Pediococcus pentosaceus strain pfp-03, composition and application thereof

Abstract

The application provides a strain of Pediococcus pentosaceus PFP-03 as well as a composition and application thereof, and belongs to the technical field of microorganisms. The preservation number of the Pediococcus pentosaceus PFP-03 is CGMCC No. 37269. The Pediococcus pentosaceus PFP-03 can treat and / or prevent intestinal diseases. The bacteria can significantly reduce the levels of key pro-inflammatory cytokines (TNF-alpha, IL-6) in colon tissues of rats, improve the overall state of IBS-D model rats, reduce the Bristol score results, the water content of feces and the AWR score, and effectively relieve the anxiety or depression-like behaviors of IBS-D model rats. Moreover, the bacteria can significantly reduce the levels of key pro-inflammatory cytokines (TNF-alpha, IL-1beta, IL-6) in serum of mice, make the body weight of the mice recover stably, reduce the phenomenon of hematochezia, improve the diarrhea symptoms, and effectively relieve the colon shortening, the increase of spleen index and the colon tissue damage caused by DSS.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a strain of Pediococcus pentosaceus PFP-03, its composition, and its application. Background Technology

[0002] Pediococcus pentosaceus ( Pediococcus pentosaceus As an important member of the lactic acid bacteria family, it is widely found in traditional fermented foods and the intestines of healthy individuals, and is recognized as a safe probiotic. Numerous in vitro and in vivo studies have confirmed that this bacterium possesses biological activities such as inhibiting the proliferation of harmful bacteria and regulating the body's immune function. These characteristics provide a solid theoretical basis and promising application prospects for its use in alleviating intestinal diseases.

[0003] There are many types of intestinal diseases, including common ones such as enteritis, irritable bowel syndrome, Crohn's disease, ulcerative colitis, and colorectal cancer.

[0004] Diarrhea-predominant irritable bowel syndrome (IBS-D) is the most common subtype of functional bowel disorder in clinical practice. It is characterized by recurrent episodes of abdominal pain and bloating, accompanied by increased bowel movement frequency, watery or pasty stools, and often accompanied by urgency and a feeling of incomplete evacuation, severely impacting patients' quality of life. In recent years, the global incidence and clinical consultation rate of IBS-D have continued to rise. Its exact pathophysiological mechanisms are not yet fully understood, but mainly involve the complex interaction of multiple factors, including genetic susceptibility, abnormal brain-gut axis interactions, visceral hypersensitivity, intestinal motility disorders, damage to the intestinal mucosal barrier, and intestinal microecological dysbiosis. Clinically, analgesics, antispasmodics, and antidepressants are commonly used to treat IBS-D, primarily focusing on symptom relief and failing to address the root cause of the disease's pathological mechanisms, leading to poor treatment efficacy.

[0005] Ulcerative colitis (UC) is a chronic, relapsing inflammatory bowel disease characterized by continuous inflammatory damage to the colonic mucosa, clinically manifesting as hematochezia, abdominal pain, and diarrhea. In recent years, the global incidence of UC has been steadily increasing. The exact pathological mechanisms remain unclear, but they primarily involve the interaction of multiple factors, including genetic susceptibility, abnormal immune regulation, environmental factors, and gut microbiota dysbiosis. Of particular note is gut microbiota dysbiosis, considered a crucial link in the development and progression of UC. Patients often exhibit decreased gut microbiota diversity, reduced beneficial microorganisms, and insufficient short-chain fatty acid metabolism, which further exacerbates intestinal mucosal barrier damage and a persistent inflammatory state. In terms of treatment, traditional drugs such as aminosalicylic acids, glucocorticoids, and immunosuppressants generally have drawbacks, including limited efficacy, significant side effects, and a high risk of inducing infections.

[0006] Therefore, providing a new strain of Pediococcus pentosaceus that can alleviate or improve enteropathy is a technical problem that urgently needs to be solved. Summary of the Invention

[0007] Therefore, the present invention aims to provide a strain of Pediococcus pentosaceus PFP-03 and its composition, wherein Pediococcus pentosaceus PFP-03 can improve enteropathy. This bacterium can significantly improve the overall condition of IBS-D model rats and effectively alleviate anxiety or depression-like behaviors in IBS-D model rats. Furthermore, this bacterium helps mice regain stable body weight, reduces rectal bleeding, improves diarrhea symptoms, and effectively alleviates colonic shortening, increased spleen index, and colonic tissue damage caused by DSS.

[0008] The objective of this invention is achieved through the following technical solution: This invention provides a strain of Pediococcus pentosaceus PFP-03, the preservation number of which is CGMCC No.37269.

[0009] The present invention provides a microbial composition comprising Pediococcus pentosaceus PFP-03 as described in claim 1.

[0010] Preferably, the dosage form of the microbial composition includes fermentation suspension, fermentation sludge, or freeze-dried powder.

[0011] Preferably, the fermentation suspension, fermentation sludge, or freeze-dried powder is obtained by fermentation of Pediococcus pentosaceus PFP-03 as described above.

[0012] Preferably, the viable count of Pediococcus pentosaceus PFP-03 in the above microbial composition is 1×10⁻⁶. 8 ~1×10 10 CFU / mL.

[0013] This invention provides the use of the above-mentioned Pediococcus pentosaceus PFP-03 or the above-mentioned microbial composition in the preparation of products for the treatment and / or prevention of enteropathy.

[0014] Preferably, the enteropathy includes prevention of diarrhea-predominant irritable bowel syndrome and / or ulcerative colitis.

[0015] The present invention provides a method for producing lactic acid and / or acetic acid, comprising the following steps: fermenting Pediococcus pentosus PFP-03 as described in claim 1 in MRS liquid medium.

[0016] Preferably, the Pediococcus pentosaceus PFP-03 or the microbial composition effectively alleviates gastrointestinal conditions, anxiety, or depression-like behaviors in IBS-D model rats; the Pediococcus pentosaceus PFP-03 or the microbial composition effectively alleviates DSS-induced colonic shortening, increased spleen index, and colonic tissue damage.

[0017] The present invention provides a medicament for treating and / or preventing enteropathy, the medicament comprising the above-mentioned Pediococcus pentosaceus PFP-03 or the above-mentioned microbial composition.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a *Pediococcus pentosaceus* strain PFP-03, with the preservation number CGMCC No. 37269. The *Pediococcus pentosaceus* PFP-03 provided by this invention can treat and / or prevent enteropathy. This bacterium can significantly reduce the levels of key pro-inflammatory cytokines (TNF-α, IL-6) in rat colon tissue, resulting in a significant improvement in the overall condition of IBS-D model rats, reducing Bristol scores, fecal water content, and AWR scores, and effectively alleviating anxiety or depression-like behaviors in IBS-D model rats. Furthermore, this bacterium significantly reduces the levels of key pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) in mouse serum, leading to stable weight recovery, reduced fecal bleeding, and improved diarrhea symptoms in mice, effectively alleviating colonic shortening, increased spleen index, and colonic tissue damage caused by DSS. *Pediococcus pentosaceus* PFP-03 exhibits strong safety.

[0019] Biological Preservation Instructions Pediococcus pentosaceus PFP-03 was named PFP-03 and classified as follows: Pediococcus pentosaceus It was deposited on January 4, 2026 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 37269. Attached Figure Description

[0020] 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.

[0021] Figure 1 This is a colony diagram of an MRS plate.

[0022] Figure 2 This is an electron microscope image with Gram staining.

[0023] Figure 3 The results of the identification of Pediococcus pentosaceus PFP-03 are shown in Figure A, which shows the homology comparison information of this bacterium; and Figure B is the phylogenetic tree.

[0024] Figure 4 This is a growth curve diagram.

[0025] Figure 5 This is a blood agar colony diagram.

[0026] Figure 6 To illustrate the ameliorative effect of Pediococcus pentosaceus PFP-03 on IBS-D rats, A represents the Bristol score of each group of rats; B represents the fecal water content of each group of rats; C represents the AWR score of each group of rats; D and E represent the open field test results; F represents the saccharide preference test results; G and H represent the contents of pro-inflammatory factors IL-6 and TNF-α in rat colon tissue, respectively.

[0027] Figure 7 To illustrate the ameliorative effect of Pediococcus pentosaceus PFP-03 on ulcerative colitis in mice, A represents the mouse experimental process; B represents the disease activity index (DAI) score of mice; C represents the mouse body weight; D represents the spleen index of each group of mice; E represents the colon length measurement of mice; F and G represent the histopathological evaluation results of colon tissue; H, I, and J represent the levels of pro-inflammatory factors IL-6, IL-1β, or TNF-α in mouse serum, respectively. Detailed Implementation

[0028] In this invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0029] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] This invention provides a strain of Pediococcus pentosaceus PFP-03, the preservation number of which is CGMCC No.37269.

[0031] The *Pediococcus pentosaceus* PFP-03 provided by this invention was isolated from fresh feces of a healthy donor; the 16S rDNA sequence of the *Pediococcus pentosaceus* PFP-03 is shown in SEQ ID. As shown in NO.1, *Pediococcus pentosaceus* PFP-03 is a Gram-positive coccus that grows well in microaerobic or anaerobic environments, does not form spores, and is non-hemolytic. The bacteria are spherical or oval, often arranged in pairs or tetrads. On MRS agar plates, it forms white, opaque, round colonies with neat edges. *Pediococcus pentosaceus* PFP-03 is non-hemolytic, sensitive to multiple antibiotics, does not exhibit antibiotic resistance, and does not produce toxins, meeting the safety standards for probiotics. Therefore, *Pediococcus pentosaceus* PFP-03 has high safety. The optimal growth temperature for *Pediococcus pentosaceus* PFP-03 is 30~37℃, and the optimal pH is 5.5~6.5. *Pediococcus pentosaceus* PFP-03 has strong gastrointestinal tolerance, tolerating acidic and bile salt environments as well as artificial gastric and intestinal fluids, which facilitates intestinal colonization and its effects. *Pediococcus pentosaceus* PFP-03 can treat and / or prevent enteropathy. This invention found that *Pediococcus pentosaceus* PFP-03 significantly reduced the levels of key pro-inflammatory cytokines (TNF-α, IL-6) in rat colon tissue, guiding the inflammatory environment towards an anti-inflammatory / repair state, resulting in a significant improvement in the overall condition of IBS-D model rats, reducing Bristol scores, fecal water content, and AWR scores, and effectively alleviating anxiety or depression-like behaviors in IBS-D model rats, specifically manifested as a significant increase in total activity distance and number of upright movements in the open field test, and an increase in the sucrose preference index. *Pediococcus pentosaceus* PFP-03 also significantly reduced the levels of key pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) in mouse serum, guiding the inflammatory environment towards an anti-inflammatory / repair state, resulting in a decrease in the DAI score in mice, specifically manifested as stable weight recovery, reduced fecal bleeding, and improved diarrhea symptoms, and effectively alleviating colonic shortening, increased spleen index, and colon tissue damage caused by DSS.

[0032] The present invention provides a microbial composition comprising the above-mentioned Pediococcus pentosaceus PFP-03.

[0033] In this invention, the dosage form of the microbial composition includes a fermentation suspension, a fermentation sludge, or a lyophilized powder. The fermentation suspension, fermentation sludge, or lyophilized powder is obtained by fermentation of *Pediococcus pentosaceus* PFP-03 as described above. The fermentation suspension, fermentation sludge, or lyophilized powder can be used alone or in combination. The viable count of *Pediococcus pentosaceus* PFP-03 in the microbial composition is 1 × 10⁻⁶. 8 ~1×10 10 CFU / mL. The viable count of the fermentation bacteria suspension is ≥1×10⁻⁶. 8 CFU / mL, the viable count of the fermented sludge is ≥1×10⁻⁶. 9 CFU / g, the viable count of the lyophilized powder is ≥1×10⁻⁶10 The freeze-dried powder has a CFU / g content and a moisture content (mass fraction) ≤5%. The freeze-dried powder may contain a protectant and / or filler as needed. The protectant is 10% skim milk powder and / or 5% sucrose (both by mass fraction) of the total mass of the freeze-dried powder. The filler may be maltodextrin. The fermentation suspension may contain a stabilizer, such as 0.05% L-cysteine ​​by mass. The microbial composition also includes any one of the following: extract, culture, culture supernatant, fermentation product, and fermentation supernatant, or any combination of two or more in any proportion. The culture or fermentation product is obtained by inoculating or fermenting *Pediococcus pentosaceus* PFP-03, centrifuging, and collecting the bacterial precipitate. The culture supernatant or fermentation supernatant is obtained by inoculating or fermenting *Pediococcus pentosaceus* PFP-03, centrifuging, and collecting the supernatant, i.e., a solution without *Pediococcus pentosaceus* PFP-03. The extract is a cell-free product obtained by using Pediococcus pentosaceus PFP-03 cells as raw material, which is broken down by any one or more of the following methods: ultrasonic lysis, enzymatic hydrolysis, freeze-thaw, and high-pressure homogenization, and then separated and purified by centrifugation, filtration, chromatography, extraction and other means.

[0034] In this invention, the microbial composition is obtained by fermenting the aforementioned Pediococcus pentosaceus PFP-03 on a culture medium. This invention does not specifically limit the type of culture medium; any conventional culture medium in the art that enables Pediococcus pentosaceus PFP-03 to grow well is acceptable. As an optional embodiment of this invention, the culture medium can be either MRS solid medium or MRS liquid medium. As an optional embodiment of the present invention, the composition of the MRS solid culture medium may be 5.0-15.0g of peptone, 5.0-15.0g of beef extract, 2.5-7.5g of yeast extract, 15.0-25.0g of glucose, 2.5-7.5g of sodium acetate, 1.5-2.5g of diammonium hydrogen citrate, 0.5-1.5g of Tween-80, 1.0-3.0g of K2HPO4, 0.15-0.25g of MgSO4, 0.03-0.07g of MnSO4, 0.3-0.7g of L-cysteine ​​hydrochloride, 10.0-20.0g of agar, and 1.0L of distilled water. As an optional embodiment of the present invention, the composition of the MRS liquid culture medium may be 5.0-15.0 g of peptone, 5.0-15.0 g of beef extract, 2.5-7.5 g of yeast extract, 15.0-25.0 g of glucose, 2.5-7.5 g of sodium acetate, 1.5-2.5 g of diammonium hydrogen citrate, 0.5-1.5 g of Tween-80, 1.0-3.0 g of K₂HPO₄, 0.15-0.25 g of MgSO₄, 0.03-0.07 g of MnSO₄, 0.3-0.7 g of L-cysteine ​​hydrochloride, and 1.0 L of distilled water. In the present invention, the culture temperature may be 30-37°C, or 30, 31, 32, 33, 34, 35, 36, or 37°C; the culture time may be 24-48 h, or 1, 1.5, or 2 days.

[0035] This invention provides the use of the above-mentioned Pediococcus pentosaceus PFP-03 or the above-mentioned microbial composition in the preparation of products for the treatment and / or prevention of enteropathy.

[0036] In this invention, the enteropathy includes prevention of diarrhea-predominant irritable bowel syndrome and / or ulcerative colitis. The Pediococcus pentosaceus PFP-03 or the microbial composition effectively alleviates gastrointestinal conditions and anxiety- or depression-like behaviors in IBS-D model rats. The Pediococcus pentosaceus PFP-03 or the microbial composition effectively alleviates DSS-induced colonic shortening, elevated spleen index, and colonic tissue damage.

[0037] The present invention provides a method for producing lactic acid and / or acetic acid, comprising the following steps: fermenting Pediococcus pentosus PFP-03 as described in claim 1 in MRS liquid medium.

[0038] In this invention, homolactic fermentation is carried out in MRS liquid medium, mainly producing lactic acid. Under glucose-limited conditions, a small amount of acetic acid may be produced, but propionic acid, butyric acid, and other short-chain fatty acids are not produced. The strain was inoculated into MRS liquid medium (containing 20 g / L glucose) and cultured at 37°C and 150 rpm for 24 h. The experimental results under these conditions were as follows: initial glucose concentration of 20.0 g / L, residual glucose after fermentation of 0.3 g / L, actual glucose consumption of 19.7 g / L, measured L-lactic acid yield of 16.2 ± 0.3 g / L, glucose consumption rate of 98.5%, corresponding to a lactic acid conversion rate >82%, with only trace amounts of acetic acid (0.12 ± 0.03 g / L) detected, and propionic acid and butyric acid not detected. Under glucose-limited conditions of 2 g / L and cultured for 36 h, the strain produced 1.48 ± 0.04 g / L of lactic acid and 0.63 ± 0.05 g / L of acetic acid, with lactic acid still being the main fermentation product.

[0039] Lactic acid conversion rate (%) = Measured lactic acid concentration (g / L) / Consumed glucose concentration (g / L) × 100% The present invention provides a medicament for treating and / or preventing enteropathy, the medicament comprising the above-mentioned Pediococcus pentosaceus PFP-03 or the above-mentioned microbial composition.

[0040] In this invention, the aforementioned *Pediococcus pentosaceus* PFP-03 or the aforementioned microbial composition can significantly reduce the levels of key pro-inflammatory cytokines (TNF-α, IL-6) in rat colon tissue, resulting in a significant improvement in the overall condition of IBS-D model rats, reducing Bristol scores, fecal water content, and AWR scores, and effectively alleviating anxiety or depression-like behaviors in IBS-D model rats. Furthermore, the aforementioned *Pediococcus pentosaceus* PFP-03 or the aforementioned microbial composition significantly reduces the levels of key pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) in mouse serum, leading to stable weight recovery, reduced fecal bleeding, and improved diarrhea symptoms in mice, effectively alleviating DSS-induced colonic shortening, increased spleen index, and colon tissue damage. The aforementioned *Pediococcus pentosaceus* PFP-03 or the aforementioned microbial composition exhibits strong safety.

[0041] In the following examples, a fecal DNA extraction kit (MP Biomedicals, catalog number 116570200), interleukin-6 (Thermo Fisher Scientific, catalog number BMS625), and tumor necrosis factor-α (Thermo Fisher Scientific, catalog number ERA57RB) were used.

[0042] The information of the healthy donor is as follows: age 28, male, BMI 22.5, healthy after physical examination and psychological testing, and has not taken antibiotics or probiotic products within the past month.

[0043] SPF-grade male Wistar rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., at 6 weeks of age and weighing 180-220g.

[0044] Male C57BL / 6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., at 6 weeks of age and weighing 20-22g.

[0045] MRS solid culture medium: 10.0g peptone, 10.0g beef extract, 5.0g yeast extract, 20.0g glucose, 5.0g sodium acetate, 2.0g diammonium citrate, 1.0g Tween-80, 2.0g K₂HPO₄, 0.2g MgSO₄, 0.05g MnSO₄, 0.5g L-cysteine ​​hydrochloride, 15g agar, 1.0L distilled water. Mix well and autoclave at 121℃ for 15 minutes. After sterilization, remove and cool to approximately 50℃. Pour 20-25ml of the medium into each sterile petri dish and allow it to solidify.

[0046] MRS liquid medium: The preparation method is the same as that of MRS solid medium, except that the agar is removed.

[0047] 50% (v / v) glycerol preservation solution: Measure 50 mL of glycerol and add 50 mL of ultrapure water, mix well. Autoclave at 121℃ for 15 min, cool and use.

[0048] Example 1 Pediococcus pentosaceus ( Pediococcus pentosaceus Screening and identification of PFP-03 bacterial suspension 1. Screening of Pediococcus pentosaceus PFP-03 Take 0.1g of fresh feces from a healthy donor, perform serial dilution, and take 10... -5 10 -6 10 -7 Three gradient dilutions (100 μL each) were spread onto MRS solid medium and anaerobically cultured at 37°C for 48 h. Observation continued until single colonies were visible to the naked eye. Typical opaque white circular single colonies were picked, inoculated onto MRS liquid medium, and cultured anaerobically at 37°C for 24–48 h.

[0049] 2. Identification of Pediococcus pentosaceus PFP-03 (1) Colony morphology After anaerobic culture at 37°C for 48 hours in MRS solid medium, this strain formed round colonies with a diameter of approximately 1-2 mm. Figure 1 As shown.

[0050] Figure 1This indicates that the colony surface is smooth and raised, with neat edges, and is opaque and milky white.

[0051] (2) Morphology under a microscope Gram staining and microscopic examination of the smear revealed that the bacterium was a Gram-positive coccus. Figure 2 As shown.

[0052] Figure 2 The bacteria are spherical or oval in shape, often arranged in pairs or tetrads, and no spores were observed.

[0053] (3) 16S rDNA identification Genomic DNA of the target strain was extracted using a fecal DNA extraction kit. The extracted genomic DNA was used as a template for PCR amplification. The upstream and downstream primers were 5'-AGAGTTTGATCMTGGCTCAG-3' (27F, SEQ ID NO.1) and 5'-GGTTACCTTGTTACGACTT-3' (1492R, SEQ ID NO.2), respectively. The PCR amplification system in Table 1 and the PCR amplification reaction procedure in Table 2 were followed for the 16S rDNA PCR experiment. After the PCR reaction was completed, the PCR product was taken for agarose gel detection and photography. The amplified fragment length was about 1500bp.

[0054] Table 1 PCR amplification system

[0055] Table 2 PCR amplification program

[0056] The PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The obtained 16S rDNA sequence is shown in SEQ ID NO:3, as follows:

[0057] A BLAST sequence comparison of the 16S rDNA shown in SEQ ID NO:1 on the NCBI website revealed that it was similar to Pediococcus pentosaceus (…). Pediococcus pentosaceus The 16S rDNA sequence homology of the sample exceeds 99.52%, such as... Figure 3 As shown in Figure A. The phylogenetic tree constructed based on this (Neighbor-Joining method) shows that PFP-03 clusters closely with the reference strain of this species, as shown in Figure A. Figure 3 As shown in Figure B. The above results collectively confirm that PFP-03 is *Pediococcus pentosaccharis*, and it is named *Pediococcus pentosaccharis* PFP-03 and biologically preserved. After identification as *Pediococcus pentosaccharis* by 16S, 0.5 mL of fresh bacterial culture was mixed with an equal volume of sterile 50% (v / v) glycerol preservation solution and frozen at -80°C to obtain cryopreserved glycerol tubes of the strain.

[0058] Figure 3 The results showed that PFP-03 was associated with Pediococcus pentosaceus (PFP-03). Pediococcus pentosaceus The reference strains of PFP-03 clustered closely, indicating that PFP-03 belongs to the group. Pediococcus pentosaceus .

[0059] Preservation Information: *Pediococcus pentosaceus* is deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 4, 2026, with accession number CGMCC No. 37269. The address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The classification name is *Pediococcus pentosaceus*. Pediococcus pentosaceus The strain name is PFP-03.

[0060] Example 2 Characterization of Pediococcus pentosaceus PFP-03 1. Growth curve determination After activating the *Pediococcus pentosaceus* PFP-03 strain preserved in Example 1, it was inoculated into MRS liquid medium at a 2% (v / v) inoculum and anaerobically cultured at 37°C for 0h, 2h, 4h, 8h, 12h, 16h, 20h, 24h, 36h, 48h, and 72h. OD values ​​were measured. 600 Values, plot growth curves, such as Figure 4 As shown.

[0061] Figure 4 This indicates that Pediococcus pentosaceus PFP-03 has good growth performance.

[0062] 2. Safety testing Hemolytic activity test: *Pediococcus pentosaceus* PFP-03 was streaked onto Columbia blood agar containing 5% defibrinated sheep blood and anaerobically cultured at 37°C for 48 hours. All samples showed γ-hemolysis, i.e., no hemolysis was observed. Figure 5 As shown.

[0063] Figure 5 This indicates that Pediococcus pentosaceus PFP-03 does not exhibit hemolysis.

[0064] Based on the results of growth curve determination and safety testing, it can be concluded that Pediococcus pentosaceus PFP-03 has good growth performance, does not exhibit hemolysis, and has strong safety.

[0065] Example 3 Application of Pediococcus pentosaceus PFP-03 in improving IBS-D model rats 1. Experimental Design (1) Animal model construction: SPF-grade male Wistar rats were used in this study. All animals were kept under a 12-hour light-dark cycle (temperature 22±2℃; humidity 50%±5%) in the absence of specific pathogens. After feeding for 1 week, they were randomly divided into a control (NC) group, a model (IBS) group, and a probiotic gavage (PP) group, with 10 rats in each group. Except for the control group rats, the other groups of rats were injected with 0.5 mL of 5% acetic acid solution into the colon daily for two weeks to obtain acetic acid enema rats. The acetic acid enema rats were placed in a restraint device and administered the solution twice a day (once in the morning and once in the afternoon) for 2 hours each time for 4 weeks to obtain IBS-D model rats.

[0066] The constructed IBS-D model rats exhibited diarrhea (increased frequency of defecation and increased fecal water content), and the Abdominal Withdrawal Reflex (AWR) score was ≥3, indicating successful model establishment.

[0067] (2) Administration and treatment methods After modeling, PP group rats were administered 200 μL of bacterial suspension (containing 1 × 10⁻⁶ live bacteria) by gavage every morning for two consecutive weeks. 9 (CFU / mL), while rats in the IBS group and NC group were given 200 μL of PBS solution by gavage every morning for two consecutive weeks, with all other feeding conditions such as temperature, humidity, light, and feed being the same.

[0068] The preparation steps for the bacterial suspension (Pediococcus pentosaceus PFP-03 suspension) in the PP group were as follows: The cryopreserved glycerol tubes of the strain preserved in Example 1 were thawed and inoculated into sterile pre-reduced MRS liquid medium at an inoculation rate of 10% (v / v). The culture was then incubated statically at 37°C under strictly anaerobic conditions for 1–2 days. The bacterial concentration was calibrated using the serial dilution plate count method (CFU method) and diluted to 1×10⁻⁶ with pre-reduced sterile PBS buffer. 9CFU / mL was used to obtain a diluted bacterial suspension. 1 mL of the diluted suspension was placed at 4°C and centrifuged at 10000 rpm for 8 min. The supernatant was discarded to obtain a bacterial pellet. Under strict anaerobic conditions, 200 μL of pre-reduced sterile PBS buffer was used to resuspend the bacterial pellet. After resuspending, the calibrated concentration was 1 × 10⁻⁶ viable cells. 9 CFU / mL was used to obtain a suspension of Pediococcus pentosaceus PFP-03.

[0069] (3) Grouping of experimental animals Table 3 Grouping of experimental animals

[0070] 2. Detection Indicators and Methods (1) Observation of general condition and defecation The mental state, activity, fur, and anal cleanliness of rats in each group were observed. The fecal Bristol grading and fecal water content were used to assess diarrhea in rats. Rats were placed in metabolic cages for 24 hours with free access to rodent food and water. They were fasted for 4 hours before the experiment (with free access to water). After fasting, the bottom of the metabolic cages was cleaned, and fresh feces were collected within 4 hours. The total weight (m0) was measured using a precision electronic balance within 30 minutes. A forced convection constant temperature oven, pre-calibrated for temperature uniformity (±1℃), was used. The feces were evenly spread on petri dishes and placed in the middle layer of the oven for continuous drying for 24 hours. After drying, the feces were removed, cooled to room temperature (25℃) in a desiccator, and immediately weighed (m1).

[0071] Fecal moisture content (%) = (m0-m1) ÷ m0 × 100.

[0072] Bristol Grading Criteria: 1 point - hard, scattered lumps like nuts; 2 points - sausage-shaped without cracks; 3 points - sausage-shaped with cracks; 4 points - smooth and soft sausage-shaped; 5 points - soft lumps with clear edges; 6 points - fluffy or pasty stools; 7 points - watery stools.

[0073] (2) Visceral sensitivity assessment Twelve hours after the last gavage, rats in each group were anesthetized and had a balloon catheter inserted through the anus. They were then given pressure stimulation of 20, 40, and 60 mm Hg for 20 seconds, with a 5-minute interval. Each stimulation was repeated three times, and the average value was taken.

[0074] The AWR score was measured by two independent observers using a double-blind method according to the following criteria: no obvious behavioral response, 0 points; slight contraction of back muscles, 1 point; contraction of abdominal muscles but not lifting off the ground, 2 points; strong contraction of abdominal muscles and lifting off the ground, 3 points; arched back with pelvic floor lifting, 4 points.

[0075] (3) Open field experiment Fourteen days after drug administration, the open field test (OFT) was performed. During the experiment, rats were first placed in a 100×100×40cm open field test chamber, with a central area (approximately 25%–30% of the area) marked at the bottom. They were allowed approximately 3 minutes to acclimatize to the environment to ensure stability. Then, the testing program was initiated, and the rats' spontaneous activity over the next 10 minutes was continuously observed and recorded. The time spent in the open field center and the total distance traveled by the rats in the last 10 minutes were calculated. After each rat's experiment, the inner walls and bottom of the open field chamber were wiped with 75% alcohol to remove animal excrement and odor.

[0076] (4) Sugar water preference experiment On day 10 of drug administration, a sucrose preference test (SPT) was conducted. The experiment consisted of two phases: a training phase and a testing phase. Before the test, the rats underwent a 3-day acclimatization training period, during which two bottles of 2% sucrose solution were placed in the cage for 24 hours, followed by a 48-hour period where one bottle of sucrose solution was randomly replaced with pure water. To prevent the rats from developing a positional preference, the water bottle positions were changed periodically. After the acclimatization period, the formal experiment began. The rats were fasted and deprived of water for 12 hours, while being given a pre-weighed bottle of 2% sucrose solution and a bottle of pure water. The water bottle positions were changed periodically, and the amount of sucrose solution and pure water ingested by the rats over 24 hours was recorded.

[0077] Sugar solution preference index (%) = sucrose solution consumption ÷ (water consumption + sucrose solution consumption) × 100.

[0078] (5) Measurement of inflammatory factors The levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in rat colon tissue were determined according to the ELISA kit instructions.

[0079] 3. Experimental Results (1) Effects of each treatment group on the general condition of rats During the acclimatization period, all rats were in good spirits, active, with smooth, glossy fur and clean anuses. After intervention, the NC group rats were in normal spirits, and their fur and activity levels remained consistent with those during the acclimatization period; the IBS group rats were in poor spirits, easily irritated, and had disheveled fur; the PP administration group rats showed improvement in spirits, reduced fighting, clean fur, and normal appetite. The Bristol scores for each group are as follows: Figure 6 As shown in Figure A, the results of the fecal water content measurement of each group of rats are as follows: Figure 6 As shown in B.

[0080] Figure 6 Figure A shows the Bristol scores of rats in each group. The results showed that compared with the NC group, the Bristol scores of rats in the IBS model group were significantly increased (p<0.01), while after PP intervention, the Bristol scores of rats in the PP group were significantly decreased (p<0.01), indicating that PP can effectively inhibit diarrhea caused by IBS. Figure 6 Figure B shows the fecal water content of rats in each group. The results showed that, compared with the NC group, the fecal water content of both the IBS group and the PP group was significantly increased before treatment (p<0.01). Compared with the IBS group after treatment, the fecal water content of rats in the PP group was significantly decreased after treatment (p<0.01).

[0081] (2) Changes in the sensitivity of rat viscera The AWR scores of rats in each group are as follows: Figure 6 As shown in C.

[0082] Figure 6 In the middle (C), the AWR scores of rats in each group are shown. Compared with the NC group, the AWR scores of rats in the IBS group were increased at 20, 40, and 60 mmHg pressures (p<0.01). Compared with the IBS group, the AWR scores of rats in the PP group were significantly decreased at 20, 40, and 60 mmHg pressures (p<0.01).

[0083] (3) Changes in anxiety or depression-like behaviors in rats in each group The results of the open field experiment are as follows Figure 6 China D and Figure 6 As shown in E.

[0084] Figure 6 China D and Figure 6 Figure E represents the results of the open field test. The results showed that, compared with the NC group, the IBS group rats exhibited significantly reduced total activity distance and number of upright movements in the open field test (p<0.01), reflecting reduced exploratory behavior or increased anxiety-like behavior in the IBS group rats. Compared with the IBS group, the PP group rats showed significantly increased total activity distance and number of upright movements (p<0.01), reflecting improved mental state.

[0085] Results of the sugar water preference experiment: Figure 6 As shown in F.

[0086] Figure 6 Figure F shows the results of the sucrose preference test. The results indicate that, compared with the NC group, the sucrose preference index of the IBS group rats was significantly decreased (p<0.01), reflecting the lack of pleasure in the IBS group rats. Compared with the IBS group, the sucrose preference index of the PP group rats was significantly increased (p<0.01).

[0087] (4) Changes in inflammatory factors in the colon tissue of rats in each group The regulatory effect of PP on the inflammatory response in IBS model rats was analyzed by detecting the levels of pro-inflammatory factors IL-6 and TNF-α in rat colon tissue. Figure 6 As shown in G and H.

[0088] Figure 6 In G and H, the levels of pro-inflammatory factors IL-6 and TNF-α in rat colon tissue were represented. The results showed that the levels of IL-6 and TNF-α in the colon tissue of rats in the NC group remained at low levels, indicating a mild inflammatory state. Compared with the NC group, the levels of these two pro-inflammatory factors in the colon of rats in the IBS group were significantly increased (P<0.01, corresponding to...). Figure 6 The results (G, H) indicate that IBS induction can promote the release of large amounts of pro-inflammatory factors; however, after PP intervention, the levels of IL-6 and TNF-α in the rat colon were significantly lower than those in the IBS group (p<0.01). Therefore, PP intervention can exert an anti-inflammatory regulatory effect on IBS-induced inflammation by reducing the levels of IL-6 and TNF-α.

[0089] The above results indicate that the IBS group had a poor overall condition, elevated Bristol scores, increased fecal water content, and elevated AWR scores, indicating successful model establishment. After intervention with Pediococcus pentosaceus PFP-03, the overall condition of the rats improved significantly, with decreased Bristol scores, significantly reduced fecal water content, and a simultaneous decrease in AWR scores. Behavioral results showed that the IBS group rats exhibited anxiety or depression-like behaviors, specifically a significant reduction in total activity distance and number of upright movements in the open field test, and a decreased sucrose preference index. These anxiety or depression-like behaviors significantly improved after Pediococcus pentosaceus PFP-03 intervention. ELISA results showed that compared with the NC group, the IBS group had significantly increased levels of IL-6 and TNF-α pro-inflammatory factors, and the inflammation level significantly decreased after Pediococcus pentosaceus PFP-03 intervention. In conclusion, Pediococcus pentosaceus PFP-03 can effectively alleviate the pathological and inflammatory state in rats with diarrhea-predominant irritable bowel syndrome.

[0090] Example 4 1. Experimental Design (1) Animal model construction: Male C57BL / 6J mice were used to construct an ulcerative colitis model by induction with sodium dextran sulfate (DSS). Mice were given a 7-day laboratory acclimatization period before the experiment. During the acclimatization period and the experiment, all animals were kept under a 12-hour light-dark cycle (temperature 22±2℃; humidity 50%±5%) in the absence of specific pathogens.

[0091] Seven-week-old C57BL / 6J mice were randomly divided into three groups using a random number table: control group (Control), dextran sulfate sodium group (DSS), and Pediococcus pentosus PFP-03 group (PP), with 10 mice in each group.

[0092] From the first day of modeling, control group mice had free access to normal water for 15 days, while mice in the dextran sodium sulfate group (DSS) and the Pediococcus pentosaceus PFP-03 group (PP) were given 3.0% (w / v) DSS solution for the first 7 days, and then switched to normal water from day 8 onwards. During the modeling period, the success of the model was monitored and confirmed by the daily Disease Activity Index (DAI) score.

[0093] (2) Administration and treatment methods From day 8 to day 15, mice in the PP group were administered 200 μL of Pediococcus pentosaceus PFP-03 bacterial suspension (containing 1 × 10⁻⁶ live bacteria) by gavage daily. 9 Mice in the DSS and Control groups were administered the same volume of PBS solution daily via gavage, while other feeding conditions such as temperature, humidity, light, and diet were kept identical. Mice were sacrificed at the end of the experiment, and tissue samples including immune organs, blood, proximal colon, feces, cecal contents, and distal colon were collected for subsequent analysis.

[0094] The bacterial suspension in the PP group (Pediococcus pentosacchari PFP-03 bacterial suspension) is the bacterial suspension (Pediococcus pentosacchari PFP-03 bacterial suspension) described in Example 3.

[0095] (3) Grouping of experimental animals Table 4 Grouping of experimental animals

[0096] 2. Detection Indicators and Methods (1) Physiological indicators Physiological indicators were used to assess the health status of mice, including water intake, food intake, body weight, and disease activity index (DAI) score.

[0097] The DAI scoring formula is: (weight loss score + fecal consistency score + fecal occult blood score) ÷ 3. Fecal occult blood in mice is detected using a fecal occult blood qualitative test kit (Solarbio, BC8270). The DAI scoring criteria are shown in the table below.

[0098] Table 5. DAI Scoring Criteria

[0099] Colon length and spleen index were measured for each group of mice. The spleen index was calculated using the following formula: spleen weight (mg) / body weight (g) to assess systemic immune response.

[0100] (2) Histopathological observation of colon tissue Distal colon tissue blocks were taken, fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin-eosin (H&E) and periodic acid Schiff reagent. Histopathological scores were calculated based on epithelial loss, crypt damage, goblet cell consumption, and inflammatory cell infiltration.

[0101] (3) Measurement of inflammatory factors The levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) in mouse serum were determined according to the ELISA kit instructions.

[0102] The specific operation is as follows: (a) Soaking the microplate: Prepare the required reagents and standards, add 300 μL of washing solution to both the standard wells and the sample wells of the microplate, let stand for 30 seconds, discard the washing solution, and pat dry.

[0103] (b) Add standards and samples: Add the corresponding volume of standards to the corresponding wells of the ELISA plate, add the appropriate volume of sample to the sample wells, and make up the volume of each well to 100 μL.

[0104] (c) Antibody incubation: Add 50 μL of the corresponding diluted antibody to the standard and sample wells, shake at 100-300 r / min, and incubate at room temperature. The incubation time for IL-6 and MPO indicators is 1.5 h, and the incubation time for IL-10 and IL-1β is 2 h.

[0105] (d) Washing: After incubation, add 300 μL of washing buffer to each well to wash the microplate, for a total of 6 washes. After each wash, discard the washing buffer and pat dry on absorbent paper.

[0106] (e) Enzyme incubation: After washing, add 100 μL of diluted horseradish peroxidase-labeled streptavidin to the standard and sample wells, shake at 100-300 r / min and incubate at room temperature.

[0107] (f) Washing: Wash the microplate 6 times in the same manner as d).

[0108] (g) Color development: After washing, add 100 μL of color development substrate to the standard wells and the sample wells, and incubate at room temperature for 5-30 min in the dark to develop color. After incubation, add 100 μL of stop solution to each well and mix thoroughly to stop color development.

[0109] (h) Reading: The reading time should be controlled within 30 minutes after adding the stop solution. Use an ELISA reader to perform dual-wavelength detection, measuring the absorbance (OD) at 450 nm and 630 nm.

[0110] 3. Experimental Results (1) General description of colonic inflammation in mice Figure 7 In the middle, A represents the mouse experiment process.

[0111] Figure 7 Figure B shows the Disease Activity Index (DAI) scores in mice, indicating that the severity of colitis in mice can be dynamically monitored using DAI scores. According to the dynamic change curves of DAI scores in the figure, the DAI scores of the different groups of mice showed significant differences throughout the experiment. On day 1, the DAI scores of all three groups of mice were close to 0, with no significant difference. As the experiment progressed, the DAI scores of the Control group mice remained at a low level, indicating stable health. In contrast, the DSS model group mice showed a rapid increase in DAI scores from day 3, reaching a peak on day 7 (p<0.001), exhibiting typical colitis symptoms such as bloody stools, diarrhea, and weight loss. This result demonstrates the successful establishment of the DSS-induced acute colitis model in mice. Although the DAI scores of the PP intervention group also showed an upward trend, the increase was significantly slower than that of the DSS group. From day 7, the scores began to decline, and by the later stages of the experiment, they were significantly lower than those of the DSS group (p<0.05), indicating that PP intervention can effectively improve the disease state of DSS-induced colitis mice, significantly reduce their DAI scores, and thus alleviate the severity of colitis.

[0112] Figure 7 The results of the body weight analysis of mice in the control, DSS, and PP groups showed significant differences in weight changes throughout the experiment. Mice in the control group exhibited stable and continuous weight gain, indicating good physiological condition. In contrast, mice in the DSS model group did not show a significant decrease in weight in the early stages of the experiment (days 1-5), but their weight gain stagnated and remained at a stable level. From day 6 onwards, their weight began to decrease significantly, accompanied by obvious disease symptoms, reaching its lowest point around day 8 (p<0.001). Although there was a slight recovery afterward, their overall weight remained significantly lower than that of the normal group. The weight change in the PP intervention group was between that of the normal and model groups, showing a certain protective effect. No mouse deaths occurred during the entire experiment.

[0113] Figure 7Figure D shows the spleen index results for each group of mice. The results indicated that, compared with the control group, the spleen index of mice in the DSS model group was significantly increased (p<0.05), suggesting that DSS-induced colitis is accompanied by a significant systemic inflammatory response, leading to compensatory splenic hypertrophy. After PP intervention, the spleen index of mice in the PP group was significantly lower than that in the DSS group (p<0.05), indicating that PP can effectively inhibit the immunopathological changes in the spleen caused by DSS and alleviate splenomegaly. Although the spleen index in the PP group was still slightly higher than that in the normal control group, the improvement trend suggests that PP has a certain protective effect in regulating the systemic immune response and alleviating colitis-related splenic lesions.

[0114] (2) Changes in colon length and tissue structure in mice Colitis may cause colon atrophy in mice, and reduced colon length is a typical characteristic of colon atrophy in colitis mice.

[0115] Figure 7 Figure E shows the colon length measurement results in mice. The results indicate that compared with the control group, the colon length in the DSS model group was significantly shortened (p<0.05), suggesting that DSS-induced colitis is accompanied by significant mucosal inflammation and tissue edema. After PP intervention, the colon length in the PP group recovered somewhat compared with the DSS group (p<0.05), indicating that PP can effectively alleviate the colon shortening phenomenon caused by DSS and has a certain protective effect on colonic tissue structure. This result further validates the positive effect of PP in improving colitis-related pathological changes at a macroscopic level.

[0116] The severity of colonic injury was further assessed through histopathological observation.

[0117] Figure 7 F and G represent the results of histopathological evaluation of colonic tissue. The results showed that, compared with the Control group, the DSS model group mice exhibited typical inflammatory lesions in their colonic tissue, characterized by mucosal structure destruction, loss of crypt structures, and lesions extending deep into the muscular layer, with significantly higher pathological scores (p<0.05). After PP intervention, the histopathological damage in the colonic tissue was significantly improved, revealing a more intact submucosal structure, and the pathological score was significantly lower than that in the DSS group (p<0.05), indicating that PP can effectively alleviate DSS-induced colonic tissue damage. Although the pathological score in the PP group was still higher than that in the normal control group, its improvement trend was consistent with the results of DAI scores, colon length, and other indicators, further confirming the protective effect of PP on colitis mice at the histological level.

[0118] (3) Changes in inflammatory factors in mouse serum The regulatory effect of PP on the inflammatory response in colitis was analyzed by detecting the levels of pro-inflammatory factors IL-6, IL-1β, and TNF-α in mouse serum. Figure 7 As shown in H, I, and J.

[0119] Figure 7 In the table, H, I, and J represent the serum levels of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α in mice. The results showed that the serum levels of IL-6, IL-1β, and TNF-α in the Control group mice remained at low levels, indicating a mild inflammatory state. Compared to the Control group, the levels of these three pro-inflammatory cytokines in the colon of mice in the DSS group were significantly increased (P<0.01), indicating that DSS induced an inflammatory response in the serum, leading to a large release of pro-inflammatory cytokines. After PP intervention, the levels of IL-6, IL-1β, and TNF-α in the colon of mice were significantly lower than in the DSS group, demonstrating the inhibitory effect of PP on these pro-inflammatory cytokines. Therefore, DSS induction significantly exacerbates the release of pro-inflammatory cytokines in mouse serum, while PP intervention can exert an anti-inflammatory regulatory effect on DSS-induced colitis by reducing the levels of IL-6, IL-1β, and TNF-α.

[0120] The above results indicate that the DSS group experienced a significant decrease in body weight and a dramatic increase in DAI scores, demonstrating the successful establishment of the model. After intervention with Pediococcus pentosaceus PFP-03, the DAI levels in mice returned to near the control group level, body weight recovered, colonic index decreased, and colonic length recovered. Pathological results showed that the colonic tissue damage in the DSS group was severe, while the damage in the PP group was significantly reduced. Simultaneously, the elevated levels of pro-inflammatory factors such as IL-6, IL-1β, and TNF-α in the DSS group were significantly reduced after intervention with Pediococcus pentosaceus PFP-03. In conclusion, Pediococcus pentosaceus PFP-03 can effectively alleviate DSS-induced colitis in mice and improve their pathological and inflammatory state.

[0121] In summary, Pediococcus pentosaceus PFP-03 can effectively alleviate the pathological and inflammatory state in rats with diarrhea-predominant irritable bowel syndrome and can also effectively alleviate DSS-induced colitis in mice, improving their pathological and inflammatory state.

Claims

1. A strain of Pediococcus pentosaceus PFP-03, characterized in that, The preservation number of the Pediococcus pentosaceus PFP-03 is CGMCC No. 37269.

2. A microbial composition, characterized in that, The microbial composition comprises Pediococcus pentosaceus PFP-03 as described in claim 1.

3. The microbial composition according to claim 2, characterized in that, The dosage forms of the microbial composition include fermentation bacterial suspension, fermentation bacterial sludge, or freeze-dried powder.

4. The microbial composition according to claim 3, characterized in that, The fermentation suspension, fermentation sludge, or freeze-dried powder is obtained by fermentation of Pediococcus pentosaceus PFP-03 as described in claim 1.

5. The microbial composition according to any one of claims 2 to 4, characterized in that, The viable count of Pediococcus pentosaceus PFP-03 in the microbial composition was 1 × 10⁻⁶. 8 ~1×10 10 CFU / mL.

6. The use of Pediococcus pentosaceus PFP-03 as described in claim 1 or the microbial composition as described in any one of claims 2 to 5 in the preparation of products for the treatment and / or prevention of enteropathy.

7. The application according to claim 6, characterized in that, The enteropathy includes prevention of diarrhea-predominant irritable bowel syndrome and / or ulcerative colitis.

8. A method for producing lactic acid and / or acetic acid, characterized in that, The method includes the following steps: fermentation of Pediococcus pentosaceus PFP-03 as described in claim 1 in MRS liquid medium.

9. The application according to claim 7, characterized in that, The Pediococcus pentosaceus PFP-03 or the microbial composition effectively alleviated gastrointestinal conditions, anxiety, or depression-like behaviors in IBS-D model rats; the Pediococcus pentosaceus PFP-03 or the microbial composition effectively alleviated DSS-induced colonic shortening, increased spleen index, and colonic tissue damage.

10. A medicine for treating and / or preventing intestinal diseases, characterized in that, The drug comprises Pediococcus pentosaceus PFP-03 as described in claim 1 or the microbial composition as described in any one of claims 2 to 5.

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