Lactobacillus reuteri and application thereof

Abstract

The invention discloses lactobacillus reuteri and application thereof. The bacterial strain is named as Limosilbacillus reuteri30, the preservation number is CCTCC (China Center for Type Culture Collection) NO: M 20241441, and the bacterial strain is preserved in the China Center for Type Culture Collection. The lactobacillus reuteri is found to be a key microorganism of walnut insoluble fiber for relieving loperamide-induced mouse constipation in the early stage. On the basis, a microbial pure culture method is utilized, and on the basis of phylogenetic analysis of the 16S rDNA gene, a plurality of lactobacillus reuteri strains are separated and identified from mouse excrement treated by WIDF. The effective strain L.reuteri30 is further screened from L.reuterii by using a constipation model mouse, and the strain remarkably reduces the first black stool time of the constipation mouse and remarkably increases the water content of stool and the ink propulsion rate, so that the strain can be used as a novel beneficial bacterium for relieving constipation.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to a Lactobacillus reuteri and an application thereof. Background Art

[0002] Constipation is a common gastrointestinal disorder in clinical practice, which has affected about 10.1% of the world's population. Constipation is caused by a variety of factors, which can be secondary to treatment, systemic diseases, secondary constipation caused by local pathology, or functional constipation caused by diet, lifestyle, or colon propulsion or rectal emptying disorders. The symptoms of functional constipation are mainly manifested as stool lumps or hard masses, incomplete emptying, anorectal obstruction or blockage during defecation, few bowel movements, and difficult defecation. Although the pathogenesis of functional constipation is not fully understood, changes in diet, enteric neurotransmitters, intestinal flora, and intestinal water balance are related to the pathogenesis of constipation. At present, some drug treatments are considered effective, but they are expensive or prone to certain side effects, such as abdominal pain and diarrhea. Therefore, effective, safe, and low-cost therapeutic drugs are still urgently needed by patients with constipation.

[0003] An increasing number of studies have shown that intestinal flora has an important impact on host health, and differences in microbial composition are associated with chronic disease states. It has been reported that patients with constipation have an imbalance in intestinal flora, such as reduced abundance of Bifidobacterium and Lactobacillus. It is worth noting that it has been clinically proven that the supplementation of some probiotics can relieve constipation.

[0004] Lactobacillus reuteri has been reported to significantly relieve constipation in children and adults, including improvements in bowel movement frequency, pain during defecation, and reductions in rectal stool volume. However, other studies have found no benefit from Lactobacillus reuteri in treating constipation in children (References: O,Sila S, I, Z, Hojsak I, S. Lack of Benefit of Lactobacillus reuteri DSM 17938 as an Addition to the Treatment of Functional Constipation. J Pediatr Gastroenterol Nutr.). These results indicate that Lactobacillus reuteri has strain-specific effect in relieving constipation. Summary of the invention

[0005] The present invention aims to provide a Lactobacillus reuteri and application thereof. The Lactobacillus reuteri can be used to improve constipation.

[0006] To solve the above technical problems, the technical solution of the present invention is as follows: a Lactobacillus reuteri, the Lactobacillus reuteri (Limosilactobacillus reuteri) is named Limosilactobacillus reuteri 30, the preservation number is: CCTCCNO: M 20241441, the preservation unit is: China Center for Type Culture Collection, the preservation address is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the preservation date is: July 1, 2024.

[0007] The present invention also provides a microbial agent containing the Lactobacillus reuteri.

[0008] The present invention also provides the use of the Lactobacillus reuteri or the microbial agent in preparing a medicine for treating or preventing constipation.

[0009] As a further description of the above scheme: the constipation is functional constipation.

[0010] As a further description of the above scheme: the constipation is functional constipation induced by loperamide.

[0011] As a further description of the above scheme: the Lactobacillus reuteri can promote the release of the neurotransmitter 5-HT.

[0012] The preliminary study of the present invention found that Limosilactobacillus reuteri (L.reuteri) is the key microorganism for walnut insoluble fiber (WIDF) to relieve loperamide-induced constipation in mice. Based on this, the present invention uses a method of pure microbial culture and phylogenetic analysis of 16S rDNA genes to isolate and identify multiple strains of Lactobacillus reuteri from WIDF-treated mouse feces. Further, constipation model mice were used to screen out the effective strain L.reuteri_30 from L.reuteri, and its laxative activity mechanism was verified.

[0013] Compared with the prior art, the present invention has the following beneficial effects: the present invention isolates Lactobacillus reuteri-30 from mouse feces, which can significantly improve constipation, specifically by significantly reducing the time of the first black stool of constipated mice, and significantly increasing the water content of feces and the ink propulsion rate. L. reuteri_30 can be used as a new type of beneficial bacteria to relieve constipation. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Flow chart of isolation and identification of key microorganisms;

[0015] Figure 2 Colony growth morphology and Gram staining (the left picture is the growth morphology picture, the right picture is the Gram staining picture);

[0016] Figure 3 Lactobacillus reuteri 30 can alleviate the symptoms of loperamide-induced constipation in mice. (A) Experimental design. (B) Time to first black stool (FBST). (C) Average weight of each stool. (D) Fecal water content (FWC). (E) Ink advancement rate (GI). (F) Representative stool photos of each group. (G) Representative pictures of ink advancement distance. All data are expressed as Mean±SEM. Compared with the CON group, #, P<0.05, ##, P<0.01, ###, P<0.001; compared with the LOP group, *, P<0.05, **, P<0.01, ***, P<0.001;

[0017] Figure 4 Effects of L. reuteri on physiological indices of loperamide-induced constipation in mice. (A) Body weight. (B) Food intake. (C) Water intake. (D) Liver index. (E) Spleen index. (F) Kidney index. (G) Cecum index. All data are expressed as Mean±SEM (n=11-12). Compared with the CON group, #, P<0.05, ##, P<0.01, ###, P<0.001; compared with the LOP group, *, P<0.05, **, P<0.01, ***, P<0.001;

[0018] Figure 5 L. reuteri can regulate serotonergic synapses and the Oprd / iNOS pathway as well as the expression of AQPs. (A) Serum 5-HT levels. (BI) Relative gene expression of 5-HT2A, Calm, Oprd, iNOS, MLC3, AQP3, AQP4, and AQP8 in mouse colon. All data are expressed as Mean±SEM. Compared with the CON group, #, P<0.05, ##, P<0.01, ###, P<0.001; compared with the LOP group, *, P<0.05, **, P<0.01, ***, P<0.001. DETAILED DESCRIPTION

[0019] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solutions. The data involved in the following examples are expressed as mean ± SEM. GraphPadPrism 8.0 version was used for statistics, and the comparison between the two groups was analyzed by unpaired Student's T test. When P < 0.05, the difference was considered significant.

[0020] Example 1 Isolation and Identification of Bacteria

[0021] Isolation of microorganisms

[0022] Previous studies have found that oral administration of WIDF can effectively relieve loperamide-induced constipation in mice. In the experiment, mice were gavaged with a mixed suspension of loperamide (10 mg / kg·bw, to establish a constipation mouse model) and WIDF (900 mg / kg·bw) at the same time, and the mouse feces were collected after continuous gavage for 13 days. Under sterile conditions, the PBS homogenate supernatant of WIDF-treated mouse feces was inoculated into MRS broth (Hopebio, Qingdao, China) at 2% and cultured anaerobically at 37°C for 24 hours. After the above bacteria were diluted (10 -4 ~10 -6 ) was spread on MRS agar medium (Hopebio, Qingdao, China) and cultured anaerobically at 37°C for 48 h. A single colony was picked and re-inoculated into MRS broth, and the above liquid and solid culture was repeated twice to obtain a purified single colony. The separation flow chart is shown in Figure 1 shown.

[0023] Identification of microorganisms

[0024] According to the manufacturer's instructions, the DNA of each colony was extracted using the Qingke DNA extraction kit (Tsingke). The above DNA was used as a template and PCR amplification was performed using 1×TSE101 gold mix (Tsingke, Beijing, China). After the PCR product was identified, it was sequenced using primers 27F / 1492R. The sequencing results were spliced ​​using Contig Express, and the inaccurate parts at both ends were removed. The spliced ​​sequences were aligned in the NCBI database, and the phylogenetic tree was constructed using MEGA 11.

[0025] The present invention isolates 9 strains of Lactobacillus reuteri, and finds that strain L.reuteri_30 (Limosilactobacillusreuteri 30) has potential laxative activity. The nucleic acid sequence of 16S rDNA of strain L.reuteri_30 is shown in SEQ ID NO.1, and the sequencing result is compared and analyzed in NCBI database (www.ncbi.nlm.gov / blast / ) using BLAST tool with the existing sequence of GenBank database, and the homology of the strain to be tested and the corresponding sequence of the known strain is analyzed. The comparison result of GenBank database shows that the homology with Lactobacillus reuteri is 100%.

[0026] Colony morphology of strain L. reuteri_30

[0027] like Figure 2As shown, after culturing L. reuteri_30 on MRS agar medium for 48 hours, the colony morphology was milky white, translucent, relatively moist, smooth, with neat edges and obvious protrusions. Under an optical microscope, the cells of the strain L. reuteri_30 were rod-shaped, non-spore-forming, and Gram-positive.

[0028] Finally, the screened strain was determined to be Lactobacillus reuteri through morphology, 16S rDNA sequencing and phylogenetic analysis, and was named Limosilactobacillus reuteri 30, which was deposited in the China Center for Type Culture Collection with the accession number: CCTCC NO: M 20241441.

[0029] Example 2 Application of Lactobacillus reuteri_30 in Alleviating Loperamide-Induced Constipation in Mice

[0030] Animal experiment design

[0031] Animals: 5-week-old C57BL / 6J male mice were provided by Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. (Beijing, China) with license number SCXK(Jing)2021-0006. Mice were housed in a controlled environment (12 h light / dark cycle) for 1 week and had free access to food and water. All experimental protocols were approved by the Life Science Ethics Committee of Yunnan Agricultural University.

[0032] 60 mice were randomly divided into 5 groups (n=12). Lactobacillus reuteri_30 and Lactobacillus reuteri_36 were isolated and identified in Example 1. Before oral administration, L.reuteri_30 and L.reuteri_36 were anaerobically cultured in MRS medium for 24 hours, the bacteria were obtained by centrifugation and then dissolved by PBS to obtain a bacterial suspension. Each mouse was gavaged twice a day. The blank control group (CON) was orally administered with sterile PBS, and the other 4 groups were given 10 mg / kg·bw of loperamide. After 1 hour, the mice in the probiotic group were orally administered with suspensions of L.reuteri_30 and L.reuteri_36 (200μL / 10 9 CFU / mL), the MAC group was orally administered with 3000 mg / kg·bw of polyethylene glycol 4000 powder, and the CON group and constipation model group (LOP) were orally administered with sterile PBS. The treatment lasted for 15 days, and the body weight, food and water intake were recorded every day.

[0033] Determination of time to first black stool and small intestinal transit in mice

[0034] The time to the first black stool and small intestinal transit in mice were determined according to existing methods. On the 14th day of the experiment, the mice were fasted overnight and provided with water. Then, each group of mice was given corresponding treatment according to the previous grouping. After 30 minutes, all mice were orally gavaged with 0.5% activated carbon solution and glue powder according to their body weight. The timing started after the mice were gavaged, and the time to the first black stool (FBST) of each mouse was recorded. The feces excreted within 6 hours of each mouse were collected, the number and wet weight were recorded, and the fecal water content (FWC) was tested.

[0035] On the 15th day of the experiment, after all mice had fasted for 12 hours, each group of mice was given corresponding treatment according to the grouping. After 30 minutes, each mouse was gavaged with activated carbon solution and glue powder according to body weight. After 20 minutes of gavage, the mice were killed by CO2. Blood was collected through the abdominal aorta, and then the entire intestine of the mouse was quickly separated, the mesentery was removed, and the distance moved by the activated carbon and the total length of the small intestine of the mouse were accurately measured. The gastrointestinal propulsion rate (GTR) of each mouse was calculated according to the previous method.

[0036] To evaluate the laxative activity of two strains of Lactobacillus reuteri, the experimental design was as follows Figure 3 A. L. reuteri_30 treatment can significantly reduce the time of the first black stool in constipated mice, significantly increase the fecal water content and ink propulsion rate, and L. reuteri_30 treatment also significantly increases the average fecal weight of mice (P < 0.05), while L. reuteri_36 has no significant effect on the defecation parameters of constipated mice ( Figure 3 BE). Representative images of mouse feces and ink propagation rate are shown in Figure 1. Figure 3 F, G. These results show that L. reuteri has strain-specific effects on relieving constipation, and L. reuteri_30, as a beneficial bacterium, can be used to relieve constipation with good results. Figure 4 It can be seen that L. reuteri_36 can significantly increase the food intake of mice (P<0.05), but L. reuteri_30 has no significant effect on food intake. In addition, the two strains of bacteria have no significant effect on the weight, water intake and organ index of mice.

[0037] Example 3 Study on the mechanism of Lactobacillus reuteri in improving constipation

[0038] To determine whether L. reuteri intervention also affected pathways such as serotonergic synapses and neuroactive ligand receptor interactions, we examined the expression levels of serotonin (5-HT) and pathway-related genes.

[0039] Enzyme-linked immunosorbent assay (ELISA) analysis

[0040] According to the manufacturer's instructions, the 5-HT content in mouse serum was detected using Mouse 5-Hydroxytryptamine ELISA Kit (CSB-E08365m, CUSABIO, China).

[0041] RT-PCR analysis

[0042] Total RNA was extracted from colon using MiniBEST Universal RNA Kit (TaKaRa). Reverse transcription was performed using PrimeScript TM RT reagent Kit (TaKaRa). Gene expression was performed using TB Green Premix Ex Taq II Kit (TaKaRa). 480System PCR instrument (Roche Diagnostics) was used for measurement. RPL19 RNA was selected as the internal reference gene, and the results were analyzed according to 2 -ΔΔCT Methods The relative expression of genes was calculated. The primer sequences used for RT-PCR are shown in Table 1.

[0043] Table 1 Primer list

[0044]

[0045] The experimental results showed that L. reuteri_30 could promote the release of neurotransmitter 5-HT and further promote the expression of 5-HT in mouse colon tissue. 2A Expression of receptors and Calm genes ( Figure 5 AC). At the same time, L.reuteri_30 intervention significantly inhibited the expression of Oprd in mouse colon tissue, and L.reuteri_30 significantly inhibited the expression of iNOS in mice ( Figure 5 D, E). MLC3 is a downstream gene of calcium ion and NO signal transduction, which is significantly upregulated under the action of L. reuteri_30 ( Figure 5 F). In addition, L. reuteri_30 significantly inhibited the expression of AQP3, AQP4, and AQP8 in mice ( Figure 5 GI). These results suggest that L. reuteri_30 may alleviate loperamide-induced constipation in mice by regulating serotonergic synapses and neuroactive ligand receptor interaction pathways.

[0046] In summary, L. reuteri_30 can relieve loperamide-induced constipation in mice, and L. reuteri_30 may relieve constipation by regulating the serotonergic synapse and neuroactive ligand receptor interaction pathway. The experiment showed that L. reuteri_30 can be used as a new type of beneficial bacteria to relieve constipation.

[0047] It should be understood that the above specific embodiments of the present invention are only used to illustrate or explain the principles of the present invention, and do not constitute a limitation of the present invention. Therefore, any modifications, equivalent substitutions, improvements, etc. made without departing from the spirit and scope of the present invention should be included in the protection scope of the present invention. In addition, the appended claims of the present invention are intended to cover all changes and modifications that fall within the scope and boundaries of the appended claims, or the equivalent forms of such scope and boundaries.

Claims

1. A Lactobacillus reuteri, characterized in that The Lactobacillus reuteri is named as Limosilactobacillus reuteri 30, with a preservation number of CCTCC NO: M 20241441, a preservation unit of China Center for Type Culture Collection, and a preservation address of Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. A microbial agent containing the Lactobacillus reuteri according to claim 1.

3. Use of the Lactobacillus reuteri according to claim 1 or the microbial agent according to claim 2 in the preparation of a drug for treating or preventing constipation.

4. The use according to claim 3, characterized in that: The constipation is functional constipation.

5. The use according to claim 3, characterized in that: The constipation is functional constipation induced by loperamide.

6. The use according to claim 3, characterized in that: The Lactobacillus reuteri can promote the release of the neurotransmitter 5-HT.

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