Lactobacillus amylovorius and use thereof

Abstract

The present application relates to the field of microorganisms, in particular, a strain of Lactobacillus amylophilus and its application. Specifically, the present application inventors have independently isolated a novel strain of Lactobacillus amylophilus, which has been deposited with the China General Microbiological Culture Collection Center (CGMCC) on May 13, 2026, under the accession number CGMCC No. 38672. Due to its unique biological characteristics and health benefits, the strain has become the focus of the present research. Specifically, the present application has clearly demonstrated through in vivo experiments that the novel strain of Lactobacillus amylophilus of the present application can effectively relieve functional constipation, and also prevent and / or treat irritable bowel syndrome (for example, irritable bowel syndrome diarrhea subtype (IBS-D)).

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to a type of Lactobacillus amyloliquefaciens and its applications. Background Technology

[0002] Functional constipation and irritable bowel syndrome (IBS) are prevalent digestive problems worldwide, especially in modern life where their incidence and prevalence are constantly rising due to changes in diet and lifestyle. Functional constipation is characterized by reduced bowel movement frequency, hard stools, or difficulty in defecation. IBS is a functional bowel disorder characterized by abdominal pain, bloating, and changes in bowel habits, with diarrhea-dominant IBS (IBS-D) being one of its major subtypes. These diseases not only affect patients' quality of life but also impose a significant economic burden.

[0003] Traditional treatments, including medication, dietary adjustments, and lifestyle modifications, can alleviate symptoms to some extent, but they often suffer from problems such as dependence, side effects, and individual variability in response. Therefore, developing new treatments to improve efficacy and patient adherence is particularly urgent.

[0004] Probiotic therapy, as an emerging treatment approach, shows potential in preventing / treating intestinal metabolic disorders due to its multiple mechanisms of action, including regulating gut microbiota balance, enhancing intestinal barrier function, and modulating immune responses. *Lactobacillus amyloliquefaciens* (… Lactobacillus amylovorus As a probiotic, Lactobacillus amyloliquefaciens has been shown to improve gut health. However, current technology has not reported any cases of Lactobacillus amyloliquefaciens relieving / treating both functional constipation and diarrhea. Summary of the Invention

[0005] Based on existing technologies, the inventors of this application have independently isolated a novel *Lactobacillus amylophilus* strain, which was deposited on May 13, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 38672. This strain, due to its unique biological characteristics and health benefits, has become the focus of this research. Specifically, this application has clearly demonstrated through in vivo experiments that the novel *Lactobacillus amylophilus* strain of this invention can effectively relieve functional constipation and also prevent and / or treat irritable bowel syndrome (e.g., irritable bowel syndrome diarrhea subtype (IBS-D)). In particular, it can increase fecal water content and / or intestinal propulsion rate in patients with functional constipation, reduce inflammation in patients with functional constipation and / or irritable bowel syndrome, and restore the intestinal barrier and intestinal homeostasis in patients with functional constipation or irritable bowel syndrome. The isolation and preservation of this strain not only ensured its uniqueness and originality, but also provided a reliable strain resource for subsequent research and application.

[0006] Functional constipation is a common digestive system disorder characterized by reduced bowel movement frequency (less than 3 times per week), hard stools, and difficulty in defecation (including straining, a feeling of incomplete evacuation, or rectal obstruction). The diagnosis is confirmed after ruling out organic lesions (such as intestinal tumors or inflammatory bowel disease) and medication-related factors. Its pathogenesis is mainly related to delayed colonic transit time, abnormal anorectal defecation motility (such as pelvic floor muscle dyssynergism), and increased visceral hypersensitivity. Pathologically, patients with functional constipation often exhibit reduced intestinal secretion (leading to decreased stool water content), weakened or disordered intestinal peristaltic waves, and intestinal microecological imbalance (such as a reduction in beneficial bacteria such as Bifidobacteria and Lactobacilli). In terms of treatment methods, the current main approach is a step-by-step treatment strategy: the first choice is lifestyle modifications (increasing dietary fiber and water intake, and moderate exercise) and biofeedback therapy; drug treatment mainly includes osmotic laxatives (such as polyethylene glycol), stimulant laxatives (such as bisacodyl), secretagogues (such as lubiprostone), and prokinetic drugs (such as prucalopride); in addition, probiotics to regulate the gut microbiota and psychological interventions (such as for patients with anxiety symptoms) are also important adjunctive treatments.

[0007] Irritable bowel syndrome with diarrhea (IBS-D) is a major subtype of irritable bowel syndrome (IBS), characterized primarily by abdominal pain or discomfort accompanied by changes in bowel habits. More than 25% of patients exhibit abnormal stool characteristics, falling into type 6 or 7 (pasty or watery stool) on the Bristol Stool Characteristics Scale, while hard stools (type 1 or 2) account for less than 25%. Its pathogenesis is complex and multifactorial, generally considered to be the result of the combined effects of dysregulation of the brain-gut axis, abnormal intestinal motility (accelerated colonic transit), visceral hypersensitivity, low-grade intestinal inflammation, and intestinal microecological dysbiosis (decreased intestinal flora diversity and altered abundance of specific flora). Pathologically, IBS-D patients often experience increased intestinal permeability (leaky gut), and intestinal mucosal immune cells are activated, releasing low levels of pro-inflammatory cytokines, leading to a persistent state of immune activation and hypersensitivity in the intestine. Treatment methods aim to alleviate symptoms and improve quality of life, including dietary interventions (such as a low-FODMAP diet to reduce the stimulation of the gut by fermentable carbohydrates), drug therapy (using antidiarrheals such as loperamide to inhibit intestinal motility, using antispasmodics such as oxytetracycline to relieve abdominal pain, using rifaximin to regulate gut microbiota, and secretagogues to regulate bowel habits), probiotic therapy, and psychotropic drugs (such as low-dose tricyclic antidepressants) to reduce visceral hypersensitivity and regulate brain-gut interaction.

[0008] For the two diseases mentioned above, the novel Lactobacillus amyloliquefaciens strain of the present invention exhibits a unique bidirectional regulatory advantage. It can improve constipation by increasing fecal water content and intestinal propulsion rate, and alleviate the symptoms of diarrhea-predominant irritable bowel syndrome by anti-inflammatory and intestinal barrier restoration, demonstrating its application prospects in regulating intestinal homeostasis.

[0009] In this regard, the technical solutions of the present invention include, but are not limited to, the following: In some respects, the present invention provides a Lactobacillus amyloliquefaciens, which was deposited on May 13, 2026 at the China General Microbiological Culture Collection Center, with accession number CGMCC NO. 38672.

[0010] In some aspects, the present invention also provides a pharmaceutical composition comprising the Lactobacillus amyloliquefaciens described herein.

[0011] In some aspects, the present invention also provides the use of the Lactobacillus amyloliquefaciens described herein in the preparation of medicaments / compositions for the prevention and / or treatment of functional constipation.

[0012] In some aspects, the present invention also provides the use of the *Lactobacillus amyloliquefaciens* described herein in the preparation of a medicament / composition for improving fecal water content and / or intestinal propulsion rate in patients with functional constipation.

[0013] In some aspects, the present invention increases the levels of 5-HT in the serum and colon of patients with functional constipation. 5-HTR3 and Tph1 The expression of mRNA increases intestinal propulsion rate.

[0014] In some respects, the present invention reduces the amount of stool in the colon of patients with functional constipation. AQP3 and AQP4 The expression of mRNA can increase the water content of feces.

[0015] In some aspects, the present invention also provides the use of the *Lactobacillus amyloliquefaciens* described herein in the preparation of a medicament / composition for reducing inflammation in patients with functional constipation and / or irritable bowel syndrome.

[0016] In some respects, the present invention reduces inflammation in patients with functional constipation by reducing serum TNF-α levels and / or reducing TNF-α levels in the colon. TNF-α Express.

[0017] In some aspects, the present invention reduces inflammation in patients with irritable bowel syndrome by reducing serum IL-6 and TNF-α levels and / or reducing colonic IL-6 and TNF-α levels. IL-6 and TNF-α The expression.

[0018] In some aspects, the present invention also provides the use of the Lactobacillus amyloliquefaciens described herein in the preparation of medicaments / compositions for the prevention and / or treatment of irritable bowel syndrome.

[0019] In some respects, the irritable bowel syndrome described in this invention presents as diarrhea-predominant.

[0020] In some aspects, the present invention also provides the use of the Lactobacillus amyloliquefaciens described herein in the preparation of a medicament / composition for reducing fecal water content, diarrhea rate and / or whole intestinal transit time in patients with diarrhea-predominant irritable bowel syndrome.

[0021] In some respects, the present invention prevents and / or treats irritable bowel syndrome (IBS) by reducing the synthesis and release of 5-HT in the colon of patients with diarrhea-predominant IBS.

[0022] In some aspects, the present invention also provides the use of the Lactobacillus amyloliquefaciens described herein in the preparation of intestinal barrier for patients with functional constipation or irritable bowel syndrome.

[0023] In some respects, the present invention improves colonic... Claudin1 Gene expression is used to restore the intestinal barrier.

[0024] In some aspects, the present invention also provides the use of the Lactobacillus amyloliquefaciens described herein in the preparation of medicaments / compositions for restoring intestinal homeostasis in patients with functional constipation or irritable bowel syndrome.

[0025] In some aspects, the patients with functional constipation and / or irritable bowel syndrome of the present invention are humans or animals. In particular, mammals, such as rats, pigs, cattle, sheep, horses, camels, alpacas, etc.

[0026] In one aspect, the mammal described in this invention is a rodent; preferably, it is a mouse.

[0027] Compared with the prior art, the present invention has the following beneficial technical effects, including but not limited to: ① The Lactobacillus amyloliquefaciens of the present invention has a functional constipation resistance effect, specifically, it increases intestinal peristalsis, improves the water content and shape of feces, thereby effectively relieving constipation symptoms; ② The Lactobacillus amyloliquefaciens of the present invention has an IBS-D alleviating effect, specifically by improving intestinal barrier function, reducing the release of inflammatory factors, regulating intestinal immune response, and alleviating IBS-D diarrhea symptoms; ③ The Lactobacillus amyloliquefaciens of the present invention has IBS-D resistance, specifically, it can enhance intestinal resistance and reduce the incidence of IBS-D; In summary, the Lactobacillus amyloliquefaciens probiotic preparation and its application of the present invention provide a safe, effective and side-effect-free new option for the prevention and treatment of functional constipation and IBS-D, which has important clinical application value and broad market prospects. Attached Figure Description

[0028] Figure 1 The growth curve of Lactobacillus amyloliquefaciens is shown.

[0029] Figure 2 The flowchart shows the experimental procedure for relieving constipation in a mouse model of constipation using Lactobacillus amyloliquefaciens.

[0030] Figure 3 This shows the flowchart of the trial on the alleviating effect of Lactobacillus amyloliquefaciens on IBS-D (prevention) model mice.

[0031] Figure 4 This shows the flowchart of the trial on the allergic effect of Lactobacillus amyloliquefaciens on IBS-D (treatment) model mice.

[0032] Figure 5 This shows the effect of *Lactobacillus amyloliquefaciens* on changes in food intake and body weight in constipated mice. Where A: Body weight; B: Average daily food intake (ADFI); CON: Control group; LOP: Constipation model group; L.amylovorus The constipation model was used in the group treated with Lactobacillus amyloliquefaciens.

[0033] Figure 6This diagram illustrates the effects of *Lactobacillus amyloliquefaciens* on fecal water content, morphology, and intestinal propulsion rate in constipated mice. Where A: fecal water content; B, D: intestinal propulsion rate; C: fecal morphology. CON: control group; LOP: constipation model group. L.amylovorus The constipation model was used in the group treated with Lactobacillus amyloliquefaciens.

[0034] Figure 7 This study demonstrates the effects of *Lactobacillus amyloliquefaciens* on serum inflammatory cytokine levels and colonic inflammatory cytokine gene expression in constipated mice. A: Serum IL-6 level; B: Serum IL-1β level; C: Serum TNF-α level; D: Colonic IL-6 mRNA expression; E: Colonic IL-1β mRNA expression; F: Colonic TNF-α mRNA expression. IL-6: Interleukin-6; IL-1β: Interleukin-1β; TNF-α: Tumor necrosis factor-α; CON: Control group; LOP: Constipation model group; L.amylovorus The constipation model was used in the group treated with Lactobacillus amyloliquefaciens.

[0035] Figure 8 This study demonstrates the effects of *Lactobacillus amyloliquefaciens* on serum gastrointestinal neurotransmitters in constipated mice. A: VIP; B: SP; C: Ach; D: GAS; E: MTL; F: PYY; G: ET-1; H: 5-HT. VIP: Vasoactive intestinal peptide; SP: Substance P; Ach: Acetylcholine; GAS: Gastrin; MTL: Motilin; PYY: Peptide; ET-1: Endothelin-1; 5-HT: Serotonin. CON: Control group; LOP: Constipation model group. L.amylovorus The constipation model was used in the group treated with Lactobacillus amyloliquefaciens.

[0036] Figure 9 This study showed the effects of *Lactobacillus amyloliquefaciens* on serum 5-HT levels and colonic 5-HT signaling pathway-related gene mRNA expression in constipated mice. A: serum 5-HT; B: colonic 5-HTR3 mRNA expression; C: colonic 5-HTR4 mRNA expression; D: colonic SERT mRNA expression; E: colonic Tph1 mRNA expression. 5-HTR3: serotonin 3 receptor; 5-HTR4: serotonin 4 receptor; SERT: serotonin transporter; Tph1: tryptophan hydroxylase 1. CON: control group; LOP: constipation model group; L.amylovorus The constipation model was used in the group treated with Lactobacillus amyloliquefaciens.

[0037] Figure 10This study demonstrates the effect of *Lactobacillus amyloliquefaciens* on the mRNA expression of genes regulating colonic water metabolism in constipated mice. A: AQP3; B: AQP4. AQP3: aquaporin 3; AQP4: aquaporin 4; CON: control group; LOP: constipation model group. L.amylovorus The constipation model was used in the group treated with Lactobacillus amyloliquefaciens.

[0038] Figure 11 This study demonstrates the effect of *Lactobacillus amyloliquefaciens* on the mRNA expression of tight junction protein-related genes in the colon of constipated mice. A: Claudin1; B: Occludin; C: ZO-1. Claudin1: tight junction protein 1; Occludin: atresia protein; ZO-1: atresia zona 1; CON: control group; LOP: constipation model group. L.amylovorus The constipation model was used in the group treated with Lactobacillus amyloliquefaciens.

[0039] Figure 12 This shows the effect of Lactobacillus amyloliquefaciens on the growth performance of IBS-D mice. A: Body weight; B: Mean daily feed intake; BW: Body weight; ADFI: Mean daily feed intake; CON: Control group; IBS-D: Diarrhea-predominant irritable bowel syndrome model group; L.amylovorus The diarrhea-predominant irritable bowel syndrome model was used in the group treated with Lactobacillus amyloliquefaciens.

[0040] Figure 13 The evaluation of IBS-D model establishment is shown. A: Body weight on days 1-7 after modeling; B: Feed intake on days 1-7 after modeling; C: Fecal water content; D: Whole intestine transit time; E: Diarrhea rate. ADFI: Mean daily feed intake; CON: Control group; IBS-D: Diarrhea-predominant irritable bowel syndrome model group; L.amylovorus The diarrhea-predominant irritable bowel syndrome model was used in the group treated with Lactobacillus amyloliquefaciens.

[0041] Figure 14 This diagram shows the effects of *Lactobacillus amyloliquefaciens* on serum inflammatory cytokine levels and colonic inflammatory cytokine gene expression in IBS-D mice. A: Serum IL-6 level; B: Serum IL-1β level; C: Serum TNF-α level; D: Colonic IL-6 mRNA expression; E: Colonic IL-1β mRNA expression; F: Colonic IL-10 mRNA expression; G: Colonic TNF-α mRNA expression. IL-6: Interleukin-6; IL-1β: Interleukin-1β; IL-10: Interleukin-10; TNF-α: Tumor necrosis factor-α; CON: Control group; IBS-D: Diarrhea-predominant irritable bowel syndrome model group; L.amylovorus The diarrhea-predominant irritable bowel syndrome model was used in the group treated with Lactobacillus amyloliquefaciens.

[0042] Figure 15 This shows the effects of *Lactobacillus amyloliquefaciens* on serum intestinal permeability indicators and colonic barrier protein-related gene mRNA expression in IBS-D mice. A: Serum D-LA content; B: Serum DAO content; C: Colonic Claudin1 gene mRNA expression; D: Colonic Occludin gene mRNA expression; E: Colonic ZO-1 gene mRNA expression. D-LA: D-lactic acid; DAO: diamine oxidase; Claudin1: tight junction protein 1; Occludin: atresia protein; ZO-1: atresia zona 1; CON: control group; IBS-D: diarrhea-predominant irritable bowel syndrome model group; L.amylovorus The diarrhea-predominant irritable bowel syndrome model was used in the group treated with Lactobacillus amyloliquefaciens.

[0043] Figure 16 This shows the effect of *Lactobacillus amyloliquefaciens* on the mRNA expression of genes related to the 5-HT metabolic pathway in the colon of IBS-D mice. A: Colonic Tph1 gene mRNA expression; B: Colonic SERT gene mRNA expression; C: Colonic Chga gene mRNA expression; D: Colonic MAOA gene mRNA expression; E: Colonic DCC gene mRNA expression. Tph1: Tryptophan hydroxylase 1; SERT: 5-hydroxytryptamine transporter; Chga: Chromogranin A; MAOA: Monoamine oxidase A; DCC: Dopa decarboxylase. CON: Control group; IBS-D: Diarrhea-predominant irritable bowel syndrome model group; L.amylovorus The diarrhea-predominant irritable bowel syndrome model was used in the group treated with Lactobacillus amyloliquefaciens.

[0044] Figure 17 The evaluation of IBS-D model establishment is shown. A: Body weight on days 0-7 during modeling; B: ADFI on days 1-7 during modeling; C: Diarrhea rate; D: Fecal water content; E: Whole intestinal transit time. ADFI: Average daily food intake; CON: Control group; IBS-D: Diarrhea-predominant irritable bowel syndrome model group; L.amylovorus The diarrhea-predominant irritable bowel syndrome model was used in the group treated with Lactobacillus amyloliquefaciens.

[0045] Figure 18 This shows the effect of *Lactobacillus amyloliquefaciens* on the growth performance of IBS-D mice. A: Body weight after modeling; B: Average daily food intake after modeling. CON: Control group; IBS-D: Diarrhea-predominant irritable bowel syndrome model group; L.amylovorus The diarrhea-predominant irritable bowel syndrome model was used in the group treated with Lactobacillus amyloliquefaciens.

[0046] Figure 19 This demonstrates the effect of *Lactobacillus amyloliquefaciens* on fecal water content in IBS-D mice. CON: Control group; IBS-D: Diarrhea-predominant irritable bowel syndrome model group; L.amylovorus The diarrhea-predominant irritable bowel syndrome model was used in the group treated with Lactobacillus amyloliquefaciens.

[0047] Figure 20 This diagram shows the effects of *Lactobacillus amyloliquefaciens* on serum inflammatory cytokine levels and colonic inflammatory cytokine gene expression in IBS-D mice. A: Serum IL-6 level; B: Serum IL-1β level; C: Serum TNF-α level; D: Colonic IL-6 mRNA expression; E: Colonic IL-1β mRNA expression; F: Colonic IL-10 mRNA expression; G: Colonic TNF-α mRNA expression. IL-6: Interleukin-6; IL-1β: Interleukin-1β; IL-10: Interleukin-10; TNF-α: Tumor necrosis factor-α; CON: Control group; IBS-D: Diarrhea-predominant irritable bowel syndrome model group; L.amylovorus The diarrhea-predominant irritable bowel syndrome model was used in the group treated with Lactobacillus amyloliquefaciens.

[0048] Figure 21 This shows the effects of *Lactobacillus amyloliquefaciens* on serum intestinal permeability indicators and colonic tight junction protein-related gene mRNA expression in IBS-D mice. A: Serum D-LA content; B: Serum DAO content; C: Colonic Claudin1 gene mRNA expression; D: Colonic Occludin gene mRNA expression; E: Colonic ZO-1 gene mRNA expression. D-LA: D-lactic acid; DAO: diamine oxidase; Claudin1: tight junction protein 1; Occludin: atresia protein; ZO-1: atresia zona 1; CON: control group; IBS-D: diarrhea-predominant irritable bowel syndrome model group; L.amylovorus The diarrhea-predominant irritable bowel syndrome model was used in the group treated with Lactobacillus amyloliquefaciens.

[0049] Figure 22 Effects of *Lactobacillus amyloliquefaciens* on mRNA expression of genes related to the 5-HT metabolic pathway in the colon of IBS-D mice. A: Colonic Tph1 gene mRNA expression; B: Colonic SERT gene mRNA expression; C: Colonic Chga gene mRNA expression; D: Colonic MAOA gene mRNA expression; E: Colonic DCC gene mRNA expression. Tph1: Tryptophan hydroxylase 1; SERT: 5-hydroxytryptamine transporter; Chga: Chromogranin A; MAOA: Monoamine oxidase A; DCC: Dopa decarboxylase. CON: Control group; IBS-D: Diarrhea-predominant irritable bowel syndrome model group; L.amylovorus The diarrhea-predominant irritable bowel syndrome model was used in the group treated with Lactobacillus amyloliquefaciens. Detailed Implementation

[0050] Example 1: Isolation, identification, culture, and preservation of Lactobacillus amyloliquefaciens 1. Strain isolation and identification Ileal digesta samples were collected from healthy growing pigs. After dilution with sterile physiological saline, the samples were plated onto MRS (De Man, Rogosa and Sharpe) solid culture plates and incubated under anaerobic conditions at 37°C and pH 6.5–7.0. After 24–48 hours of incubation, the colony morphology on the culture medium was observed, and colonies exhibiting characteristics of *Lactobacillus amyloliquefaciens* were selected. The selected colonies were then purified to ensure the acquisition of a single strain.

[0051] The strain was identified at the molecular level using 16S rRNA gene sequencing technology. The sequencing results were compared with known *Lactobacillus amyloliquefaciens* 16S rRNA gene sequences to confirm the strain's identity. The sequence alignment results showed that the strain isolated in this application had a 99.52% similarity to existing *Lactobacillus amyloliquefaciens* 16S rRNA sequences, thus confirming that the strain isolated in this application is a *Lactobacillus amyloliquefaciens* strain. Lactobacillus amylovorus ).

[0052] The identified Lactobacillus amyloliquefaciens strain was mixed with a 40% glycerol solution in equal proportion, then added to cryovials and stored in an ultra-low temperature freezer at -80°C to maintain its activity and stability.

[0053] 16S rRNA sequencing information: 2. Strains culture and growth curve determination Cultured under anaerobic conditions at 37°C for 24 h in De Man, Rogosa and Sharpe (MRS) medium.

[0054] The formula for MRS liquid culture medium is as follows (per liter): 10g peptone, 10g beef extract, 5g yeast extract, 20g glucose, 5g sodium acetate, 2g diammonium hydrogen citrate, 1 mL Tween-80, 2g dipotassium hydrogen phosphate, 0.2g magnesium sulfate heptahydrate, and 0.05g manganese sulfate tetrahydrate.

[0055] The formula for MRS solid culture medium is as follows (per liter): 10g peptone, 10g beef extract, 5g yeast extract, 20g glucose, 5g sodium acetate, 2g diammonium hydrogen citrate, 1 mL Tween-80, 2g dipotassium hydrogen phosphate, 0.2g magnesium sulfate heptahydrate, 0.05g manganese sulfate tetrahydrate, and 20g agar.

[0056] The growth curve of *Lactobacillus amyloliquefaciens* was determined using the Finnish Bioscreen fully automated growth curve analyzer. The results are as follows: Figure 1 As shown.

[0057] The growth curve shows that this strain is highly active, reaching the logarithmic growth phase in about 4 hours of culture and rapidly reaching the plateau phase in about 9 hours of culture.

[0058] 3. Study on strain characteristics 3.1 Antibiotic susceptibility testing In vitro drug susceptibility testing determines the degree of inhibition or tolerance of bacteria to different types of antibiotics. According to standards such as CLSI, strains are classified as sensitive (S, effective at conventional doses), intermediate (I, potentially effective at high doses or local concentrations), or resistant (R, ineffective at conventional doses) to evaluate the drug resistance, safety, and drug potential of strains.

[0059] Operating steps: (1) Adjust the bacterial suspension to 10 8 Take 100 μL of CFU / mL and spread it evenly on an MRS solid medium plate. After spreading, let it stand at room temperature for 3-5 minutes to allow the moisture on the plate surface to be absorbed to prevent the drug sensitivity paper from moving; (2) Use sterile forceps to pick up the drug sensitivity paper containing the quantitative antibiotic and gently press it on the agar surface to ensure no gaps in contact. Place the drug sensitivity paper evenly on the dry MRS medium surface; (3) Invert the plate and place it in a 37℃ constant temperature incubator for 24 h. After the incubation, take out the plate and use a vernier caliper or a special reading instrument to measure the diameter of the inhibition zone (including the diameter of the paper), in millimeters (mm). Calculate the sensitivity rate based on the size of the inhibition zone. The results are shown in Table 1 below.

[0060] Table 1. Results of drug resistance testing of the novel Lactobacillus amyloliquefaciens strain in this application.

[0061] Antibiotic susceptibility assessment criteria: inhibition zone diameter ≤12 mm is resistant (R), inhibition zone diameter 13-16 mm is intermediate (I), inhibition zone diameter ≥17 mm is sensitive (S).

[0062] According to the results of the antibiotic susceptibility test in Table 1, the strain showed susceptibility to all tested antibiotics, with a low overall risk of resistance and good safety.

[0063] 3.2 Hydrophobicity Test Determining whether a bacterial cell surface is more hydrophilic or hydrophobic reflects its molecular structure and is directly related to its adhesion, self-aggregation, and colonization abilities. Specific operational steps: 1. Centrifuge the Lactobacillus amyloliquefaciens culture for 24 hours at 3000×g for 10 min and discard the supernatant; 2. Resuspend in 1×PBS buffer, 3000×g, 10min, wash 2-3 times, and measure absorbance at 600nm: approximately 0.8. 3. Mix 1 mL of hydrophobic agent with 3 mL of Lactobacillus amyloliquefaciens suspension thoroughly, let stand at room temperature for 20 min to separate the organic and aqueous phases, and measure the absorbance of the aqueous phase at 600 nm using 1×PBS buffer as a control.

[0064] The results are shown in Table 2 below.

[0065] Table 2. Results of surface hydrophobicity determination of the novel Lactobacillus amyloliquefaciens strain of this application.

[0066] Criteria for judging hydrophobicity: Low: 0-29%; Medium: 30%-59%; High: 60%-100%.

[0067] As shown in Table 2, the surface hydrophobicity of this strain is at a medium to high level, indicating that the surface hydrophobic properties of the bacteria are good, which is conducive to enhancing the bacterial self-aggregation ability and improving the potential for adhesion and colonization of the intestinal epithelium.

[0068] 3.3 Self-agglomeration ability test In vitro experiments measuring the ability of bacteria of the same species to aggregate, clump together, and settle are commonly used indicators for evaluating bacterial adhesion and colonization abilities. Specific operational steps: 1. Centrifuge the Lactobacillus amyloliquefaciens culture for 24 hours at 3000×g for 10 min and discard the supernatant; 2. Resuspend in 1×PBS buffer, 3000×g, 10min, wash 2-3 times, and measure absorbance at 600nm: approximately 0.8. 3. Take 4 mL of bacterial suspension, mix thoroughly, and incubate at room temperature for 5 h and 24 h. Use 1×PBS buffer as a control and measure the absorbance at 600 nm.

[0069] The results are shown in Table 3 below: Table 3. Results of the self-agglutination ability test of the novel Lactobacillus amylophilis strain in this application.

[0070] As shown in Table 3, the strain exhibits a self-agglutination rate of over 90%, demonstrating a strong self-agglutination ability. This indicates that the strain has significant hydrophobic properties on its cell surface and excellent potential for intestinal adhesion and colonization.

[0071] 4. Strain preservation The novel Lactobacillus amylovorus strain isolated above was deposited on May 13, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 38672.

[0072] Example 2: Preventive and therapeutic effects of Lactobacillus amyloliquefaciens on functional constipation and diarrhea-predominant irritable bowel syndrome. 1. Strain preparation Prior to oral administration, the culture medium was centrifuged at 4000 × g and 4°C for 5 minutes. After centrifugation, the supernatant was removed, and the bacterial pellet was washed twice with sterile physiological saline buffer. The bacterial cells were then resuspended in sterile physiological saline buffer to obtain 10-1 9 The bacterial solution at CFU / mL was used for subsequent animal experiments via gavage.

[0073] 2. Animal Model Establishment and Experimental Design 2.1 Establishment of a functional constipation model Thirty healthy female C57BL / 6 mice (6-8 weeks old, weighing 17.27 ± 0.206 g, purchased from Spifor (Beijing) Biotechnology Co., Ltd.) were selected for the experiment and divided into 3 groups of 10 mice each. The experimental period was 21 days. For the first 14 days, the control group (CON) and the model group (LOP) were gavaged with 0.2 mL of sterile saline, while the experimental group ( L.amylovorusMice were administered 0.2 mL of Lactobacillus amyloliquefaciens solution via gavage. Modeling began in the final week of the experiment, with both the model and experimental groups receiving 0.2 mL of loperamide hydrochloride solution (Sigma, catalog number PHR1162) via gavage. The experimental group continued to receive 0.2 mL of Lactobacillus amyloliquefaciens solution via gavage. Blood (blood was collected by enucleation at the end of the experiment, and serum was separated) and colon tissue were collected from mice on day 21. Mouse weight, food intake, and fecal samples were recorded throughout the experiment. A basal maintenance diet was provided throughout the experiment, ensuring free access to food and water. For detailed experimental design, please refer to [link to experimental design]. Figure 2 .

[0074] 2.2 Establishment of a Diarrhea-Predominant Irritable Bowel Syndrome (IBS-D) Model (Preventive Effect) Thirty healthy male mice (6-8 weeks old, weighing 19.88 ± 0.315 g, purchased from Spifor (Beijing) Biotechnology Co., Ltd.) were selected for the experiment and divided into three groups of 10 mice each. Specifically, they were randomly divided into a healthy control group (CON), a model group (IBS-D), and an experimental group (…). L.amylovorus The experiment lasted 21 days. For the first 14 days, the control group (CON) and the model group (IBS-D) were gavaged with 0.2 mL of sterile saline, while the experimental group (CON) was gavaged with 0.2 mL of sterile saline. L.amylovorus Mice were administered 0.2 mL of Lactobacillus amyloliquefaciens solution via gavage. Modeling began in the final week of the experiment. Mice in both the model and experimental groups were given an enema with 4% acetic acid solution (≥99% acetic acid to physiological saline in a volume ratio of 4:96). The specific procedure was as follows: Under ether inhalation anesthesia, (200±20) μL of 4% acetic acid solution was injected into the colon 3–4 cm from the anus. The skin near the anus was held closed for 10 seconds, followed by flushing with (200±20) μL of sterile physiological saline. The mice were then suspended until they awoke. This process was repeated once after 3 days. The model group mice underwent two restraint stress events, each lasting 2 hours, while the control group mice were not subjected to restraint stress. Blood (blood was collected from the eyes at the end of the experiment, and serum was separated) and colon tissue were collected from the mice on day 21. Mouse weight, food intake, and fecal samples were recorded throughout the experiment. All mice were fed a basal maintenance diet with free access to food and water. For detailed experimental design, please refer to [link to experimental design]. Figure 3 .

[0075] 2.3 Establishment of a Diarrhea-Predominant Irritable Bowel Syndrome (IBS-D) Model (Treatment Efficacy) Thirty healthy male mice (6-8 weeks old, weighing 22.95 ± 0.116 g, purchased from Spifort (Beijing) Biotechnology Co., Ltd.) were selected for the experiment and divided into three groups of 10 mice each. These groups were randomly assigned to a healthy control group (CON), a model group (IBS-D), and an experimental group (…). L.amylovorusThe experiment involved several groups of mice, including the CON group (no modeling) and the IBS group (modeling) and the experimental group (modeling) and the experimental group (lactobacillus amyloliquefaciens). The experiment lasted 21 days. For the first 7 days, modeling was performed. Mice in both the model group (IBS-D) and the experimental group were given an enema with 4% acetic acid solution (≥99% acetic acid to saline solution volume ratio 4:96). The procedure was as follows: under ether inhalation anesthesia, (200 ± 20) μL of 4% acetic acid solution was injected into the colon 3–4 cm from the anus. The skin near the anus was held closed for 10 seconds, followed by flushing with (200 ± 20) μL of saline solution. The mice were then suspended until they recovered. This process was repeated once after 3 days. During this period, the model group mice underwent restraint stress twice, each time for 2 hours, while the control group mice were not subjected to restraint stress. Starting from day 7, the control group (CON) and the model group (IBS-D) were given 0.2 mL of sterile saline solution, while the experimental group (… L.amylovorus Mice were administered 0.2 mL of Lactobacillus amyloliquefaciens solution via gavage. Blood (blood was collected by enucleation at the end of the experiment, and serum was separated) and colon tissue were collected from mice on day 21. Mouse weight, food intake, and fecal samples were recorded throughout the experiment. A basal maintenance diet was provided throughout the experiment, with free access to food and water. For detailed experimental design, please refer to [link to experimental design]. Figure 4 .

[0076] 3. Sample Collection and Analysis 3.1 Fecal moisture content and form For the functional constipation model experiment, feces from mice in each group were collected on days 19, 20, and 21. After recording the wet weight, the feces were dried at 65°C, and the dry weight was recorded to evaluate the effect of probiotics on improving fecal moisture content in constipated mice. Furthermore, the morphology (size, consistency) and quantity of feces were observed and recorded to evaluate the effect of probiotics on improving defecation function in constipated mice.

[0077] Calculation formula: Fecal moisture content (%) = (Wet weight - Dry weight) / Wet weight × 100% 3.2 Intestinal propulsion rate For the functional constipation model experiment, on day 21, mice in each group were fasted overnight but not restricted in water. The next day, they were given 0.2 mL of activated charcoal solution by gavage. The mice were sacrificed 20 minutes later, and gastrointestinal tissue was collected. The total length of the small intestine and the distance the activated charcoal was propelled were measured to evaluate the effect of probiotics on improving intestinal peristalsis.

[0078] Calculation formula: Intestinal propulsion rate (%) = Activated charcoal propulsion distance / Total small intestine length × 100% 3.3 Whole intestinal transit time After IBS-D modeling, the fecal water content and intestinal transit time of mice in each group were observed and measured. Before evaluation, all mice were fasted for 12 h but allowed free water. 0.2 mL of 3% phenol red suspension was administered orally to each mouse. The time from gavage to the excretion of the first red feces was recorded within 2 h.

[0079] 3.4 Diarrhea rate IBS-D modeling begins with daily observation of stool characteristics and a diarrhea score. The diarrhea score is based on the following 0-4 point scale: 0 points: formed, granular stool; 1 point: sticky stool, adhering to the walls of microtubules; 2 points: pasty stool, possibly with mucus; 3 points: watery stool, possibly with mucus; 4 points: stool with blood.

[0080] Calculation formula: Diarrhea rate (%) = Total number of diarrhea episodes / (Total number of experimental mice × Number of days for modeling) × 100 3.5 Serum Biochemical Analysis Serum levels of interleukin-6 (IL-6), interleukin-1β (IL-β), tumor necrosis factor-α (TNF-α), motilin (MTL), vasoactive intestinal peptide (VIP), substance P (SP), serotonin (5-HT), gastrin (GAS), tyrosine kinase (PYY), acetylcholine (ACH), endothelin-1 (ET-1), diamine oxidase (DAO), and D-lactate (D-LA) were detected using an enzyme-linked immunosorbent assay (ELISA) kit. The kits were purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd., and the assay methods were performed according to the kit's instructions.

[0081] 3.6 Gene Expression Total RNA was extracted from colon samples using TRIzol reagent (purchased from Aidlab Biotechnologies Co., Ltd., Beijing, China) according to the manufacturer's instructions. RNA concentration and the A260 / A280 ratio were determined using an Epoch microplate spectrophotometer (BioTek Instruments, Inc., VT, USA), with ratios between 2.0 and 2.2 reserved for further analysis. Reverse transcription was performed using the M5 Sprint qPCR RT kit and gDNA removal agent (Mei5 Biotechnology, Co., Ltd., Beijing, China) according to the manufacturer's protocol. β-actin was used as an internal reference gene, and the relative expression level of the target gene was calculated using the 2^-ΔΔCt method. Amplification specificity was verified by melting curve analysis after amplification. Quantitative RT-qPCR was performed using the QuantStudio™ RT-PCR system (Biorad) and SYBR Green reagent (Biorad, CA, USA). Primer sequences are shown in Table 4.

[0082] Table 4 RT-qPCR Primer Sequence List

[0083] 3.7 Statistical Analysis The experimental data were statistically analyzed using SAS 9.4 software. One-way ANOVA was used, and multiple comparisons were performed using the Tukey method. Independent samples t-tests were used for analysis during the modeling phase of the IBS-D (treatment) trial. P <0.05 indicates a significant difference, 0.05 ≤ P <0.10 indicates a trend. P >0.05 indicates that the difference is not significant.

[0084] 4. Experimental Results 4.1 The efficacy of Lactobacillus amyloliquefaciens in preventing and / or treating functional constipation (1) Growth performance like Figure 5 As shown in Figure A, compared with the control group (CON), the body weight of mice in the model group (LOP) was significantly reduced on day 21. P <0.05). However, there was no significant difference on day 0, day 7, and day 14 ( P >0.05). Compared with the model group (LOP), the group that was orally administered Lactobacillus amylophilis ( L.amylovorus Mice with significantly increased body weight on day 21 ( P <0.05), and there were no significant differences on days 0, 7, and 14. P>0.05). Additionally, such as Figure 5 As shown in Figure B, compared with the control group (CON), the food intake of mice in the model group (LOP) was not significantly different. Compared with the model group (LOP), the amount of food consumed by mice orally administered Lactobacillus amyloliquefaciens (Lactobacillus amyloliquefaciens) was significantly higher. L.amylovorus There was no significant difference in food intake among mice. P >0.05).

[0085] (2) Fecal water content and form, and intestinal propulsion rate like Figure 6 As shown in Figure A, compared with the control group (CON), the water content of the model group (LOP) was significantly reduced. P <0.05%. Compared with the model group, the group fed with Lactobacillus amylophilis ( L.amylovorus The fecal water content was significantly increased ( ) P <0.05). For example... Figure 6 As shown in Figure C, the stool morphology also reveals that, compared to the control group, the constipation model group (LOP) had less stool, and the stool was dry and smaller. This was achieved through gavage with Lactobacillus amyloliquefaciens (Lactobacillus amyloliquefaciens). L.amylovorus It can alleviate changes in stool consistency caused by constipation to some extent. For example... Figure 6 As shown in B and 6D, compared with the control group (CON), the intestinal propulsion rate of the model group (LOP) was significantly reduced. P <0.05). Similarly, compared with the model group (LOP), gavage with Lactobacillus amylophilis ( L.amylovorus The intestinal propulsion rate was significantly increased ( ) P <0.05).

[0086] (3) Inflammatory cytokines like Figure 7 As shown in B and 7C, compared with the control group (CON), the serum levels of IL-1β and TNF-α in mice in the model group (LOP) were significantly increased. P <0.05), but had no significant effect on IL-6 ( P >0.05; Figure 7 A). Compared with the model group (LOP), gavage with Lactobacillus amyloliquefaciens ( L.amylovorus It can significantly reduce the level of TNF-α in mouse serum ( P <0.05; Figure 7 C), but had no significant effect on IL-6 and IL-1β. P >0.05; Figure 7 (A and 7B). For example... Figure 7 As shown in Figure F, compared with the control group (CON), the colon of the model group (LOP) mice contained... TNF-α Gene expression was significantly increased ( P <0.05), but for IL-6 and IL-1βThere was no significant effect on mRNA expression. P >0.05; Figure 7 D and 7E). Compared with the model group (LOP), gavage with Lactobacillus amylophilus (D and 7E) L.amylovorus It can significantly reduce the amount of protein in the colon. TNF-α mRNA expression ( P <0.05; Figure 7 F), but for IL-6 and IL-1β There was no significant effect on mRNA expression. P >0.05; Figure 7 D and 7E).

[0087] (4) Gastrointestinal neuroendocrine regulation like Figure 8 As shown, compared with the control group (CON), the serum levels of VIP, Ach, GAS, and MTL in the model group (LOP) mice were significantly reduced. P <0.05; Figure 8 A, 8C, 8F, and 8G). However, it has no effect on SP, PYY, and ET-1. P >0.05; Figure 8 B, 8D, and 8E). Compared to the model group (LOP), gavage with Lactobacillus amylophilis ( L.amylovorus It can significantly increase the levels of VIP, GAS and MTL in mouse serum. P <0.05; Figure 8 A, 8F, and 8G), but showed no significant difference in serum SP, Ach, PYY, and ET-1 levels in mice ( P >0.05; Figure 8 B, 8C, 8D, and 8E).

[0088] VIP (vasoactive intestinal peptide) is a neuropeptide with broad biological effects, playing a key role in regulating intestinal motility. VIP promotes relaxation of intestinal smooth muscle, increases intestinal blood flow, and accelerates the propulsion of intestinal contents, thereby relieving constipation symptoms. Therefore, a significant increase in VIP levels helps improve intestinal transit function and alleviate constipation in mice. GAS (motilin) ​​is a gastrointestinal hormone mainly produced in the stomach and duodenum, which stimulates gastric acid secretion and intestinal motility. Increased motilin levels enhance gastric emptying and intestinal propulsion in mice, helping to relieve constipation symptoms. MTL (gastrin-releasing peptide) is a peptide hormone that stimulates gastrin secretion and also participates in regulating intestinal motility. Increased MTL levels further promote gastric acid secretion and intestinal motility by increasing gastrin secretion, thus having a positive effect on relieving constipation.

[0089] The levels of SP (substance P), Ach (acetylcholine), PYY (tyrosine acetylcholine), and ET-1 (endothelin-1) did not show significant changes in this study. These hormones and neuropeptides also play a role in regulating intestinal motility and secretion, but in this study, gavage administration of *Lactobacillus amyloliquefaciens* did not induce significant changes in these indicators. This may indicate that *Lactobacillus amyloliquefaciens* has a weaker regulatory effect on these factors, or that these factors play a less significant role than VIP, GAS, and MTL in the pathophysiology of constipation.

[0090] Gavage administration of Lactobacillus amyloliquefaciens significantly increased the levels of VIP, GAS, and MTL in mouse serum. These changes may work together to relieve constipation symptoms through mechanisms such as promoting intestinal motility, increasing intestinal blood flow, and regulating gastric acid secretion.

[0091] (5) 5-HT signal path like Figure 9 As shown in Figure A, compared with the control group (CON), the serum 5-HT level in the model group (LOP) mice was significantly reduced ( P <0.05%. Compared with the model group, oral administration of Lactobacillus amylophilis ( L.amylovorus It can significantly increase the 5-HT content in mouse serum ( P <0.05).

[0092] like Figure 9 As shown in B and 9E, compared with the control group (CON), the model group (LOP) 5-HTR3 and Tph1 Significantly reduced ( P <0.05), but for SERT and 5-HTR4 There was no significant effect on mRNA gene expression. P >0.05; Figure 9 C and 9D). Compared with the model group (LOP), gavage with Lactobacillus amylophilis (C and 9D) L.amylovorus It can significantly increase 5-HTR3 and Tph1 mRNA expression ( P >0.05; Figure 9 (B and 9E), but for 5-HTR4 and SERT There was no significant effect on mRNA expression. P >0.05; Figure 9 C and 9D).

[0093] 5-HT, a key neurotransmitter, plays a central role in regulating mood, sleep, and gut motility and sensory function. Lactobacillus amyloliquefaciens (… L. amylovorus By significantly increasing the level of 5-HT in mouse serum and upregulating the level of 5-HT in the colon... 5-HTR3 and Tph1 The expression of *Lactobacillus amyloliquefaciens* mRNA enhances the activity of the 5-HT signaling pathway, thereby alleviating constipation symptoms. 5-HT receptor 3, as one of the 5-HT receptors, participates in regulating intestinal motility and secretion; while Tph1, a key enzyme in 5-HT synthesis, has its expression upregulated, contributing to increased 5-HT synthesis. *Lactobacillus amyloliquefaciens* promotes intestinal motility and improves intestinal secretion and sensory function by regulating these key molecules, thus playing a role in relieving constipation.

[0094] (6) Regulation of colonic water metabolism like Figure 10 As shown in A and 10B, compared with the control group (CON), the model group (LOP) AQP3 and APQ4 Significantly increased ( P <0.05). Compared with the model group (LOP), gavage with Lactobacillus amylophilis ( L.amylovorus It can significantly reduce AQP3 and AQP4 mRNA expression ( P <0.05).

[0095] AQP3 and AQP4 are aquaporins that play a crucial role in regulating water absorption and secretion in the colon. In the model group (LOP), AQP3 and AQP4 The significant increase in these aquaporins is associated with increased water absorption related to constipation, leading to harder stools and slower intestinal transit. Lactobacillus amyloliquefaciens softens stools, accelerates intestinal transit, and relieves constipation symptoms by reducing the expression of these aquaporins, thereby decreasing water absorption in the colon and increasing the water content of the stool.

[0096] (7) Expression of colonic tight junction protein-related mRNA like Figure 11 As shown in Figure A, compared to the control group (CON), the model group (LOP) Claudin1 Significantly reduced ( P <0.05), but for Occludin and ZO-1 No significant effect ( P >0.05; Figure 11 B and 11C). Compared with the model group (LOP), *Lactobacillus amyloliquefaciens* (B and C) was significantly reduced. L.amylovorus It can significantly improve Claudin1 mRNA expression ( P <0.05; Figure 11 A), but for Occludin and ZO-1 No significant effect ( P >0.05; Figure 11 B and 11C).

[0097] Claudin1, a member of the tight junction protein family, plays a crucial role in maintaining the integrity of the intestinal barrier and regulating intestinal permeability. In the model group (LOP), significant reductions in Claudin1 were associated with intestinal barrier dysfunction, leading to increased intestinal permeability, inflammatory responses, and abnormal intestinal motility. Lactobacillus amyloliquefaciens, by increasing Claudin1 expression, enhances intestinal barrier function, reduces inflammation, and improves intestinal motility, thus positively impacting constipation symptoms.

[0098] 4.2 The effect of Lactobacillus amyloliquefaciens in preventing diarrhea-predominant irritable bowel syndrome (IBS-D) (1) Growth performance like Figure 12 As shown in A and 12B, prior to modeling, gavage administration of Lactobacillus amylophilis for two weeks before the experiment had no significant effect on the body weight and average daily food intake of mice. P >0.05).

[0099] (2) Modeling and evaluation like Figure 13 As shown in Figure A, compared with the control group (CON), the model group (IBS-D) experienced a significant decrease in body weight from day 18 to 21. P <0.05, but there was no significant change from day 15 to day 17 ( P >0.05). Similarly, on days 17 and 19–21, the feed intake of the control group (CON) was significantly higher than that of the model group (IBS-D). P <0.05%, with no significant change on days 15-16 and 18. P >0.05; Figure 13 B). Compared with the model group (IBS-D), oral administration of Lactobacillus amylophilis ( L.amylovorus It can significantly increase the body weight and food intake of mice at 19-21 days. P <0.05; Figure 13 (A and 13B). For example... Figure 13 As shown in C and 13E, compared with the control group (CON), the fecal water content and diarrhea rate of the model group (IBS-D) were significantly increased. P <0.05). Similarly, the total intestinal transit time of control (CON) mice was significantly longer than that of the model group (IBS-D). P <0.05; Figure 13 D). Compared with the model group (IBS-D), oral administration of Lactobacillus amyloliquefaciens (D) L.amylovorus It can significantly reduce fecal water content and diarrhea rate, as well as increase whole-intestinal transit time. P <0.05; Figure 13 (CE). The results above indicate that the IBS-D model was successfully established.

[0100] (3) Inflammatory markers like Figure 14 As shown in A and 14C, compared with the control group (CON), the serum levels of IL-6 and TNF-α in mice in the model group (IBS-D) were significantly increased. P <0.05%. Compared with the model group (IBS-D), oral administration of Lactobacillus amylophilis ( L.amylovorus It can significantly reduce serum levels of IL-6 and TNF-α. P <0.05), but had no significant effect on IL-1β ( P >0.05; Figure 14 B). For example... Figure 14 As shown in D and 14F, compared with the control group (CON), the colon of mice in the model group (IBS-D) was significantly different. IL-6 and TNF-α Increased mRNA expression ( P <0.05%, compared with the model group (IBS-D), oral administration of Lactobacillus amylophilis ( L.amylovorus It can significantly reduce colon cancer in mice. IL-6 and TNF-α mRNA expression ( P <0.05). But for IL-1β and IL-10 There was no significant effect on mRNA expression. P >0.05; Figure 14 E and 14G).

[0101] (4) Intestinal barrier related indicators like Figure 15 As shown in A and 15B, compared with the control group (CON), the serum levels of D-LA and DAO in mice in the model group (IBS-D) were significantly increased. P <0.05); compared with the model group (IBS), gavage with Lactobacillus amylophilis ( L.amylovorus The levels of D-LA and DAO in the serum of mice were significantly reduced. Figure 15 As shown in Figure C, compared with the control group (CON), the colon of mice in the model group (IBS-D) was... Claudin1 mRNA expression was significantly reduced ( P <0.05%, compared with the model group (IBS-D), gavage with Lactobacillus amylophilis ( L.amylovorus mouse colon Claudin1 mRNA expression was significantly increased ( P <0.05), but for Occludin and ZO-1 There was no significant effect on mRNA expression. P >0.05; Figure 15Elevated serum D-LA and DAO levels reflect increased intestinal permeability, while decreased levels indicate improved intestinal barrier function. Increased Claudin1 mRNA expression in the colon indicates enhanced intestinal barrier function. Therefore, these results suggest that *Lactobacillus amyloliquefaciens* (D and 15E) L. amylovorus By reducing serum D-LA and DAO levels and increasing Claudin1 mRNA expression in the colon, it helps improve intestinal barrier function and reduce inflammatory response in IBS-D mouse models, and has a potential positive effect on alleviating IBS-D symptoms.

[0102] (5) Expression of mRNAs related to the colonic 5-HT metabolic pathway like Figure 16 As shown in A and 16C, compared with the control group (CON), the colon of mice in the model group (IBS-D) was... Tph1 and Chga mRNA expression was significantly increased ( P <0.05), but for SERT , MAOA and DCC There was no significant effect on mRNA expression. P >0.05; Figure 16 B, 16D, and 16E). Compared to the model group (IBS-D), gavage with Lactobacillus amylophilis (B, 16D, and 16E) L.amylovorus It can significantly reduce Tph1 mRNA expression ( P <0.05; Figure 16 A), regarding SERT , MAOA , Chga and DCC mRNA expression was not affected ( P >0.05; Figure 16 B, 16C, 16D, and 16E).

[0103] The mRNA expression levels of genes related to the 5-HT (serotonin) metabolic pathway in the colon were altered in IBS-D model mice, with significantly increased mRNA expression of Tph1 (tryptophan hydroxylase 1) and Chga (chromogranin A), which is associated with increased 5-HT synthesis in IBS-D. 5-HT is a neurotransmitter that plays a crucial role in regulating intestinal motility and sensory transmission, and changes in its metabolic pathway are associated with IBS-D symptoms. Compared to the model group, gavage administration of *Lactobacillus amyloliquefaciens* (…) L. amylovorusThe expression of Tph1 mRNA in the colon of mice was significantly reduced, indicating that *Lactobacillus amyloliquefaciens* alleviates IBS-D symptoms by reducing 5-HT synthesis. Meanwhile, *Lactobacillus amyloliquefaciens* had no significant effect on the mRNA expression of SERT (5-HT transporter), MAOA (monoamine oxidase A), and DCC (dicortin), suggesting that its main function is focused on regulating 5-HT synthesis. These results indicate that *Lactobacillus amyloliquefaciens* has a positive impact on the symptoms of an IBS-D mouse model by regulating the expression of genes related to the 5-HT metabolic pathway in the colon.

[0104] 4.3 Efficacy of Lactobacillus amyloliquefaciens in the treatment of diarrhea-predominant irritable bowel syndrome (1) Modeling and evaluation like Figure 17 As shown in Figure A, compared with the model group (IBS-D), the control group (CON) showed a significant increase in body weight on days 1–3 and 5–7. P <0.05, but there was no significant change on day 4 ( P >0.05). Similarly, on days 2 and 5-7, the feed intake of the control group was significantly higher than that of the model group ( P <0.05%, with no significant change on day 1 and days 3-4. P >0.05; Figure 17 B). For example... Figure 17 As shown in C and 17E, compared with the control group (CON), the fecal water content and diarrhea rate of the model group (IBS-D) were significantly increased. P <0.05. Similarly, the total intestinal transit time of mice in the control group was significantly higher than that in the model group ( P <0.05; Figure 17 D). The results above indicate that the IBS-D model was successfully established.

[0105] (2) Growth performance like Figure 18 As shown in Figure A, compared with the control group (CON), the mice in the model group (IBS-D) had significantly lower body weight on days 7 and 14. P <0.05%. Compared with the model group (IBS-D), oral administration of Lactobacillus amylophilis (Lactobacillus amylophilis) to animals... L.amylovorus Mice showed a significant increase in body weight on days 7 and 14. P <0.05; Figure 18 A). However, there were no significant changes in body weight and food intake on day 0 ( P >0.05; Figure 18 A and 18B).

[0106] (3) Fecal water content like Figure 19As shown, compared with the control group (CON), the fecal water content of mice in the model group (IBS-D) was significantly increased. P <0.05%. Compared with the model group (IBS-D), oral administration of Lactobacillus amylophilis (Lactobacillus amylophilis) to animals... L.amylovorus The water content of mouse feces was significantly reduced. P <0.05).

[0107] (4) Inflammatory markers like Figure 20 As shown in A and 20C, compared with the control group (CON), the serum levels of IL-6 and TNF-α in mice in the model group (IBS-D) were significantly increased. P <0.05%. Compared with the model group (IBS-D), oral administration of Lactobacillus amylophilis ( L.amylovorus It can significantly reduce serum levels of IL-6 and TNF-α. P <0.05), but had no significant effect on IL-1β ( P >0.05; Figure 20 B). For example... Figure 20 As shown in D and 20F, compared with the control group (CON), the colon of mice in the model group (IBS-D) was... IL-6 and TNF-α Increased mRNA expression ( P <0.05%, compared with the model group (IBS-D), oral administration of Lactobacillus amylophilis ( L.amylovorus It can significantly reduce colon cancer in mice. IL-6 and TNF-α mRNA expression ( P <0.05), but for IL-1β and IL-10 There was no significant effect on mRNA expression. P >0.05; Figure 20 E and 20G).

[0108] (5) Intestinal barrier related indicators like Figure 21 As shown in A and 21B, compared with the control group (CON), the serum levels of D-LA and DAO in mice in the model group (IBS-D) were significantly increased. P <0.05%, compared with the model group (IBS-D), gavage with Lactobacillus amylophilis ( L.amylovorus The level of DAO in the serum of mice was significantly reduced, but had no significant effect on D-LA. P >0.05). For example... Figure 21 As shown in Figure C, compared with the control group (CON), the colon of mice in the model group (IBS-D) was... Claudin1 mRNA expression was significantly reduced ( P<0.05%, compared with the model group (IBS-D), gavage with Lactobacillus amylophilis ( L.amylovorus mouse colon Claudin1 mRNA expression was significantly increased ( P <0.05), but for Occludin and ZO-1 There was no significant effect on mRNA expression. P >0.05; Figure 21 D and 21E).

[0109] DAO is a biomarker of intestinal inflammation and barrier dysfunction. Compared with the model group, mice gavaged with *Lactobacillus amyloliquefaciens* showed significantly lower serum DAO levels, indicating that *Lactobacillus amyloliquefaciens* helps alleviate intestinal inflammation and improve intestinal barrier function. Claudin1 is a key component of tight junction proteins and is essential for maintaining intestinal barrier integrity. Compared with the model group, mice gavaged with *Lactobacillus amyloliquefaciens* showed significantly lower levels of DAO in their colon. Claudin1 The significantly increased mRNA expression indicates that *Lactobacillus amyloliquefaciens* improves intestinal barrier function by enhancing the expression of tight junction proteins. In summary, *Lactobacillus amyloliquefaciens* improves intestinal barrier function by reducing serum DAO levels and upregulating colonic DAO expression. Claudin1 mRNA expression helps improve intestinal barrier function and reduce inflammatory response in IBS-D mouse models.

[0110] (6) Expression of mRNAs related to the colonic 5-HT metabolic pathway like Figure 22 As shown in A and 22C, compared with the control group (CON), the colon of mice in the model group (IBS-D) was... Tph1 and Chga mRNA expression was significantly increased ( P <0.05), but for SERT , MAOA and DCC There was no significant effect on mRNA expression. P >0.05; Figure 22 B, 22D, and 22E). Compared to the model group (IBS-D), gavage with Lactobacillus amylophilis (B, 22D, and 22E) L.amylovorus It can significantly reduce Tph1 and Chga mRNA expression ( P <0.05; Figure 22 A and 22C), for SERT , MAOA and DCC mRNA expression was not affected ( P >0.05; Figure 22 B, 22D, and 22E).

[0111] Tph1 is a key enzyme in 5-HT synthesis, and Chga is responsible for packaging 5-HT into secretory granules. Compared with the model group, mice gavaged with Lactobacillus amyloliquefaciens had significantly higher levels of 5-HT in their colons. Tph1 and Chga The significantly reduced mRNA expression indicates that *Lactobacillus amyloliquefaciens* alleviates IBS-D symptoms by inhibiting 5-HT synthesis. Although *Lactobacillus amyloliquefaciens* significantly affects the expression of genes related to 5-HT synthesis, it has no significant effect on the mRNA expression of the 5-HT transporter SERT, the 5-HT degrading enzyme MAOA, or the 5-HT receptor DCC. This suggests that *Lactobacillus amyloliquefaciens* primarily functions by regulating the 5-HT synthesis pathway, rather than 5-HT transport and signal transduction. *Lactobacillus amyloliquefaciens* alleviates IBS-D symptoms by reducing... Tph1 and Chga mRNA expression was reduced, decreasing the synthesis and release of 5-HT in the colon, thus helping to alleviate IBS-D-related intestinal hypersensitivity and motility dysfunction. In summary, *Lactobacillus amyloliquefaciens* positively impacts the symptoms of an IBS-D mouse model by specifically regulating the expression of genes related to the 5-HT metabolic pathway in the colon.

Claims

1. A type of Lactobacillus amyloliquefaciens, characterized in that, It was deposited at the China General Microbiological Culture Collection Center (CGMCC) on May 13, 2026, with accession number CGMCC NO. 38672.

2. A pharmaceutical composition, characterized in that, It contains Lactobacillus amyloliquefaciens as described in claim 1.

3. Use of the Lactobacillus amyloliquefaciens according to claim 1 in the preparation of a medicament / composition for the prevention and / or treatment of functional constipation.

4. Use of the Lactobacillus amyloliquefaciens according to claim 1 in the preparation of a medicament / composition for improving fecal water content and / or intestinal propulsion rate in patients with functional constipation.

5. Use of the Lactobacillus amyloliquefaciens according to claim 1 in the preparation of a medicament / composition for reducing inflammation in patients with functional constipation and / or irritable bowel syndrome.

6. Use of the Lactobacillus amyloliquefaciens according to claim 1 in the preparation of a medicament / composition for the prevention and / or treatment of irritable bowel syndrome.

7. The use according to claim 6, characterized in that, The irritable bowel syndrome is characterized by diarrhea.

8. The use of the Lactobacillus amyloliquefaciens according to claim 1 in the preparation of a product that restores the intestinal barrier in patients with functional constipation or irritable bowel syndrome.

9. Use of the Lactobacillus amyloliquefaciens according to claim 1 in the preparation of a medicament / composition for restoring intestinal homeostasis in patients with functional constipation or irritable bowel syndrome.

10. Use of the Lactobacillus amyloliquefaciens according to claim 1 in the preparation of a medicament / composition for reducing fecal water content, diarrhea rate and / or whole intestinal transit time in patients with diarrhea-predominant irritable bowel syndrome.

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