Compound microbial inoculant capable of preventing and treating ulcerative colitis and application thereof

Abstract

The application provides a compound microbial agent capable of preventing and treating ulcerative colitis and an application thereof, and belongs to the technical field of microbial agents. The compound microbial agent capable of preventing and treating ulcerative colitis provided by the application is composed of Clostridium leptum and Akkermansia muciniphila. The compound microbial agent provided by the application can exert a synergistic effect, has strong intestinal colonization ability, good anti-inflammatory and mucosal repair effects, has a significant prevention and treatment effect on ulcerative colitis, specifically, can reduce colon shortening caused by ulcerative colitis, can reduce weight loss caused by ulcerative colitis, and can improve the severity of ulcerative colitis.

Description

Technical Field

[0001] This invention belongs to the field of microbial preparation technology, specifically relating to a compound bacterial agent that can prevent and treat ulcerative colitis and its application. Background Technology

[0002] Ulcerative colitis (UC) is a recurrent chronic inflammatory bowel disease with a complex pathogenesis, long course, high relapse rate, and high risk of complications, severely impacting patients' quality of life. Currently, clinically available treatments for ulcerative colitis mainly include aminosalicylic acids, glucocorticoids, immunosuppressants, and biologics. However, these treatments generally suffer from limited applicability, significant adverse reactions, the potential for drug resistance or dependence with long-term use, and high cost for some medications, making it difficult to meet the clinical needs for safe, effective, and long-term use.

[0003] Disruptions in the gut microbiota and impaired intestinal mucosal barrier are key factors in the development and progression of ulcerative colitis. Prevention and treatment strategies based on gut microbiota regulation have become a research hotspot in this field, but existing microecological preparations still have significant shortcomings in practical applications. Existing probiotic preparations generally suffer from problems such as unreasonable strain compatibility, single target of action, weak intestinal colonization ability, and limited anti-inflammatory and mucosal repair effects. They are generally ineffective in improving intestinal flora imbalance, blocking the progression of inflammation, and repairing damaged mucosal barriers. They are unable to effectively break the vicious cycle of "flora disorder - barrier damage - inflammation amplification" in the course of ulcerative colitis. Their effects on the prevention, relief, and recurrence control of ulcerative colitis are not ideal, and their clinical application is greatly limited.

[0004] Based on the above situation, developing novel compound microecological preparations that can efficiently regulate the intestinal microecology, synergistically repair the mucosal barrier, and inhibit intestinal inflammatory responses for the prevention and treatment of ulcerative colitis has important practical significance and application value. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a compound bacterial agent that can prevent and / or treat ulcerative colitis, which can exert a synergistic effect, has strong intestinal colonization ability, good anti-inflammatory and mucosal repair effects, and has a significant preventive and therapeutic effect on ulcerative colitis.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a compound bacterial agent capable of preventing and treating ulcerative colitis, composed of Clostridium leptum and Akkermansia muciniphila.

[0007] Preferably, the ratio of viable bacteria of Clostridium perfringens to Akkermansia myxophilus is 1:1.

[0008] Preferably, the viable bacteria count in the compound microbial agent is 2 × 10⁻⁶. 9 CFU / g or higher or 2×10 9 CFU / mL or higher.

[0009] Preferably, the Clostridium truncatum strain is a strain with preservation number ATCC 29065 or DSM 753.

[0010] Preferably, the Akkermansia myxophilus strain is a strain with accession number ATCC BAA-835 or DSM 22959.

[0011] This invention also provides the application of the above-mentioned compound microbial agent in the preparation of products for the prevention and / or treatment of ulcerative colitis.

[0012] Preferably, the product includes a drug.

[0013] Preferably, the compound microbial agent can reduce colonic shortening caused by ulcerative colitis, reduce weight loss caused by ulcerative colitis, and improve the severity of ulcerative colitis.

[0014] Preferably, the ulcerative colitis is ulcerative colitis caused by sodium dextran sulfate.

[0015] The beneficial effects of this invention are: The compound bacterial agent provided by this invention can exert a synergistic effect, has strong intestinal colonization ability, good anti-inflammatory and mucosal repair effects, and has a significant preventive and therapeutic effect on ulcerative colitis. Specifically, it can reduce colonic shortening caused by ulcerative colitis, reduce weight loss caused by ulcerative colitis, and improve the severity of ulcerative colitis. Attached Figure Description

[0016] Figure 1 The graph shows the statistical distribution of mouse body weight and DAI score. A represents the DAI score of the mixed bacterial culture, B represents the body weight of the mixed bacterial culture, C represents the DAI score of a single *Clostridium leptum* bacterium, D represents the body weight of a single *Clostridium leptum* bacterium, E represents the DAI score of a single *Akkermansia muciniphila* bacterium, and F represents the body weight of a single *Akkermansia muciniphila* bacterium. This indicates that compared with the 3% DSS group, p < 0.01; This indicates that compared with the 3% DSS group, p < 0.05.

[0017] Figure 2 A comparison of colon length in mice from different groups. Detailed Implementation

[0018] This invention provides a compound bacterial agent capable of preventing and treating ulcerative colitis, composed of Clostridium tenuifolium and Akkermansia myxophilus.

[0019] Existing commercially available probiotics mainly consist of Lactobacillus and Bifidobacterium, which are not specifically designed for patients with ulcerative colitis. The combined action of the two probiotics provided in this invention better matches the intestinal flora characteristics of ulcerative colitis patients, making it suitable for individualized treatment. In this invention, *Clostridium leptum* is preferably a strain with accession numbers ATCC 29065 or DSM 753, and *Akkermansia muciniphila* is preferably a strain with accession numbers ATCC BAA-835 or DSM 22959. *Clostridium leptum* is a bacillus, including motile and non-motile bacilli. Motile species move via peritrichous flagella, and most species can form oval or spherical spores. *Akkermansia muciniphila* is Gram-negative, non-motile, and strictly anaerobic.

[0020] In this invention, the live count ratio of *Clostridium tenuifolium* and *Ackermania pseudomallei* is preferably 1:1; when the compound microbial agent is a solid preparation, the live count in the compound microbial agent is preferably 2 × 10⁻⁶. 9 CFU / g or higher; when the compound microbial agent is a liquid preparation, the viable count in the compound microbial agent is preferably 2 × 10⁻⁶. 9 CFU / mL or higher.

[0021] This invention also provides the application of the above-mentioned compound microbial agent in the preparation of products for the prevention and / or treatment of ulcerative colitis. In this invention, the product preferably comprises a drug, and is preferably an oral product. In this invention, the compound microbial agent can reduce colonic shortening caused by ulcerative colitis, can reduce weight loss caused by ulcerative colitis, and can improve the severity of ulcerative colitis; the ulcerative colitis is preferably ulcerative colitis caused by sodium dextran sulfate (DSS).

[0022] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0023] Unless otherwise specified, the following embodiments are all conventional methods.

[0024] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0025] The Clostridium leptum strains used in the following examples are those with accession numbers ATCC 29065 or DSM753, and the Akkermansia muciniphila strains are those with accession numbers ATCC BAA-835 or DSM 22959, purchased from Ningbo Mingzhou Biotechnology Co., Ltd.

[0026] Example 1 Culture and identification of Clostridium perfringens and Akkermansia myxophilus The carbohydrate broth (CMC) basal medium was purchased from Ningbo Mingzhou Biotechnology Co., Ltd. 5.75 g of CMC medium was weighed and diluted to 100 mL with distilled water. An appropriate amount of ground beef granules was added, filling the liquid to 1 / 3 of its height. After mixing, the mixture was autoclaved at 121℃ for 30 min, cooled to below 50℃, and under aseptic conditions, one vial of 0.5 mg heme chloride and one vial of 5 mg vitamin K1 were added per 100 mL. The mixture was then placed in an anaerobic chamber containing a mixture of 80% N2, 10% H2, and 10% CO2 for overnight deoxygenation to obtain the deoxygenated CMC medium.

[0027] The purchased lyophilized powders of *Clostridium perfringens* and *Ackermania marcescens* were transferred to deoxygenated CMC medium, fully activated, and cultured for 24–72 h. After streaking onto plates in an anaerobic chamber, and culturing under anaerobic conditions for another 24–72 h, single colonies were picked for identification. According to Hangzhou Microbiotechnology Co., Ltd., the 16S rRNA nucleotide characteristic sequence of *Ackermania marcescens* is: AACGAACGCTGGCGGCGTGGATAAGACATGCAAGTCGAACGAGAGAATTGCTAGCTTGCTAATAATTCTCTAGTGGCGCACGGGTGAGTAACACGTGAGTAACCTGCCCCCGAGAGCGGGATAGCCCTGGGAAACTGGGATTAATACCGCATAGTATCGAAAGATTAAAGCAGCAATGCGCTTGGGGATGGGCTCGCGGCCTA. TTAGTTAGTTGGTGAGGTAACGGCTCACCAAGGCGATGACGGGTAGCCGGTCTGAGAGGATGTCCGGCCACACTGGAACTGAGACACGGTCCAGACACCTACGGGTGGCAGCAGTCGAGAATCATTCA CAATGGGGGAAACCCTGATGGTGCGACGCCGCGTGGGGGAATGAAGGTCTTCGGATTGTAAACCCCTGTCATGTGGGAGCAAATTAAAAAGATAGTACCACAAGAGGAAGAGACGGCTAACTCTG (SEQ ID NO.1), 16S of Clostridium tenellae The characteristic rRNA nucleotide sequence is GACGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAACGGAGTTAAATTCGACACCCGAGTATCCGGCCGGGAGGCGGGGTGCTGGGGGTTGGATTTAACTTAGTGGCGGACGG GTGAGTAACGCGTGAGTAACCTGCCTTTCAGAGGGGGATAACGTTCTGAAAAGAACGCTAATACCGCATAACATCAATTTATCGCATGATAGGTTGATCAAAGGAGCAATCCGCTGGAAGATGGACTCG The sequence CGTCCGATTAGCCAGTTGGCGGGGTAACGGCCCACCAAAGCGACGATCGGTAGCCGGACTGAGAGGTTGAACGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGGATATTGCACAATGGGGGAAACCCTGATGCAGCAACGCCGCGTGAGGGAAGAAGGTTTTCGGATTGTAAACCTCTGTTCTTAGTGACGATAATGACGGTAGCTAAGGAGAAAGCTCCGGCTAACTACG (SEQ ID NO. 2) indicates that the bacteria obtained from the activated culture are indeed *Clostridium tenuifolium* and *Ackermania sinensis*.

[0028] Example 2 (1) Cultivation and preparation of bacterial suspension of Clostridium truncatum and Akkermansia myxophila After 24–72 h of anaerobic culture, *Clostridium tenuifolium* and *Ackermania pseudomallei* showed turbidity and white flocculent precipitate in the liquid culture medium. After mixing by pipetting, 200 μL of the mixture was transferred to a plate, spread evenly with an L-shaped spreader, and then incubated anaerobically for another 24–72 h. *Clostridium tenuifolium* formed a transparent, dense bacterial film on blood agar plates, while *Ackermania pseudomallei* formed pinhead-sized, transparent colonies. Bacterial counting was performed using a McFarland turbidimeter. 4 mL of sterile anaerobic phosphate-buffered saline (PBS) was added to a sterile tube, the McFarland turbidimeter was zeroed, and colonies were scraped from the plate with a sterile swab, transferred to the aforementioned sterile tube, shaken to dislodge the colonies, and the turbidity was measured again to obtain the specific McFarland concentration (McF). Based on the formula 0.5 McFarland = 1.5 × 10⁻⁶, the McFarland concentration was determined. 8 The conversion formula for CFU / mL can be used to obtain the specific bacterial concentration.

[0029] Using the above method, the concentrations of Clostridium tenuifolium and Akkermansia myxophilus were quantified to 2 × 10⁻⁶. 9 CFU / mL was mixed thoroughly by pipetting in a new EP tube at a 1:1 volume ratio to prepare a dual-cell mixture of *Clostridium tenuifolium* and *Ackermania maughanii* with concentrations of 1×10⁻⁶ CFU / mL. 9 A mixed bacterial solution of CFU / mL was administered to mice via gavage.

[0030] (2) Combined prevention / treatment of mouse ulcerative colitis model with Clostridium perfringens and Akkermansia myxophilus Six-week-old male C57BL / 6J mice were pre-fed for one week and then divided into five groups: PBS group (Con), 3% DSS group, 3% DSS + Clostridium leptum + Akkermansia muciniphila group (also known as 3% DSS + dual-bacteria combination group), 3% DSS + Clostridium leptum group, and 3% DSS + Akkermansia muciniphila group.

[0031] Mice in the PBS group were given 200 μL / d of PBS by gavage for 17 days. Starting from day 10, their drinking water was changed to sterile water.

[0032] Mice in the 3% DSS group were given 200 μL / d of PBS by gavage for 17 days. Starting from day 10, their drinking water was changed to 3% DSS solution.

[0033] The group receiving 3% DSS + Clostridium leptum + Akkermansia muciniphila was treated with 200 μL / d of the mixed bacterial solution by gavage for 17 days. Starting from day 10, the drinking water was changed to 3% DSS solution.

[0034] The 3% DSS + Clostridium leptum group was given 200 μL / d of Clostridium leptum bacterial culture (concentration of 2×10⁻⁶). 9 The patient was given oral gavage (CFU / mL) for 17 days, and starting from day 10, the drinking water was changed to 3% DSS solution.

[0035] The 3% DSS + Akkermansia muciniphila group was given 200 μL / d of Akkermansia muciniphila bacterial suspension (concentration of 2×10⁻⁶). 9 The patient was given oral gavage (CFU / mL) for 17 days, and starting from day 10, the drinking water was changed to 3% DSS solution.

[0036] Starting from day 10 of the experiment, changes in body weight and disease activity index (DAI score based on the article "Chemically induced mouse models of acute and chronic intestinal inflammation") were recorded for each group of mice. After the experiment, anatomical sampling was performed to measure the colon length of the mice in their natural state. The results are as follows: Figure 1 and Figure 2 As shown, the mixed bacterial solution of the present invention can significantly improve the degree of weight loss and colon shortening in mice caused by DSS modeling, and can significantly reduce the DAI score.

[0037] The Bliss independent model method is introduced. This model quantifies the difference between the "sum of independent effects" and the "actual effect" by calculating the difference between the "theoretical combined effect" and the "actual combined effect," which can determine whether the beneficial effects of *Clostridium tenuifolium* combined with *Ackermania muscaria* in the prevention / treatment of ulcerative colitis are synergistic. According to the Bliss model, the theoretical combined effect of independent actions is: I 联合（理论） =I C.l +I Akk -I C.l ×I Akk , where I C.l This refers to the group receiving Clostridium leptum via gavage alone, I Akk This refers to the Akkermansia muciniphila group that received oral gavage alone.

[0038] By analyzing the effects of exogenously added probiotics in each group, the inhibition rate of weight loss in each group can be calculated and denoted as I. 组别体重， The inhibition rate of DAI in each group is denoted as I. 组别D ;I 组别体重 The calculation formula = (W) 组别终点 -W DSS组终点 ) / (W DSS组终点 )×100%, I 组别D The calculation formula is: (DAI) DSS组 -DAI 组别 ) / (DAI DSS组 )×100%.

[0039] according to Figure 2 The inhibition rates of weight loss and DAI in the Clostridium leptum monotherapy group, Akkermansia muciniphila group, and the combined group can be calculated separately: I C.l体重 = (90.356% - 86.461%) / (86.461%) × 100% = 4.505%; I Akk体重 = (83.9678% - 83.582%) / (83.582%) × 100% = 0.462%; I 双菌联合体重 = (83.013% - 78.406%) / (78.406%) × 100% = 5.876%; I 理论联合体重损失抑制率 =I C.l体重 +I Akk体重 -I C.l体重 ×I Akk体重 =4.505% + 0.462% - 4.505% × 0.462% = 4.946%; I 双菌联合体重 >I 理论联合体重损失抑制率 ; Similarly: I C.l D = (8.78 - 8.1) / 8.78 × 100% = 7.745%; I Akk D = (9.9-10) / 9.9×100%=-1.01%; I 双菌联合D = (9.9 - 8.8) / 9.9 × 100% = 11.111%; I 理论联合DAI抑制率 =I C.l D +I Akk D -I C.l D ×I Akk D =7.745% + (-1.01%) - 7.745% × (-1.01%) = 6.813%; I 双菌联合D >I 理论联合DAI抑制率 .

[0040] The calculation results above show that when Clostridium tenuifolium is combined with Akkermansia myxophilus, it has a synergistic effect in the prevention and / or treatment of ulcerative colitis, and can synergistically improve weight loss and disease severity in DSS-induced colitis mice.

[0041] Example 3 A compound bacterial agent for the prevention and treatment of ulcerative colitis comprises Clostridium tenuifolium and Akkermansia myxophilus in a 1:1 live bacteria ratio, wherein the total live bacteria count of the compound bacterial agent is 2 × 10⁻⁶. 9 CFU / g.

[0042] Example 4 A compound bacterial agent for the prevention and treatment of ulcerative colitis comprises Clostridium tenuifolium and Akkermansia myxophilus in a 1:1 live bacteria ratio, wherein the total live bacteria count of the compound bacterial agent is 2 × 10⁻⁶. 9CFU / mL or higher.

[0043] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A compound bacterial agent capable of preventing and treating ulcerative colitis, characterized in that, It is composed of Clostridium truncatum and Akkermansia myxophila.

2. The compound microbial agent according to claim 1, characterized in that, The ratio of viable bacteria of Clostridium perfringens to Akkermansia myxophilus was 1:

1.

3. The compound microbial agent according to claim 1, characterized in that, The viable bacteria count in the compound microbial agent is 2 × 10⁻⁶. 9 CFU / g or higher or 2×10 9 CFU / mL or higher.

4. The compound microbial agent according to claim 1, characterized in that, The Clostridium truncatum strain is a strain with preservation numbers ATCC29065 or DSM 753.

5. The compound microbial agent according to claim 1, characterized in that, The *Ackermania* strain mentioned is a strain with accession number ATCC BAA-835 or DSM 22959.

6. The use of the compound microbial agent according to any one of claims 1 to 5 in the preparation of products for the prevention and / or treatment of ulcerative colitis.

7. The application according to claim 6, characterized in that, The products include pharmaceuticals.

8. The application according to claim 6, characterized in that, The compound microbial agent can reduce colonic shortening caused by ulcerative colitis, reduce weight loss caused by ulcerative colitis, and improve the severity of ulcerative colitis.

9. The application according to claim 6, characterized in that, The ulcerative colitis mentioned is ulcerative colitis caused by sodium dextran sulfate.

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