CN118421496A - Lactobacillus casei rhamnosus with effects of preventing colitis and relieving constipation - Google Patents

Abstract

The present invention discloses a Lactobacillus rhamnosus strain having the efficacy of preventing colitis and relieving constipation, and the deposit number thereof is CGMCC NO.27364. The Lactobacillus rhamnosus strain provided by the present invention can improve the weight loss of mice induced by DSS, reduce the disease activity index of mice, improve the shortening of the colon of mice, and alleviate the damage of colon tissue. The strain also has the functions of increasing the water content of mouse feces, promoting the propulsion rate of the small intestine, and relieving the damage of the small intestinal villi and colon mucosa.

Description

Lactobacillus rhamnosus with effects of preventing colitis and relieving constipation

Technical Field

The invention belongs to the technical field of probiotic screening and application, and particularly relates to Lactobacillus rhamnosus with the effects of preventing colitis and relieving constipation.

Background technique

Ulcerative colitis (UC) is an inflammatory bowel disease, with lesions mainly occurring in the base of the rectum or part of the rectum. Since the cause of the disease is unknown and there is no specific treatment, there is a lack of effective prevention methods. Traditional treatments for UC include four types of drugs: aminosalicylic acid, hormones, antibiotics, and immunosuppressants. However, these drugs are prone to relapse after discontinuation and have significant side effects, which limits their clinical use.

With the continuous improvement of people's living standards and changes in dietary structure, constipation has gradually become one of the important factors affecting people's quality of life. Currently, the treatment of constipation mainly adopts four methods: drug therapy, biofeedback therapy, changing eating habits and lifestyle, and intestinal microecological therapy. However, drug therapy causes adverse reactions, biofeedback therapy is limited in the applicable group, and a healthy lifestyle is difficult to maintain. Therefore, intestinal microecological therapy has become a hot research direction for constipation treatment.

Summary of the invention

The purpose of the present invention is to provide Lactobacillus rhamnosus with the efficacy of preventing colitis and relieving constipation, thereby preventing the occurrence of colitis and relieving constipation.

The Lactobacillus rhamnosus (LGG) AFY03 strain provided by the present invention is a strain for alleviating colitis and constipation. The preservation number of the AFY03 strain is CGMCC No. 27364, and the preservation date is May 17, 2023; the preservation unit is the General Microbiology Center of the China Culture Collection Administration, and the address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

The strain provided by the invention is used for preventing colitis and relieving constipation.

The Lactobacillus rhamnosus AFY03 strain provided by the invention can be used to prepare products for preventing and treating colitis and constipation.

The culture temperature of the Lactobacillus rhamnosus AFY03 strain is preferably 37°C.

The Lactobacillus rhamnosus AFY03 strain screened and obtained by the present invention has been confirmed by mouse model experiments to be able to alleviate mouse colitis induced by DSS and mouse constipation induced by ice water activated carbon; the use of the strain can significantly reduce the incidence of colitis and constipation in model mice.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Effect of Lactobacillus rhamnosus AFY03 on body weight of colitis mice;

Figure 2: Effect of Lactobacillus rhamnosus AFY03 on disease activity index;

Figure 3: Effect of Lactobacillus rhamnosus AFY03 on colon length in colitis mice;

Figure 4: Effects of Lactobacillus rhamnosus AFY03 on colon histopathological morphology of colitis mice;

Figure 5: Changes in basic body indicators of mice;

Figure 6: Fecal moisture content of each group of mice during the experiment;

Figure 7: Time of discharge of the first red stool in each group of mice;

Figure 8: Graphs of small intestine length and small intestine propulsion rate of mice in each group;

Figure 9: Histopathological morphology of small intestine;

Figure 10: Colon histopathological morphology.

Detailed ways

The present invention is described in detail below in conjunction with specific embodiments and drawings.

Example 1: Isolation and purification of strains

40 mL of naturally fermented yogurt was collected from the Altay region of Xinjiang. The yogurt was placed in a sterile centrifuge tube, placed in a food sampling box, and stored in a 4°C refrigerator in the laboratory for later use.

1. Isolation and identification of lactic acid bacteria

1.1 Isolation and purification of lactic acid bacteria

Take 1mL of naturally fermented yogurt sample, dilute it 10 times with sterile saline to 10 ^-6 , then take 100% of bacterial solution of 10 ^-4 , 10 ^-5 , and 10 ^-6 to plate, culture at 37℃ for 24-48h, observe and record the colony morphology. Pick colonies of different morphologies on the plate for streaking separation, and after culturing at 37℃ for 48h, pick single colonies of different morphologies on the plate for streaking separation again, and repeat this 2 to 3 times until pure single colonies with consistent morphology are obtained.

1.2 Lactic acid bacteria DNA extraction

Inoculate the purified suspected target strain into MRS broth, culture at 37℃ for 18-24h, and then use a bacterial genomic DNA extraction kit to extract DNA. Number the extracted DNA and store it in a -20℃ freezer for later use.

1.4 PCR amplification of genomic DNA and agarose gel electrophoresis detection

The extracted DNA was subjected to PCR amplification, including upstream primer 27F (5'-AGA GTT TGA TCC TGGCTCAG-3') 1', downstream primer 1495R (5'-CTA CGG CTA CCTTGT TAC GA-3') 1A, 2A CGG CTACCTTGT TAC GA, and template DNA 1G. The system was supplemented to 25 with sterile dd H ₂ O. Sterile ultrapure water was used to replace template DNA as a negative control. The amplification conditions were: 94℃5min; 94℃30s, 55℃30s, 72℃1min, a total of 29 cycles, and finally 72℃ extension for 5min.

Then, 5 amplified products were taken for agarose gel electrophoresis detection, the agarose concentration was 1.5%, the electrophoresis conditions were 110V, 45min. The successfully detected PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing, and the sequenced sequences were compared and analyzed using the BLAST (Basic Local Alignment Search Tool) program in NCBI to determine that the screened strain was Lactobacillus rhamnosus.

The screened strain was named Lactobacillus rhamnosus AFY03 strain for preservation, with the preservation number CGMCC No. 27364 and the preservation date May 17, 2023; the preservation unit is the General Microbiology Center of China Culture Collection Administration, with the address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

Example 2: Effect of Lactobacillus rhamnosus AFY03 strain on colitis in mice

The animal experiment design is shown in Table 1: 40 6-week-old male C57BL/6 mice, weighing (20±3g), were fed adaptively for 1 week. All groups were given normal drinking water and feed during the adaptation period. After the adaptation period, the mice were divided into 4 groups (n=10), namely normal group, model group, SASP group, and AFY03 group. After the end of adaptive feeding, except for the normal group, the other three groups of mice were allowed to drink 2% DSS water freely for 7 consecutive days. The SASP group was gavaged with 100 mg/kg sulfasalazine daily, and the AFY03 group was gavaged with 10 ⁹ CFU/kg rhamnosus Lactobacillus AFY03 daily. After 28 days, the mice were fasted for 12 hours but not water, and then the mice were dissected. After blood was collected from the orbital vein, the mice were killed by dislocation of the neck and dissected, and blood samples were collected. The mice were dissected and the colon tissue (cecum to anus) was collected and the length was measured. A portion (0.5 cm) of the mouse colon tissue was fixed in 4% paraformaldehyde for histopathological analysis, and the remaining portion was stored at -80°C and used for colon tissue-related gene and cytokine expression analysis.

Table 2: Animal experiment design plan

2.1 Effect of Lactobacillus rhamnosus AFY03 on body weight changes in mice

After the adaptive feeding, the body weight of mice was measured daily during 0-7 days, and every other day during 8-21 days, and the changes were recorded and observed.

During the experiment, the weight changes of mice are shown in Figure 1. Compared with the normal group, the model group had a significant decrease in weight (P<0.05); compared with the model group, the SASP and AFY03 groups had a certain degree of weight loss in the early stage, but with the treatment of SASP and Lactobacillus rhamnosus AFY03, the weight began to recover on the seventh day compared with the model group. The weight of the model group recovered on the 10th day, but the effect was not as good as that of the AFY03 group. It was confirmed that Lactobacillus rhamnosus AFY03 significantly improved the weight loss induced by DSS.

2.2 Effect of Lactobacillus rhamnosus AFY03 on disease activity index

During the experiment, DAI was scored every two days. The fecal occult blood was determined by the highly sensitive o-tolidine method, and the specific operation process was carried out according to the kit instructions. The scoring details are shown in Table 2.

Table 2: Mouse DAI scoring standard

As shown in Figure 2, the DAI score of the model group began to increase after modeling, and the DAI of mice increased significantly during free drinking of 2% DSS solution, and decreased during free drinking of UP water, but the score was still higher than that of the other three groups (p<0.05). Compared with the model group, the DAI score of the rhamnosus Lactobacillus casei AFY03 group was significantly reduced until the end of the experimental observation period. This result confirms that rhamnosus Lactobacillus casei AFY03 significantly improved the weight loss, diarrhea and bloody stools of mice induced by DSS.

2.3 Effect of Lactobacillus rhamnosus AFY03 strain on colon length in mice

As shown in Figure 3, in different groups, the colon of mice in the normal group was the longest (8.35cm), while that of mice in the model group was the shortest (5.55cm). The colon lengths of mice in the SASP group (6.35cm) and the rhamnosus AFY03 group (6.65cm) were medium, which was longer than that of mice in the model group. Compared with the model group, SASP treatment and rhamnosus Lactobacillus AFY03 treatment significantly improved the shortening of the mouse colon induced by DSS (p<0.05).

2.4 Effects of Lactobacillus rhamnosus AFY03 strain on pathological changes in mouse colon tissue

H&E staining was used to prepare sections, and the pathological changes of colon tissue were observed under a microscope.

Colon tissue sections were prepared by HE staining and observed under a microscope. As shown in Figure 4, the colon morphology of the normal group mice was relatively good, the structure of the intestinal epithelium and crypts was relatively normal, the mucosa was also normal, and there were many complete goblet cells, almost no inflammatory infiltration, and no obvious tissue damage. In the DSS-induced colitis mice (model group), the colon epithelial cell damage was incomplete, the crypts were deformed or incomplete or even disappeared, the number of goblet cells was greatly reduced and damaged, the glandular was damaged, and the infiltration of inflammatory cells was seen to be serious. Compared with the model group, the intestinal mucosal tissue of the SASP group was intact, with a large range of normal epithelial tissue, a small range of goblet cell loss, no other significant damage, broken intestines, and a small number of inflammatory cells, but no other significant pathological changes compared with the model group. In the colon tissue of the AFY03 group, the infiltration of inflammatory cells was rare or not significant, and the number of intestinal glands was basically normal, the morphology of epithelial cells was normal, only a few goblet cells were deformed or lost, and there was no obvious crypt damage or loss.

Under microscopic observation, the model group showed extremely significant pathological changes, and the SASP group showed a small amount of inflammatory cell infiltration, but the pathological changes were not obvious. In the AFY03 group, there was almost no inflammatory cell infiltration and no obvious pathological changes. This confirmed that Lactobacillus rhamnosus AFY03 has a certain improvement effect on colon tissue damage caused by DSS-induced ulcerative colitis and can effectively inhibit inflammation.

Example 3: Effect of Lactobacillus rhamnosus AFY03 strain on constipation in mice

Experimental animal treatment: 50 female Kunming mice aged 4 weeks and weighing between 25±5g were housed in an animal room, where the room temperature was maintained at 25±2℃, the relative humidity was maintained at 50±5%, the light/dark cycle was adjusted every 12 hours, and the mice were given standard feed and drinking water. The experiment began after one week of adaptive feeding. After the adaptive feeding, the mice were randomly divided into 5 groups, with 10 mice in each group, namely: normal group, constipation group, bisacodyl drug treatment group, rhamnosus lactobacillus AFY03 high-dose group and rhamnosus lactobacillus AFY03 low-dose group. During the 14-day experimental period, normal saline was given to the normal group and constipation group by gavage every day, bisacodyl solution (concentration of 100 mg/kg) was given to the bisacodyl group every day, and the high-dose group of Lactobacillus rhamnosus AFY03 was given a bacterial suspension with a concentration of 1×10 ⁹ CFU/kg every day, and the low-dose group was given a bacterial suspension with a concentration of 1×10 ⁸ CFU/kg every day; after an interval of two hours, except for the normal group mice, the other four groups of mice were gavaged with ice water activated carbon (mass concentration of 100 g/L, 0°C) every day. During the experiment, the body weight, food intake, water intake, feces mass, feces water content of all mice were measured every day, and gavage operations were performed. After gavage on the 14th day, all groups of mice were fasted but not watered for 24 hours. On the 15th day, all groups of mice were gavaged with 0.2 mL of red ink. After 30 minutes, half of the mice in each group (5 mice) were blooded from the orbital vein and then killed. The mice were dissected and the small intestine was removed. The propulsion distance of the red ink in the small intestine was observed and recorded, and the small intestine propulsion rate was calculated based on this. The remaining 5 mice in each group were observed and recorded for the time of the first red stool discharge, and then blood was collected and dissected.

3.1 Effects of Lactobacillus rhamnosus AFY03 on basic body parameters of constipated mice

The effect of Lactobacillus rhamnosus AFY03 on the basic body parameters of constipated mice is shown in Figure 5. As time goes by, the weight of each group of mice increases. Compared with the weight of each group of mice measured on the first day of the experiment, the weight of the normal group, constipation group, bisacodyl group, and high- and low-dose groups of Lactobacillus rhamnosus AFY03 increased by 17.0%, 16.8%, 14.5%, 12.1%, and 14.8% on the 14th day, respectively. However, the analysis found that there was no significant difference in the weight of each group of mice (p>0.05). In addition, there was no significant difference in the food intake and water intake of each group of mice during the experiment (p>0.05). This shows that Lactobacillus rhamnosus AFY03 and the test substances did not have a significant effect on the basic body parameters of mice.

3.2 Effect of Lactobacillus rhamnosus AFY03 on fecal moisture content in constipated mice

As shown in Figure 6, there was no significant difference in the fecal moisture content of mice in each group from the 1st to the 9th day of the experiment. On the 10th day of the experiment, the fecal moisture content of mice in the constipation group began to be significantly lower than that of the other four groups. On the 14th day of the experiment, the fecal moisture content of mice in the constipation group was significantly lower than that of the normal group (p<0.05), proving that the mouse constipation model induced by ice water activated carbon was successfully established. Although ice water activated carbon can weaken intestinal peristalsis, the fecal moisture content of mice in the bisacodyl group did not change much overall, ranging from 58% to 63%. Although the fecal moisture content of mice in the high- and low-dose groups of Lactobacillus rhamnosus AFY03 decreased during the experiment, it began to approach that of the bisacodyl group from the 10th day. After 14 days of oral gavage, the fecal moisture content of the high- and low-dose groups of Lactobacillus rhamnosus AFY03 and the bisacodyl group was significantly higher than that of the constipation group (p<0.05). There was no significant difference in the fecal moisture content between the high-dose group and the bisacodyl group (p>0.05), confirming that the ability of the high-dose group of Lactobacillus rhamnosus AFY03 to increase the fecal moisture content of mice was roughly the same as that of the bisacodyl group.

3.3 Effect of Lactobacillus rhamnosus AFY03 on the time to first red stool in constipated mice

As shown in Figure 7, compared with the normal group, the time of first red stool discharge in the constipation group increased by 59.0% (p<0.05), which again shows that the constipation mouse model induced by ice water activated carbon was successfully established. Compared with the constipation group, the time of first red stool discharge in the bisacodyl group, the high-dose and low-dose groups of Lactobacillus rhamnosus AFY03 were 36.1%, 31.4%, and 13.9% earlier, respectively, which were significantly different from the constipation group (p<0.05).

3.4 Effect of Lactobacillus rhamnosus AFY03 on small intestinal propulsion rate in constipated mice

As shown in Figure 8, there was no significant difference in the length of the small intestine of each group of mice measured in the experiment (p>0.05), indicating that the constipation model of mice induced by ice water activated carbon does not affect the length of the small intestine of mice. By comparing the propulsion rate of red ink in the small intestine of mice, it can be seen that the small intestine propulsion rate of red ink in the constipation group is significantly lower than that in the normal group (p<0.05). After the constipated mice were gavaged with rhamnosus lactobacillus casei AFY03, their small intestine propulsion speed was significantly faster than that of the constipation group in the same period of time, and the small intestine propulsion rate was significantly higher than that of the constipation group (p<0.05), confirming that rhamnosus lactobacillus casei AFY03 can promote small intestinal peristalsis, accelerate the propulsion speed of red ink in the small intestine of mice, and relieve constipation.

3.5 Effects of Lactobacillus rhamnosus AFY03 on the small intestine and colon histomorphology of constipated mice

The small intestine and colon were fixed in 4% paraformaldehyde solution and then dehydrated with 95% ethanol. The alcohol in the tissue was then replaced with xylene to form a transparent tissue, which was then embedded in paraffin. After cooling, the tissue was sliced using a microtome and stained with hematoxylin-eosin (H&E) to produce pathological sections of the small intestine and colon for observation under an upright microscope.

As shown in Figure 9, the villi of the small intestine of mice in the normal group were arranged neatly, without wrinkles, with thick intestinal walls and evenly distributed goblet cells, while the villi of the small intestine of the constipation group were severely damaged, with severe wrinkles and destroyed goblet cells. Although the villi of the small intestine of mice in the high-dose and low-dose groups of Lactobacillus rhamnosus AFY03 also showed some wrinkles and breaks, they were more complete than those in the constipation group, confirming that Lactobacillus rhamnosus AFY03 had a certain improvement effect on the villi of the small intestine, and the morphology and arrangement of the villi of the small intestine of mice in the high-dose group were close to those in the bisacodyl group.

As shown in Figure 10, the colon tissue morphology of mice in the normal group was good, the mucosal epithelium and crypt structure were intact, there were a large number of goblet cells, and there was no inflammatory cell infiltration. The crypts of the colon tissue of mice in the constipation group were shortened, the number of goblet cells decreased, and there was inflammatory cell infiltration. Although there were a small number of lymphocytes aggregated in the high-dose and low-dose groups of Lactobacillus rhamnosus AFY03, and there was a certain amount of inflammatory cell infiltration, the colon morphology of the high-dose and low-dose groups of Lactobacillus rhamnosus AFY03, and the bisacodyl group was improved compared with the constipation group, and the integrity of the mucosal epithelium and crypt structure was similar to that of the normal group, which still confirmed that Lactobacillus rhamnosus AFY03 can improve the pathological damage of the colon mucosa.

Claims (8)

1. The plant lactobacillus casei is characterized in that the preservation number of the lactobacillus rhamnosus is CGMCC No.27364.

2. The use of lactobacillus rhamnosus as claimed in claim 1 for the prophylactic treatment of colitis or constipation.

3. Use of lactobacillus rhamnosus as claimed in claim 1 in the manufacture of a product for the prophylactic treatment of colitis or constipation.

4. The use according to claim 3, wherein the product is a functional food, pharmaceutical or health product.

5. The use of claim 3, wherein the article is a probiotic formulation.

6. A probiotic preparation, characterized in that the probiotic preparation comprises the live strain of lactobacillus rhamnosus according to claim 1.

7. Use of a probiotic formulation according to claim 5 for the preparation of a product for the prophylactic treatment of colitis or constipation.

8. A method of culturing lactobacillus rhamnosus as claimed in claim 1, wherein said method is carried out at 37 ℃.