Pediococcus acidilactici with functions of improving emotion and intestinal health and application of pediococcus acidilactici

Abstract

The invention provides pediococcus acidilactici with functions of improving emotion and intestinal health and application of the pediococcus acidilactici. The pediococcus acidilactici A25-4 is preserved in Guangdong Microbial Culture Collection Center on May 23, 2025, and the preservation number of the pediococcus acidilactici A25-4 is GDMCC NO: 66384. The pediococcus acidilactici A25-4 has better cholate resistance and artificial gastrointestinal fluid resistance and can produce indole lactic acid ILA at high yield, and the optimal carbon source is fructo-oligosaccharide and galactooligosaccharide. The pediococcus acidilactici A25-4 in-vivo intervention can significantly improve the symptoms of an irritable bowel syndrome model mouse, specifically, the anxiety mood of a tested object is significantly improved; the viscera hypersensitivity of a tested object is obviously relieved; excessive activation of mast cells of a tested object is obviously inhibited; the ILA level in a tested object is obviously improved.

Description

Technical Field

[0001] This invention relates to the fields of microbial applications and biotechnology, specifically to a type of Pediococcus lactis that improves mood and gut health, and its applications. Background Technology

[0002] Irritable bowel syndrome (IBS) is a chronic functional gastrointestinal disorder characterized by abdominal pain and altered bowel patterns. Its onset is often related to psychological factors and significantly impacts patients' quality of life. Current diagnosis is based on the Rome IV symptom criteria and is further divided into four subtypes: diarrhea-predominant IBS (IBS-D), constipation-predominant IBS (IBS-C), mixed IBS (IBS-M), and unclassified IBS (IBS-U). IBS affects 12%–20% of the global population, with 18%–23% of the population in my country affected. IBS-D is the most prevalent, accounting for 40%–65% of IBS cases. Current treatments are mainly limited to using antidiarrheal drugs or laxatives to control symptoms. However, due to the complex pathogenesis, the treatment scope for IBS is limited, with poor efficacy, significant side effects, and a high relapse rate. Therefore, a more moderate treatment strategy is needed.

[0003] Recent studies have revealed differences in the gut microbiota of IBS patients compared to healthy individuals. These differences can lead to variations in gut metabolites, with dysregulation of multiple tryptophan metabolism pathways potentially linked to IBS. Indole lactic acid (ILA) is a metabolite produced by the breakdown of tryptophan by gut microbiota. Studies have shown that ILA possesses anti-inflammatory, immunomodulatory, nerve cell protective, and neurodevelopment-promoting functions. Furthermore, certain strains derived from fermented foods may improve IBS by increasing ILA levels in the body. Therefore, screening for ILA-producing strains from fermented foods that can metabolize tryptophan and utilize these strains to increase ILA levels in the body could be a novel strategy for treating IBS.

[0004] Some probiotics of the Lactobacillus genus are known to produce ILA (intra-lactamase) and play an important role in the prevention and treatment of some inflammatory diseases. However, no studies have yet demonstrated the effect of these ILA-producing probiotics in alleviating irritable bowel syndrome (IBS). Therefore, screening for a high-ILA-producing probiotic and conducting functional evaluation of its effect on alleviating IBS would be helpful in developing novel probiotic products and could provide new solutions for the prevention and treatment of IBS. Summary of the Invention

[0005] Technical Problem to be Solved: To address the aforementioned technical problems, the purpose of this invention is to provide a *Pediococcus lactis* strain with functions of improving mood and gut health, and its application. The *Pediococcus lactis* A25-4 strain of this invention was isolated from *Cula*, a fermented food characteristic of the Qinghai-Tibet Plateau. It can metabolize tryptophan to produce ILA, is tolerant to bile salts and gastrointestinal fluids, and its optimal carbon source is fructooligosaccharides and galactooligosaccharides. Its growth time is 464.4 min. Studies have shown that its use can significantly improve the symptoms of irritable bowel syndrome in mice. Specifically, it significantly improves the anxiety of the subjects. 2) Alleviate visceral hypersensitivity in subjects; 3) Inhibit excessive activation of mast cells in subjects; 4) Increase ILA levels in subjects.

[0006] Technical solution: A type of Pediococcus lactis with functions of improving mood and gut health, wherein the Pediococcus lactis is Pediococcus lactis ( Pediococcus acidilactici Accession number A25-4 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 23, 2025, with accession number GDMCC NO:66384.

[0007] Furthermore, the lactic acid cocci A25-4 was isolated from Qula, a fermented food product from the Qinghai-Tibet Plateau.

[0008] Furthermore, the *Pediococcus lactis* A25-4 produces high levels of indolelactone (ILA) through the metabolism of tryptophan.

[0009] Furthermore, the amount of ILA produced by the *Pediococcus lactis* A25-4 metabolizing tryptophan is 15.05 μg / mL.

[0010] Furthermore, the *Pediococcus lactis* A25-4 proliferated in a culture medium containing 0.1% (w / v) bile salts and grew well in artificial gastrointestinal fluid.

[0011] Furthermore, the optimal carbon source for the *Pediococcus lactis* A25-4 is fructooligosaccharide and galactooligosaccharide.

[0012] Furthermore, the growth time of the lactic acid cocci A25-4 is 464.4 min.

[0013] This invention also provides the application of Pleurotus ostreatus A25-4 in the preparation of functional foods, health products and drugs for the prevention and treatment of irritable bowel syndrome.

[0014] Furthermore, the prevention and treatment of irritable bowel syndrome specifically includes: 1) Significantly improved the anxiety level of the subjects; 2) Alleviate visceral hypersensitivity reactions in test subjects; 3) Inhibits excessive activation of mast cells in test subjects; 4) Increase the level of ILA in the subjects.

[0015] This invention also provides the application of Pleurotus ostreatus A25-4 in the preparation of functional foods, health products, and pharmaceuticals that improve mood.

[0016] Furthermore, the Pediococcus lactis A25-4 in the functional foods, health products, and drugs is a live strain, a dry strain, or a strain metabolite.

[0017] Furthermore, the drug includes pharmaceutically acceptable carriers.

[0018] Furthermore, the dosage form of the drug is one of the following: powder, tablet, granule, capsule, solution, suspension, emulsion, and lyophilized preparation. Beneficial effects

[0019] The *Pediococcus lactis* A25-4 strain of this invention was isolated from *Cula*, a fermented food native to the Qinghai-Tibet Plateau. It can metabolize tryptophan to produce ILA, is tolerant of bile salts and gastrointestinal fluids, and its optimal carbon source is fructooligosaccharides and galactooligosaccharides. Its growth time is 464.4 min. Studies have shown that its use can significantly improve the symptoms of irritable bowel syndrome in mice. Specifically, it significantly improves the anxiety of the subjects. 2) Alleviate visceral hypersensitivity in subjects; 3) Inhibit excessive activation of mast cells in subjects; 4) Increase ILA levels in subjects.

[0020] 2. The functional evaluation results of Pyocortisone lactis A25-4 in this invention are good, and the efficacy is significant. Attached Figure Description

[0021] Figure 1 A flowchart for screening and identifying probiotics that produce high levels of tryptophan metabolites from Qula, a fermented food from the Qinghai-Tibet Plateau. Figure 2 The graphs show the bile salt tolerance, gastrointestinal fluid tolerance, and optimal carbon source screening results for Pediococcus lactis A25-4; where A represents the growth curve in 0.1% bile salt medium; B represents the survival rate in simulated gastrointestinal fluid; and C represents the optimal carbon source screening results. Figure 3 The study aimed to improve anxiety levels with Pediococcus lactis A25-4; where A was the dwell time in the open arm of the elevated cruciate maze, and B was the dwell time in the central area of ​​the open field.

[0022] Figure 4 The test result was the rectal balloon dilation test for Pediococcus lactis A25-4; where A represents the AWR score with a volume of 0.1 mL of air; B represents the AWR score with a volume of 0.2 mL of air; and C represents the AWR score with a volume of 0.3 mL of air. Figure 5This assay demonstrates the inhibition of mast cell overactivation by *Pediococcus lactis* A25-4. A represents the results of fluorescent staining for Tpsab1 and COX2 in the colon; B represents the quantitative results for Tpsab1 in the colon; C represents the quantitative results for COX2 in the colon; D represents the expression results for Tpsab1 mRNA in the colon; E represents the expression results for COX2 mRNA in the colon; and F represents the quantitative results for PGE2 in the colon. Figure 6 This invention provides an experiment to increase the level of ILA in vivo using Pseudococcus lactis A25-4; where A represents the fecal ILA content and B represents the serum ILA content. Detailed Implementation

[0023] This invention proposes a type of Pediococcus lactis with functions of improving mood and gut health, and its applications. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the following will provide a more detailed description of the invention with reference to specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0024] The raw materials and reagents used in the embodiments of this invention are all commercially available. In the following examples, the dosage of *Pediococcus lactis* A25-4 is 2 × 10⁻⁶. 10 CFU / mL / d.

[0025] All experimental data were analyzed using GraphPad Prism 10.1 software for ANOVA or unpaired t-tests. All results are expressed as mean ± standard deviation. p < 0.05 was considered statistically significant. Each experiment was repeated three times.

[0026] Example 1 Isolation and identification of Pediococcus lactis A25-4 1. Sample collection The isolated samples were derived from fermented foods characteristic of the Qinghai-Tibet Plateau. Samples were collected into sterile sample tubes and brought back to the laboratory for sorting. For detailed screening and identification procedures, please refer to [link to relevant documentation]. Figure 1 .

[0027] 2. Separation and purification Freshly collected samples were transferred to a clean bench. 1 g / 1 mL of sample was added to 15 mL of sterile PBS (phosphate-buffered saline), thoroughly vortexed, and then serially diluted and plated. The culture medium used was MRS medium with the following composition (g / L): peptone 10.0, beef extract 8.0, yeast extract 4.0, glucose 20.0, dipotassium hydrogen phosphate 2.0, diammonium hydrogen citrate 2.0, sodium acetate 5.0, magnesium sulfate 0.2, manganese sulfate 0.04, Tween 80 1.0, pH 5.7±0.2, 25℃. After incubation at 37℃ for 48 h, single colonies were picked and streaked to obtain pure cultures of each bacterial strain.

[0028] 3. Preservation of microbial strains The obtained pure culture strain was cultured until the concentration was approximately 10. 9 CFU / mL, take 500 μL of bacterial culture and add 500 μL of 40% glycerol to make the glycerol concentration reach 20%, and then store at -80℃.

[0029] 4. 16S rDNA identification Pediococcus acidilactici With the closest kinship and a similarity of 99%, this fungus belongs to the genus [of the family / organism]. Pediococcus Species.

[0030] Example 2 In vitro functional evaluation test of Pyotrophic cephalosporin A25-4 1. Materials and Methods 1.1 Experimental Materials Strain: Pediococcus lactis A25-4. Main reagents: MRS medium, ILA standard, chromatographic methanol, sodium dihydrogen phosphate, glacial acetic acid, porcine bile salts (Macklin, BR), artificial gastrointestinal fluid.

[0031] 1.2 Experimental Methods The selected probiotics were cultured in MRS medium for 24-48 h and then centrifuged. The ILA content of the strains was determined by Shimadzu LC-16 high performance liquid chromatography, thereby determining the ability of the strains to metabolize tryptophan and produce ILA.

[0032] The bile salt tolerance and gastrointestinal fluid tolerance of probiotics were determined. The strains were cultured in MRS liquid medium at 37°C for 18 h. The bacterial cultures were then inoculated into media containing 0.1%, 0.2%, and 0.3% bile salts, as well as into media without bile salts. The OD values ​​of the culture media were measured every 1 h using a BioScreen growth curve analyzer. 600 The absorbance value was measured at nm, and a growth curve was plotted based on the measured absorbance values ​​to determine the effect of different concentrations of bile salts on the growth of *Pediococcus lactis* A25-4. The strain was cultured at 37℃ for 18 h, and the cells were counted (h0). The cells were collected by centrifugation at 8000×g for 10 min and resuspended in an equal volume of artificial gastric fluid. After incubation at 37℃ for 3 h, the cells were counted again and centrifuged at 8000×g for 10 min. The cells were resuspended in an equal volume of artificial intestinal fluid and incubated at 37℃ for 4 h, and the cells were counted (h1). The survival rate of the strain was calculated as logh1 / h0×100%. (Artificial gastric fluid: composed of pepsin and hydrochloric acid, pH 3, sterile solution, simulating the human gastric environment; artificial intestinal fluid: composed of trypsin and phosphate, pH approximately 6.8, sterile solution, simulating the human small intestine environment.) Using carbon-free MRS medium as a control, *Pediococcus lactis* A25-4 was inoculated into media containing different prebiotics and cultured for 24 h. The OD of the media was measured every 1 h using a BioScreen growth curve analyzer. 600 The absorbance value was measured in nm, and a growth curve was plotted based on the measured absorbance value to determine the effect of prebiotics on the growth of Pediococcus lactis A25-4.

[0033] Pediococcus lactis A25-4 was inoculated into MRS medium at a rate of 5% (v / v) and cultured at 37°C. Starting from the 3rd hour of culture, samples of the Pediococcus lactis A25-4 suspension were taken every 2 hours to measure the absorbance at 600 nm (OD600) until 12 hours of culture. The generation time (G) of Pediococcus lactis A25-4 was calculated using the following formula: P t =P0×2 n n = logP t -logP0 / log2 G = t / n P0 and P t t represents the initial OD600 of Pyotrophic lateral sclerosis A25-4 and the OD600 at the end of the selected time period, respectively. t represents time and n represents the generation number.

[0034] Whole-genome sequencing analysis was performed on Pediococcus lactis A25-4. The sequencing results were compared and annotated using databases such as NR. By comparing all gene sequences with genes related to tryptophan metabolism and ILA production, it was determined whether the strain carried genes related to tryptophan metabolism and ILA production, thus re-determining the function of this strain in metabolizing tryptophan and producing ILA at the gene level. Test results

[0035] The ability of Pediococcus lactis A25-4 to metabolize tryptophan and produce ILA The amount of ILA produced by *Pediococcus lactis* A25-4 through metabolizing tryptophan was 15.05 μg / mL.

[0036] The comparison results of relevant gene databases for metabolizing tryptophan to produce ILA are shown in Table 1. The analysis results indicate that *Pediococcus lactis* A25-4 carries the aromatic amino acid transaminase (ArAT) and L-lactate dehydrogenase (LDH) genes. ArAT metabolizes tryptophan to indole-3-pyruvate, and LDH metabolizes indole-3-pyruvate to ILA. Therefore, it is further confirmed that *Pediococcus lactis* A25-4 has the ability to metabolize tryptophan to produce ILA.

[0037] Table 1. Tryptophan metabolism-related genes in Pediococcus lactis A25-4 strain number strain name Tryptophan metabolism-related enzymes Substrate A25-4 Pediococcus acidilactici Aromatic amino acid transaminase (ArAT) and L-lactate dehydrogenase (LDH) Tryptophan indole-3-pyruvate 2. Results of bile salt and gastrointestinal fluid resistance analysis Depend on Figure 2 It was found that *Pediococcus lactis* A25-4 grew well in MRS medium containing 0.1% bile salts, and the survival rate of *Pediococcus lactis* A25-4 in simulated gastric and intestinal fluids was higher than 95%. This indicates that *Pediococcus lactis* A25-4 has the ability to tolerate bile salts and gastrointestinal fluids.

[0038] 3. Results of the optimal carbon source screening analysis Depend on Figure 2 It was found that the growth of *Pediococcus lactis* A25-4 was evaluated in media containing fructooligosaccharides, barley β-glucan, inulin, galactooligosaccharides, xylan, mannose oligosaccharides, quinoa β-glucan, yeast glucan, chitosan, glucose, and carbon-free media. The results showed that the growth of *Pediococcus lactis* A25-4 inoculated into media supplemented with fructooligosaccharides and galactooligosaccharides was not significantly different from that inoculated with glucose. Therefore, the optimal carbon source for *Pediococcus lactis* A25-4 is fructooligosaccharides and galactooligosaccharides.

[0039] 4. Results of growth generation time measurement The growth time of P. lactis A25-4 was 464.4 min.

[0040] In summary, *Pediococcus lactis* A25-4 exhibits good proliferation ability, the capacity to produce ILA, with fructooligosaccharides and galactooligosaccharides being the optimal carbon sources. It is also resistant to bile salts and gastrointestinal fluids.

[0041] Example 3 Lactococcus A25-4 improves anxiety associated with irritable bowel syndrome. This invention uses animal experiments to evaluate the efficacy of the *Pediococcus lactis* A25-4 strain, in order to demonstrate the effectiveness of this strain.

[0042] 1. Grouping of laboratory animals Twenty-one 8-week-old male C57BL / 6J mice were selected for the experiment and divided into three groups: a control group, an irritable bowel syndrome (IBS) model group, and a model supplemented with Pediococcus lactis A25-4 group (IBS+A25-4), with seven mice in each group. The mice underwent 4 weeks of control and Pediococcus lactis A25-4 intervention.

[0043] 2. Processing methods for each group Control group: Normal drinking water, supplemented with 200 μL of PBS by gavage daily.

[0044] Probiotic treatment: The cultured Pediococcus lactis A25-4 was suspended in an appropriate amount of PBS until the viable count reached 2 × 10⁻⁶. 10 CFU / mL, prepare fresh before use. Administer 200 μL of bacterial suspension daily via gavage.

[0045] 3. Animal behavioral testing methods Behavioral analysis was performed on mice after 4 weeks of feeding to evaluate their anxiety levels.

[0046] 3.1 Elevated Cross Maze Test The elevated plus-maze (EPM) is an internationally recognized classic method for measuring anxiety response. The EPM consists of two open arms (30 cm long × 8 cm wide), two closed arms (30 cm long × 8 cm wide), and a central area (8 cm × 8 cm), at a height of 70 cm above the ground. The experiment used software to detect the time mice spent in the open and closed arms within 5 minutes and calculated the proportion of time spent in the open arms.

[0047] 3.2 Open Field Test The activity level of mice was assessed using an open field test. A cube-shaped box (length × width × height; 40 × 40 × 40 cm) was prepared. At the start of the experiment, the mice were placed in the center of the area and allowed to move freely within the open field for 5 minutes. During this time, the total distance traveled by the mice was recorded using software. Test results

[0048] Depend on Figure 3 The results showed that, compared to control mice, IBS mice exhibited more pronounced anxiety, manifested as reduced time spent in the open arm and open field central region. However, after 4 weeks of supplementation with Pediococcus lactis A25-4, the time spent in the open arm and open field central region of IBS mice significantly increased. These results indicate that Pediococcus lactis A25-4 can alleviate anxiety in irritable bowel syndrome mice.

[0049] Example 4 Lactococcus A25-4 alleviates visceral hypersensitivity reactions in irritable bowel syndrome 1. The experimental grouping and treatment were the same as in Example 3.

[0050] 2. Rectal balloon dilation (CRD) Mice were fed for 4 weeks before undergoing CRD analysis to evaluate visceral hypersensitivity. Mice were fasted for 12–16 hours prior to the experiment. On the day of the experiment, mice were briefly anesthetized with isoflurane (4%), followed by continuous anesthesia with 2%. A pediatric urinary catheter (6 Fr, 2 mm outer diameter) lubricated with liquid paraffin was inserted into the descending colon of the mice. The location was 1 cm from the anus. The urethral catheter was secured to the mouse's tail with tape. The mice were placed in a plastic box, only able to walk forward and backward, and unable to turn. After 30 minutes of acclimatization, colonic and rectal dilation was performed stepwise, with pressures increasing from 0 μL, 100 μL, 200 μL, and 300 μL, each pressure increasing for 20 seconds. Gas was expelled when changing pressures. A 5-minute rest period was allowed after each 20-second dilation. After evaluation, the balloon was deflated and withdrawn. The abdominal withdrawal reflex scoring criteria were as follows: 0 points, no obvious response during rectal balloon dilation; 1 point, cessation after brief head movement. 2 points, abdominal muscle contraction. 3 points, abdominal contraction and lifting. 4 points, arching the back and lifting the pelvis.

[0051] Mice were sacrificed after the CRD experiment, and colon tissue was collected and fixed in 4% paraformaldehyde. After steps such as embedding, sectioning, dewaxing, permeabilization, antigen retrieval, inactivation of endogenous enzyme activity, blocking, primary antibody incubation, secondary antibody incubation, and DAPI mounting, the colon tissue was photographed under a fluorescence microscope to observe the expression of Tpsab1 and COX2. Test results

[0052] The CRD test measures the abdominal withdrawal reflex (AWR) in the colon of adult mice and the CRD threshold pressure that induces rapid contraction of abdominal muscle tissue. Figure 3 It was found that the AWR score of IBS mice was significantly increased with air volumes of 0.1 mL, 0.2 mL and 0.3 mL, while the AWR score was significantly reduced after 4 weeks of intervention with Pediococcus lactis A25-4.

[0053] Overactivation of mast cells is one of the causes of visceral hypersensitivity in irritable bowel syndrome. Figure 4 It was found that Tpsab1, COX2 and PGE2 were significantly increased in the colon of IBS mice, indicating excessive activation of mast cells. However, intervention with Pediococcus lactis A25-4 for 4 weeks could significantly inhibit the excessive activation of mast cells, thereby reducing visceral hypersensitivity.

[0054] Example 5 Pleurotus ostreatus A25-4 increases ILA levels in the body. 1. The experimental grouping and treatment were the same as in Example 3.

[0055] 2. Experimental Methods Mice were treated for 4 weeks, and their feces were collected. Serum was then collected after sacrifice. High-performance liquid chromatography (HPLC) was used to detect ILA levels in both feces and serum. The specific methods are as follows: Fecal sample preparation: Weigh 0.10 g of mouse feces, add 1 mL of methanol, vortex mix, and incubate in a water bath at 40℃ for 20 min, mixing once every 5 min. After the water bath, place in a -20℃ refrigerator for 20 min, then remove and centrifuge at 12000 r / min for 10 min at 4℃. Filter the supernatant after centrifugation through a 0.22 µm microporous organic filter membrane into a brown vial and store at 4℃ for analysis.

[0056] Serum sample preparation: Take 30 μL of serum, add methanol to 300 μL, and let stand at -20℃ for 20 min. Then centrifuge the sample at 4℃ and 12000 r / min for 10 min. Filter the supernatant through a 0.22 μm organic filter membrane into a brown vial and place it in a 4℃ refrigerator for testing.

[0057] Liquid chromatography: A ZORBAX XDB C18 column (4.6×250 μm, 5 μm) was used as the analytical column. Mobile phase A: chromatographic methanol, mobile phase B: 15 mmol / L sodium dihydrogen phosphate solution (pH 2.8), flow rate: 1.0 ml / min, gradient elution program: 0-12 min, A:B=42:58, 12-28 min, A:B=50:50, 28-35 min, A:B=85:15, column temperature: 30℃, fluorescence detector detection wavelength (excitation wavelength / emission wavelength): 282 / 352 nm, injection volume: 10 μL. Test results

[0058] Depend on Figure 5 It can be seen that after 4 weeks of intervention, compared with the IBS group, the supplementation of Pyotrophic Lateral Sclerosis A25-4 significantly increased the ILA levels in mouse feces and serum.

[0059] Example 6 The preparation method of vacuum lyophilized Powder of Pediococcus lactis A25-4 is as follows: The ampoules and preservative were autoclaved and set aside for later use. The overnight culture was streaked and incubated at 37°C for 24 hours. After confirming no contamination, the following steps were performed: The culture was collected by centrifugation and washed twice with sterile physiological saline. An equal volume of 10%–30% skim milk preservative was added to suspend the culture and prepare a suspension with a colony count of 10-1. 8 ~10 10The bacterial suspension was prepared at a concentration of 100 cells / mL and dispensed into sterile ampoules. The ampoules were pre-frozen at -80 °C for 1–2 hours. Following the standard operating procedure for freeze dryers, the ampoules were freeze-dried for 8–20 hours until completely freeze-dried. After freeze-drying, the sample ampoules were removed, and the area below the cotton plug at the neck of the ampoule was sealed using a strong flame, following the standard operating procedure for vacuum ampoule sealing machines.

[0060] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A type of Pediococcus lactis with functions of improving mood and gut health, characterized in that, The *Pediococcus lactis* mentioned is *Pediococcus lactis* A25-4, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 23, 2025, with accession number GDMCCNO:66384.

2. The *Pediococcus lactis* strain with mood-improving and gut health-enhancing functions according to claim 1, characterized in that, The *Pediococcus lactis* A25-4 produces high levels of indole-lactic acid by metabolizing tryptophan.

3. The application of the lactic acid cocci with mood-improving and gut health-enhancing functions as described in claim 1 or 2 in the preparation of functional foods, health products, and drugs for the prevention and treatment of irritable bowel syndrome.

4. The application according to claim 3, characterized in that, The prevention and treatment of irritable bowel syndrome specifically includes: 1) Significantly improved the anxiety of the subjects; 2) Alleviate visceral hypersensitivity reactions in test subjects; 3) Inhibits excessive activation of mast cells in test subjects; 4) Increase the level of ILA in the subjects.

5. The use of the lactic acid bacteria with mood-improving and gut health-enhancing functions as described in claim 1 or 2 in the preparation of mood-improving functional foods, health products, and pharmaceuticals.

6. The application according to claim 3 or 5, characterized in that, The Pediococcus lactis A25-4 in the functional foods, health products, and drugs mentioned above is a live strain, a dry strain, or a strain metabolite.

7. The application according to claim 3 or 5, characterized in that, Drugs include pharmaceutically acceptable carriers.

8. The application according to claim 3 or 5, characterized in that, The dosage form of the drug is one of the following: powder, tablet, granule, capsule, solution, suspension, emulsion, and lyophilized preparation.

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