but also provides an important strain foundation and scientific basis for developing functional foods, probiotic preparations, or feed additives with regional characteristics and clear health benefits. This has significant theoretical research value and market application prospects. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a Pediococcus pentosaceus strain and its application. This strain is a microbial individual with a specific genetic background and functional characteristics isolated from traditional dairy products in eastern Inner Mongolia, which has a unique microbial ecology. It also possesses a variety of excellent characteristics, such as high cholesterol-lowering efficiency, strong gastrointestinal tolerance, stable broad-spectrum antibacterial activity, and good intestinal colonization potential.

[0006] To achieve the above objectives, the present invention provides a *Pediococcus pentosaceus* (… Pediococcus acidilactici Strain L27 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252576.

[0007] Furthermore, its 16S rDNA sequence contains the nucleotide sequence shown in SEQ ID No. 1.

[0008] The present invention also provides the application of the above-mentioned Pediococcus pentosaceus strain L27 in the preparation of products with cholesterol-lowering function.

[0009] Furthermore, the product is a pharmaceutical, functional food, food additive, or feed additive.

[0010] The present invention also provides a fermented dairy product, which is made by fermenting raw milk with a starter culture containing the above-mentioned Pediococcus pentosaceus strain L27.

[0011] The present invention also provides a composition comprising the above-described Pediococcus pentosaceus strain L27 and a pharmaceutically or food-grade acceptable carrier.

[0012] Further, the composition is a live bacterial preparation, inactivated preparation, culture, metabolite, or mixture thereof of the strain.

[0013] The present invention also provides a fermentation agent comprising the above-mentioned Pediococcus pentosaceus strain L27 as an active ingredient, wherein the viable count of the strain in the fermentation agent is not less than 1 × 10⁻⁶. 9 CFU / mL.

[0014] The present invention also provides the use of the Pediococcus pentosaceus strain L27 as described above in the preparation of products for inhibiting or preventing infection or colonization caused by Escherichia coli O157:H7, Salmonella or Shigella.

[0015] The present invention also provides a probiotic microcapsule comprising the cells of the above-mentioned Pediococcus pentosaceus strain L27, and an encapsulation material for encapsulating the cells, the encapsulation material comprising alginate and / or chitosan.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects: (1) Excellent cholesterol-lowering function: In in vitro fermentation experiments, strain L27 showed a cholesterol removal rate of up to 38.98%. This significant effect indicates that the strain can effectively assimilate or co-precipitate cholesterol, and has core application value in the development of probiotic products that regulate blood lipids and prevent cardiovascular and cerebrovascular diseases.

[0017] (2) Unique and stable broad-spectrum antibacterial activity: Strain L27 produced distinct inhibition zones of 14-17 mm in diameter against a variety of important foodborne and enteric pathogens, including Escherichia coli O157:H7, Salmonella enteritidis, and Shigella, confirming its broad-spectrum antibacterial activity. Characterization experiments of the antibacterial substances revealed that its antibacterial activity completely disappeared after neutralization treatment, while the activity remained unchanged after heat treatment, proteinase K treatment, and catalase treatment. This clearly reveals that the antibacterial effect of L27 mainly originates from the organic acids produced by its metabolism, rather than bacteriocins, hydrogen peroxide, or heat-sensitive proteins. This characteristic makes its antibacterial activity more stable and less susceptible to the effects of processing or storage conditions.

[0018] (3) Good potential for adapting to the gastrointestinal environment: Bile salt tolerance: The survival rate remains above 85% at a bile salt concentration of 0.1%-0.3%, proving that it can tolerate the bile environment in the small intestine and is expected to reach the intestine in a live state to play a role.

[0019] (4) Adhesion and colonization competition: The strain exhibited 21.71% cell surface hydrophobicity and 26.07% self-aggregation ability. More importantly, it showed coagulation rates of 18.75% to 43.22% against all tested pathogens, with the highest coagulation rate against Salmonella typhimurium. This characteristic indicates that L27 can compete with pathogens for adhesion sites in the intestine through a "competitive exclusion" mechanism, thereby inhibiting pathogen colonization and playing a biological barrier role.

[0020] (5) Basic safety characteristics: The hemolysis rate of strain L27 was only 0.040%, which was much lower than that of the positive control, indicating that it had no hemolytic toxicity and met the basic safety requirements of probiotics. L27 showed resistance to some antibiotics such as ampicillin and gentamicin.

[0021] In summary, the L27 lactic acid bacteria strain of this invention combines highly effective cholesterol-lowering properties, broad-spectrum antibacterial activity mediated by organic acids, certain tolerance to gastrointestinal fluids, and potential competitive inhibition of pathogens. These synergistic effects make it suitable not only for developing probiotic health products for people with high blood lipids, but also as a biological preservative for food preservation, or for preparing microecological preparations to regulate intestinal flora and prevent intestinal infections. Attached Figure Description

[0022] 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.

[0023] Figure 1This is a diagram of the inhibition zone of the fermentation broth of lactic acid bacteria L27; where A represents the indicator pathogen. Shigella sonnei The inhibition zone diagram; B represents the indicator pathogen. Salmonella Enteritidis The inhibition zone diagram; C represents the indicator pathogen. Salmonella Paratyphi The inhibition zone diagram; D represents the indicator pathogen. Shigella flexneri The inhibition zone diagram; E represents the indicator pathogen. Escherichia coli The inhibition zone diagram of O157:H7; the numbers 1-5 in each diagram represent no treatment, heat treatment, catalase treatment, proteinase K treatment and neutralization treatment, respectively; the English letters on the plates indicate the pathogens. Figure 2 The results of antibiotic susceptibility testing for L27 are shown in the figure. Among them, A shows the results of antibiotic susceptibility testing for tetracycline (TE), ampicillin (AMP), ceftriaxone (CTR), gentamicin (GEN), and penicillin (P); B shows the results of antibiotic susceptibility testing for erythromycin (E), trimethoprim (TMP), lincomycin (MY), ciprofloxacin (CIP), and chloramphenicol (C). Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] All raw materials used in this invention are not particularly limited in their source; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0026] There are no particular restrictions on the purity of any of the raw materials used in this invention. However, this invention preferably uses raw materials of analytical grade or purity commonly used in the field of chemical synthesis.

[0027] Example 1: Isolation, screening and identification of strain L27 The strain L27 of this invention was isolated from traditional fermented dairy product samples collected in eastern Inner Mongolia, China. The specific isolation, screening, and identification process is as follows: 1. Sample processing and strain isolation: Take 5 g of traditional fermented dairy product sample, add it to 45 mL of sterile physiological saline, shake thoroughly to mix, and prepare 10... -1 The sample was homogenized. Then, a gradient dilution (10⁻⁶) was performed. -2 Up to 10 -7A suitable dilution of the bacterial suspension was spread onto MRS agar plates (containing 0.5% CaCO3 for preliminary identification of acid-producing bacteria) and incubated anaerobically at 37°C for 48 hours. Single colonies with a clear clear zone (indicating dissolved calcium salts and acid production) were picked for purification, yielding approximately 120 pure cultures of suspected lactic acid bacteria.

[0028] 2. Initial Screening for Cholesterol-Lowering Function: To rapidly screen strains with cholesterol-lowering potential, the above-mentioned purified strains underwent initial screening for their in vitro cholesterol-lowering ability. Each strain was anaerobically activated in MRS broth at 37°C for 24 hours. A 4% (v / v) suspension of the activated bacteria was inoculated into MRS-CHOL medium containing water-soluble cholesterol (final concentration 100 μg / mL) and anaerobically cultured at 37°C for 48 hours. After culture, 1 mL of fermentation broth was centrifuged at 8000×g for 10 minutes, and the residual cholesterol in the supernatant was determined using a total cholesterol assay kit (purchased from Nanjing Jiancheng Biotechnology Institute). The culture medium without inoculated strains served as a blank control.

[0029] The percentage reduction in cholesterol is calculated using the following formula: %.

[0030] Initial screening results showed that some strains exhibited cholesterol-lowering rates of 10%-25%, with strain L27 showing the most outstanding cholesterol-lowering effect, with a preliminary measurement value exceeding 35%, and therefore it was selected as a key research subject.

[0031] 3. Molecular biological identification of the strain: To further confirm the taxonomic position of strain L27, 16S rDNA sequence analysis was performed. Genomic DNA was extracted from strain L27 and PCR amplification was performed using universal primers for bacterial 16S rDNA (forward SEQ ID NO.2: 5′-agagtttgatcctggctcag-3′; reverse SEQ ID NO.3: 5′-aaggaggtgatccagccgca-3′).

[0032] The total volume of the PCR reaction system is 50 μl, which includes 25 μl of 2×M5 Hiper plus Taq HiFi PCR premix, 22 μl of deionized water, 1 μl each of forward and reverse primers, and 1 μl of DNA template.

[0033] The amplification program was as follows: pre-denaturation at 94°C for 8 minutes; followed by 32 cycles, each cycle consisting of denaturation at 94°C for 30 seconds, annealing at 52°C for 30 seconds, extension at 72°C for 30 seconds; and a final extension at 72°C for 7 minutes.

[0034] After verification by 1.5% agarose gel electrophoresis, the PCR products were sent to a sequencing company for bidirectional sequencing. The obtained sequencing results were compared with the NCBI database using BLAST, and the results showed that they were consistent with... Pediococcus acidilactici The sequence showed the highest homology (>99%) with *Pediococcus pentosaceus*. Phylogenetic analysis based on this sequence further confirmed its classification. The sequence has been submitted to GenBank and has been granted accession number OQ455802.

[0035] L27 (OQ455802, SEQ ID NO.1): 1 gacgtgcttg cactgaatga gattttaaca cgaagtgagt ggcggacggg tgagtaacac 61 gtgggtaacc tgcccagaag caggggataa cacctggaaa cagatgctaa taccgtataa 121 cagagaaaac cgcctggttt tcttttaaaa gatggctctg ctatcacttc tggatggacc 181 cgcggcgcat tagctagttg gtgaggtaac ggctcaccaa ggcgatgatg cgtagccgac 241 ctgagagggt aatcggccac attgggactg agacacggcc cagactccta cgggaggcag 301 cagtaggggaa tcttccacaa tggacgcaag tctgatggag caacgccgcg tgagtgaaga 361 agggtttcgg ctcgtaaagc tctgttgtta aagaagaacg tgggtgagag taactgttca 421 cccagtgacg gtatttaacc agaaagccac ggctaactac gtgccagcag ccgcggtaat 481 acgtaggtgg caagcgttat ccggatttat tgggcgtaaa gcgagcgcag gcggtctttt 541 aagtctaatg tgaaagcctt cggctcaacc gaagaagtgc attggaaact gggagacttg 601 agtgcagaag aggacagtgg aactccatgt gtagcggtga aatgcgtaga tatatggaag 661 aacaccagtg gcgaaggcgg ctgtctggtc tgtaactgac gctgaggctc gaaagcatgg 721 gtagcgaaca ggattagata ccctggtagt ccatgccgta aacgatgatt actaagtgtt 781 ggagggtttc cgcccttcag tgctgcagct aacgcattaa gtaatccgcc tggggagtac 841 gaccgcaagg ttgaaactca aaagaattga cgggggcccg cacaagcggt ggagcatgtg 901 gtttaattcg aagctacgcg aagaacctta ccaggtcttg acatcttctg ccaacctaag 961 agattaggcg ttcccttcgg ggacagaatg acaggtggtg catggttgtc gtcagctcgt 1021 gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttattactagttgccagca 1081 ttcagttggg cactctagtg agactgccgg tgacaaaccg gaggaaggtggggacgacgt 1141 caaatcatca tgccccttat gacctgggct acacacgtgc tacaatggatggtacaacga 1201 gtcgcgaaac cgcgaggttt agctaatctc ttaaaaccat tctcagttcggactgtaggc 1261 tgcaactcgc ctacacgaag tcggaatcgc tagtaatcgc ggatcagcatgccgcggtga 1321 atacgttccc gggccttgta cacaccgccc gtcacaccat gagagtttgtaacacccaaa 1381 gccggtgggg taacctttta ggagctagcc gtctaaggtg ggacagatga Preservation Information: Strain L27 was deposited on November 17, 2025, at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M 20252576, at Wuhan University, Wuhan, China. Its taxonomic name is... Pediococcus sp. L27.

[0036] Example 2: Quantitative determination of the in vitro cholesterol-lowering ability of strain L27 To accurately assess the cholesterol-lowering efficacy of strain L27, quantitative experiments were conducted based on the initial screening.

[0037] Experimental Methods: The activated L27 strain was inoculated into MRS-CHOL medium at a 4% (v / v) inoculum and cultured anaerobically at 37°C for 48 hours. Three replicates were set up for each group. After culture, the supernatant was collected by centrifugation, and cholesterol concentration was measured strictly according to the cholesterol reagent kit instructions. A blank control was also set up with uninoculated medium. Results showed that the L27 strain of this invention achieved an in vitro cholesterol-lowering rate of up to 38.98%.

[0038] Example 3: Assessment of Gastrointestinal Environment Tolerance Probiotics must withstand the harsh environment of gastric acid and intestinal bile after oral ingestion in order to reach the intestines and colonize in a viable state. This embodiment systematically evaluated the tolerance of strain L27 to low pH and bile salts, and conducted a detailed investigation at different bile salt concentration gradients.

[0039] 1. Acid Resistance Test: The activated L27 strain was collected by centrifugation, washed twice with sterile PBS (pH 7.2), and resuspended in MRS broth at pH values ​​of 2.5, 3.0, 3.5, and 6.6 (control), adjusting the initial OD600 value to approximately 0.1. After static incubation at 37°C under anaerobic conditions for 3 hours, samples were serially diluted and plated onto MRS agar plates. After incubation at 37°C for 48 hours, the viable cell count was determined. Viability (%) = (Viable cells after treatment / Viable cells before treatment) × 100%. The results showed that the survival rates of the L27 strain were 5.2% and 11.6% at pH 3.0 and 3.5, respectively, indicating that it possesses a certain degree of acid resistance and can partially survive in a simulated acidic gastric environment.

[0040] 2. Bile salt tolerance test: Activated bacterial strains were inoculated into MRS broth (pH 6.6) containing different concentrations (0.1%, 0.2%, 0.3%, 0.5%, w / v) of bovine bile salts, with a control of MRS broth without bile salts. After anaerobic incubation at 37℃ for 24 hours, the OD600 value was measured. Survival rate (%) = (OD600 value of bile salt-containing medium / OD600 value of control medium) × 100%. The results are shown in Table 1. Table 1 Results analysis: The survival rate of strain L27 remained above 85% at bile salt concentrations of 0.1%-0.3%, and even exceeded 50% at a high concentration of 0.5%, indicating that it has strong bile salt tolerance and can adapt to the bile environment in the small intestine.

[0041] 3. Simulated Gastrointestinal Digestion Survival Test: To further simulate the real-world process after oral administration, the survival of strain L27 after continuous treatment with gastric and intestinal fluids was evaluated. The viable bacterial count was approximately 10-1. 9 L27 bacterial suspension (CFU / mL) was prepared. First, the bacterial suspension was mixed with simulated gastric fluid (containing 0.3% pepsin, pH 3.0) at a ratio of 1:9 (v / v) and incubated anaerobically at 37°C for 0, 0.5, 1, 2, and 3 hours, respectively, and samples were taken to determine the viable bacterial count. Subsequently, the mixture treated with gastric fluid for 3 hours was adjusted to pH 6.8 with 1M NaOH and mixed with simulated intestinal fluid (containing 0.1% pancreatin and 0.3% bile salts) at a ratio of 1:1, and incubated anaerobically at 37°C. Viable bacterial counts were determined at 0, 3, and 6 hours, respectively. The results are shown in Table 2. Table 2 The results showed that after 3 hours of gastric fluid treatment, the viable bacterial count of strain L27 decreased from approximately 3.75 × 10⁻⁶ initially. 9CFU / mL decreased to 3.20 × 10⁻⁶ 8 The viable bacterial count was approximately 8.5% (CFU / mL). Subsequently, the viable bacterial count in the intestinal fluid rebounded slightly in the first 3 hours, and remained at around 10 at 6 and 8 hours. 8 The CFU / mL level demonstrates that L27 can tolerate the gastrointestinal digestive process and enter the lower part of the intestine as a considerable number of live bacteria.

[0042] Example 4: Study on antibacterial activity and its mechanism of action To evaluate the ability of strain L27 to inhibit pathogens and to explore its antimicrobial material basis, the following experiments were conducted in this embodiment.

[0043] 1. Broad-spectrum antibacterial activity assay: The agar diffusion method was used. The indicator pathogen (… Escherichia coli O157:H7, Salmonella Paratyphi , Salmonella Enteritidis , Shigella sonnei , Shigella flexneri Fresh culture medium (approximately 10) 8 The L27 strain (CFU / mL) was evenly spread onto LB agar plates. The L27 strain was cultured in MRS broth for 24 hours, and the cell-free supernatant (CFS) was collected by centrifugation (6000×g, 10 min) and filtered through a 0.22 μm filter for sterilization. 100 μL of L27 CFS was added to each well of the plate containing the pathogen. Sterile MRS broth was used as a negative control. After incubation at 37°C for 18–24 hours, the diameter of the inhibition zone was measured. L27 CFS produced significant inhibition zones (14–17 mm in diameter) against all tested foodborne pathogens, indicating broad-spectrum antibacterial activity.

[0044] 2. Characterization of antimicrobial substances: To identify the main antimicrobial substances produced by strain L27, its CFS was treated as follows: (1) Untreated (control); (2) pH adjusted to 7.0 (neutralization treatment); (3) heated at 100℃ for 10 minutes (heat treatment); (4) treated with catalase (1 mg / mL); (5) treated with proteinase K (1 mg / mL). The antimicrobial activity of the treated samples against the above-mentioned indicator pathogens was tested by the agar diffusion method described above.

[0045] result( Figure 1 As shown in Table 3, the antibacterial activity completely disappeared after neutralization treatment, while the antibacterial activity after heat treatment, catalase treatment, and proteinase K treatment was not significantly different from that of the untreated control group. This indicates that the antibacterial effect of strain L27 mainly originates from organic acids (such as lactic acid and acetic acid) produced by its metabolism, rather than bacteriocins, hydrogen peroxide, or heat-sensitive proteins. This characteristic makes its antibacterial activity more stable during food processing and storage.

[0046] Table 3 Example 5: Analysis of cell surface properties and co-aggregation ability The hydrophobicity of probiotic cell surface, self-aggregation ability, and co-aggregation ability with pathogens are important indicators for assessing their intestinal adhesion potential and competitive rejection effect.

[0047] 1. Hydrophobicity and Self-aggregation Determination: Hydrophobicity was determined by the microbial adhesion hydrocarbon method (MATH) with xylene as the organic phase. Self-aggregation was calculated by measuring the change in absorbance of the bacterial suspension after standing for different times. Specific methods are described in the background section. The results showed that the cell surface hydrophobicity of strain L27 was (21.71 ± 0.12)%, and the self-aggregation rate was (26.07 ± 1.85)%, exhibiting certain surface hydrophobic properties and self-aggregation ability.

[0048] 2. Determination of co-aggregation ability with pathogens: Equal volumes of strain L27 and each indicator pathogen were mixed separately (bacterial concentration approximately 10⁻⁶). 8 (CFU / mL), after vortexing, the mixture was incubated at 37°C for 3 hours. The absorbance of the supernatant was measured at 600 nm, and the co-aggregation percentage was calculated. The results are shown in Table 4. Strain L27 showed varying degrees of co-aggregation with all tested pathogens, among which... Salmonella Enteritidis The highest co-aggregation rate was achieved, reaching (43.22 ± 0.34)%. This characteristic indicates that L27 can "capture" or "encapsulate" pathogens through physical co-aggregation, thereby potentially interfering with their adhesion and colonization in the intestine and playing a biological barrier role.

[0049] Table 4 Example 6: Security Assessment 1. Hemolytic activity assay: An equal volume of CFS from strain L27 was mixed with 5% (v / v) human erythrocyte suspension. PBS and 0.1% Triton X-100 solution were used as negative and positive controls, respectively. After incubation at 37°C for 1 hour, the supernatant was centrifuged, and the absorbance was measured at 540 nm. Hemolysis rate (%) = [(sample OD...] 540 - Negative control OD 540 ) / (Positive control OD 540 - Negative control OD 540 The result showed that the hemolysis rate of strain L27 was 0.040%, which was much lower than the 100% of the positive control, indicating that it had no hemolytic toxicity.

[0050] 2. Antibiotic susceptibility testing: The Kirby-Bauer disk diffusion method was used to test the susceptibility of strain L27 to 10 common antibiotics. Pediococcus acidilactici Not belonging to the category of common pathogens, and with no mention of MIC criteria in CLSI M100, we assessed its resistance according to the standards for enterococci. Enterococci and *Pediococcus lactis* do not belong to the same genus, and their specific resistance profiles may differ. For *Enterococcus*, aminoglycosides (except in high-level resistance tests), cephalosporins, lincomycin, and trimethoprim-sulfamethoxazole may show in vitro activity but are ineffective in clinical treatment and should not be reported as susceptible. Furthermore, enterococci are naturally resistant to gentamicin. Results are as follows... Figure 2 As shown in Table 5, strain L27 was sensitive to penicillin, ampicillin, and chloramphenicol, resistant to erythromycin and ciprofloxacin, and intermediately resistant to tetracycline. Other antibiotics such as ceftriaxone and lincomycin showed inhibition zones, but only the diameter of the inhibition zone was observed, while gentamicin and trimethoprim did not show inhibition zones. This resistance spectrum is consistent with the inherent characteristics of some lactic acid bacteria, but it requires attention during application.

[0051] Table 5 Example 7: Preparation of cholesterol-lowering fermented dairy products This embodiment aims to illustrate how to prepare a functional fermented dairy product using the strain L27 of the present invention.

[0052] The steps are as follows: 1. Strain activation and starter culture preparation: The preserved L27 strain was inoculated into MRS liquid medium and anaerobically activated at 37°C for two generations (18-24 hours per generation). The activated bacterial solution was centrifuged to collect the bacterial cells, washed with sterile physiological saline, and a high-concentration starter culture suspension (approximately 10 μL) was prepared. 10 (CFU / mL).

[0053] 2. Raw milk processing: Take fresh milk or reconstituted milk and standardize the fat content to about 3.5%. Pasteurize the milk at 90-95℃ for 5-10 minutes, and then quickly cool it to 37℃.

[0054] 3. Inoculation and Fermentation: Inoculate the prepared L27 starter culture into the treated raw milk at an inoculation rate of 1%-3% (v / v) and mix thoroughly. Conduct anaerobic fermentation at a constant temperature of 37℃. Monitor the pH value regularly during fermentation; terminate fermentation when the pH drops to 4.5-4.6 (usually requiring 8-12 hours).

[0055] 4. Post-fermentation ripening and storage: The fermented curd is rapidly cooled to approximately 4°C and then post-fermented at this temperature for 12-24 hours to obtain a better flavor and texture, resulting in cholesterol-lowering yogurt rich in active L27 strains. The product contains up to 10 live bacteria. 8 -10 9 CFU / mL.

[0056] 5. Product Function Validation (In Vitro): Take 10g of the fermented milk product prepared above, dilute and homogenize with PBS, and determine the cholesterol removal rate of its cell-free supernatant after culturing in MRS-CHOL medium for 48 hours, following the method in Example 2. Comparative Experiment Setup: (a) Fermented milk prepared from unfermented raw milk using the same process. The results showed that the fermented milk containing L27 exhibited significant in vitro cholesterol-lowering activity (approximately 30%), significantly higher than that of the raw milk (<5%).

[0057] Example 8: Preparation of Probiotic Microcapsules To enhance the L27 strain's tolerance to gastric acid and achieve targeted release into the intestine, this embodiment provides a method for preparing probiotic microcapsules.

[0058] The steps are as follows: 1. Prepare a 2% (w / v) aqueous solution of sodium alginate, sterilize and cool.

[0059] 2. Add a high concentration of L27 cells (≥10) 11 The bacterial cell-sodium alginate suspension was prepared by mixing the bacterial cell (CFU / g) with the sodium alginate solution at a ratio of 1:10 (w / v) and dispersing them thoroughly.

[0060] 3. Using a dropping device (such as a syringe), the mixed suspension is added drop by drop into a vigorously stirred 2% (w / v) calcium chloride solution to form gel beads.

[0061] 4. After the gel beads have solidified in the calcium chloride solution for 20 minutes, remove them and rinse them with sterile water.

[0062] 5. To further enhance the protective effect, sodium alginate gel beads can be immersed in 0.5% (w / v) chitosan (dissolved in 1% acetic acid solution) for 10 minutes to form a sodium alginate-chitosan composite coating. After rinsing, freeze-dry to obtain L27 probiotic microcapsule powder.

[0063] Efficacy validation: The microcapsules and an equal volume of free L27 bacterial powder were treated with simulated gastric fluid (pH 2.0, 2 hours) and then transferred to simulated intestinal fluid (pH 6.8, 4 hours). The viable bacterial count was measured at each stage. The results showed that microencapsulation significantly improved the survival rate of the L27 strain in simulated gastric fluid (from <1% of free bacteria to >70%) and ensured its effective release in the intestinal environment.

[0064] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A type of Pediococcus pentosaceus ( Pediococcus acidilactici strain L27, characterized in that, It is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M 20252576.

2. The Pediococcus pentosaceus strain L27 according to claim 1, characterized in that, Its 16S rDNA sequence contains the nucleotide sequence shown in SEQ ID NO:

1.

3. The use of the Pediococcus pentosaceus strain L27 as described in claim 1 or 2 in the preparation of a product with cholesterol-lowering function.

4. The application according to claim 3, characterized in that, The product is a pharmaceutical, functional food, food additive, or feed additive.

5. A fermented dairy product, characterized in that, It is made by fermenting raw milk with a starter culture containing Pediococcus pentosus strain L27 as described in claim 1 or 2.

6. A composition, characterized in that, It comprises the Pediococcus pentosaceus strain L27 as described in claim 1 or 2, and a pharmaceutically or food-grade acceptable carrier.

7. The composition according to claim 6, characterized in that, The composition is a live bacterial preparation, inactivated preparation, culture, metabolite, or mixture thereof of the strain.

8. A fermenting agent, characterized in that, The active ingredient is Pediococcus pentosaceus strain L27 as described in claim 1 or 2, and the viable count of the strain in the fermentation agent is not less than 1 × 10⁻⁶. 9 CFU / mL.

9. The use of the Pediococcus pentosaceus strain L27 as described in claim 1 or 2 in the preparation of products for inhibiting or preventing infection or colonization caused by Escherichia coli O157:H7, Salmonella or Shigella.

10. A probiotic microcapsule, characterized in that, The mixture comprises the cell of Pediococcus pentosaceus strain L27 as described in claim 1 or 2, and an embedding material encapsulating the cell, the embedding material comprising alginate and / or chitosan.

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