A strain of Pediococcus pentosaceus, FREE-U AP01, and its application

Abstract

This invention discloses a strain of *Pediococcus pentosaceus*, FREE-U AP01, and its applications. The strain, FREE-U AP01, has the accession number CGMCC No. 37267. This invention screened and obtained a strain of *Pediococcus pentosaceus*, FREE-U AP01, which, through its excellent temperature adaptability, high survival rate in acid and bile salt tolerance, rapid acid production capacity, and significant antibacterial activity, solves the problems of low survival rate and unstable antibacterial effect of existing probiotics, providing a powerful and high-quality strain resource for microecological preparations and animal feed additives.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, specifically relating to a strain of Pediococcus pentosaceus, FREE-U AP01, and its applications. Background Technology

[0002] Lactic acid bacteria are key probiotics that can replace antibiotics, inhibiting pathogens and maintaining gut health by producing organic acids and bacteriocins. Pediococcus pentosaceus, an important member of the lactic acid bacteria family, has advantages such as rapid growth and abundant metabolites.

[0003] The functions of probiotics are well understood, such as regulating the gut microbiota, alleviating gastrointestinal motility disorders, and improving inflammation. However, the practical applications of probiotics (including human health and gut microbiota regulation in livestock farming) currently face the following main shortcomings: 1. Poor environmental tolerance: Many probiotic strains show probiotic potential in vitro, but they cannot tolerate the low pH environment of gastric acid and the erosion of high concentrations of bile salts in the intestine. As a result, a large number of them die before reaching the intestine, leading to an extremely low actual colonization rate. 2. Lack of high-quality strains with multiple effects: Excellent probiotics not only need to reach the intestines "alive", but also need to have strong acid-producing ability and direct inhibition of pathogenic bacteria.

[0004] In summary, there is still a relative scarcity of bacterial strains on the market that combine high survival rate, rapid reproduction, and strong broad-spectrum antibacterial ability. Summary of the Invention

[0005] Purpose of the invention: This invention addresses the problems existing in the prior art by screening and obtaining a strain of Pediococcus pentosaceus, FREE-U AP01. Through its excellent temperature adaptability, high survival rate in acid and bile salts, rapid acid production capacity, and significant antibacterial activity, this invention solves the problems of low survival rate and unstable antibacterial effect of existing probiotics, providing a powerful and high-quality strain resource for microecological preparations and animal feed additives.

[0006] Technical solution: A strain of Pediococcus pentosaceus FREE-U AP01, with accession number CGMCC No.37267.

[0007] Biological characteristics of a strain of Pediococcus pentosaceus FREE-U AP01: Excellent industrial propagation characteristics: Pediococcus pentosaceus FREE-U AP01 has an optimal growth temperature of 37℃, a short environmental adaptation period, enters the logarithmic growth phase within 2 hours of inoculation, and can reach an OD600 of about 1.4 within 24 hours of fermentation, thus having extremely high industrial fermentation capacity.

[0008] Excellent gastrointestinal tolerance: After incubation for 2 hours in strongly acidic environments at pH 3.0 and pH 4.0, the viable count of Pediococcus pentosaceus FREE-U AP01 remained at 10. 7 It exhibits a high level of CFU / mL and demonstrates good tolerance to high concentrations of bile salts up to 0.3%, ensuring that it can pass through the gastrointestinal tract as a live bacterium.

[0009] Powerful metabolic acid production and antibacterial capabilities: After entering the intestine, it can rapidly produce acid (the pH value drops to about 4.1 within 12 hours), and form obvious inhibition zones against a variety of indicator pathogens in the plate antagonism experiment. It improves the host's intestinal microecology through a dual mechanism of physical occupation and chemical antibacterial action.

[0010] An inoculum containing the aforementioned Pediococcus pentosaceus FREE-U AP01.

[0011] A method for preparing a microbial agent, wherein the above-mentioned Pediococcus pentosacchari FREE-U AP01 is inoculated into a fermentation medium for fermentation to obtain a fermentation broth; The fermentation broth is mixed with a protectant to obtain a microbial agent.

[0012] A microbial agent prepared by any one of the methods described above.

[0013] The application of Pediococcus pentosaceus FREE-U AP01 or any of the above-mentioned bacterial agents in the preparation of microecological preparations for maintaining intestinal health, fermented foods, functional foods, and animal probiotic feed additives.

[0014] Beneficial effects: The Pediococcus pentosaceus strain FREE-U AP01 disclosed in this invention and its application have the following beneficial effects: 1. "Living" colonization: Compared with ordinary lactic acid bacteria, Pediococcus pentosaceus FREE-U AP01 has excellent acid and bile salt resistance, which ensures that it is a "live" bacteria, which is the material basis for its probiotic effects in the body. 2. Antibacterial and gut-protective: Pediococcus pentosaceus FREE-U AP01 has a strong ability to produce acid and lower pH value, as well as a direct antagonistic ability against pathogenic microorganisms, which can effectively repel harmful bacteria and optimize the host's intestinal flora structure.

[0015] 3. Easy to mass produce: The optimal growth temperature of Pediococcus pentosaceus FREE-U AP01 is close to that of conventional fermentation conditions. It grows rapidly, has a large biomass, and has a high survival rate after being prepared into freeze-dried powder, making it highly valuable for industrial promotion. Attached Figure Description

[0016] Figure 1 This is a colony morphology diagram of strain AP01.

[0017] Figure 2 This is a Gram-stained microscopic image of strain AP01.

[0018] Figure 3 Phylogenetic tree of strain AP01 based on the 16S rRNA gene.

[0019] Figure 4 A bar chart annotating the COG functional classification of the whole genome of Pediococcus pentosaceus FREE-U AP01.

[0020] Figure 5 A statistical diagram of the KEGG pathway classification of the whole genome of Pediococcus pentosaceus FREE-U AP01.

[0021] Figure 6 This is a growth curve of Pediococcus pentosaceus FREE-U AP01.

[0022] Figure 7 The growth curves of Pediococcus pentosaceus FREE-U AP01 at different temperatures are shown.

[0023] Figure 8 This is a graph evaluating the acid resistance of Pediococcus pentosaceus FREE-U AP01.

[0024] Figure 9 This is a graph evaluating the bile salt tolerance of Pediococcus pentosaceus FREE-U AP01.

[0025] Figure 10 This is a diagram showing the inhibition zone of Pediococcus pentosaceus FREE-U AP01 against Escherichia coli.

[0026] Figure 11 This is a diagram showing the inhibition zone of Pediococcus pentosaceus FREE-U AP01 against Salmonella.

[0027] Figure 12 This is a diagram showing the inhibition zone of Pediococcus pentosaceus FREE-U AP01 against Staphylococcus aureus.

[0028] Figure 13 This is a schematic diagram of the acid production curve of Pediococcus pentosaceus FREE-U AP01.

[0029] The detailed preservation information for a strain of Pediococcus pentosaceus FREE-U AP01 is as follows: The accession number is: CGMCC No. 37267 The storage period is from January 4, 2026. Its classification name is: Pediococcus pentosaceus Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections; Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China. Detailed Implementation

[0030] The specific embodiments of the present invention are described in detail below.

[0031] The "range" disclosed in this invention is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if a range of 10–50 is listed for a specific parameter, it is also expected that ranges of 10–40 and 20–50 are also included. Furthermore, if the minimum range values ​​are 1 and 2, and the maximum range values ​​are 3, 4, and 5, then the following ranges are all expected: 1–3, 1–4, 1–5, 2–3, 2–4, and 2–5. In this application, unless otherwise stated, the numerical range "a–b" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0–5" means that all real numbers between "0–5" have been listed herein; "0–5" is merely a shortened representation of these numerical combinations.

[0032] Unless otherwise specified, all embodiments and optional embodiments of this application can be combined to form new technical solutions.

[0033] Unless otherwise specified, all technical features and optional technical features of this application may be combined to form new technical solutions.

[0034] Unless otherwise specified, all steps in this application may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the method may also include step (c), indicating that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0035] Unless otherwise specified, the terms "comprising" and "including" as used in this application can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other components not listed may also be included, or that only the listed components may be included.

[0036] Unless otherwise specified, the reaction will proceed under normal temperature and pressure conditions.

[0037] Unless otherwise specified, all parts or percentages are by weight or by weight percentage.

[0038] In this invention, all the substances used are known substances that can be purchased or synthesized by known methods.

[0039] In this invention, all the devices or equipment used are conventional devices or equipment known in the art and are readily available.

[0040] A strain of Pediococcus pentosaceus FREE-U AP01, with accession number CGMCC No. 37267.

[0041] The detailed preservation information for a strain of Pediococcus pentosaceus FREE-U AP01 is as follows: The accession number is: CGMCC No. 37267 The storage period is from January 4, 2026. Its classification name is: Pediococcus pentosaceus Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections; Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China.

[0042] Biological characteristics of a strain of Pediococcus pentosaceus FREE-U AP01: Excellent industrial propagation characteristics: Pediococcus pentosaceus FREE-U AP01 has an optimal growth temperature of 37℃, a short environmental adaptation period, enters the logarithmic growth phase within 2 hours of inoculation, and can reach an OD600 of about 1.4 within 24 hours of fermentation, thus having extremely high industrial fermentation capacity.

[0043] Excellent gastrointestinal tolerance: After incubation for 2 hours in strongly acidic environments at pH 3.0 and pH 4.0, the viable count of Pediococcus pentosaceus FREE-U AP01 remained at 10. 7 It exhibits a high level of CFU / mL and demonstrates good tolerance to high concentrations of bile salts up to 0.3%, ensuring that it can pass through the gastrointestinal tract as a live bacterium.

[0044] Powerful metabolic acid production and antibacterial capabilities: After entering the intestine, it can rapidly produce acid (the pH value drops to about 4.1 within 12 hours), and form obvious inhibition zones against a variety of indicator pathogens in the plate antagonism experiment. It improves the host's intestinal microecology through a dual mechanism of physical occupation and chemical antibacterial action.

[0045] An inoculum containing the aforementioned Pediococcus pentosaceus FREE-U AP01.

[0046] A method for preparing a microbial agent, wherein the above-mentioned Pediococcus pentosacchari FREE-U AP01 is inoculated into a fermentation medium for fermentation to obtain a fermentation broth; The fermentation broth is mixed with a protectant to obtain a microbial agent.

[0047] Furthermore, the fermentation medium is composed of the following components: 2.0% glucose, 1.0% peptone, 1.0% beef extract, 0.5% yeast extract, 0.5% sodium acetate, 0.2% diammonium citrate, 0.1% Tween-80, 0.2% dipotassium hydrogen phosphate, 0.058% magnesium sulfate, and 0.025% manganese sulfate.

[0048] Furthermore, the *Pediococcus pentosaceus* FREE-U AP01 is inoculated using a seed culture, the preparation method of which is as follows: The above-mentioned Pediococcus pentosaceus FREE-U AP01 was inoculated into seed culture medium and cultured to obtain seed liquid.

[0049] Furthermore, the viable count of Pediococcus pentosaceus FREE-U AP01 in the seed culture was 1.0 × 10⁻⁶. 8- 5.0×10 9 CFU / mL, for example, can be 1.0 × 10⁻⁶. 8 CFU / mL, 2.0×10 8 CFU / mL, 5.0×10 8 CFU / mL, 1.0×10 9 CFU / mL, 3.0×10 9 CFU / mL, 5.0×10 9 CFU / mL, etc.

[0050] Furthermore, based on the total volume of the seed culture medium, the inoculation amount of the seed liquid is 2-5% by volume, for example, it can be 2%, 3%, 4%, 5%, etc.

[0051] Furthermore, the seed culture medium consists of the following components: 2.0% glucose, 1.0% peptone, 1.0% beef extract, 0.5% yeast extract, 0.5% sodium acetate, 0.2% diammonium citrate, 0.1% Tween-80, 0.2% dipotassium hydrogen phosphate, 0.01% magnesium sulfate, and 0.005% manganese sulfate.

[0052] Furthermore, the fermentation process conditions are as follows: the culture temperature is 37℃, the rotation speed is 100 r / min, no aeration is allowed, the tank pressure is maintained at 0.05Mpa, the pH value of the fermentation broth is adjusted to 5.5-6.0 (for example, the pH value of the fermentation broth can be 5.5, 5.6, 5.8, 6.0, etc.), and the culture time is 48h.

[0053] Further, the protective agent is at least one selected from starch, sucrose, lactose, trehalose, sodium citrate, skim milk powder, and maltodextrin. In another embodiment, the protective agent is starch. In another embodiment, the protective agent is sucrose. In another embodiment, the protective agent is lactose. In another embodiment, the protective agent is trehalose. In another embodiment, the protective agent is sodium citrate. In another embodiment, the protective agent is skim milk powder. In another embodiment, the protective agent is maltodextrin. In another embodiment, the protective agent is a mixture of starch, sucrose, lactose, trehalose, sodium citrate, skim milk powder, and maltodextrin in equal mass ratios.

[0054] A microbial agent prepared by any one of the methods described above.

[0055] The application of Pediococcus pentosaceus FREE-U AP01 or any of the above-mentioned bacterial agents in the preparation of microecological preparations for maintaining intestinal health, fermented foods, functional foods, and animal probiotic feed additives.

[0056] Example 1: Isolation, screening and identification of Pediococcus pentosaceus FREE-U AP01 1. Isolation and purification of the strain: In October 2025, 1 ml of a sample from bee intestines was placed in a test tube containing 9 ml of physiological saline, shaken to mix, and 10 strains were obtained. -1 Diluent, and so on, perform a series of 10-fold serial dilutions to prepare 10... -2 10 -3 10 -4 10 -5 10 -6 Different gradients of dilution; The obtained diluted solution was spread onto MRS medium and incubated at 37°C for 72 hours. Single colonies were picked and purified by streaking. Sterilized MRS medium is poured into petri dishes to obtain MRS petri dishes. After solidification, the purified bacterial suspension is spread on the MRS petri dishes, with each MRS petri dish controlled to have about 50-80 colonies.

[0057] 2. Screening of strains: Single strains were picked from the culture dish and inoculated into MRS liquid medium. After culturing for 24 hours, the OD value was measured.

[0058] Sterilized MRS medium was poured into petri dishes to obtain MRS culture dishes. A fast-growing bacterial strain (i.e., a strain with an OD600 value ≥ 1.2 after 24 hours of culture) was selected, and an appropriate amount of bacterial suspension was spread onto the MRS culture dish. The strain was then incubated at 37℃ for 24 hours to obtain the candidate strain AP01. Its colony morphology is shown in the figure below. Figure 1 As shown. Figure 1 The macroscopic morphology of a single colony of this strain on a solid culture medium plate is shown (it appears as a milky white, round granular structure).

[0059] 3. Morphological and physiological / biochemical identification: A small amount of bacterial culture of candidate strain AP01 was subjected to Gram staining and microscopic examination, including: The strain AP01, which is in the logarithmic growth phase and has a stable colony size, has a single colony size ≤1mm, and is round, milky white, smooth, raised, with neat edges and opaque.

[0060] Gram staining is performed on a glass slide using standard procedures including crystal violet, iodine mordant, and basic fuchsin. Under a light microscope, the stain appears purple, indicating Gram-positive cocci (such as...). Figure 2 (As shown).

[0061] Gram staining and microscopic observation, along with morphological descriptions based on Bergey's Manual of Systematic Bacteriology (Second Edition), were used to screen similar strains for subsequent molecular identification.

[0062] 4.16S rRNA phylogenetic identification: Genomic DNA was extracted from strain AP01, and the 16S rRNA gene sequence was amplified. Then, 16S rRNA was measured in strain AP01. The full-length 16S rRNA sequence of strain AP01 was compared with relevant type strains in the NCBI database, and a phylogenetic tree based on 16S rRNA was constructed using phylogenetic tree software (e.g., Figure 3 (As shown). The results showed that strain AP01 had the highest homology with the type strain of Pediococcus pentosaceus. Based on morphological identification, strain AP01 was identified as Pediococcus pentosaceus and named FREE-UAP01.

[0063] Example 2: Genome-wide characterization of Pediococcus pentosaceus FREE-U AP01 Methods and Results: Whole genome sequencing and assembly were performed on Pediococcus pentosaceus FREE-U AP01.

[0064] Data analysis shows that the CleanData obtained from sequencing has a Q30 base quality ratio as high as 97.99%, a GC content of 37.88%, and a whole genome size of approximately 2.39 Mb (total splicing length 2398740 bp).

[0065] Genome prediction results showed that the strain contained 2119 coding sequences (CDS), covering approximately 53.896% of the genome, and included 6 rRNAs and 57 tRNAs. This clear genomic information provides underlying genetic data support for the probiotic characteristics of this strain.

[0066] Example 3: Genomic mechanisms of carbohydrate metabolism and acid production in Pediococcus pentosaceus FREE-U AP01 Methods and Results: To reveal the molecular mechanism of efficient acid production by Pediococcus pentosus FREE-UAP01, high-level functional annotation of its genome was performed.

[0067] 1. COG functional classification and CAZy analysis: Genomic COG annotation results show (see...) Figure 4The gene clusters involved in the "Carbohydrate transport and metabolism" category (Category G) are extremely abundant, indicating that *Pediococcus pentosaceus* FREE-UAP01 possesses a strong foundation for substrate uptake and carbohydrate utilization. Simultaneously, 38 carbohydrate-active enzyme (CAZy) related genes were annotated within the genome, including key enzyme systems such as carbohydrate esterases (CEs, such as CE1, CE7, CE10, etc.) and helper oxidoreductases (AAs, such as AA1). These enzyme systems endow this strain with powerful polysaccharide degradation and transformation capabilities. Figure 4 The meaning of the x-axis in the figure is as follows: A: RNA processing and modification; B: Chromatin structure and dynamics; C: Energy production and conversion; D: Cell cycle control, cell division, chromosome partitioning; E: Amino acid transport and metabolism; F: Nucleotide transport and metabolism; G: Carbohydrate transport and metabolism; H: Coenzyme transport and metabolism; I: Lipid transport and metabolism; J: Translation, ribosomal structure and biogenesis; K: Transcription; L: Replication, recombination, and repair; M: Cell wall / membrane / envelope biogenesis; N: Cell motility; O: Posttranslational modification, protein turnover, molecular chaperones P: Inorganic ion transport and metabolism Q: Secondary metabolites biosynthesis, transport, and catabolism; R: General function prediction only; S: Function unknown; T: Signal transduction mechanisms; U: Intracellular trafficking, secretion, and vesicular transport; V: Defense mechanisms; W: Extracellular structures; Y: Nuclear structure; Z: Cytoskeleton.

[0068] 2. KEGG metabolic pathway analysis: Pathway enrichment analysis showed (see...) Figure 5 A total of 545 genes were successfully annotated into the KEGG database. Among them, the glycolysis / gluconeogenesis (ko00010) pathway was enriched with up to 26 key metabolic genes, and the pentose phosphate pathway (ko00030) was enriched with 19 genes. This explains the strong acid-producing capacity exhibited by this strain in in vitro fermentation at the molecular level.

[0069] Example 4: Growth curve determination of Pediococcus pentosaceus FREE-U AP01 Method: The prepared AP01 seed culture was inoculated into MRS liquid medium and cultured at 37℃. OD600 was measured every 2 hours.

[0070] For detailed results, please see [link to results]. Figure 6 .from Figure 6 It can be seen that strain AP01 exhibits a lag phase from 0-2 hours after inoculation, rapidly enters the logarithmic growth phase after 2 hours, and reaches the stationary phase around 12 hours, with a maximum OD600 of approximately 1.4. This indicates that *Pediococcus pentosaceus* FREE-U AP01 has a short environmental adaptation period and strong reproductive capacity.

[0071] Example 5: Determination of the optimal growth temperature for Pediococcus pentosaceus FREE-U AP01 Methods: The prepared AP01 seed culture was inoculated into MRS liquid medium and cultured at 25℃, 30℃, 37℃, 40℃ and 45℃ respectively. The OD600 value of the fermentation broth was measured at 0h, 4h, 8h, 12h and 24h.

[0072] For detailed results, please see [link to results]. Figure 7 .from Figure 7 It can be seen that the growth rate of Pediococcus pentosaceus FREE-U AP01 varies significantly at different temperatures.

[0073] The strain grew most rapidly at 37℃ and 40℃, with the fastest biomass accumulation at 37℃, reaching an OD600 of approximately 1.4 after 24 hours, which is the optimal growth temperature. 30℃ was the next most suitable temperature. Growth was somewhat limited at 25℃ and 45℃. This indicates that *Pediococcus pentosaceus* FREE-U AP01 exhibits the best proliferative capacity at the host core temperature (37℃).

[0074] Example 6: Determination of Acid-Producing Capacity of Pediococcus Pentosacchari FREE-U AP01 Methods: The pH change of *Pediococcus pentosaceus* FREE-U AP01 was monitored during 24 h of fermentation in MRS liquid medium. Fermentation conditions were constant temperature static incubation at 37℃.

[0075] The results are as follows Figure 13 As shown, the pH of the fermentation broth of *Pediococcus pentosaceus* FREE-U AP01 decreased rapidly, dropping to around 4.12 within 12 hours and eventually stabilizing at 3.85. This strong and rapid acid-producing capacity helps *Pediococcus pentosaceus* FREE-U AP01 quickly establish an acidic microenvironment in the gut, synergistically inhibiting harmful bacteria.

[0076] Example 7: Evaluation of the acid resistance of Pediococcus pentosaceus FREE-U AP01 Methods: The prepared AP01 seed culture was inoculated into MRS medium with pH values ​​of 2.0, 3.0, and 4.0, respectively. The gastric acid environment was simulated at 37℃, and the viable cell count (Log) was measured at 0 h, 0.5 h, 1.0 h, and 2.0 h. 10 (CFU / mL). The results are as follows: Figure 8 As shown, *Pediococcus pentosaceus* FREE-UAP01 exhibited extremely strong tolerance under acidic conditions of pH 3.0 and pH 4.0. After 2 hours of cultivation, the viable cell count curve of *Pediococcus pentosaceus* FREE-UAP01 remained relatively stable at approximately 7.0 Log0.0. 10 The high level of (CFU / mL) did not show a significant decrease. Even under extremely acidic conditions at pH 2.0, *Pediococcus pentosaceus* FREE-U AP01 maintained a high survival rate for 0.5 hours. This demonstrates that *Pediococcus pentosaceus* FREE-U AP01 can effectively resist the bactericidal effects of the acidic environment in the digestive tract.

[0077] Example 8: Evaluation of bile salt tolerance of Pediococcus pentosaceus FREE-U AP01 Methods: The prepared AP01 seed culture was inoculated into MRS medium containing 0.1%, 0.2%, and 0.3% ox bile salts, respectively, and cultured at 37℃. Viable cell counts were measured at 0h, 0.5h, 1.0h, and 2.0h. The results are as follows: Figure 9 As shown, the survival rate of *Pediococcus pentosaceus* FREE-U AP01 decreased in a concentration-dependent manner with increasing bile salt concentration, but *Pediococcus pentosaceus* FREE-U AP01 successfully survived at all concentrations. Particularly at a bile salt concentration of 0.1%, the viable count of *Pediococcus pentosaceus* FREE-U AP01 remained above 6.0 after 2 hours of incubation. Even in a high bile salt concentration environment of 0.3%, the viable count of *Pediococcus pentosaceus* FREE-U AP01 remained stable after a decrease, without complete lysis and death. This indicates that *Pediococcus pentosaceus* FREE-U AP01 can resist the toxicity of high concentrations of bile salts in the small intestine and possesses the physiological basis for colonization in the intestine.

[0078] Example 9: Evaluation of the antibacterial activity of Pediococcus pentosaceus FREE-U AP01 Methods: The antibacterial activity of Pediococcus pentosaceus FREE-U AP01 was evaluated using the perforation method.

[0079] Negative control (NC) and AP01 bacterial suspensions were added to indicator bacterial plates containing Escherichia coli, Salmonella, and Staphylococcus aureus, respectively. After incubation at a suitable temperature, the formation of inhibition zones was observed. The results are as follows: Figure 10-12As shown in the photographs of the antibacterial plate experiment, no inhibition zone was observed around the negative control (NC), while clear, well-defined inhibition zones with neat edges were formed around the areas where Pediococcus pentosaceus FREE-UAP01 was added. The diameters of the inhibition zones reached 2.00 cm (Escherichia coli), 2.10 cm (Salmonella), and 2.10 cm (Staphylococcus aureus), respectively. This strongly demonstrates that Pediococcus pentosaceus FREE-UAP01 can metabolize and produce a large amount of antibacterial substances, exhibiting significant antagonistic and bactericidal effects against the tested pathogens. Escherichia coli and Salmonella are major pathogens causing intestinal diseases and diarrhea in livestock and poultry (such as piglets and poultry). Pediococcus pentosus FREE-U AP01 exhibits potent and broad-spectrum killing ability against the aforementioned pathogens. Combined with its strong resistance to gastric acid and high concentrations of bile salts (0.3%), it demonstrates that it can enter the animal intestine in live form and exert its antagonistic function against pathogens, providing a solid foundation for its use as a probiotic feed additive to maintain animal intestinal health.

[0080] Example 10: Preparation of Pediococcus pentosaceus FREE-U AP01 freeze-dried bacterial powder 1. Strain activation and expansion: Pure Pediococcus pentosaceus FREE-U AP01 colonies were activated using MRS liquid medium (37℃, 24h). After activation, purity and viable count were tested. The results met the criteria (no contaminating bacteria, viable count reached 1.0 × 10⁻⁶). 8 After being transferred to a 500L fermenter for further culture (CFU / mL), the culture was expanded.

[0081] Fermentation conditions: culture temperature 37℃, rotation speed 100r / min, no aeration, tank pressure maintained at 0.05Mpa, pH of fermentation broth adjusted to 5.5, cultured for 48h to ensure cell concentration meets production requirements.

[0082] 2. Collection of bacterial sludge and addition of preservative: Place the fermentation broth in an ultracentrifuge and centrifuge at 10,000 rpm for 10 minutes. Remove the supernatant and collect the bacterial sludge from the bottom (the temperature should be maintained at 4°C during centrifugation). Add a compound freeze-drying preservative (such as 25% skim milk powder, 7.5% trehalose, and 0.75% glycerol) to the bacterial sludge and mix thoroughly to prepare a bacterial suspension.

[0083] 3. Freeze-drying and pulverization: The bacterial suspension containing the preservative was dispensed into freezing plates and pre-frozen using a gradient cooling method to form uniform ice crystals. It was then placed in a freeze dryer for vacuum drying. After drying, it was physically pulverized to obtain *Pediococcus pentosaceus* FREE-U AP01 freeze-dried bacterial powder.

[0084] Results: The Pediococcus pentosaceus FREE-U AP01 freeze-dried bacterial powder prepared in Example 10 was a milky white powder with good resolubility.

[0085] Plate count analysis showed that, thanks to the strain's excellent stress resistance, the viable count of *Pediococcus pentosaceus* FREE-U AP01 lyophilized bacterial powder remained stable at 1.0 × 10⁻⁶. 11 CFU / g or higher.

[0086] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention.

Claims

1. A strain of Pediococcus pentosaceus FREE-U AP01, with accession number CGMCC No. 37267.

2. A microbial agent, characterized in that, This bacterial agent contains Pediococcus pentosaceus FREE-U AP01 as described in claim 1.

3. A method for preparing the microbial agent according to claim 2, characterized in that, The steps are as follows: The *Pediococcus pentosaceus* FREE-U AP01 of claim 1 is inoculated into a fermentation medium for fermentation to obtain a fermentation broth; then the fermentation broth is mixed with a protectant to obtain a bacterial agent.

4. The method for preparing a microbial agent as described in claim 3, characterized in that, The fermentation medium consists of the following components: 2.0% glucose, 1.0% peptone, 1.0% beef extract, 0.5% yeast extract, 0.5% sodium acetate, 0.2% diammonium citrate, 0.1% Tween-80, 0.2% dipotassium hydrogen phosphate, 0.058% magnesium sulfate, and 0.025% manganese sulfate.

5. The method for preparing a microbial agent as described in claim 4, characterized in that, The *Pediococcus pentosaceus* FREE-UAP01 was inoculated using a seed culture, the preparation method of which is as follows: The above-mentioned Pediococcus pentosaceus FREE-U AP01 was inoculated into seed culture medium and cultured to obtain seed liquid.

6. The method for preparing a microbial agent as described in claim 5, characterized in that, The viable count of Pediococcus pentosaceus FREE-U AP01 in the seed culture was 1.0 × 10⁻⁶. 8 - 5.0×10 9 CFU / mL, and / or Based on the total volume of the seed culture medium, the inoculation amount of the seed solution is 2-5% by volume, and / or The seed culture medium consists of the following components: 2.0% glucose, 1.0% peptone, 1.0% beef extract, 0.5% yeast extract, 0.5% sodium acetate, 0.2% diammonium citrate, 0.1% Tween-80, 0.2% dipotassium hydrogen phosphate, 0.01% magnesium sulfate, and 0.005% manganese sulfate.

7. The method for preparing a microbial agent as described in claim 3, characterized in that, The fermentation process conditions are as follows: the culture temperature is 37℃, the rotation speed is 100 r / min, no aeration is allowed, the tank pressure is maintained at 0.05Mpa, the pH value of the fermentation broth is adjusted to 5.5-6.0, and the culture time is 48h.

8. The method for preparing a microbial agent as described in claim 3, characterized in that, The protective agent is at least one of starch, sucrose, lactose, trehalose, sodium citrate, skim milk powder, and maltodextrin.

9. A microbial agent, characterized in that, Prepared by the method described in any one of claims 3-8.

10. The application of the Pediococcus pentosaceus strain FREE-U AP01 as described in claim 1 or the bacterial agent as described in claim 2 or 9 in the preparation of microecological preparations for maintaining intestinal health, fermented foods, functional foods, and probiotic feed additives for animals.

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