Coahndrikia coagulatum AACE9 and application of ahndrikia coagulatum AACE9

Abstract

The invention discloses Heynickia coagulans AACE9 and application thereof, and relates to the technical field of biological medicine, the Heynickia coagulans AACE9 is preserved in China General Microbiological Culture Collection Center on July 15, 2024, the preservation number is CGMCCNO.31302, and the preservation address is No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing. The invention also discloses application of the strain in preparation of products for preventing or treating colitis. The strain has an immune regulation function, can reduce the disease activity index and relieve colon and intestinal injury, and has a relatively great application prospect in the aspect of treating and preventing colitis.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a Bacillus heindrix coagulans AACE9 and an application thereof. Background Art

[0002] Ulcerative colitis (UC) is an inflammatory disease, including ulcerative colitis and Crohn's disease. Its pathogenic mechanism involves multiple factors, including immune system disorders, genetic susceptibility, imbalance of intestinal microbiota, and environmental factors. Under normal circumstances, the immune system in the intestine maintains the integrity of the intestinal mucosa through a series of complex signaling pathways to prevent the invasion of pathogens. However, in patients with colitis, the intestinal immune system is abnormal, resulting in an overreaction to harmless bacteria or food antigens in the intestine. As the inflammation continues, patients will experience symptoms such as abdominal pain, diarrhea, blood in the stool, and weight loss. In severe cases, it may also lead to complications such as intestinal perforation and cancer. The incidence of colitis is increasing worldwide. According to statistics, the annual incidence of ulcerative colitis in European and American countries is 10-20 / 100,000 respectively. In Asian countries such as China, although the incidence is relatively low, with the significant changes in eating habits, pace of life and environmental factors, the incidence of colitis in China has risen sharply, and it has become one of the common and difficult diseases in my country's digestive system. Relevant domestic surveys show that the incidence rate of ulcerative colitis in my country is about 3-8 / 100,000 people, and is showing a trend of becoming younger.

[0003] At present, the treatments for colitis mainly include drug therapy, surgical treatment, and adjuvant therapy. Commonly used drugs include 5-aminosalicylic acid drugs (such as mesalazine), immunosuppressants (such as azathioprine), biological agents (such as anti-TNF-α antibodies), and corticosteroids. These drugs control the disease by inhibiting inflammatory responses or regulating the immune system, but long-term use may bring a series of side effects, such as immunosuppression, increased risk of infection, and osteoporosis. In addition, some refractory patients may need surgical resection of the affected intestinal segment to relieve symptoms. However, surgery cannot cure the disease, and there is a risk of postoperative recurrence.

[0004] In recent years, with the in-depth study of the pathogenesis of colitis, new treatment methods have emerged. Probiotic therapy has become a popular treatment method due to its relative safety and few side effects. Probiotics regulate the intestinal microbiome, enhance the intestinal barrier function, and inhibit the growth of pathogenic microorganisms, thereby achieving the purpose of reducing inflammation, preventing or treating colitis. Among them, Heyndrickxia coagulans has become a popular probiotic due to its unique biological characteristics and potential anti-inflammatory effects.

[0005] Bacillus coagulans is a facultative anaerobic Gram-positive bacillus with strong acid and heat resistance, which can survive and function in the harsh environment of the gastrointestinal tract. Compared with other probiotics, a notable feature of Bacillus coagulans is its ability to form spores with high stress resistance, which makes it more stable during processing, storage and use.

[0006] Therefore, there is an urgent need to find a probiotic strain of Bacillus coagulans that has a good therapeutic effect on ulcerative colitis, which can be used to replace traditional drugs to prevent or treat ulcerative colitis and provide a safe and effective treatment option for patients with colitis to meet the growing market demand. Summary of the invention

[0007] In order to solve the above technical problems, the purpose of the present invention is to provide a coagulant Bacillus heindrix AACE9 and its application, which has immunomodulatory function, can reduce the disease activity index, alleviate colon intestinal damage, and has great application prospects in the treatment and prevention of colitis.

[0008] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: Provided is a Heyndrickxia coagulans AACE9, which was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on July 15, 2024, with a deposit number of CGMCC NO.31302, and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0009] The above-mentioned Heyndrickxia coagulans AACE9 was isolated from the contents of the colon sample of Nandan Yao chicken from a farm in Kunming City. It is a facultative anaerobic Gram-positive bacterium. After sequencing analysis, the nucleotide sequence of its genome is shown in SEQ ID NO.1. Through sequence comparison in the NCBI database, the results showed that the strain was Heyndrickxia coagulans.

[0010] The present invention also provides the use of the above-mentioned Bacillus heindrix coagulans AACE9 in preparing products for preventing or treating colitis.

[0011] Furthermore, the above-mentioned products are microbial agents, medicines or health products.

[0012] The present invention also provides a microbial agent for preventing colitis, comprising the above-mentioned Hyndrickella coagulans AACE9.

[0013] The present invention has the following beneficial effects:

[0014] 1. The coagulant Bacillus heindrix AACE9 of the present invention can improve the physiological symptoms such as decreased appetite, weight loss, colon shortening and intestinal damage caused by colitis.

[0015] 2. The AACE9 of the present invention has an immunomodulatory function, which can reduce the levels of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, and IL-8, and increase the levels of anti-inflammatory cytokines IL-10 and TGF-β.

[0016] 3. This strain also has the effects of improving intestinal barrier function, promoting colon mucosal repair, reducing colon inflammatory cell infiltration and maintaining the integrity of crypt structure. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The disease activity index DAI score results of each group of mice;

[0018] Figure 2 The graphs are the changes in food intake of mice in each group;

[0019] Figure 3 The graphs are the changes in water drinking of mice in each group;

[0020] Figure 4 The weight changes of mice in each group after 7 days are shown;

[0021] Figure 5 The colon length comparison chart of mice in each group;

[0022] Figure 6 This is a comparison chart of organ indexes of mice in each group;

[0023] Figure 7 The images are of colon tissue sections of mice in each group;

[0024] Figure 8 This is a comparison chart of the anti-inflammatory factor IL-10 levels in the serum of mice in each group;

[0025] Fig. 9 This is a comparison chart of the anti-inflammatory factor TGF-β levels in the serum of mice in each group;

[0026] Fig.10 This is a comparison of the levels of pro-inflammatory factor IL-1β in the serum of mice in each group;

[0027] Fig.11 This is a comparison chart of the pro-inflammatory factor IL-6 levels in the serum of mice in each group;

[0028] Fig.12 This is a comparison chart of the pro-inflammatory factor IL-8 levels in the serum of mice in each group;

[0029] Fig.13This is a comparison chart of the levels of pro-inflammatory factor TNF-α in the serum of each group of mice. DETAILED DESCRIPTION

[0030] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0031] Example 1 Isolation, identification and preservation of AACE9 of Bacillus heindrix coagulans

[0032] (1) Isolation of AACE9 from Bacillus heindrix

[0033] Weigh 1g of the colon sample of Nandan Yao chicken from a farm in Kunming, suspend it in PBS buffer, spread it on LB solid medium after gradient dilution, pick typical single colonies for expansion culture; take 100μL of the bacterial solution of each single colony and spread it on MRS solid medium containing 5% CaCO3 for culture; pick single colonies with calcium-dissolving rings and expand them in LB liquid medium. The bacterial solution was purified by partitioning and streaking for strain identification, and another 0.5mL of bacterial solution was centrifuged at 3000r / min for 10min, the supernatant was discarded, 30% glycerol was added and stored in a -80℃ refrigerator.

[0034] (2) Identification of AACE9 of Bacillus heindrix

[0035] Using TSINGKE TSP701-50 Bacteria Genomic DNA Kit (Beijing Qingke Biotechnology Co., Ltd.) was used to extract bacterial DNA, and then universal primers 27F and 1492R (shown in SEQ ID NO.2 and SEQ ID NO.3, respectively) were used to amplify the 16S rRNA gene. The PCR product was sent to Wuhan Qingke Biotechnology Co., Ltd. for sequencing. The sequencing result is shown in SEQ ID NO.1. The sequence was aligned using BLAST in NCBI GenBank and was determined to be Heyndrickxia coagulans.

[0036] (3) Preservation of strains

[0037] The strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 15, 2024, with the deposit registration number CGMCC NO.31302, and the deposit location is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0038] Example 2 Preparation of liquid bacterial agent of coagulating Bacillus hyendriculariae AACE9

[0039] The AACE9 stock solution of Hendrix coagulans was streaked and inoculated into LB solid medium and cultured at 37°C for 24 hours. Then a single colony was picked and inoculated into LB liquid medium and cultured at 37°C for 18 hours. After the culture was completed, centrifuged at 3000r / min for 5 minutes, the supernatant was discarded, PBS buffer was added for washing, and centrifuged for another 5 minutes to obtain bacterial sludge for use. PBS buffer was then added and mixed evenly. The bacterial concentration was adjusted using a spectrophotometer to form a bacterial concentration of 2.0×10 9 CFU / mL of bacterial suspension is the liquid bacterial agent of coagulating Bacillus subtilis AACE9.

[0040] Example 3

[0041] Twenty-four 6-week-old SPF male C57BL / 6J mice weighing 19-22 g were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. These mice were free to drink water and eat during the experiment. The experimental environment temperature was 23-25°C, the humidity was 40%-60%, and the day and night cycle was 12h (the animal experiment plan was reviewed and approved by the Animal Protection and Utilization Committee of Kunming University of Science and Technology). The mouse ulcerative colitis model was induced by adding 3% dextran sulfate sodium salt (DSS) to the drinking water.

[0042] The experimental period was 14 days. After 7 days of adaptive feeding, the mice were randomly divided into 4 groups (n=6): normal control group (NC), model group (DSS), compound Lactobacillus acidophilus tablets group (CL), and coagulant Hyendrickella group (HC). In the last 7 days, except for the NC group, the mice were given sterile water supplemented with 3% DSS. The treatment methods of each group were as follows: NC group was gavaged with 200 μL PBS daily; DSS group was gavaged with 200 μL PBS daily; CL group was gavaged with 200 μL compound Lactobacillus acidophilus tablets (2.0×10 9 CFU / mL); the HC group was gavaged daily with 200 μL of liquid bacterial agent of Hyndrickella heindrix AACE9 (2.0×10 9 CFU / mL). The mice were fasted but not watered for 12 h at 9 pm on the 14th day of the experiment, and euthanized on the morning of the 15th day. After euthanasia, the spleen, liver, kidney, serum, and colon of the mice were collected to measure the organ index, inflammatory factor content, and colon length, and H&E staining sections were performed for imaging.

[0043] In the mouse colitis model, the Disease Activity Index (DAI) can be used to assess the severity of colitis. The Disease Activity Index score is usually combined with weight loss, stool characteristics, and the amount of fecal bleeding for a comprehensive assessment. The fecal occult blood qualitative detection kit (Beijing Regen Biotechnology Co., Ltd.) was used to detect the blood content in mouse feces. The criteria for DAI scoring are shown in Table 1.

[0044] Table 1 DAI scoring criteria

[0045]

[0046] The final DAI score is the sum of the above three scores. The higher the score, the more severe the colitis.

[0047] (1) Effects of AACE9 of Bacillus heindritis on basic physiological parameters of mice

[0048] The treatment effect was evaluated by monitoring the weight change, fecal formation and fecal occult blood volume of mice. The basic physiological index data of mice are shown in Table 2.

[0049] Table 2 Basic physiological index data of mice in each group

[0050]

[0051] Note: Different lowercase letters indicate significant differences among treatment groups (p<0.05).

[0052] The disease activity index (DAI) scores of mice in each group were as follows: Figure 1 The results showed that the disease activity index of mice in the DSS group was significantly higher than that in the control group, indicating that the ulcerative colitis model was successfully established in mice, while the disease activity index of mice in the CL and HC groups decreased significantly after taking the bacterial agent.

[0053] The changes in food intake and water intake of mice in each group were as follows: Figure 2 and Figure 3 As shown, the food and water intake of mice in the CL and HC groups decreased more slowly, indicating that their symptoms of anorexia were alleviated.

[0054] The weight changes of mice in each group after 7 days were as follows Figure 4 As shown in the figure, different lowercase letters indicate significant differences among the treatment groups (p<0.05). The results showed that the body weight of mice in the DSS group decreased by 9.42±3.74% after modeling, while the body weight of mice in the NC group, CL group, and HC group increased by 4.46±1.40%, 2.64±2.82%, and 2.91±1.67%, respectively. The body weight of mice in the CL group and HC group recovered well.

[0055] The colon length of each group of mice was Figure 5 As shown in the figure, different lowercase letters indicate significant differences among the treatment groups (p<0.05). The results showed that the colon length of the DSS group was significantly shortened, while the colon length of the HC group mice was significantly prolonged to 5.18±0.38cm, which was close to the level of the colon length of the NC group mice.

[0056] The organ indexes of mice in each group are as follows Figure 6 As shown in the figure, different lowercase letters indicate significant differences among the treatment groups (p<0.05). The results showed that compared with the DSS group, the spleen index of mice in the CL and HC groups was significantly reduced, and the severity of the inflammatory response was milder than that in the DSS group; there was no significant difference in the liver and kidney indexes of mice in each group.

[0057] From the above, we can see that AACE9 of Bacillus heindritis coagulans significantly improved DSS-induced symptoms such as loss of appetite, diarrhea, and colon bleeding in mice, enabled the mice to recover their weight faster, and inhibited colon shortening, demonstrating its significant preventive potential.

[0058] (2) Effect of AACE9 of Bacillus heindritis on colon histopathology in mice

[0059] Colon tissue section images of mice in each group are shown in the figure below. Figure 7 As shown. Figure 7 It can be seen that the colon tissue of mice in the DSS group showed obvious histological damage, including large-area ulcers in intestinal tissue, excessive proliferation of connective tissue, loss of crypts, goblet cell damage, inflammatory cell infiltration, and large-scale edema in the submucosal layer. The degree of tissue damage in mice in the CL group was mild, mainly manifested as local small focal ulcers and mild expansion of crypts, accompanied by more inflammatory cell infiltration, mild edema of the mucosal layer and muscular layer, and loose connection. In the colon sections of mice in the HC group, tissue damage almost disappeared, the mucosal epithelium remained intact, the propria goblet cells were abundant, the crypt structure was good, there were few small focal inflammatory cell infiltrations, the mucosal layer and the muscular layer were tightly connected, the degree of damage was milder than that of the CL group, and its tissue structure was almost the same as that of the NC group.

[0060] From the above, we can see that AACE9 of Bacillus heindritis coagulans improved the stress resistance of mice and significantly alleviated DSS-induced colon tissue damage by protecting the mucosal epithelium, maintaining the crypt structure, restoring the function of goblet cells, reducing inflammatory cell infiltration, and maintaining the tight structure of the mucosal layer and muscular layer.

[0061] (3) Effect of AACE9 of Bacillus heindritis on serum inflammatory factors in mice

[0062] After the experiment, the inflammatory factors in the serum of each group of mice were detected, and the levels of IL-10, TGF-β, IL-1β, IL-6, IL-8 and TNF-α were as follows Figure 8-13 Different lowercase letters indicate significant differences among treatment groups (p<0.05).

[0063] Depend on Figure 8-13 It can be seen that compared with the NC group, the DSS group significantly reduced the expression of anti-inflammatory factors TGF-β and IL-10 in serum, and significantly increased the expression of pro-inflammatory factors IL-1β, IL-6, IL-8 and TNF-α in serum. Compared with the DSS group, the HC group can significantly increase the expression of anti-inflammatory factors TGF-β and IL-10 in serum, and significantly reduce the expression of pro-inflammatory factors IL-1β, IL-6, IL-8 and TNF-α. The content of anti-inflammatory factors in mice in the HC group was significantly increased, and the pro-inflammatory factors were significantly reduced, indicating that AACE9 of Bacillus heindritus can effectively inhibit the inflammatory response and enhance the anti-inflammatory ability.

[0064] From the above, we can see that AACE9 of Bacillus heindrix coagulans can reduce the inflammatory response of ulcerative colitis by regulating the balance of inflammatory factors in the body, showing a significant immunomodulatory effect.

[0065] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Heyndrickxia coagulans AACE9, characterized in that It was deposited in the General Microbiology Center of China Culture Collection Administration on July 15, 2024, with the deposit number CGMCCNO.31302, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. Use of the AACE9 of Bacillus coagulans according to claim 1 in the preparation of a product for preventing or treating colitis.

3. The use according to claim 2, characterized in that The product is a microbial agent, a medicine or a health product.

4. A microbial agent for preventing colitis, characterized in that: It comprises the coagulans bacteria AACE9 as described in claim 1.

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