Clostridium butyricum as well as composition and application thereof

Abstract

The invention relates to the technical field of microorganisms, and provides Witzia ciliaris KY009 (preservation number is CGMCC (China General Microbiological Culture Collection Center) NO. 33480) and Clostridium butyricum KY002 (preservation number is CGMCC NO. 33478), and the two strains are separated from an excrement sample. Experiments prove that the two strains can obviously improve the sperm quality and quantity, regulate the sex hormone level, improve the intestinal inflammation symptom and regulate the expression of related inflammatory factors. The invention also provides a composition containing the two strains, wherein the composition comprises a food composition, a pharmaceutical composition, a health-care product composition, a functional microbial agent and the like. The compositions are useful for improving reproductive health, modulating sex hormone levels, treating intestinal inflammation and promoting mucosal repair, and alleviating metabolic and immune related diseases. The strain and the composition provided by the invention have wide application prospects in the fields of medicines, foods and health-care products.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, in particular to Clostridium butyricum. Background Art

[0002] In addition to environmental factors and lifestyle factors, male infertility is primarily caused by abnormal development of the reproductive system. It is well known that normal male fertility depends on a healthy male reproductive system, neuroendocrine regulation, and normal spermatogenesis. Sperm mature in the epididymis, are discharged through the vas deferens and ejaculatory ducts, and then mix with seminal vesicle fluid and prostatic fluid to form semen. Abnormalities in any of these processes can lead to fertility problems. A growing number of studies have found a close relationship between the gut microbiome and male reproductive health. The gut microbiome can influence testicular function, spermatogenesis, and sex hormone metabolism in various ways.

[0003] Inflammatory bowel disease (IBD) is a nonspecific, chronic, relapsing inflammatory disease affecting the gastrointestinal tract. It primarily includes ulcerative colitis (UC) and Crohn's disease (CD). Crohn's disease typically affects the entire gastrointestinal tract, manifesting as multiple, discrete granulation cysts within the intestine. Ulcerative colitis, on the other hand, primarily develops in the terminal colon and gradually spreads throughout the entire colon, characterized by extensive ulcerative lesions within the colonic tissue. Patients with IBD often experience symptoms such as abdominal pain, diarrhea, bloody stools, and weight loss, which can severely impact their physical function and daily life. Factors contributing to the development of IBD primarily include individual genetic susceptibility, environmental factors, immune responses, and microbial imbalances, but the specific pathogenic mechanisms remain unclear. Recent studies have demonstrated that the pathogenesis of IBD is closely linked to intestinal microbial dysbiosis, and that restoring intestinal microbial balance can significantly alleviate IBD.

[0004] Probiotics are active bacteria that can colonize the human body, alter the microbial composition of a specific part of the host, and produce beneficial effects on the host. Because of their ability to modulate mucosal and systemic immune function, maintain intestinal flora balance, promote nutrient absorption, and enhance intestinal health, researchers are increasingly interested in the impact of probiotics on various diseases, particularly intestinal disorders. An increasing number of people are choosing to improve their intestinal health through probiotic supplementation. Probiotic supplementation primarily involves single-strain and mixed-strain supplementation. Single-strain probiotics use only one strain and can treat specific conditions, while mixed-strain probiotics are composed of multiple strains and can synergize across multiple therapeutic areas. Research in these areas requires increased attention and investment. Summary of the Invention

[0005] Based on the above issues, the inventors isolated two new strains from healthy human feces: Weizmannella coagulans KY009 (CGMCC NO. 33480) and Clostridium butyricum KY002 (CGMCC NO. 33478). These strains have shown significant potential in improving reproductive health, metabolic immunity, and intestinal diseases by regulating sex hormone levels, repairing the intestinal barrier, and inhibiting inflammation, providing a new microbial intervention strategy for the treatment of related diseases. The details are as follows: The first aspect of the present invention is to provide Weizmannella coagulans KY009, Clostridium butyricum KY002 Weizmannia coagulans KY009 was deposited in the General Microbiology Center of the China Culture Collection Administration on January 20, 2025. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC NO.33480, and the classification name is Weizmannia coagulans.

[0006] Its 16S rDNA is shown in SEQ ID NO: 3 Clostridium butyricum KY002 was deposited in the General Microbiology Center of the China Culture Collection Administration on January 20, 2025. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC NO. 33478, and the classification name is Clostridium butyricum.

[0007] Its 16S rDNA is shown in SEQ ID NO: 4 The Weizmannella coagulans and Clostridium butyricum were isolated from feces.

[0008] The second aspect of the present invention provides a composition comprising Weizmannella coagulans KY009 and / or Clostridium butyricum KY002 A composition comprises Weizmannella coagulans KY009. The composition can be a food composition, a pharmaceutical composition, a health product composition, a functional bacterial agent, etc.

[0009] The pharmaceutical composition may be a composition comprising Weizmannella coagulans KY009 and a pharmaceutical excipient, or a composition comprising Clostridium butyricum KY002 and a pharmaceutical excipient, or a composition comprising both Weizmannella coagulans KY009 and Clostridium butyricum KY002 and a pharmaceutical excipient.

[0010] The excipients in the pharmaceutical composition can be diluents / fillers selected from microcrystalline cellulose, starch (corn / potato), lactose, mannitol, and calcium carbonate; binders selected from hydroxypropyl methylcellulose (HPMC), gelatin, and polyvinylpyrrolidone (PVP); and disintegrants such as cross-linked sodium carboxymethylcellulose (CCNa) and low-substituted hydroxypropyl cellulose (L-HPC). Lubricants can be selected from magnesium stearate, silicon dioxide, and talc. Coating materials can be selected from gastric / enteric coatings (such as acrylic resins and shellac); and protective agents used to maintain the stability of the bacterial agent can be selected from trehalose, skim milk powder, glycerin, and the like. The pharmaceutical composition can further include a targeted delivery component (such as a pH-sensitive polymer for colonic release). The food composition further includes prebiotics selected from fructooligosaccharides (FOS), galacto-oligosaccharides (GOS), inulin, and resistant dextrin (to promote bacterial colonization); nutrients selected from B vitamins, zinc (to enhance immunity), and dietary fiber (such as polydextrose); flavorings / correctives selected from citric acid, steviol glycosides, and natural fruit powder (to improve palatability); and stabilizers selected from pectin and xanthan gum (to prevent bacterial sedimentation).

[0011] Functional bacterial agents primarily include live bacteria and, optionally, culture medium compositions, such as yeast extract and soy peptone. They may also include other probiotics for enhanced efficacy, such as Lactobacillus (e.g., Lactobacillus acidophilus and Lactobacillus rhamnosus); Bifidobacterium (e.g., Bifidobacterium infantis and Bifidobacterium longum); and yeasts such as Saccharomyces boulardii. Furthermore, functional bacterial agents may also include carrier materials such as diatomaceous earth and corn starch (for solid-state fermentation agents); and synergists such as short-chain fatty acid salts (e.g., sodium butyrate) and plant extracts (e.g., curcumin, for anti-inflammatory synergy).

[0012] The bacterial agent can be in various forms such as capsules, tablets, powders, drops, granules, microencapsulated particles, etc. Capsules and tablets are preferred when used as pharmaceutical compositions, powders are preferred when used as food compositions, and microencapsulated particles are preferred when used as functional bacterial agents. More preferably, sodium alginate is added for embedding to enhance acid and heat resistance.

[0013] The route of administration can be oral, gavage / tube feeding, local administration, etc. Oral administration is the most preferred recommendation. Suppositories can also be used when needed for female microecological regulation.

[0014] In the composition, the concentration of the Weizmannella coagulans KY009 or Clostridium butyricum KY002 in the probiotic composition is 1×109 CFU / g, Clostridium butyricum is 1×10 9 CFU / g.

[0015] When the composition contains both Weizmannella coagulans and Clostridium butyricum, the ratio of the number of live bacteria of Weizmannella coagulans to Clostridium butyricum is 7:3, 6:4, 5:5, preferably 5:5, and preferably the number of live bacteria of Weizmannella coagulans and Clostridium butyricum is 0.5×10 9 CFU / g.

[0016] The third aspect of the present invention is to provide the use of Weizmannella coagulans KY009, Clostridium butyricum KY002 and the composition The invention relates to the use of Weizmannella coagulans KY009, Clostridium butyricum KY002 and a composition containing the same in the preparation of a medicine for improving reproductive health, improving sex hormone imbalance, alleviating intestinal inflammation and mucosal repair, and immune-related diseases; the invention relates to the use of Weizmannella coagulans KY009, Clostridium butyricum KY002 and a composition containing the same in improving reproductive health, sex hormone regulation, alleviating intestinal inflammation and mucosal repair, and immune-related diseases.

[0017] Weizmannella coagulans KY009, Clostridium butyricum KY002, and compositions containing them, which improve reproductive health and regulate sex hormones, are suitable for treating infertility and regulating sex hormone imbalance. Infertility includes male infertility and female infertility. Specifically, they can be used to increase sperm count and motility, regulate testosterone levels, and improve oligospermia and asthenospermia. Regulating sex hormone imbalances can specifically increase serum testosterone, follicle-stimulating hormone (FSH), and estradiol levels. They are also suitable for treating delayed puberty, sexual dysfunction, or menopausal hormone disorders.

[0018] Weizmannella coagulans KY009, Clostridium butyricum KY002, and compositions containing them can be used to treat intestinal inflammation and mucosal repair, and are suitable for alleviating inflammatory bowel disease, irritable bowel syndrome, and antibiotic-associated diarrhea. Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease, can specifically be used to alleviate colon tissue damage and weight loss. Irritable bowel syndrome (IBS), characterized by alternating symptoms such as intestinal flora imbalance, diarrhea, and constipation, and antibiotic-associated diarrhea (AAD), characterized by intestinal microecology and overgrowth of pathogenic bacteria such as Clostridium difficile, can also be alleviated and improved by Weizmannella coagulans KY009, Clostridium butyricum KY002, and compositions containing them.

[0019] Beneficial effects The Clostridium butyricum KY002 and its composition provided by the present invention have multiple beneficial effects. Clostridium butyricum KY002 can regulate the intestinal flora-gonadal axis by producing short-chain fatty acids (such as butyric acid), effectively increasing serum testosterone, FSH and estradiol levels, thereby improving male oligospermia, asthenospermia symptoms and female menopausal hormone disorders. When used in combination with Weizmannella coagulans KY009, the two can synergistically enhance the ability to regulate sex hormones, and have a significant improvement effect on reproductive health problems such as puberty stunting, sexual dysfunction and infertility. In terms of intestinal health, both Clostridium butyricum KY002 and Weizmannella coagulans KY009 can inhibit the release of pro-inflammatory factors and promote the expression of anti-inflammatory factors, and have the function of significantly alleviating inflammatory bowel disease. The combination of Weizmannella coagulans and the two can work synergistically to effectively alleviate the development of intestinal inflammation.

[0020] Furthermore, the acid-resistant properties of Weizmannella coagulans KY009 complement the colonization ability of Clostridium butyricum KY002, significantly improving strain survival and therapeutic efficacy after optimizing the formulation. The composition can be formulated as microencapsulated particles, capsules, or powders, with enhanced stability achieved through encapsulation technology, making it suitable for various administration methods, including oral administration and tube feeding. Overall, this invention not only provides a new microbial intervention strategy for reproductive health issues but also offers a safe and effective treatment option for intestinal inflammatory diseases, promising broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Figure 2 shows the colony formation after bacterial liquid coating. Left: Colony characteristics of Weizmannella coagulans; Right: Colony characteristics of Clostridium butyricum.

[0022] Figure 2 Mouse testis and epididymis weights.

[0023] Figure 3 Mouse sperm count assay.

[0024] Figure 4 Mouse sperm motility assay.

[0025] Figure 5 Detection of testosterone concentration in mouse serum.

[0026] Figure 6 Detection of FSH concentration in mouse serum.

[0027] Figure 7 Estradiol concentration in mouse serum.

[0028] Figure 8Changes in colon length in mice from different groups. From left to right: NC group, DSS group, KY009 group, KY002 group, KY-29 group (KY002:KY009 = 7:3), KY-30 group (KY002:KY009 = 6:4), and KY-31 group (KY002:KY009 = 5:5).

[0029] Figure 9 Comparison of tissue section staining of mice treated in different groups.

[0030] Figure 10 CXCL1 gene expression in mouse colon tissue.

[0031] Figure 11 IL-1β gene expression in mouse colon tissue.

[0032] Figure 12 IL10 gene expression in mouse colon tissue.

[0033] Figure 13 TNF-α gene expression in mouse colon tissue. DETAILED DESCRIPTION

[0034] Example 1 Isolation, Screening and Identification of Weizmannella coagulans and Clostridium butyricum 1. Strain isolation and screening The fecal sample was evenly mixed with PBS, heated in an 80°C water bath for 10 minutes, and then graded diluted from 10-2 to 10-6. 100 μL of each was evenly spread on MRS and TSN culture media, and incubated anaerobically at 37°C for 24-48 hours. When colonies formed, the morphological characteristics of the colonies were observed, and single colonies that met the morphological characteristics of Weizmannella coagulans and Clostridium butyricum were selected. Results are shown in the table. Figure 1 The left picture shows the colony characteristics of Weizmannella coagulans; the right picture shows the colony characteristics of Clostridium butyricum.

[0035] 2. Molecular Identification Universal primer sequences for 16S rDNA amplification: 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO: 1) 1492R: 5'-TACGGYTACCTTGTTACGACTT-3' (SEQ ID NO: 2) Table 1 PCR amplification reaction system

[0036] Prepare a 1% agarose gel (1g agarose plus 100mL deionized water). Bring to a boil 2-3 times. When cooled to approximately 55°C, add 10μL of 4S Green buffer, mix thoroughly, and pour into a gel casting tank. Once solidified, add 1μL of loading buffer and 5μL of the PCR product mixture to the gel wells. Run gel electrophoresis at 220V for 30 minutes. If a band appears at the target band (1500bp), send the sample to the Shanghai branch of Beijing Liuhe BGI Genomics Co., Ltd. for Sanger sequencing. Compare the sequencing results on the ezbiocloud website.

[0037] PCR product sequencing results: The sequencing results were compared on the ezbiocloud website, and strain KY009 was identified as Weizmannia coagulans and strain KY002 as Clostridium butyricum.

[0038] Weizmannella coagulans KY009 16S rDNA sequence: Clostridium butyricum KY002 16S rDNA sequence: Example 2: Sex Hormone Detection of Animals Fed with Strain This study selected adult male mice aged 8 weeks and randomly divided them into 4 groups, with 10 mice in each group. Weizmannella coagulans and Clostridium butyricum powder were resuspended in physiological saline and used as the KY009 experimental group, KY002 experimental group, and the KY-29 experimental group of the two bacteria mixture. The group that was gavaged with normal saline alone served as the NC (Negative Control) control group. The gavage bacterial suspension should be prepared and used once a day, with a total concentration of 10 9 CFU / mL. In the mixed bacterial group KY-29, the ratio of viable Weizmannella coagulans to Clostridium butyricum was 7:3. The inventors also isolated strains of Bacillus coagulans and Clostridium butyricum from commercially available probiotic products and used them as the control groups for KY009 and KY002, respectively.

[0039] Bacterial strains: KY009 and KY002 were isolated from healthy human feces in the inventor's laboratory; Bacillus coagulans and Clostridium butyricm were purchased from Xi'an Peptide Kang Bioengineering Co., Ltd.

[0040] Control group (NC): normal mice, gavage with 0.2 mL of 0.85% saline; Weizmannella coagulans group (Bacillus coagulans and KY009): Weizmannella coagulans was administered orally at a dose of 10 9 CFU / bird / day.

[0041] Clostridium butyricum group (Clostridium butyricum and KY002): Clostridium butyricum was administered orally at a dose of 10 9 CFU / mouse / day.

[0042] The intervention group (KY-29) was given a mixture of Weizmannella coagulans and Clostridium butyricum by gavage at a dose of 10 9 CFU / mouse / day.

[0043] At the end of the feeding experiment, the animals were anesthetized and sacrificed, and their reproductive tracts were dissected. The testicles and epididymis were removed, isolated, and washed with saline. The left cauda epididymis was crushed and placed in an F12 dish containing 2 ml of DMEM and 5% FBS. The dish was incubated at 37°C for 30 minutes. To liquefy the sperm suspension, 0.5 ml of the sperm suspension was added to 10 μl of 10% formaldehyde (1:10).

[0044] 1. Epididymis and Testis Weight Measurement The testicles and epididymis were removed and weighed. Figure 2 As shown in the data, the weights of the testicles and epididymis of the KY002, KY009 and KY-29 groups were significantly higher than those of the NC group, and the combined strain of the KY-29 group was superior to the single strain effect of the KY002 and KY009 groups; compared with the Bacillus coagulans control group, the weights of the testicles and epididymis of the mice in the KY002 group were higher (p<0.001); compared with the Clostridium butyricum control group, the weights of the testicles and epididymis of the mice in the KY009 group were also significantly heavier (P<0.001). This indicates that KY002 and KY009 have a more obvious effect on promoting the development of mouse sexual organs than other strains of bacteria in the control group.

[0045] 2. Sperm count Observe spermatozoa using an optical microscope. Use a Neubauer slide to observe spermatozoa in four small corner squares (0.2 mm 2 ) and a large center square (1 mm 2 Sperm were counted in 5 squares (i.e., 250 small squares on a Neubauer slide, × 400). Finally, the number of sperm in 5 squares × dilution factor / number of counting squares (mm) was calculated and reported. 2 ) × the depth of the Neubauer chamber”, and the unit is million sperm / ml. To improve the accuracy, the sperm count in each suspension was repeated three times. The experimental results are shown in Figure 3 As shown, the sperm counts of the KY002, KY009 and KY-29 groups were significantly higher than that of the NC group, and the combined strain of the KY-29 group was better than the single strain effect of the KY002 and KY009 groups.

[0046] 3. Sperm motility Sperm motility is measured using Eosin Y in a specific volume of sperm suspension. First, a 40 μl suspension of epididymal sperm is added to 10 μl of 0.5% Eosin Y and examined at 40× LM magnification. Dead sperm absorb Eosin Y into their heads and necks due to damage to the sperm plasma membrane caused by free radicals.

[0047] However, live sperm do not take up Eosin Y. To determine the viability of sperm, the average percentage of live sperm (%) / total number of sperm was calculated in three replicates for each sample. Figure 4 As shown in the data, the sperm motility of the KY002, KY009 and KY-29 groups was significantly higher than that of the NC group, and the combined strain of the KY-29 group was more effective than the single strain of the KY002 and KY009 groups.

[0048] 4. Serum testosterone (T), FSH and estradiol levels were determined by ELISA.

[0049] At the end of the experimental period, blood samples were obtained from the facial vein of awake mice and immediately centrifuged at 500 × g for 10 min at 4°C to collect serum. Serum samples were immediately stored and kept at −20°C until analysis.

[0050] (1) Testosterone The testosterone in mouse serum was determined using an ELISA kit. Figure 5 Compared with the NC group, the testosterone concentrations in the serum of mice in the three experimental groups increased significantly, and the testosterone concentration in the KY-29 group was higher than that in the KY002 and KY009 groups, indicating that the mixed bacterial formula of KY009 and KY002 is more conducive to the expression of testosterone in mice.

[0051] (2) FSH The FSH content in mouse serum was determined using an ELISA kit. Figure 6 Compared with the NC group, the serum FSH concentrations of the three experimental groups increased significantly, and the serum FSH concentrations of the KY-29 group were higher than those of the KY002 and KY009 groups, indicating that the mixed bacterial formula of KY009 and KY002 is more conducive to the expression of follicle-stimulating hormone (FSH) in mice.

[0052] (3) Estradiol The elution kit was used to measure the estradiol in mouse serum. Figure 7 Compared with the NC group, the serum estradiol concentrations of the three experimental groups increased significantly, and the KY-29 group had a higher estradiol concentration than the KY002 and KY009 groups, indicating that the mixed bacterial formula of KY009 and KY002 is more conducive to the expression of estradiol in mice.

[0053] The above data show that both Clostridium butyricum KY002 and Weizmannella coagulans KY009 can promote the expression of sex hormones in mice, and the synergistic use of Clostridium butyricum KY002 and Weizmannella coagulans KY009 has a better effect.

[0054] Example 3 Detection of Inflammatory Bowel Disease in Animals Fed with Bacteria A DSS-induced colitis model was used. Before model establishment, mice were weighed and randomly divided into seven groups of 10 mice each. After grouping, mice in each group (except the NC group) were given drinking water containing 2.5% DSS. Mice were weighed daily before oral gavage until the weight loss rate in the DSS group exceeded 20%, indicating successful model establishment. The formula for mouse weight loss is as follows: Mouse weight loss rate (%) = (initial weight - current weight) / initial weight * 100%. A mixed powder of Weizmannella coagulans and Clostridium butyricum was suspended or dissolved in normal saline, and the bacterial suspension for oral gavage was prepared fresh. Dosing was once daily.

[0055] Group settings: Control group (NC): normal mice, gavage with 0.2 mL of 0.85% saline; DSS model group (DSS): mice were induced with DSS and gavaged with 0.2 mL of 0.85% saline; DSS-coagulated Weizmannella group (KY009): mice were induced with DSS and orally gavaged with Weizmannella coagulated at a dose of 10 9 CFU / mouse / day; DSS Clostridium butyricum group (KY002): mice were induced with DSS and gavaged with Clostridium butyricum at a dose of 10 9 CFU / mouse / day; The DSS model Weizmannella coagulans and Clostridium butyricum intervention group (KY-29): mice were induced with DSS and orally administered with a mixture of Weizmannella coagulans and Clostridium butyricum at a dose of 10 9 CFU / mouse / day, the ratio of live bacteria counts of Weizmannella coagulans and Clostridium butyricum was 7:3.

[0056] The DSS model Weizmannella coagulans and Clostridium butyricum intervention group (KY-30): mice were induced with DSS and gavaged with a mixture of Weizmannella coagulans and Clostridium butyricum at a dose of 10 9 CFU / mouse / day, the ratio of live bacteria counts of Weizmannella coagulans and Clostridium butyricum was 6:4.

[0057] The DSS model Weizmannella coagulans and Clostridium butyricum mixture intervention group (KY-31): mice were induced with DSS and gavaged with Weizmannella coagulans and Clostridium butyricum mixture at a dose of 10 9 CFU / mouse / day, the ratio of live bacteria count of Weizmannella coagulans and Clostridium butyricum was 5:5.

[0058] The average weight of mice in the blank control group (NC) was 19 g on the first day and 19.54 g on the 14th day; the average weight of mice in the DSS model (DSS) was 18.91 on the first day and 14.98 g on the 14th day. A weight loss rate of >20% indicated that the model was successfully established.

[0059] 1. Colon length and colon histopathological examination The experiment lasted 14 days, and the weight and condition of the mice were recorded daily. On the 15th day, all mice were sacrificed, and colon tissue was removed, photographed, and measured. After measuring colon length, the cecum was removed, and the colon contents were flushed clean with PBS using a syringe. 4% PFA was then injected into the colon and flushed along the inside with a syringe. The colon was then immersed in 1% PFA for 30 minutes. The colon was cut longitudinally, and the colon was slowly and carefully rolled up from the distal end to form a Swiss roll. This roll was carefully placed in an embedding frame, and the embedding frame containing the small intestinal Swiss roll was placed in 4% PFA fixative and fixed overnight at 4°C. Hematoxylin-eosin staining (HE) of the intestinal tissue was performed by Wuhan Saiweier Biotechnology Co., Ltd., and the sections were subjected to pathological examination.

[0060] Table 2 Tissue damage scoring criteria Score Severity of inflammation Accumulation and depth of inflammation Degree of crypt damage Lesion extent (%) 0 none none none none 1 Mild mucosal layer Damage to the basal third of the crypts 1～25 2 Moderate Mucosa and submucosa Damage to the basal 2 / 3 crypts 26～50 3 severe Inflammation accumulates throughout the entire layer All crypts are lost 51～75 4 / / Complete destruction of crypts and intestinal epithelium 76～100 The results showed that although the colon length of the experimental group was higher than that of the DSS group and similar to that of the NC group, there was no significant difference between the experimental group and the DSS group. Figure 8 H&E staining of colon sections was performed to assess colon mucosal damage. Colon sections from the control group showed changes in intestinal structure and barrier function, specifically mucosal layer destruction, crypt damage, and inflammatory cell infiltration (e.g. Figure 9 In contrast, treatment with the probiotic strains had a protective effect on the colon, as evidenced by intact crypts and less inflammatory cell infiltration.

[0061] 2. Expression of colon tissue-related genes Colonic tissue stored at -80°C was lysed with TriZol lysis buffer, and total RNA was extracted using chloroform, isopropanol, and 75% ethanol. Absorbance at the A260 / A280 and A260 / A230 ratios was measured using an ultra-microspectrophotometer to determine RNA concentration and purity. Acceptable RNA was reverse-transcribed into cDNA. Real-time quantitative PCR (qPCR) reactions were performed using the TaqPro Universal SYBR qPCR Master Mix kit (Nanjing Novozymes Biotech Co., Ltd.). A 20 μl reaction volume contained 1 μl of cDNA, 1 μl of each 10 μmol / L upstream and downstream primers, and 7 μl of ddH2O. PCR was performed on a CFX Duet real-time fluorescence quantitative analyzer (Bio-Rad) using the following conditions: 95°C denaturation for 3 min, followed by 39 cycles of denaturation at 95°C for 10 s and annealing at 60°C for 30 s. TBP was used as an internal control, and the 2-ΔΔCt method was used for analysis. Gene expression was calculated using the formula: 2Cttarget (control) – Cttarget (treatment) / 2CtTBP (control) – CtTBP (treatment). qPCR was used to analyze the expression of CXCL1, TNF-α, IL-1β, and IL-10 genes in the colon.

[0062] (1) Proinflammatory factor CXCL1 The expression of CXCL1 gene in colon was analyzed by qPCR. Figure 10 Compared with the NC group, the expression of chemokine CXCL1 in the colon tissue of mice in the NC group was very low, and the expression of chemokine CXCL1 in the DSS group was significantly higher than that in the NC group. The expression of chemokine CXCL1 in the five groups with probiotic intervention decreased significantly, indicating that the use of probiotics Weizmannella coagulans, Clostridium butyricum, and a mixture of the two bacteria can inhibit the expression of inflammatory factor CXCL1.

[0063] (2) Proinflammatory cytokine IL-1β The expression of IL-1β gene in colon was analyzed by qPCR. Figure 11 Compared with the NC group, the expression of IL-1β in the colon tissue of mice in the NC group was very low, and the expression of IL-1β in the DSS group was significantly higher than that in the NC group. However, the expression of IL-1β in the five groups with probiotic intervention decreased significantly, indicating that the use of probiotics Weizmannella coagulans, Clostridium butyricum, and a mixture of the two bacteria can inhibit the expression of the inflammatory factor IL-1β.

[0064] (3) Anti-inflammatory factor IL10 The expression of IL10 gene in colon was analyzed by qPCR. Figure 12 As shown. IL10 is involved in the regulation of inflammation during the immune response and has the effect of inhibiting excessive inflammation. Compared with the DSS group, the expression of IL10 in the colon of mice treated with oral administration of KY002, KY009, and a mixture of KY002 and KY009 in different ratios significantly increased. Furthermore, IL10 levels reached the highest level in mice treated with a mixture of KY002 and KY009 (7:3). This suggests that intervention with the probiotics Weizmannella coagulans, Clostridium butyricum, and a mixture of these two bacteria can promote the expression of anti-inflammatory factors.

[0065] (4) Proinflammatory factor TNF-α The expression of TNF-α gene in colon was analyzed by qPCR. Figure 13 Compared with the NC group, the expression of TNF-α in the colon tissue of mice in the NC group was very low, the expression of TNF-α in the DSS group was significantly higher than that in the NC group, and the expression of TNF-α in the five groups with probiotic intervention decreased significantly, indicating that the use of probiotics Weizmannella coagulans, Clostridium butyricum, and a mixture of the two bacteria can inhibit the expression of the inflammatory factor TNF-α.

[0066] According to the above experimental results, the levels of inflammatory factors in the colon tissue of each group of mice were as follows: Figure 10-13 As shown, it shows that adding probiotics can relieve intestinal inflammation in mice, both single bacteria and mixed bacteria can be used, and the effect of mixed bacteria is better than that of single bacteria. In our mixed bacteria combination group, we conducted mixed bacteria combination optimization, and the experimental results showed that the dosage of the mixture of Weizmannella coagulans and Clostridium butyricum was 10 9 CFU / mouse / day, when the ratio of coagulant Weizmannella: butyricum live bacteria count is 5:5, it can better alleviate the inflammation in the colon tissue.

Claims

1. A Clostridium butyricum KY002, with a deposit number of CGMCC NO. 33478, was deposited on January 20, 2025, at the General Microbiology Center of the China Culture Collection Administration, at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. The Clostridium butyricum according to claim 1, characterized in that: Its 16S rDNA is shown in SEQ ID NO:

3.

3. A composition comprising the Clostridium butyricum according to claim 1, wherein the composition is selected from a food composition, a pharmaceutical composition, a health product composition, and a functional bacterial agent.

4. The composition according to claim 3, further comprising Weizmannella coagulans KY009, which was deposited on January 20, 2025 at the General Microbiology Center of China Culture Collection Administration, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC NO. 33480. The composition according to claim 4 , wherein the 16S rDNA of Weizmannella coagulans is represented by SEQ ID NO:

4.

6. The composition according to any one of claims 3 to 5, wherein the pharmaceutical composition further comprises a pharmaceutical excipient; the food composition further comprises a prebiotic, a nutrient, a dietary fiber, a flavoring agent and / or a flavoring agent, and a stabilizer; and the functional bacterial agent further comprises a culture medium composition, co-cultured probiotics, a carrier material, and a synergist.

7. The composition according to claim 6, wherein the co-cultured probiotics in the functional bacterial agent are at least one selected from the genus Lactobacillus, the genus Bifidobacterium, and the genus Saccharomyces.

8. Use of the Clostridium butyricum according to any one of claims 1-2, the Weizmannella coagulans according to any one of claims 4-5, or the composition according to any one of claims 3-7 in the preparation of a medicament for improving reproductive health, treating sex hormone imbalance, treating intestinal inflammation and mucosal repair, and metabolic and immune-related diseases; or Use in the preparation of food compositions or functional bacterial agents for improving reproductive health, regulating sex hormones, relieving intestinal inflammation and mucosal repair, and relieving metabolic and immune-related diseases.

9. A composition comprising Weizmannella coagulans and Clostridium butyricum according to claims 1-2, wherein the ratio of the viable bacteria count of Weizmannella coagulans to Clostridium butyricum is 7:3, 6:4, or 5:5, preferably 5:

5. 10 . The composition according to claim 9 , wherein the Weizmannella coagulans is the Weizmannella coagulans described in any one of claims 4 to 5 , and the Clostridium butyricum is the Clostridium butyricum described in any one of claims 1 to 2 .

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