Paracaseicilus colonicum for improving laxative colon and its use

Abstract

The application discloses a strain of paracasei for improving laxative colon and application thereof, and belongs to the field of microorganisms. The paracasei CCFM1379 provided by the application can significantly shorten the time of the first black stool of a laxative colon mouse, increase the water content of feces, increase the number of fecal particles within 5 hours, and thus relieve constipation of the laxative colon mouse. The paracasei CCFM1379 can increase the content of excitatory neurotransmitters Ach and 5-HT in the colon tissue of the laxative colon mouse, reduce the release of inhibitory neurotransmitter NO, and thus promote intestinal peristalsis. The paracasei CCFM1379 can increase the content of short-chain fatty acids in the feces of the laxative colon mouse, and thus increase intestinal secretion. The paracasei CCFM1379 can reduce the content of IL-1 beta in the colon tissue of the laxative colon mouse, and thus reduce intestinal inflammatory response, and finally maintain intestinal homeostasis. Therefore, the paracasei CCFM1379 has considerable application prospect for slow transit constipation including laxative colon.

Description

Technical Field

[0001] This invention relates to a strain of *Lactobacillus paracasei* that improves laxative colon function and its applications, belonging to the field of microbiology. Background Technology

[0002] Laxative colon is a condition caused by long-term use of laxatives, leading to decreased responsiveness to laxatives and dependence. As the condition progresses, the dosage and effectiveness of laxatives become inversely proportional, eventually resulting in the loss of laxative effect and the formation of laxative colon. Laxative colon is a type of slow-transit constipation, and its main symptoms include decreased colonic motility, slowed peristalsis leading to difficulty in defecation, reduced defecation frequency, and hard stools.

[0003] Currently, the treatment for laxative colon is largely the same as for slow transit constipation, starting with lifestyle modifications and laxative therapy, followed by the use of secretagogues or prokinetic agents if necessary. For more severe cases, when conventional treatments are ineffective, colectomy may be necessary to relieve symptoms. Despite this, the clinical treatment of laxative colon remains challenging. Most laxatives have minimal effect and may even worsen intestinal nerve damage in patients with laxative colon. Furthermore, secretagogues and prokinetic agents can cause adverse reactions such as diarrhea, nausea, abdominal pain, and flatulence, presenting certain side effects. In recent years, using probiotics and other gut microbiota preparations to relieve constipation has become a new research hotspot. To date, an increasing number of animal studies have demonstrated the significant alleviating effects of probiotics on constipation or intestinal motility disorders, achieving good efficacy in numerous clinical studies with relatively few adverse reactions. Therefore, research on the effects of probiotics on laxative colon relief holds promise for making probiotics a gentler and more effective method for relieving constipation or intestinal motility disorders, and also provides a model for future treatment of other diseases using microbiota preparations. Summary of the Invention

[0004] Technical issues The technical problem to be solved by the present invention is to provide a strain of Lactobacillus paracasei CCFM1379 that improves laxative colon and to provide the application of this strain.

[0005] Technical solution To address the aforementioned technical problems, this invention provides a strain of *Lactobacillus paracasei* (…). Lacticaseibacillus paracaseiCCFM1379 can alleviate constipation in patients with laxative-induced colonic diarrhea, as demonstrated by shortening the time to first black stool in mice with laxative-induced colonic diarrhea, increasing fecal water content, and increasing the number of fecal pellets within 5 hours. Furthermore, this strain can significantly increase the content of ACh and 5-HT in colonic tissue, significantly reduce the level of inflammation in colonic tissue, and increase the content of short-chain fatty acids in feces. Compared with CCFM1164, this strain has a better effect on relieving constipation, indicating that it can effectively alleviate constipation and thus help patients with laxative-induced colonic diarrhea.

[0006] This invention provides a strain of Lactobacillus paracasei ( Lacticaseibacillus paracasei The Lactobacillus paracasei CCFM1379 was deposited on January 11, 2024, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 64270.

[0007] The Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1379 was derived from a fecal sample from a male in Xining, Qinghai Province. Sequencing analysis of this strain, followed by nucleic acid sequence alignment using NCBI Standard Nucleotide BLAST, showed 100% similarity to the nucleic acid sequences of *Lactobacillus paracasei*. Therefore, the strain was identified as *Lactobacillus paracasei* and named *Lactobacillus paracasei*. Lacticaseibacillus paracasei ) CCFM1379.

[0008] The *Lactobacillus paracasei* CCFM1379 possesses the following biological characteristics: (1) Bacterial characteristics: Gram-positive, non-spore-forming, non-motile bacteria.

[0009] (2) Colony characteristics: round, raised, smooth, with neat edges.

[0010] (3) Growth characteristics: Under constant temperature of 37℃, it reaches the end of the logarithmic phase after about 18 h of culture in MRS medium.

[0011] (4) It has a strong tolerance to simulated gastrointestinal fluid.

[0012] (5) It can effectively relieve laxative colon, significantly increase the content of 5-HT and Ach in colon tissue, significantly reduce the inflammation level of colon tissue, and increase the content of short-chain fatty acids in feces.

[0013] The present invention also provides a microbial agent, wherein the microbial agent contains the above-mentioned *Lactobacillus paracasei* (…). Lacticaseibacillus paracasei CCFM1379 or its fermentation broth, or its freeze-dried powder.

[0014] The present invention also provides a medicine containing the above-mentioned *Lactobacillus paracasei* (…). Lacticaseibacillus paracasei CCFM1379 or the above-mentioned microbial agents.

[0015] In one embodiment of the invention, the pharmaceutical product comprises Lactobacillus paracasei CCFM1379 and a pharmaceutically permissible carrier.

[0016] In one embodiment of the present invention, the carrier includes one or more of the following commonly used in medicine: fillers, adhesives, wetting agents, disintegrants, lubricants, and flavoring agents.

[0017] In one embodiment of the present invention, the dosage form of the medicine includes granules, capsules, tablets, pills, or oral liquid.

[0018] In one embodiment of the present invention, the *Lactobacillus paracasei* ( Lacticaseibacillus paracasei The minimum addition level of CCFM1379 to the drug is 10. 8 CFU / mL or 10 8 CFU / g.

[0019] The present invention also provides the above-mentioned Lactobacillus paracasei ( Lacticaseibacillus paracasei Application of CCFM1379 or the above-mentioned microbial agents in the preparation of health products that help with bowel movements.

[0020] The present invention also provides the above-mentioned Lactobacillus paracasei ( Lacticaseibacillus paracasei Application of CCFM1379 or the above-mentioned microbial agents in the preparation of drugs to improve slow-transit constipation.

[0021] In one embodiment of the present invention, the slow transit constipation includes, but is not limited to, laxative colon, characterized in that the improvement of slow transit constipation includes at least one of the following: (a) Shortened the time to first black stool in mice with laxative colon, increased fecal water content, and increased the number of fecal pellets within 5 hours; (b) Increase the Ach content in the colon tissue of mice treated with laxatives; (c) Increased serotonin 5-HT levels in colon tissue of mice treated with laxatives; (d) Reduces the level of neuronal nitric oxide synthase (nNOS) in the colon tissue of mice treated with laxatives; (e) Reduces inflammation levels in the colonic tissue of mice treated with laxatives; (f) Increase the content of short-chain fatty acids (SCFAs) in the feces of mice with laxative colonization.

[0022] The present invention also provides Lactobacillus paracasei (… Lacticaseibacillus paracaseiCCFM1379 bacterial agent.

[0023] In one embodiment of the present invention, the bacterial agent contains *Lactobacillus paracasei* (… Lacticaseibacillus paracasei The number of viable bacteria in CCFM1379 is ≥10. 8 cfu / g or 10 8 cfu / mL.

[0024] In one embodiment of the present invention, the bacterial agent is a mixture containing *Lactobacillus paracasei* (…). Lacticaseibacillus paracasei The number of viable bacteria obtained by drying the CCFM1379 bacterial solution was ≥10. 8 cfu / g or 10 8 Powder with cfu / mL.

[0025] In one embodiment of the present invention, the drying refers to vacuum freeze drying.

[0026] Beneficial effects (1) The *Lactobacillus paracasei* GDMCC No: 64270 of this invention exhibits good activity. Compared to the model group, intervention with *Lactobacillus paracasei* CCFM1379 shortened the time to first black stool in laxative-treated colonic mice to 60.95% of the model group (p<0.01), increased the number of fecal pellets at 5 hours by 57.14% (p<0.05), and increased fecal water content by 17.27% (p<0.0001), indicating that gavage administration of *Lactobacillus paracasei* CCFM1379 can alleviate constipation symptoms in laxative-treated colonic mice. Furthermore, after intervention with *Lactobacillus paracasei* CCFM1379, the Ach content increased by 146.03% (p<0.01) and the 5-HT content increased by 88.51% (p<0.001) compared to the model group, and the SCFAs content also increased significantly. Importantly, *Lactobacillus paracasei* CCFM1379 showed significant effects on three constipation indicators: time to first black stool, stool water content, and number of stool particles in 5 hours. In contrast, *Lactobacillus paracasei* CCFM1164 was only effective on two of these indicators. Furthermore, *Lactobacillus paracasei* CCFM1379 was also more effective in increasing ACh, 5-HT, and SCFA levels. These results indicate that *Lactobacillus paracasei* CCFM1379 has a superior overall effect on relieving constipation compared to *Lactobacillus paracasei* CCFM1164.

[0027] (2) This invention can be regarded as a drug for improving slow transit constipation, and can also be applied to pharmaceuticals, thereby playing a wide role and having a very valuable application prospect.

[0028] Preservation of biological materials A strain of Lactobacillus paracasei ( Lacticaseibacillus paracaseiCCFM1379, its taxonomic name is: Lacticaseibacillus paracasei It was deposited on January 11, 2024 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 64270. Attached Figure Description

[0029] Figure 1 Lactobacillus paracasei (Lacticaseibacillus paracasei The effects of CCFM1379 on constipation relief were measured by: (a) time to first black stool; (b) stool water content; (c) number of stool particles in 5 hours; and (d) small intestinal propulsion rate.

[0030] Figure 2 Lactobacillus paracasei ( Lacticaseibacillus paracasei The effect of CCFM1379 on Ach content in colon tissue.

[0031] Figure 3 Lactobacillus paracasei ( Lacticaseibacillus paracasei Effects of CCFM1379 on 5-HT content in colon tissue.

[0032] Figure 4 Lactobacillus paracasei ( Lacticaseibacillus paracasei The effects of CCFM1379 on the relative expression and content of nNOS in colon tissue: (a) relative expression of nNOS; (b) content of nNOS.

[0033] Figure 5 Lactobacillus paracasei ( Lacticaseibacillus paracasei The effect of CCFM1379 on inflammation levels: (a) relative expression of IL-1β; (b) IL-1β content.

[0034] Figure 6 Lactobacillus paracasei ( Lacticaseibacillus paracasei The effects of CCFM1379 on short-chain fatty acids: (a) acetic acid content; (b) propionic acid content; (c) butyric acid content; (d) isobutyric acid content; (e) valeric acid content; (f) isovaleric acid content. Detailed Implementation

[0035] The male C57BL / 6J mice used in the following examples were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.

[0036] The strain information involved in the following examples is as follows: Lactobacillus paracasei ( Lacticaseibacillus paracasei (CCFM1379, categorized as follows) Lacticaseibacillus paracaseiIt was deposited on January 11, 2024 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 64270.

[0037] Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1164, its taxonomic name is: Lacticaseibacillus paracasei It has been published in patent CN112877260A, with accession number GDMCC No: 61479.

[0038] The culture media involved in the following examples are as follows: MRS liquid culture medium: 10g beef extract; 10g tryptone; 5g yeast extract; 20g glucose; 5g anhydrous sodium acetate; 0.1g MgSO4·7H2O; 0.05g MnSO4·H2O; 2g diammonium hydrogen citrate; 2.6g K2HPO4·3H2O; 1mL Tween 80; adjust pH to 6.8±0.2; bring volume to 1L. Autoclave at 115℃ for 20min.

[0039] MRS solid medium: 2% agar powder is added to the MRS liquid medium.

[0040] Preparation of Lactobacillus paracasei suspension in the following examples Lactobacillus paracasei CCFM1379 and Lactobacillus paracasei CCFM1164 were inoculated into MRS solid medium and cultured at 37°C for 48 h to obtain single colonies. The obtained single colonies were then inoculated into MRS liquid medium and cultured at 37°C for 24 h to activate them.

[0041] After activation for three generations, the bacterial culture was inoculated into 1 L of MRS liquid medium at a 2% (v / v) inoculation rate. After shaking to mix, the culture was incubated at 37 °C for 18 h. The culture was then centrifuged at 8000 g / min at 4 °C for 10 min. After discarding the supernatant, the cells were washed twice with sterile physiological saline, and centrifuged again under the same conditions. After discarding the supernatant, the cells were resuspended in 10% sterile defatted emulsion to prepare a final concentration of 5 × 10⁻⁶ cells / mL. 9 CFU / mL of *Lactobacillus paracasei* CCFM1379 and CCFM1164 bacterial suspensions were obtained to prepare the bacterial suspensions for gavage administration. These suspensions were then frozen at -80°C for one week.

[0042] Before conducting animal experiments, the frozen bacterial culture was removed from the freezer, and the number of viable bacteria was determined using the plate spread method at the initial stage and after one week of freezing. The order of magnitude of the initial and one-week viable bacterial counts did not change, indicating that freezing the bacterial culture did not affect the experiment and it could be used for animal experiments.

[0043] The following examples illustrate the methods for detecting the levels of Ach, 5-HT, nNOS, and IL-1β in colonic tissue. The expression levels of Ach, 5-HT, nNOS, and IL-1β in colon tissue were quantified using ELISA. The specific method was as follows: Colon tissue was rinsed with pre-cooled PBS to remove residual blood and surrounding adipose tissue. After weighing, the tissue was minced. The minced tissue was then mixed with PBS solution at a weight-to-volume ratio of 1:9 and homogenized using a high-throughput tissue homogenizer. Finally, the homogenate was centrifuged at 5000×g for 5 minutes. After 10 minutes, collect the supernatant for testing. Perform the experiment according to the instructions of the corresponding reagent kit, and calculate the levels of Ach, 5-HT, nNOS, and IL-1β in the tissue based on the standard curve.

[0044] Example 1: Lactobacillus paracasei ( Lacticaseibacillus paracasei Obtaining CCFM1379 1. Isolation and screening of Lactobacillus paracasei: (l) A fecal sample was collected from a male in Xining, Qinghai Province, using a disposable sterile fecal collection device and incubated at 37°C for 72 hours under anaerobic conditions. The colony morphology was observed and recorded, and colonies were picked and streaked for purification. The colonies were then incubated in MRS liquid medium at 37°C for 48 hours. The obtained colonies were Gram-stained, and the colony morphology was recorded. Gram-negative bacterial strains and Gram-positive cocci were discarded from the colonies, and Gram-positive bacilli were selected.

[0045] (2) After catalase analysis, discard catalase-positive strains and retain catalase-negative strains.

[0046] 2. Molecular biological identification of Lactobacillus paracasei: (l) Single-cell genome extraction: The catalase-negative strains screened in step 1 were cultured overnight. 1 mL of the overnight bacterial suspension was placed in a 1.5 mL centrifuge tube and centrifuged at 10,000 rpm for 2 min. The supernatant was discarded to obtain bacterial cells. The bacterial cells were washed with 1 mL of sterile water and centrifuged at 10,000 rpm for 2 min. The supernatant was discarded to obtain bacterial cells. 200 μL of SDS lysis buffer was added, and the mixture was incubated at 80℃ for 30 min. 200 μL of phenol-chloroform solution was added to the bacterial lysis buffer. The composition and volume ratio of the phenol-chloroform solution were Tris-saturated phenol:chloroform:isoamyl alcohol = 25:24:1. After inverting and mixing, the mixture was centrifuged at 12,000 rpm for 5-10 min. 200 μL of the supernatant was collected. 400 μL of ice-cold ethanol or ice-cold isopropanol was added to 200 μL of the supernatant. The mixture was incubated at -20℃ for 1 h and centrifuged at 12,000 rpm for 5-10 min. The supernatant was discarded. 500 μL of SDS lysis buffer was added to the supernatant. Resuspend the precipitate in 70% (v / v) ice-cold ethanol, centrifuge at 12000 rpm for 1-3 min, discard the supernatant; dry in a 60℃ oven or air dry; redissolve the precipitate in 50 μL ddH2O for PCR preparation.

[0047] (2) 16S rDNA PCR: A. Bacterial 16S rDNA 50μL PCR reaction system: 10×Taq buffer, 5μL; dNTP, 5μL; 27F, 0.5μL; 1492R, 0.5μL; Taq enzyme, 0.5μL; template, 0.5μL; ddH2O, 38μL.

[0048] B. PCR conditions: 95℃ for 5 min; 95℃ for 10 s; 55℃ for 30 s; 72℃ for 30 s; step 2-4 30×; 72℃ for 5 min; 12℃ for 2 min.

[0049] C. Prepare a 1% agarose gel, then mix the PCR product with 10000× loading buffer, load 2 μL, run at 120V for 30 min, and then perform gel imaging.

[0050] D. The obtained PCR product was sent to a professional sequencing company. The sequencing results were compared with those obtained by searching and similarity analysis in GeneBank using BLAST. It was identified as *Lactobacillus paracasei* and named *Lactobacillus paracasei*. Lacticaseibacillus paracasei CCFM1379, deposited at Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCC No: 64270, stored at -80℃ for future use.

[0051] Example 2: The effect of Lactobacillus paracasei CCFM1379 on relieving constipation in mice with laxative colon The specific steps are as follows: (1) Lactobacillus paracasei ( Lacticaseibacillus paracasei Preparation of bacterial suspension After the Lactobacillus paracasei CCFM1379 strain was taken out of the -80℃ freezer, it was streaked onto MRS solid medium and cultured at 37℃ for 48h. Single colonies were picked and cultured on MRS liquid medium at 37℃ for 24h to prepare seed culture. The prepared seed culture was inoculated into a new MRS liquid medium at an inoculation rate of 2% (v / v) and cultured at 37°C for 18 h. The culture was repeated for another generation in the same manner to prepare the fermentation broth of Lactobacillus paracasei CCFM1379. The prepared Lactobacillus paracasei CCFM1379 fermentation broth was then centrifuged at 8000 r / min and 4℃ for 10 min, and then resuspended in a 10% (w / w) skim milk aqueous solution to obtain a bacterial suspension for animal experiments.

[0052] (2) Thirty healthy male C57BL / 6J mice aged 8 weeks were acclimatized to the environment for 1 week and randomly divided into 5 groups: control group, model group, prucalopride group (positive drug group), Lactobacillus paracasei CCFM1164 group (positive bacteria group, as described in patent CN112877260A), and Lactobacillus paracasei CCFM1379 group. Each group contained 6 mice. Gavage was administered to each mouse starting at 9:00 AM every day, with each mouse receiving 0.2 mL of a concentration of 5×10⁻⁶. 9 CFU / mL bacterial suspension.

[0053] The grouping and treatment methods for experimental animals are shown in Table 1: Table 1 Grouping of experimental animals

[0054] After gavage in week 15, mice were placed individually in cages lined with absorbent paper, and their feces were collected and weighed to obtain the wet weight. After freeze-drying, the dry weight was obtained. The water content of the feces was calculated using the following formula.

[0055] Fecal moisture content = (fecal wet weight - fecal dry weight) / fecal wet weight × 100%; Each mouse was given 0.2 mL of ink by gavage. The time of the first black stool and the number of black stools excreted within 5 hours were recorded from the start of gavage.

[0056] Before sacrifice, each mouse was given 0.2 mL of ink by gavage. After 30 minutes, the mice were sacrificed and dissected. The upper part of the small intestine from the lower end of the pylorus to the cecum was cut off. The total length of the small intestine was measured as the "total length of the small intestine", and the length from the pylorus to the front of the ink was measured as the "ink propulsion length". The propulsion rate of the small intestine was calculated according to the following formula.

[0057] Small intestinal propulsion rate = (length of ink propulsion (cm)) / (total length of small intestine (cm)) × 100%; Results of the time to first black stool, stool water content, number of stool particles in 5 hours, and small intestinal propulsion rate are as follows: Figure 1 As shown in the figure, compared with the control group, the time to first black feces in mice after modeling (199.75 min) was prolonged to 1.38 times that of the blank control group (144.50 min) (p<0.01), the fecal water content (54.87% after modeling) decreased by 10.98% (p<0.01), the number of fecal particles within 5 hours (7 after modeling) decreased by 30% (p<0.01), while the small intestinal propulsion rate (87.29% after modeling) did not change significantly. This indicates that the model mice have colonic transit disorder, and the laxative colon mouse model was successfully established.

[0058] Compared to the model group, gavage administration of Lactobacillus paracasei CCFM1379 shortened the time to first black stool in laxative-treated colon mice (121.75 min) to 60.95% of the model group (p<0.01), increased the number of fecal pellets at 5 hours (11 pellets in the CCFM1379 group) by 57.14% (p<0.05), and increased the fecal water content (64.35% in the CCFM1379 group) by 17.27% (p<0.0001). This indicates that gavage administration of Lactobacillus paracasei CCFM1379 can alleviate constipation symptoms in laxative-treated colon mice.

[0059] Furthermore, CCFM1379 showed significant effects on three constipation indicators: time to first black stool, stool moisture content, and number of stool particles in 5 hours. In contrast, CCFM1164 was only effective on two constipation indicators: time to first black stool and number of stool particles in 5 hours. This indicates that Lactobacillus paracasei CCFM1379 has a better overall effect on relieving constipation than Lactobacillus paracasei CCFM1164.

[0060] Therefore, the above results indicate that Lactobacillus paracasei CCFM1379 can effectively improve laxative colon, and its effect is better than that of Lactobacillus paracasei CCFM1164.

[0061] Example 3: Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1379 can increase the content of ACh in the colon tissue of mice treated with laxatives. The grouping, modeling, and treatment methods for C57BL / 6J mice were the same as in Example 2. In addition, a group was added treated with *Lactobacillus paracasei* CCFM132. *Lactobacillus paracasei* CCFM1321 was cultured according to the method disclosed in patent CN117143766A to obtain a bacterial suspension, and the experiment was conducted according to the experimental setup in Table 1. The expression level of Ach in colon tissue was quantified using ELISA. The specific method is as follows: The colon tissue was rinsed with pre-cooled PBS to remove residual blood and surrounding adipose tissue. After weighing, the tissue was minced. The minced tissue was then mixed with PBS solution at a weight-to-volume ratio of 1:9 using a high-throughput tissue homogenizer. The homogenate was centrifuged at 5000×g for 5-10 minutes, and the supernatant was collected for analysis. The experiment was performed according to the instructions of the corresponding kit, and the Ach content in the colon tissue was calculated based on the standard curve. Results are as follows: Figure 2 As shown. Ach can significantly excite gastrointestinal smooth muscle, increasing its contraction amplitude, tension, and peristalsis, and can promote gastric and intestinal secretion, causing symptoms such as defecation.

[0062] like Figure 2 As shown, the Ach content in the colonic tissue of mice treated with laxatives (21.66 pmol·pg⁻¹) was significantly higher than that in the control group (48.63 pmol·pg⁻¹). -1 The Ach content in the mouse intestine was significantly reduced by 55.46% (p<0.01), indicating that long-term treatment with senna leaves leads to a decrease in Ach content in the mouse intestine. After gavage administration of *Lactobacillus paracasei* CCFM1379, the Ach content was 53.29 pmol·pg. -1 The efficacy was 146.03% higher than the model group (p<0.01), and 34.33 pmol·pg higher than the Lactobacillus paracasei CCFM1164 group. -1 The level of ACh in mice increased by 55.23%, which was superior to the positive control group. Simultaneously, CCFM1321 was used as a control strain. After gavage administration of *Lactobacillus paracasei* CCFM1321, the ACh content in the colon of mice was 44.28 pmol·pg. -1 CCFM1379 showed a greater effect than CCFM1321 in increasing intestinal Ach levels in mice with laxative colon.

[0063] Therefore, the results show that Lactobacillus paracasei CCFM1379 can alleviate laxative colon by increasing the Ach content in colonic tissue.

[0064] Example 4: Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1379 can increase the content of 5-HT in the colonic tissue of mice treated with laxatives. The grouping, modeling, and treatment methods for C57BL / 6J mice were the same as in Example 2. In addition, a group was added treated with *Lactobacillus paracasei* CCFM132. *Lactobacillus paracasei* CCFM1321 was cultured according to the method disclosed in patent CN117143766A to obtain a bacterial suspension, and the experiment was conducted according to the experimental setup in Table 1. The expression level of 5-HT in colon tissue was quantified using ELISA. The specific method was the same as in Example 3, and the results are as follows: Figure 3 As shown. Serotonin 5-HT can regulate intestinal motility, gastrointestinal secretion, and coordinate intestinal sensory functions. It is also an important neurotransmitter involved in the brain-gut axis. For example... Figure 3 As shown, the 5-HT content in the colonic tissue of mice treated with laxatives was 20.63 ng·g after modeling. -1 Compared to the control group (30.53 ng·g), -1 The levels of 5-HT in the mouse intestines decreased significantly by 32.43% (p<0.05), indicating that long-term treatment with senna leaves leads to a decrease in 5-HT content in the mouse intestines.

[0065] Following oral administration of Lactobacillus paracasei CCFM1379, the 5-HT content was 38.89 ng·g. -1 The efficacy was 88.51% higher than the model group (p<0.001); compared with the *Lactobacillus paracasei* CCFM1164 group (23.78 ng·g), it was higher. -1 The level of 5-HT in mice increased by 63.54%, which was superior to the positive control group. Simultaneously, CCFM1321 was used as a control strain. After gavage administration of *Lactobacillus paracasei* CCFM1321, the 5-HT content in the colon of mice was 33.19 ng·g⁻¹. -1 CCFM1379 was superior to CCFM1321 in increasing the intestinal 5-HT content in mice with laxative colon.

[0066] Therefore, the results show that Lactobacillus paracasei CCFM1379 can relieve laxative colon by increasing the 5-HT content in colonic tissue.

[0067] Example 5: Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1379 can reduce the expression of neuronal nitric oxide synthase (nNOS) in the colon tissue of mice treated with laxatives. The grouping, modeling, and treatment methods for C57BL / 6J mice are the same as in Example 2.

[0068] The expression level of nNOS in colon tissue was quantified using ELISA. The specific method was the same as in Example 3, and the results are as follows: Figure 4 As shown in b.

[0069] Real-time quantitative polymerase chain reaction (RT) was used The expression level of the nNOS gene was determined by qPCR. First, RNA was extracted from fresh colon tissue. The specific method was as follows: 0.2g of fresh colon tissue taken from dissected mice was repeatedly ground in a mortar (180℃, 4 h high-temperature enzyme inactivation) with liquid nitrogen. Then, 1 mL of TrizoL reagent was added to the mortar, and grinding continued until the liquid was basically clear. The mixture was then collected into a 1.5 mL enzyme-free centrifuge tube and allowed to stand at room temperature for 15 min. 200 μL of chloroform solution was added to the centrifuge tube, gently shaken for 15 s, allowed to stand at room temperature for 10 min, and centrifuged at 4℃, 12000 r / min for 15 min. 600 μL of the colorless upper aqueous phase was transferred to another enzyme-free centrifuge tube, and 500 μL of isopropanol was added. Invert the tube to mix thoroughly, let stand at room temperature for 10 min. After standing, centrifuge at 12000 r / min for 10 min at 4℃, discard the supernatant, leaving the white precipitate formed at the bottom of the centrifuge tube containing RNA. Add 1 mL of 75% ethanol solution prepared with DEPC water, vortex to resuspend, centrifuge at 7500 r / min for 5 min at 4℃, discard the supernatant, and allow to evaporate and dry at room temperature. Add 30 μL of RNase-free water to the dried RNA. After the RNA dissolves, determine the RNA concentration and purity using Nanodrop, and assess the RNA quality by agarose gel electrophoresis. Using the extracted total RNA as a template, reverse transcribe cDNA according to the instructions of the HiScript III RTSuperMix for qPCR reverse transcription kit from Novizan Biosciences Co., Ltd., and store at -20℃.

[0070] mice nNOS Genes and internal reference genes GAPDH Gene primers are shown in Table 2. Table 2: Mice nNOS Genes and GAPDH Gene primer sequence

[0071] qRT-PCR reaction system and conditions: PCR amplification was performed using a Bio-Rad® CFX96™ real-time quantitative PCR instrument, and the fluorescence signal was read. nNOS Genes and GAPDH The qRT-PCR reaction system for the gene is as follows: Table 3: qRT-PCR reaction system

[0072] The qRT-PCR reaction conditions were: 95℃ for 30 s; 95℃ for 10 s, 60℃ for 30 s, for a total of 40 cycles. mGAPDHThe gene was used as an internal reference gene, and the results were analyzed using CFX96Manager software. The experimental results are as follows: Figure 4 As shown in a.

[0073] NO is an inhibitory neurotransmitter that can stimulate VIP release, inhibit intestinal motility, and induce smooth muscle relaxation. Due to its reactive chemical properties, it is easily oxidized in the body. Therefore, we characterized changes in NO levels by detecting neuronal nitric oxide synthase (nNOS).

[0074] Depend on Figure 4 As shown in a / b, the nNOS content in the colonic tissue of mice treated with laxatives was 0.80 μmol·g⁻¹. -1 The levels of NO and its gene expression (2.50) were significantly increased by 90.48% (p<0.05) and 131.48% (p<0.01) compared to the control group, indicating excessive release of NO in the intestine of laxative-treated mice.

[0075] Following oral administration of Lactobacillus paracasei CCFM1379, the nNOS content was 0.69 μmol·g⁻¹. -1 The expression levels of the positive strain and the gene expression level (p<0.001) were reduced by 13.75% and 83.60% respectively compared with the model group. Compared with the Lactobacillus paracasei CCFM1164 group, the nNOS gene expression level of Lactobacillus paracasei CCFM1379 group was reduced by 40.58%, which was better than the positive strain control group.

[0076] Therefore, the above results indicate that Lactobacillus paracasei CCFM1379 can alleviate constipation by reducing the expression of nNOS in the colon.

[0077] Example 6: Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1379 reduces interleukin-1β (IL-1β) expression in colon tissue of mice treated with laxatives. The grouping, modeling, and treatment methods for C57BL / 6J mice are the same as in Example 2.

[0078] The expression level of IL-1β in colon tissue was quantified using ELISA. The specific method was the same as in Example 3, and the results are as follows. Figure 5 As shown in b.

[0079] Real-time quantitative polymerase chain reaction (RT) was used qPCR) to determine IL-1β Gene expression levels, RNA extraction, and reverse transcription methods were the same as in Example 5, and the experimental results are as follows: Figure 5 As shown in a.

[0080] mice IL-1β Genes and internal reference genes GAPDH Gene primers are shown in Table 3. Table 4: Mice IL-1β Genes and GAPDH Gene primer sequence

[0081] IL-1β has strong pro-inflammatory activity, can induce a variety of pro-inflammatory mediators, and ultimately lead to widespread inflammatory events.

[0082] Depend on Figure 5 According to a / b, the IL-1β content in the colonic tissue of mice treated with laxatives was 33.05 ng / g. -1 The expression levels of the virus and gene (p<0.0001) were significantly increased by 91.37% and 46.60% (p<0.01) respectively compared to the control group, indicating that the intestines of the laxative-treated mice were severely inflamed.

[0083] Following oral administration of Lactobacillus paracasei CCFM1379, the IL-1β level was 14.74 ng / g. -1 The expression levels of *Lactobacillus paracasei* (CCFM1164) and *Lactobacillus paracasei* (CCFM1379) were reduced by 55.40% (p<0.0001) and 58.94% (p<0.01) respectively compared to the model group. Compared to the *Lactobacillus paracasei* CCFM1379 group, the expression levels of *Lactobacillus paracasei* CCFM1379 were significantly lower. IL-1β The content was reduced by 39.86% compared with the CCFM1164 group, which was better than the positive strain control group.

[0084] Therefore, the above results indicate that Lactobacillus paracasei CCFM1379 can alleviate constipation by reducing the expression of IL-1β in the colon and lowering the level of colonic inflammation.

[0085] Example 7: Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1379 increases the content of short-chain fatty acids in the feces of mice with laxative colon. The grouping, modeling, and treatment methods for C57BL / 6J mice are the same as in Example 2.

[0086] The method for determining short-chain fatty acids in mouse feces is as follows: After freeze-drying to remove moisture, weigh 25 mg of mouse feces and place it in a 2 mL centrifuge tube. Add 500 μL of saturated NaCl solution to the centrifuge tube, let stand for 10 min, and then thoroughly disrupt the tissue using a tissue homogenizer (60 Hz, 30 s, 3 times). Next, add 20 μL of 10% sulfuric acid solution (volume concentration) and vortex to mix. Then add 1 mL of anhydrous diethyl ether and mix thoroughly. Centrifuge at 12000 × g for 15 min at 4℃ to completely separate the organic phase. Transfer the upper organic phase to a new 2 mL centrifuge tube. Add 0.25 g of anhydrous sodium sulfate to remove residual moisture and centrifuge at 12000 × g for 5 min at 4℃. Then transfer the upper organic phase to a sample vial for analysis. The gas chromatography-mass spectrometry (GC-MS) system used an Rtx-Wax column with a length of 30 m and an inner diameter of 0.25 μm; the carrier gas was He, with a flow rate of 2 mL / min; the injection volume was 1 μL, and the split ratio was 10:1; the injection temperature was set to 240 °C, and the temperature was increased according to the following program: the initial temperature was 100 °C, increased to 140 °C at 7.5 °C / min, then increased to 200 °C at 60 °C / min, held for 3 min, and the ionization temperature was 220 °C; the analysis was performed using selected ion scanning mode, and the concentrations (μmol / g) of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid were calculated using the external standard method.

[0087] Short-chain fatty acids (SCFAs) are saturated fatty acids with chains ranging from 1 to 6 carbon atoms, and are the main products of dietary fiber fermentation in the gut. SCFAs play various local roles in improving gut health. For example, SCFAs can maintain the integrity of the intestinal wall barrier and prevent intestinal inflammation. SCFAs also affect mucus production in the gastrointestinal tract, increasing fecal water content. SCFAs can also affect gastrointestinal motility, possibly by activating SCFA receptors, releasing the intestinal hormone peptide YY (PYY), or inducing serotonin release from enterochromaffin cells.

[0088] Depend on Figure 6 Compared with the control group, the fecal contents of the model group mice showed a decrease of 35.54% (p<0.001), 49.31% (p<0.001), 33.26% (p<0.01), 39.83% (p<0.01), 43.39% (p<0.01), and 28.71% (p<0.05) in acetic acid, propionic acid, and isovaleric acid, respectively. This indicates that long-term senna leaf treatment significantly reduced the content of short-chain fatty acids in the fecal contents of mice with laxative colonization.

[0089] After intervention with *Lactobacillus paracasei* CCFM1379, compared with the model group, the acetic acid content increased by 60.59% (p<0.01), returning to the control group level; the propionic acid content increased by 115.47% (p<0.0001), returning to the control group level; the butyric acid content increased by 65.27% (p<0.01), returning to the control group level; the isobutyric acid content increased by 76.70% (p<0.01), returning to a level slightly higher than the control group level; the valerate content increased by 117.27% (p<0.001), returning to the control group level; and the isovaleric acid content increased by 48.95%, returning to the control group level. Compared with the *Lactobacillus paracasei* CCFM1164 group, the *Lactobacillus paracasei* CCFM1379 group was superior to the positive strain control group in both the effect on the types of short-chain fatty acids and the recovery of short-chain fatty acid content.

[0090] Therefore, the results show that intervention with Lactobacillus paracasei CCFM1379 can significantly increase the level of short-chain fatty acids in the colon, provide motility to the colon, enhance colonic peristalsis, increase fecal water content, and thus relieve laxative colitis.

[0091] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1379, characterized in that, The Lactobacillus paracasei CCFM1379 was deposited on January 11, 2024, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 64270.

2. A microbial inoculant, characterized in that, The microbial agent contains Lactobacillus paracasei CCFM1379 as described in claim 1, or its fermentation broth, or its freeze-dried powder.

3. A medicine, characterized in that, The drug contains Lactobacillus paracasei CCFM1379 as described in claim 1 or the microbial agent as described in claim 2.

4. The medicine as described in claim 3, characterized in that, The amount of *Lactobacillus paracasei* CCFM1379 added to the pharmaceutical product is at least 10. 8 CFU / mL or 10 8 CFU / g.

5. The medicine as described in claim 4, characterized in that, The dosage forms of the medicine include granules, capsules, tablets, pills, or oral liquids.

6. The use of Lactobacillus paracasei CCFM1379 as described in claim 1 or the microbial agent as described in claim 2 in the preparation of health products that help with bowel movements.

7. The use of Lactobacillus paracasei CCFM1379 as described in claim 1 or the microbial agent as described in claim 2 in the preparation of laxative colon medicines.

8. The application as described in claim 7, characterized in that, The laxative for improving colon function has at least one of the following functions: (a) Shortened the time to first black stool in mice with laxative colon, increased fecal water content, and increased the number of fecal pellets within 5 hours; (b) Increase the content of acetylcholine (ACh) in the colon tissue of mice treated with laxatives; (c) Increased serotonin 5-HT levels in colon tissue of mice treated with laxatives; (d) Reduces the level of neuronal nitric oxide synthase (nNOS) in the colon tissue of mice treated with laxatives; (e) Reduces inflammation levels in the colonic tissue of mice treated with laxatives; (f) Increase the content of short-chain fatty acids in the feces of mice with laxative colonization.

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