Application of lysinibacillus fusiformis in preparation of medicine for preventing and treating constipation

Abstract

The invention discloses an application of lysinibacillus fusiformis in preparation of a medicine for preventing and treating constipation, and particularly, the lysinibacillus fusiformis can promote digestion, dredge intestines and moisten bowel and play a role in preventing constipation; the traditional Chinese medicine composition also can inhibit weight loss and colon shortening, increase the water content of excrement, enhance the intestinal transport capacity and relieve colitis injury caused by constipation, realizes treatment of constipation, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the application of *Bacillus fusiformis* in the preparation of drugs for the prevention and treatment of constipation. It belongs to the field of microbial application technology. Background Technology

[0002] Constipation is a bowel motility disorder characterized by reduced bowel movement frequency, difficulty or pain during defecation, often accompanied by bloating, abdominal discomfort, and a feeling of incomplete evacuation. Chronic constipation lasting for months can significantly reduce quality of life and may lead to complications such as fecal impaction, hemorrhoids, and rectal prolapse. The pathophysiological mechanism of constipation is multifactorial, primarily due to impaired colonic motility, resulting in prolonged fecal retention time and delayed transit.

[0003] In modern society, constipation has become an increasingly common ailment due to factors such as fast-paced lifestyles and excessive workloads. Clinically, magnesium sulfate and mosapride are commonly used to assist bowel movements. However, long-term reliance on medication can lead to decreased intestinal motility in constipated patients, thus worsening their symptoms. Numerous studies have revealed the relationship between constipation and gut microbiota dysbiosis, suggesting that regulating the gut microbiota in constipated patients may be a novel approach to aiding bowel movements.

[0004] *Lysinibacillus fusiformis* is a group of Gram-positive bacteria with acid- and heat-resistant properties. It exists in the environment and animal intestines and is a beneficial bacterium used in plant and environmental research. Currently, it is unclear whether *Lysinibacillus fusiformis* can alleviate constipation symptoms. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of Bacillus fusiformis in the preparation of drugs for the prevention and treatment of constipation.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: 1. Application of Bacillus fusiformis in the preparation of drugs for the prevention and treatment of constipation.

[0007] As one of the preferred technical solutions, the spindle-shaped lysine spore-forming bacillus is CCTCAB 94030, purchased from the China Center for Type Culture Collection.

[0008] As one of the preferred technical solutions, prevention includes: promoting digestion and relieving constipation; treatment includes: inhibiting weight loss and colon shortening, increasing fecal water content, enhancing intestinal transit capacity, and reducing colonic inflammation damage caused by constipation.

[0009] 2. The use of compositions containing *Bacillus fusiformis* in the preparation of medicaments for the prevention and treatment of constipation.

[0010] As one of the preferred technical solutions, the spindle-shaped lysine spore-forming bacillus is CCTCAB 94030, purchased from the China Center for Type Culture Collection.

[0011] As one of the preferred technical solutions, prevention includes: promoting digestion and relieving constipation; treatment includes: inhibiting weight loss and colon shortening, increasing fecal water content, enhancing intestinal transit capacity, and reducing colonic inflammation damage caused by constipation.

[0012] 3. A drug for the prevention and treatment of constipation, wherein the active ingredient is Bacillus fusiformis or a composition containing Bacillus fusiformis.

[0013] As one of the preferred technical solutions, the spindle-shaped lysine spore-forming bacillus is CCTCAB 94030, purchased from the China Center for Type Culture Collection.

[0014] As one of the preferred technical solutions, prevention includes: promoting digestion and relieving constipation; treatment includes: inhibiting weight loss and colon shortening, increasing fecal water content, enhancing intestinal transit capacity, and reducing colonic inflammation damage caused by constipation.

[0015] As one of the preferred technical solutions, the effective component of the drug is a bacterial suspension of *Bacillus fusiformis*, which is prepared by the following method: *Bacillus fusiformis* is inoculated onto LB agar plates using the streak plating method and grown in an incubator at 37°C; after 24 hours, a single colony is picked and placed in 5 mL of LB agar, and cultured at 37°C and 200 rpm for 10 hours; then, 0.1 mL of the bacterial suspension is inoculated into 9.9 mL of LB agar at a volume ratio of 1:100, and cultured at 37°C and 200 rpm for 8-10 hours; when the OD600 reaches 0.8-1.0, the culture is stopped; the cultured bacterial suspension is centrifuged at 8000 rpm for 10 minutes, the supernatant is discarded, and the collected bacterial cells are dissolved in 1.5 mL of 2% PBS solution (pH=7.2) to obtain the final product.

[0016] The beneficial effects of this invention are as follows: This invention discloses the application of *Bacillus fusiformis* in the preparation of drugs for the prevention and treatment of constipation. Specifically, *Bacillus fusiformis* can promote digestion, lubricate the intestines and prevent constipation; it can also inhibit weight loss and colon shortening, increase fecal water content, enhance intestinal transit capacity, reduce colonic inflammation damage caused by constipation, and achieve the treatment of constipation, showing good application prospects.

[0017] This invention cultured a suspension of spindle-shaped lysine-containing Bacillus spp. and administered the bacterial suspension to constipation model mice by gavage.

[0018] The OD600 value of the bacterial solution administered by gavage was 0.8–1.0, and the frequency of gavage was 0.2 mL per day. This invention provides histopathological analysis to evaluate the effect of the *Bacillus fusiformis* suspension in alleviating the pathological symptoms caused by constipation. The results showed that *Bacillus fusiformis* could inhibit weight loss in constipation model mice, reduce colonic shortening in constipation model mice, increase fecal water content in constipation model mice, enhance intestinal transit capacity by accelerating whole intestinal transit time and small intestinal transit rate in constipation model mice, and alleviate colonic inflammatory damage caused by constipation by reducing inflammatory cell infiltration in the colon and improving crypt and goblet cell morphological abnormalities in constipation model mice. Therefore, the *Bacillus fusiformis* has the effect of alleviating the pathological symptoms caused by constipation.

[0019] In other words, this invention, through gavage administration of *Bacillus fusiformis* to constipation model mice, observed a slowdown in the rate of weight loss and colonic shortening, an increase in fecal water content, an acceleration in whole-intestinal transit time and small intestinal transit rate, and a reduction in colonic inflammatory damage. This indicates that *Bacillus fusiformis* can alleviate constipation-induced intestinal motility disorders, reduce intestinal pathological damage, and alleviate constipation-related pathological symptoms, thus exhibiting a good alleviating effect on constipation symptoms.

[0020] The spindle-shaped lysine-containing Bacillus of the present invention can be used in the preparation of drugs that promote digestion / intestinal lubrication or improve constipation. It can also be used in drugs that inhibit weight loss and colon shortening, increase fecal water content, enhance intestinal transit capacity, and alleviate colonic inflammation and related symptoms caused by constipation. This provides theoretical support for the development of probiotic preparations for the prevention and treatment of constipation and related symptoms, and provides new ideas for the clinical prevention and treatment of constipation. Attached Figure Description

[0021] Figure 1 shows a comparison of changes in body weight and colon length in constipation model mice after intervention with suspensions of Bacillus fusiformis, Bacillus amyloliquefaciens, and Enterococcus spp.; where A represents the weight gain rate and B represents the colon length.

[0022] Figure 2 shows a comparison of the changes in Bristol fecal score and fecal water content in constipation model mice after intervention with suspensions of Bacillus fusiformis, Bacillus amyloliquefaciens, and Enterococcus spp.; where A is the Bristol fecal score and B is the fecal water content.

[0023] Figure 3 is a comparison of intestinal transport capacity in constipation model mice after intervention with suspensions of Bacillus fusiformis, Bacillus amyloliquefaciens, and Enterococcus spp.; where A is the total intestinal transport time and B is the small intestinal transport rate.

[0024] Figure 4 shows the HE staining image and inflammation damage score of the colon in a constipation model mouse after intervention with Bacillus fusiformis; where A is HE staining and B is inflammation damage score and crypt damage score. Detailed Implementation

[0025] The present invention will be further described below with reference to the accompanying drawings and embodiments. It should be noted that the following description is only for explaining the present invention and does not limit its content.

[0026] The spindle-shaped lysine spore-bearing bacillus involved in this invention is CCTCAB 94030, which was purchased from the China Center for Type Culture Collection.

[0027] Experiments were designed using Bacillus amyloliquefaciens (CGMCC 1.3450, purchased from China General Microbiological Culture Collection Center) and Enterococcus spp. (CGMCC 1.997, purchased from China General Microbiological Culture Collection Center) as technical comparisons.

[0028] Example 1: Cultivation of bacterial strains and preparation of bacterial suspension: Bacillus fusiformis, Bacillus amyloliquefaciens, or Enterococcus smallus were inoculated onto LB agar plates (purchased from Sangon Biotech) using the streak plating method and grown at 37°C. After 24 hours, a single colony was picked and incubated in 5 mL of LB agar at 37°C and 200 rpm for 10 hours. Then, 0.1 mL of the bacterial suspension was inoculated into 9.9 mL of LB agar at a 1:100 volume ratio and incubated at 37°C and 200 rpm for 8-10 hours. Incubation was stopped when the OD600 reached 0.8–1.0. The cultured bacterial suspension was centrifuged at 8000 rpm for 10 minutes, the supernatant was discarded, and the collected bacterial cells were dissolved in 1.5 mL of 2% PBS solution (pH 7.2) to obtain a suspension of Bacillus fusiformis, Bacillus amyloliquefaciens, or Enterococcus smallus.

[0029] Example 2: Intervention of different bacterial strain suspensions on constipation model mice: Seven-week-old male Kun-Ming mice (purchased from Hunan Slack Jingda Experimental Animal Co., Ltd.) were purchased and acclimatized for one week. Loperamide hydrochloride (purchased from RHAWN) was diluted in phosphate-buffered saline (PBS) to a final concentration of 20 mg / kg. After the acclimatization period, mice were administered 0.2 mL of loperamide hydrochloride solution (20 mg / kg) by gavage at fixed times daily for four days to establish a constipation model.

[0030] A suspension of Bacillus fusiformis, Bacillus amyloliquefaciens, and Enterococcus spp. was prepared according to the method in Example 1, and a sterile PBS solution with a pH of 7.2 and a mass concentration of 2% was prepared.

[0031] The drug administration details for each group were arranged according to the experimental grouping (Table 1).

[0032]

[0033] The improvement effect was assessed based on changes in body weight, fecal water content, intestinal transit capacity, and colonic inflammation in each group of mice.

[0034] Example 3: Bacillus fusiformis can inhibit weight loss in constipation model mice: In the animal testing phase of Example 2, the weight of mice in each group was weighed and recorded at the beginning and end of the experiment. As shown in Figure 1A, compared with the constipation group, the weight of mice in the Ba and Lf groups recovered after intervention with the suspensions of the three strains, and the Lf group showed the highest weight gain rate, indicating that the intervention of Bacillus fusiformis can significantly inhibit the weight loss caused by constipation.

[0035] Example 4: Bacillus fusiformis can inhibit colonic shortening caused by constipation: After the animal experiments, each mouse was dissected, colon tissue was collected, and the length was measured and recorded. As shown in Figure 1B, after intervention with the suspensions of the three strains, only the Lf group showed a significantly inhibited trend of colon length shortening compared to the constipation group, indicating that intervention with Bacillus fusiformis can significantly restore colon shortening caused by constipation.

[0036] Example 5: *Bacillus fusiformis* can increase the fecal water content in constipation model mice: Feces were collected from two groups of mice, and the appearance, shape, and luster of the feces were scored according to the Bristol Fecal Score Scale and recorded. As shown in Figure 2A, after intervention with the suspensions of the three strains, only the Bristol Fecal Score of constipated mice in the Lf group was significantly increased, indicating that the intervention of Bacillus fusiformis can gradually transform feces into a normal form with a soft texture and glossy surface.

[0037] The wet weight of the feces was then weighed and recorded. Afterward, the feces were dried at 100°C for 60 minutes, and the dry weight was weighed and recorded. The water content of the feces was calculated using the formula (wet weight - dry weight) / wet weight. Figure 2As shown in Figure B, after intervention with the suspensions of the three strains, the fecal water content of constipated mice in the Ba and Lf groups gradually increased. However, the intervention with the suspension of Bacillus fusiformis showed a better effect on restoring the fecal water content, indicating that the intervention of Bacillus fusiformis could improve the phenomenon of dry and hard feces.

[0038] Example 6: *Bacillus fusiformis* can increase intestinal transit capacity in constipation model mice: Methylcellulose red dye was prepared by dissolving 0.2 g methylcellulose and 0.6 g carmine in 10 mL PBS. Methylcellulose and carmine were purchased from Shanghai Maclean Biochemical Co., Ltd. Each mouse was placed in an individual cage and administered 0.2 mL of methylcellulose red dye via gavage. Feces were collected, and the presence of red dye was tested by rubbing the feces on white paper. The time elapsed from the start of gavage to the first excretion of feces containing red dye was defined as the whole-intestinal transit time. As shown in Figure 3A, after intervention with the three strain suspensions, only the Lf group of constipated mice showed a significantly shortened whole-intestinal transit time, indicating that the intervention with *Bacillus fusiformis* could shorten the retention time of food in the gastrointestinal tract.

[0039] Each mouse was fasted but allowed free access to water for 24 hours. Each mouse was placed in an individual cage and administered 0.2 mL of Indian ink (purchased from Bioharp, Lanjieke Technology Co., Ltd.) via gavage. After 30 minutes, the mice were sacrificed, and the gastrointestinal tract (from stomach to rectum) was removed, straightened on a white waterproof board, and the small intestine length and the distance the Indian ink was propelled were measured. The small intestinal transit rate was calculated by dividing the Indian ink propulsion distance by the small intestine length. As shown in Figure 3B, intervention with the suspensions of all three strains increased the small intestinal transit rate in constipated mice, but the Lf group showed the best small intestinal transit rate, indicating that intervention with *Bacillus fusiformis* accelerated the small intestinal transit capacity.

[0040] Example 7: Bacillus fusiformis can improve colitis caused by constipation: After the animal experiments, colonic tissue from mice was removed, cleaned, and preserved in a 4% paraformaldehyde solution. Based on a comprehensive evaluation of the results of Examples 3-6, the intervention effect of the *Bacillus fusiformis* suspension was the best among the three strains. Therefore, colonic tissue from the Lf group mice was selected for histopathological analysis. HE staining and pathological sections were used to observe the inflammatory damage in the colon. As shown in Figures 4A and 4B, the HE staining results of the colon indicate that constipation led to diffuse infiltration of inflammatory cells in the mouse colon, resulting in obvious inflammatory symptoms, along with abnormal morphology of crypts and goblet cells. After intervention with *Bacillus fusiformis*, the degree of inflammation and crypt damage in the constipated mice decreased, indicating that intervention with *Bacillus fusiformis* can protect the mouse colon.

[0041] In summary, the results showed that *Bacillus fusiformis* had the best effect on improving constipation symptoms in mice. Specifically, it significantly inhibited weight loss and colon shortening in constipation model mice, increased fecal water content, enhanced intestinal transit capacity, reduced colonic inflammation caused by constipation, and had a good alleviating effect on intestinal motility disorders caused by constipation.

[0042] While the specific embodiments of the present invention have been described above in conjunction with the accompanying drawings, this is not intended to limit the scope of protection of the present invention. Based on the technical solutions of the present invention, various modifications or variations that can be made by those skilled in the art without creative effort are still within the scope of protection of the present invention.

Claims

1. Application of Bacillus fusiformis in the preparation of drugs for the prevention and treatment of constipation.

2. The application according to claim 1, characterized in that, The spindle-shaped lysine spore-forming bacillus was CCTCAB94030, purchased from the China Center for Type Culture Collection.

3. The application according to claim 1, characterized in that, Prevention includes: Promotes digestion and relieves constipation; Treatment includes: inhibiting weight loss and colon shortening, increasing stool water content, enhancing intestinal transit capacity, and reducing colonic inflammation damage caused by constipation.

4. The use of compositions containing *Bacillus spindleii* in the preparation of medicaments for the prevention and treatment of constipation.

5. The application according to claim 4, characterized in that, The spindle-shaped lysine spore-forming bacillus was CCTCAB94030, purchased from the China Center for Type Culture Collection.

6. A medicine for preventing and treating constipation, characterized in that, Its active ingredient is Bacillus fusiformis or a composition containing Bacillus fusiformis.

7. The application according to claim 6, characterized in that, The spindle-shaped lysine spore-forming bacillus was CCTCAB94030, purchased from the China Center for Type Culture Collection.

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