Lactobacillus paracasei for prevention and / or treatment of digestive system diseases

Abstract

The present invention relates to a novel Lactobacillus paracasei BGI-N2, the preservation number of which is CCTCC (China Center For Type Culture Collection) NO: M20221486, a composition containing the novel Lactobacillus paracasei BGI-N2 as an active ingredient, and an application of the novel Lactobacillus paracasei BGI-N2. The lactobacillus paracasei comprises a 16S rDNA (ribosomal deoxyribonucleic acid) sequence as shown in SEQ ID NO: 1. The lactobacillus paracasei BGI-N2 provided by the embodiment of the invention has good safety and excellent gastrointestinal tract environment tolerance, and can effectively promote intestinal tract movement and defecation capacity and prevent or improve constipation.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to a Lactobacillus paracasei for preventing or treating digestive system diseases, a composition containing the Lactobacillus paracasei as an effective ingredient, and application thereof. Background Art

[0002] Constipation is a common digestive system disease, and its symptoms mainly include poor defecation, reduced bowel movement, dry stool, difficulty in defecation, discomfort in defecation, pain during defecation, or obvious abdominal distension and pain during defecation. These symptoms not only seriously affect the patient's quality of life, but may also cause a series of health problems, such as indigestion, hemorrhoids, colon cancer and other digestive system diseases. Modern people are particularly prone to constipation problems due to irregular living habits, unhealthy diet, lack of exercise, mental stress, side effects of drugs, intestinal diseases, pregnancy and other reasons. Currently, more than 1 / 3 of the world's population is affected by constipation.

[0003] Relevant methods to improve constipation include drug therapy, biofeedback therapy, and surgical treatment. Drug therapy is to promote intestinal peristalsis or increase intestinal secretions through oral or anal administration, thereby promoting defecation and softening feces. Common drugs used for drug treatment include laxatives, lubricants, saline laxatives, and stimulant laxatives. Biofeedback therapy trains patients through electrophysiological instruments and biofeedback technology to improve patients' perception of pelvic floor muscles and defecation, thereby improving defecation function. Surgical treatment mainly restores defecation function by surgically removing or repairing the anal sphincter or rectum. However, the use of drug treatment will cause drug dependence problems. Once constipated patients stop taking the medicine, they often cannot defecate normally or even have aggravated constipation symptoms. Long-term use of drugs can easily lead to increased intestinal tolerance to drugs, making the drug efficacy weakened or even ineffective; biofeedback therapy requires professional technical support, the treatment process is long, and it will cause discomfort; surgical treatment is expensive, has high risks, and requires a long recovery period, and is only suitable for a small number of people.

[0004] Therefore, a safe and effective method for preventing or treating digestive system diseases, particularly constipation, is urgently needed. Summary of the invention

[0005] The present invention aims to solve one of the technical problems in the related art at least to a certain extent. To this end, the present invention provides a Lactobacillus paracasei for preventing or treating digestive system diseases, a composition containing the Lactobacillus paracasei as an effective ingredient and its application.

[0006] The embodiment of the first aspect of the present invention provides a Lactobacillus paracasei comprising the 16S rDNA sequence shown in SEQ ID NO:1.

[0007] In some embodiments, the strain is Lactobacillus paracasei BGI-N2 with a deposit number of CCTCC NO: M20221486.

[0008] The second aspect of the present invention provides a composition for preventing and / or treating digestive system diseases, comprising an effective amount of Lactobacillus paracasei as described in any embodiment of the first aspect. In some embodiments, the composition further comprises a pharmaceutically or food acceptable auxiliary material.

[0009] In some embodiments, the composition further comprises other drugs for preventing and / or treating digestive system diseases;

[0010] In some embodiments, the composition further comprises prebiotics and / or probiotics.

[0011] In some embodiments, the composition is administered orally or rectally.

[0012] In some embodiments, the composition is in the form of a powder, granules, capsule, tablet, solution, suspension, syrup, ointment, plaster or suppository.

[0013] In some embodiments, the composition comprises 1.0×10 7 to 1.0×10 12 CFU / mL or CFU / g of Lactobacillus paracasei BGI-N2.

[0014] In some embodiments, 5.0×10 7 Up to 5.0×10 11 Lactobacillus paracasei BGI-N2.

[0015] The embodiments of the third aspect of the present invention provide the use of the Lactobacillus paracasei described in any embodiment of the first aspect or the composition described in any embodiment of the second aspect in the preparation of a medicament for preventing and / or treating digestive system diseases.

[0016] In some embodiments, the digestive system disease is selected from the group consisting of constipation, dyspepsia, hemorrhoids, and colon cancer.

[0017] In some embodiments, the composition regulates gastrointestinal motility by increasing motilin secretion, regulating serotonin synthesis and transport, and reducing nitric oxide levels.

[0018] The embodiments of the present invention achieve the following beneficial effects:

[0019] (1) Lactobacillus paracasei BGI-N2 provided by the embodiment of the present invention can be used to effectively promote intestinal peristalsis and defecation ability, prevent or improve constipation, and has a laxative effect. Lactobacillus paracasei BGI-N2 is not only comparable to the constipation treatment effect of traditional constipation treatment drug domperidone, but also avoids the drug dependence and side effect problems caused by traditional drug intervention. In addition, the supplementation of probiotics helps to maintain the balance of intestinal microecology, can bring long-term benefits to intestinal health, reduce the risk of constipation, and achieve the effect of preventing constipation.

[0020] (2) The Lactobacillus paracasei BGI-N2 provided in the embodiment of the present invention is an excellent probiotic strain of local origin, which has no potential harm to the body and has good safety. It can stay in the gastrointestinal environment for a long time and maintain a high survival rate. It has excellent gastrointestinal environment tolerance and is suitable for preventing or improving constipation.

[0021] (3) The Lactobacillus paracasei BGI-N2 provided in the embodiment of the present invention can improve gastrointestinal health by regulating indicators of gastrointestinal health, such as regulating the secretion of motilin, the synthesis and transport of neurotransmitters such as serotonin and NO, to regulate gastrointestinal motility, and act on interstitial cells of Cajal (ICC) to control gastrointestinal rhythm, ultimately achieving multi-dimensional effective prevention or improvement of constipation. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the present invention or related technologies, the following briefly introduces the drawings required for use in the embodiments or related technical descriptions. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 Schematic diagram of the colony morphology of Lactobacillus paracasei BGI-N2 in an embodiment of the present invention;

[0024] Figure 2 This is a schematic diagram of the whole genome evolutionary tree result of Lactobacillus paracasei BGI-N2 in the embodiment of the present invention;

[0025] Figure 3 Schematic diagram of the constipation treatment results of Lactobacillus paracasei BGI-N2 in the embodiment of the present invention (the experiment was performed in triplicate; * indicates p<0.05 relative to the model control group; ** indicates p<0.01 relative to the model control group; *** indicates p<0.001 relative to the model control group; tested by ONE-WAY ANOVA);

[0026] Figure 4Schematic diagram of the constipation prevention results of Lactobacillus paracasei BGI-N2 in the embodiment of the present invention (the experiment was performed in triplicate; * indicates p<0.05 relative to the model control group; ** indicates p<0.01 relative to the model control group; *** indicates p<0.001 relative to the model control group; tested by ONE-WAY ANOVA);

[0027] Figure 5 (A)-(D) are schematic diagrams showing the results of Lactobacillus paracasei BGI-N2 regulating gastrointestinal motility regulatory factors in the embodiments of the present invention. Figure 5 (A) is a schematic diagram of the results of regulating motilin. Figure 5 (B) is a schematic diagram of the results of regulating the expression of the kitb gene. Figure 5 (C) is a schematic diagram of the results of regulating the expression of the kitla gene. Figure 5 (D) is a schematic diagram of the results of regulating the expression of the kitlb gene (the experiment was performed in triplicate; * indicates p < 0.05 relative to the model control group; ** indicates p < 0.01 relative to the model control group; *** indicates p < 0.001 relative to the model control group; tested by ONE-WAY ANOVA);

[0028] Figure 6 Schematic diagram of the results of regulating nitric oxide by Lactobacillus paracasei BGI-N2 in the embodiments of the present invention (the experiment was performed in triplicate; * indicates p<0.05 relative to the model control group; ** indicates p<0.01 relative to the model control group; *** indicates p<0.001 relative to the model control group; tested by ONE-WAY ANOVA);

[0029] Figure 7 (A)-(F) are schematic diagrams showing the results of Lactobacillus paracasei BGI-N2 regulating serotonin and related genes in the embodiments of the present invention. Figure 7 (A) is a schematic diagram showing the results of regulating serotonin. Figure 7 (B) is a schematic diagram of the results of regulating tph2 gene expression. Figure 7 (C) is a schematic diagram showing the results of regulating tph1b gene expression. Figure 7 (D) is a schematic diagram of the results of regulating tph1a gene expression. Figure 7 (E) is a schematic diagram of the results of regulating the expression of the sert gene. Figure 7 (F) Schematic diagram of the results of regulating htr1a gene expression (the experiment was performed in triplicate; * indicates p < 0.05 relative to the model control group; ** indicates p < 0.01 relative to the model control group; *** indicates p < 0.001 relative to the model control group; tested by ONE-WAY ANOVA). DETAILED DESCRIPTION

[0030] The present invention is further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art and are not intended to limit the present invention in any way.

[0031] The present invention is made based on the following knowledge of the inventors:

[0032] Constipation is a common digestive system disease, which not only seriously affects the patient's quality of life, but also causes a series of digestive system diseases, such as indigestion, hemorrhoids, colon cancer, etc. Relevant methods to improve constipation include drug therapy, biofeedback therapy, and surgical treatment. Drug therapy is to use drugs to promote intestinal peristalsis or increase intestinal secretions through oral or anal administration, thereby promoting defecation and softening stool. Common drugs used for drug treatment include laxatives, lubricants, saline laxatives, and stimulant laxatives. Biofeedback therapy trains patients through electrophysiological instruments and biofeedback technology to improve patients' perception of pelvic floor muscles and defecation, thereby improving defecation function. Surgical treatment mainly restores defecation function by surgically removing or repairing the anal sphincter or rectum.

[0033] However, the use of drug treatment will lead to the problem of drug dependence. Once constipated patients stop taking the drug, they often cannot defecate normally or even their constipation symptoms worsen. In addition, long-term use of drugs can easily lead to increased intestinal tolerance to the drugs, which weakens the efficacy of the drugs or even makes them ineffective. Biofeedback treatment requires professional technical support, has a long treatment process, and can cause discomfort. Surgical treatment is expensive, has high risks, and requires a long recovery period, and is only suitable for a small number of people.

[0034] Gastrointestinal flora plays an important role in the occurrence and development of constipation. People with constipation are often accompanied by an imbalance of gastrointestinal flora. Supplementing with appropriate probiotics can not only produce organic acids and / or a variety of bioactive substances that are beneficial to intestinal peristalsis to promote defecation, but also reduce residues and increase defecation volume by breaking down food components that are difficult for the human body to digest. In addition, probiotics can also inhibit the growth and reproduction of harmful bacteria, while increasing the number of beneficial bacteria to maintain the balance of intestinal microecology.

[0035] In the present embodiment, "probiotics" should be understood as "live microorganisms that have a beneficial effect on the health of the host by improving the intestinal microbial environment of the host in the gastrointestinal tract of animals including humans". When live microorganisms with probiotic activity are provided in the form of dried cells or fermentation products in the form of a single or composite strain, the probiotics can have a beneficial effect on the intestinal flora of the host.

[0036] However, probiotics are strain-specific. Different strains may have differences in colony morphology, growth characteristics, metabolites, physiological functions, etc., and their therapeutic efficacy for patients with functional constipation is naturally different. Therefore, it is necessary to provide strains at the strain level that can effectively prevent and treat digestive system diseases, especially constipation, and have good safety and excellent gastrointestinal environmental tolerance.

[0037] According to the above practical needs, a first aspect of an embodiment of the present invention provides a Lactobacillus paracasei comprising a 16S rDNA sequence as shown in SEQ ID NO: 1.

[0038]

[0039] In some embodiments, the strain is Lactobacillus paracasei BGI-N2 with a deposit number of CCTCC NO: M20221486.

[0040] The embodiment of the present invention provides a kind of Lactobacillus paracasei, such as Figure 1 As shown, it belongs to the genus Lactobacillus (Lactobacillus), and it is named Lactobacillus paracasei BGI-N2 (Lactobacillus paracasei BGI-N2); the preservation registration number is CCTCC NO:M20221486, the preservation institution is: China Center for Type Culture Collection; the address of the preservation unit: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province; No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province; the postal code is 430072; the preservation time is September 26, 2022.

[0041] It should be noted that according to the update of strain naming caused by the changes in the international taxonomic status of relevant strains, Lactobacillus paracasei and Lacticaseibacillus paracasei can be used interchangeably in this application.

[0042] In an embodiment of the present invention, the lactobacillus paracasei BGI-N2 with the sequence shown in SEQ ID NO:1 can be understood by those skilled in the art as the original strain. It is understandable that bacterial strains such as lactobacillus paracasei BGI-N2 can undergo spontaneous mutation or be artificially cultured to form variant strains, such as the deletion of nucleotides, the addition of nucleotides or the replacement of nucleotides. "Variant strain" has a highly identical gene sequence and extremely similar biological functions with "lactobacillus paracasei BGI-N2", and the mutated gene does not affect the conservative sequence of lactobacillus paracasei BGI-N2 substantially, thereby does not affect the genetic stability of lactobacillus paracasei BGI-N2. More specifically, this "variant strain" is also a strain of BGI-N2, showing the physiological activity and functional characteristics of BGI-N2.

[0043] The Lactobacillus paracasei BGI-N2 and variants thereof provided by the embodiment of the present invention can be used to effectively promote intestinal peristalsis and defecation ability, prevent or improve constipation. Lactobacillus paracasei BGI-N2 is not only comparable to the constipation treatment effect of traditional constipation treatment drug domperidone, but also avoids the drug dependence and side effect problems brought by traditional drug intervention. In addition, the supplementation of probiotics helps to maintain the balance of intestinal microecology, can bring long-term benefits to intestinal health, reduce the risk of constipation, and reach the effect of preventing constipation. BGI-N2 has no potential harm to body, has good safety, in addition it can stay for a long time in the gastrointestinal environment, and maintains a high survival rate, has excellent gastrointestinal environmental tolerance, and is suitable for preventing or improving constipation.

[0044] The second aspect of the present invention provides a composition for preventing and / or treating digestive system diseases, comprising an effective amount of Lactobacillus paracasei as described in any embodiment of the first aspect. In some embodiments, the composition further comprises a pharmaceutically or food acceptable auxiliary material.

[0045] In some embodiments, the composition further comprises other drugs for preventing and / or treating digestive system diseases;

[0046] In some embodiments, the composition further comprises prebiotics and / or probiotics.

[0047] In some embodiments, the composition is administered orally or rectally.

[0048] In some embodiments, the composition is in the form of a powder, granules, capsule, tablet, solution, suspension, syrup, ointment, plaster or suppository.

[0049] In some embodiments, the pharmaceutically acceptable excipient is, for example, a carrier, an excipient, a diluent, an adjuvant, a vehicle, an excipient, a carrier or a combination thereof, and / or other substances having a concentration that does not affect the activity of the microorganism, and the present invention is not limited thereto.

[0050] The term "pharmaceutically acceptable carrier" refers to any preparation or carrier medium that can deliver an effective amount of the active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects on the host or patient. Representative carriers include water, oil, vegetables and minerals, cream bases, lotion bases, ointment bases, etc. These bases include suspending agents, viscosity enhancers, transdermal enhancers, etc. Their preparations are well known to those skilled in the art of cosmetics or topical drugs. For additional information about the carrier, reference can be made to Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams & Wilkins (2005), the contents of which are incorporated herein by reference.

[0051] In some embodiments, the composition comprises 1.0×10 7 to 1.0×10 12 CFU / mL or CFU / g of Lactobacillus paracasei BGI-N2.

[0052] It is understood that probiotics are prepared into bacterial powder through conventional processes in the microbial industry such as high-density fermentation, filtration centrifugation, freeze drying or spray drying, in which the number of live bacteria is 1.0×10 7 to 1.0×10 12 CFU / g. The bacterial powder can be prepared into the above-mentioned various forms when applied. In some embodiments, the bacterial powder is dissolved in water to obtain different bacterial concentrations (such as 50 million CFU / ml).

[0053] The embodiments of the third aspect of the present invention provide the use of the Lactobacillus paracasei described in any embodiment of the first aspect or the composition described in any embodiment of the second aspect in the preparation of a medicament for preventing and / or treating digestive system diseases.

[0054] In some embodiments, the digestive system disease is selected from the group consisting of constipation, dyspepsia, hemorrhoids and colon cancer.

[0055] In some embodiments, the composition regulates gastrointestinal motility by increasing motilin secretion, regulating serotonin synthesis and transport, and reducing nitric oxide levels.

[0056] In some embodiments, the composition regulates gastrointestinal motility regulatory factors and / or neurotransmitters and related genes to regulate gastrointestinal motility, specifically, through at least one of motilin, kitla, kitlb, kitb, nitric oxide, serotonin, tph1a, tph1b, tph2, sert, and htr1a.

[0057] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0058] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0059] Example 1 Isolation, Identification and Preservation of Lactobacillus paracasei BGI-N2

[0060] In this example, Lactobacillus paracasei BGI-N2 was isolated, identified and preserved.

[0061] (1) Isolation of strains

[0062] A stool sample from a healthy Chinese male was collected and placed in an anaerobic chamber. 0.2 g of stool sample was mixed in 1 mL of sterile phosphate buffered saline (PBS, item number B640011, purchased from Shanghai Biotech Co., Ltd.), and then graded diluted. 4 Take 100ul from the sample dilution of 100 times, spread it on PYG medium (Cat. No. 027340, Huankai Microbiology Technology Co., Ltd.), and culture it anaerobically at 37℃ for 48 to 72h. Then select a single colony for streaking to obtain a pure culture strain. Figure 1 This is the colony morphology of the strain. It can be seen that the colonies of this strain on the plate are round, with clear edge boundaries, white or light yellow, and the texture of the colonies is smooth, moist and has a distinct luster.

[0063] (2) Identification of strains

[0064] DNA was extracted from the isolated pure culture strain using a bacterial genomic DNA extraction kit (article number DP302, purchased from Tiangen Biochemical Technology Beijing Co., Ltd.). The extracted genomic DNA was used as a template for PCR amplification of its 16S rDNA gene, wherein the sequences of the primers used are shown in SEQ ID NO: 2 and SEQ ID NO: 3 (SEQ ID NO: 2, 27F-AGAGTTTGATCATGGCTCAG, SEQ ID NO: 3, 1492R-TAGGGTTACCTTGTTACGACTT), and the PCR amplification program is shown in Table 1, and the cycle was repeated 30 times. The PCR amplification product was sequenced using the Sanger method to obtain the 16S rDNA sequence of the strain (SEQID NO: 1). By comparing the NCBI database, it was found that the strain with the highest homology was Lactobacillus paracasei, with a similarity of 99.9%. The strain belongs to the genus Lactobacillus (Lactobacillus), and it was named Lactobacillus paracasei BGI-N2 (Lactobacillus paracasei BGI-N2).

[0065] Table 1 PCR amplification program

[0066]

[0067] (3) Whole genome sequencing of strains

[0068] The whole genome sequence of the BGI-N2 strain was obtained by whole genome sequencing of the genomic DNA extracted in step (2), and evolutionary analysis was performed based on single copy genes and related strains (mostly model strains), such as Figure 2 As shown, according to the branch distance information in the schematic diagram of the whole genome evolutionary tree results, it can be seen that Lactobacillus paracasei BGI-N2 has a relatively distant relationship with known strains, and therefore can be identified as a new strain.

[0069] (4) Preserved strains

[0070] The BGI-N2 strain is preserved in the China Center for Type Culture Collection; the preservation registration number is CCTCCNO: M20221486; the address of the preservation unit is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province; No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province; the postal code is 430072; the preservation time is September 26, 2022.

[0071] Example 2 Gastrointestinal tolerance of Lactobacillus paracasei BGI-N2

[0072] In this example, the intestinal environment tolerance of Lactobacillus paracasei BGI-N2 was evaluated by simulating the human gastrointestinal environment.

[0073] Activate the strain and count it. The bacterial liquid was placed in artificial gastric juice (pH = 2.0, pH = 3.0), artificial intestinal juice (pH = 6.8), and MRS liquid culture medium containing 0.3% bile salt (Cat. No. 027312, purchased from Huankai Microbiological Technology Co., Ltd.), and incubated at 37°C for 2 hours. The initial viable count value was taken as the survival rate of 100%. The viable counts were performed on each group at the completion of the experiment. The survival rate (%) = 100% × the number of viable bacteria after 2 hours / the initial number of viable bacteria.

[0074] Table 2 Gastrointestinal environmental tolerance of Lactobacillus paracasei BGI-N2

[0075]

[0076] The gastrointestinal environment tolerance of Lactobacillus paracasei BGI-N2 is shown in Table 2. The survival rates of Lactobacillus paracasei BGI-N2 after 2 hours under gastric acid pH 2.0 and pH 3.0 were 94.1% and 97.8% respectively, the survival rate after 2 hours under simulated small intestinal fluid was 99.5%, and the survival rate of the strain under 0.3% concentration of bile salt was 90.4%. The above results show that the Lactobacillus paracasei BGI-N2 in the embodiment of the present invention has excellent tolerance to gastric acid, intestinal fluid and bile salt in the gastrointestinal system, can survive in the gastrointestinal tract, and is suitable for preventing or improving constipation.

[0077] Example 3: Intestinal retention capacity of Lactobacillus paracasei BGI-N2

[0078] This example simulates the human gastrointestinal environment to affect the retention capacity of Lactobacillus paracasei BGI-N2 in the intestine, so as to further evaluate the strain's tolerance to the intestinal environment.

[0079] The BGI-N2 strain was labeled with CM-Dil fluorescence (Cat. No. 2174897, purchased from Thermo Fisher Scientific China Co., Ltd.), and wild-type AB strain zebrafish 5 days after fertilization (5dpf) were selected and placed in a 6-well plate, with 30 zebrafish in each well. 500 million CFU of BGI-N2 strain were added to each well for 24 hours. At the same time, commercial control bacteria Lactobacillus rhamnosus LGG, Bifidobacterium animalis BB12 and Lactobacillus casei YLD were used as control strains for synchronous experiments. The zebrafish were then transferred to water without strain addition for 24 hours. Each group of zebrafish was placed under a fluorescence microscope for intestinal fluorescence intensity measurement to evaluate the strain's ability to stay in the intestine. The test results are expressed as mean ± standard deviation.

[0080] Table 3 Intestinal retention ability of strains

[0081]

[0082] The intestinal retention ability of the strains is shown in Table 3. The 24h retention rate of Lactobacillus paracasei BGI-N2 in the zebrafish intestine is (83.37±5.26)%, and the intestinal retention ability is significantly better than that of Lactobacillus casei YLD, and there is no significant difference with the strains reported to be able to stay in the intestine, such as Lactobacillus rhamnosus LGG and Bifidobacterium animalis subspecies BB12. The above results show that the Lactobacillus paracasei BGI-N2 of the embodiment of the present invention can effectively stay in the intestine for a long time, thereby exerting its probiotic effects such as regulating the intestine, and is suitable for preventing or improving constipation.

[0083] Example 4: Safety of Lactobacillus paracasei BGI-N2

[0084] This example evaluates the safety of Lactobacillus paracasei BGI-N2 by determining the minimum toxic concentration (MTC) test.

[0085] Based on the previously obtained BGI-N2 genome sequence information, the Virulence Factor Database (VFDB) and the Comprehensive Antibiotic Resistance Database (CARD) were used to compare with it to complete the preliminary strain safety assessment. The evaluation results showed that no virulence factors and antibiotic resistance genes were found in the BGI-N2 genome of Lactobacillus paracasei. No obvious safety risks were found in the genome level of this strain, and it has good safety.

[0086] The minimum toxic concentration (MTC) test was performed using zebrafish. 5dpf wild-type AB strain zebrafish were randomly assigned to 6-well plates, with 30 zebrafish per well. 125, 250, 500, 1000, and 2000 μg / mL of BGI-N2 powder were added to each well, and a normal control group was set up for 24 hours. The growth of zebrafish was then observed, and the minimum toxic concentration (MTC) of the sample to zebrafish was determined.

[0087] Table 4 Minimum toxic concentration (MTC) test results of Lactobacillus paracasei BGI-N2

[0088]

[0089] The minimum toxic concentration (MTC) test results of Lactobacillus paracasei BGI-N2 are shown in Table 4. The results show that Lactobacillus paracasei BGI-N2 has no toxic reaction below the dose of 2000ug / mL and does not affect the health of zebrafish. This shows that Lactobacillus paracasei BGI-N2 has good safety and no potential harm to the body.

[0090] Example 5: Therapeutic effect of Lactobacillus paracasei BGI-N2 on zebrafish constipation symptoms

[0091] The present invention example evaluates the therapeutic effect of Lactobacillus paracasei BGI-N2 on constipation symptoms through a zebrafish constipation model.

[0092] 5dpf wild-type AB strain zebrafish were selected in a culture dish, and aluminum sulfate solution (Cat. No. RH424739, Shanghai Yien Chemical Technology Co., Ltd.) was added to establish a zebrafish constipation model. At the same time, Nile Red (Cat. No. SLBP9326V, purchased from Sigma) was used as a fluorescent indicator of intestinal contents. Zebrafish were randomly assigned to 6-well plates, with 30 tails per well. 500 million CFU / mL, 100 million CFU / mL, and 200 million CFU / mL of BGI-N2 bacterial powder were added to each well, and the positive control was 50.0 μg / mL of domperidone (Cat. No. 190104499, purchased from Xi'an Janssen Pharmaceutical Co., Ltd.). A normal control group and a model control group were set up at the same time. After 4.5 hours of treatment, the Nile Red fluorescence intensity data of the zebrafish intestine in each group was measured under a fluorescence microscope.

[0093] Constipation treatment results of Lactobacillus paracasei BGI-N2 Figure 3 As shown in the figure, after the constipation zebrafish model was constructed with aluminum sulfate, the Nile red fluorescence intensity of the zebrafish intestine in the model control group increased significantly, indicating that the discharge of intestinal contents was blocked and the constipation zebrafish model was successfully established. After intervention treatment with the positive drug domperidone and medium and high doses of Lactobacillus paracasei BGI-N2, the fluorescence intensity in the zebrafish was significantly reduced (p<0.001). The Nile red fluorescence intensity in the constipated zebrafish treated with medium and high doses of Lactobacillus paracasei BGI-N2 was reduced to normal levels. Compared with the Nile red fluorescence intensity in the normal control group, there was no statistically significant difference between the two, which indicates that Lactobacillus paracasei BGI-N2 has a significant promoting effect on the emptying of intestinal contents and can effectively improve constipation symptoms.

[0094] Example 6: Preventive effect of Lactobacillus paracasei BGI-N2 on zebrafish constipation symptoms

[0095] The present invention example evaluates the preventive effect of Lactobacillus paracasei BGI-N2 on constipation symptoms through a zebrafish constipation model.

[0096] 5dpf wild-type AB strain zebrafish were randomly assigned to 6-well plates, with 30 zebrafish per well. 500 million CFU / mL, 100 million CFU / mL, and 200 million CFU / mL of Lactobacillus paracasei BGI-N2 powder were added, respectively. The positive control was 50.0 μg / mL domperidone. A normal control group and a model control group were set up at the same time. After 24 hours of treatment, the samples were removed, and Nile red was added to each group as a fluorescent indicator of intestinal contents. After 3 hours, aluminum sulfate was added to the water solution in all experimental groups except the normal control group to establish a constipation model. After 6 hours of treatment, the Nile red fluorescence intensity of the zebrafish intestine in each group was measured under a fluorescence microscope.

[0097] The results of Lactobacillus paracasei BGI-N2 on the prevention of constipation symptoms in zebrafish Figure 4 As shown. When the positive drug domperidone and medium and high doses of Lactobacillus paracasei BGI-N2 were used for advance prevention, the fluorescence intensity in the zebrafish was significantly reduced compared with the model control group without any preventive measures (p<0.001). Compared with the Nile red fluorescence intensity in the normal control group, the Nile red fluorescence intensity in the zebrafish constipation model using medium and high doses of Lactobacillus paracasei BGI-N2 was at a normal level, and there was no statistically significant difference between the two. The above results show that Lactobacillus paracasei BGI-N2 has a significant promoting effect on the emptying of intestinal contents and can effectively prevent the occurrence of constipation.

[0098] Example 7: Effects of Lactobacillus paracasei BGI-N2 on gastrointestinal motility regulatory factors and their gene expression levels

[0099] The embodiment of the present invention evaluates the effect of Lactobacillus paracasei BGI-N2 on regulating gastrointestinal motility regulatory factors and their gene expression levels through a zebrafish constipation model.

[0100] According to the steps shown in Example 6, zebrafish of 500 million CFU / mL, 100 million CFU / mL, and 200 million CFU / mL Lactobacillus paracasei BGI-N2 powder treatment groups, positive control domperidone group, normal control group, and model control group were obtained. Motilin levels were measured in the above zebrafish using a motilin ELISA detection kit, and qPCR measurement and data analysis of the expression levels of genes related to the development of interstitial cells of Cajal (ICC) were performed.

[0101] It should be noted that motilin (MTL) is a gastrointestinal motility regulator, which is secreted by Mo cells and can promote gastrointestinal motility and the passage of intestinal contents. ICC cells are pacemaker cells that control rhythmic electrical activity in the intestine and are associated with gastrointestinal motility disorders. ICC can express c-kit, a tyrosine protein kinase receptor encoded by the c-kit gene; the receptor coordinates the rhythmic contraction of the intestine by binding to ligand proteins (encoding genes include kitla, kitb and kitlb, etc.). By measuring the expression levels of the above-mentioned related regulatory genes, the function of BGI-N2 in regulating intestinal rhythms is evaluated.

[0102] The results of Lactobacillus paracasei BGI-N2 regulating gastrointestinal motility regulatory factors and their gene expression levels are as follows Figure 5 (A)-(D) Compared with the normal control group, the MTL level in the model control group using aluminum sulfate modeling was significantly reduced ( Figure 5 (A)), the relative expression levels of kitla, kitb, and kitlb genes were significantly decreased ( Figure 5 (B)-(D)), indicating that the constipation model with limited ICC cell development, insufficient MLT secretion level, and low gastrointestinal motility was successfully constructed. After intervention with the positive drug domperidone and different doses of Lactobacillus paracasei BGI-N2, the MTL level in the body and the relative expression levels of kitla, kitb, and kitlb genes were positively correlated with the dose. Among them, medium and high doses of BGI-N2 can significantly increase the MTL level in constipated zebrafish, and kitla, kitb, and kitlb also showed significant increases. The above results show that Lactobacillus paracasei BGI-N2 can effectively regulate Lactobacillus paracasei BGI-N2 to regulate gastrointestinal motility regulatory factors, promote ICC cell development, and increase the secretion level of motilin MLT, thereby effectively promoting intestinal peristalsis and improving constipation symptoms.

[0103] Example 8: Effect of Lactobacillus paracasei BGI-N2 on the level of inhibitory neurotransmitter nitric oxide

[0104] The embodiment of the present invention evaluates the effect of Lactobacillus paracasei BGI-N2 in regulating nitric oxide through a zebrafish constipation model.

[0105] According to the steps shown in Example 6, zebrafish of 500 million CFU / mL, 100 million CFU / mL, and 200 million CFU / mL Lactobacillus paracasei BGI-N2 powder treatment groups, positive control domperidone group, normal control group, and model control group were obtained. Each group was water-soluble and added with NO-specific fluorescent dye (Cat. No. D3320050, purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.) to stain and measure NO in zebrafish.

[0106] It should be noted that nitric oxide (NO) is one of the most important inhibitory neurotransmitters in the enteric nervous system. NO can increase the concentration of cyclic guanosine monophosphate in gastrointestinal smooth muscle, leading to smooth muscle relaxation, weakened gastrointestinal motility, gastrointestinal motility disorders and thus constipation.

[0107] The results of regulating nitric oxide by Lactobacillus paracasei BGI-N2 are as follows Figure 6 As shown. Compared with the normal control group, the NO level in the zebrafish in the constipation model group increased significantly. After treatment with the positive drug domperidone and different doses of Lactobacillus paracasei BGI-N2, the NO level in the zebrafish in the corresponding groups showed an extremely significant decrease (p<0.001) and showed a dose-dependent response. The above results indicate that Lactobacillus paracasei BGI-N2 can maintain the rhythmic movement of the gastrointestinal tract by reducing the concentration of inhibitory neurotransmitter NO acting on the gastrointestinal tract, thereby achieving the effect of improving constipation symptoms.

[0108] Example 9: Effects of Lactobacillus paracasei BGI-N2 on 5-HT levels and expression of its regulatory genes

[0109] The present invention example evaluates the effect of Lactobacillus paracasei BGI-N2 on regulating the expression of serotonin (5-HT) and its related genes through a zebrafish constipation model.

[0110] According to the steps shown in Example 6, zebrafish of 500 million CFU / mL, 100 million CFU / mL, and 200 million CFU / mL Lactobacillus paracasei BGI-N2 powder treatment groups, positive control domperidone group, normal control group, and model control group were obtained. Then, the 5-HT level was determined using a serotonin ELISA detection kit (Cat. No. 202306, purchased from Shanghai ELISA Biotechnology Co., Ltd.), and the expression level of 5-HT related regulatory genes was determined using qPCR.

[0111] It should be noted that serotonin (5-HT) mainly plays a role in the central nervous system and gastrointestinal tract. Beneficial intestinal bacteria can directly secrete 5-HT or secrete short-chain fatty acids to stimulate enterochromaffin cells (EC) to secrete 5-HT and increase the level of intestinal 5-HT secretion. 5-HT binds to multiple 5-HT receptors in the intestine, thereby promoting intestinal smooth muscle contraction, increasing intestinal peristalsis and the frequency of defecation. This process is regulated by multiple genes in the body.

[0112] The results of regulating the expression of serotonin (5-HT) and its related genes by Lactobacillus paracasei BGI-N2 are shown in Figure 7 (A)-(F) Compared with the normal control group, the 5-HT level in zebrafish in the constipation model group was significantly reduced ( Figure 7(A)), 5-HT synthesis-related genes tph1a, tph1b, tph2 ( Figure 7 (B)-(D)) and the relative expression levels of 5-HT receptor-related gene htr1a ( Figure 7 (F)) was significantly decreased, and the relative expression level of sert gene, a 5-HT transporter regulatory gene ( Figure 7 (E)) was significantly increased. After treatment with the positive drug domperidone and different doses of Lactobacillus paracasei BGI-N2, the 5-HT level in the zebrafish of the corresponding group increased with the increase of the dose, and the relative expression levels of the synthesis regulatory genes tph1a, tph1b, tph2, and the 5-HT receptor regulatory gene htr1a increased significantly, and the 5-HT transporter regulatory gene sert decreased significantly. The above results show that Lactobacillus paracasei BGI-N2 can promote the synthesis of 5-HT and the action of intestinal receptors in the body, reduce 5-HT transport, maintain the content of 5-HT in the intestine, thereby promoting the contraction of intestinal smooth muscle, increasing intestinal peristalsis and the frequency of defecation, and playing an effective role in improving constipation symptoms.

[0113] The results of Examples 7-9 collectively show that the Lactobacillus paracasei BGI-N2 in the examples of the present invention can improve gastrointestinal health by regulating indicators of gastrointestinal health, such as regulating the secretion of motilin, the synthesis and transport of neurotransmitters such as serotonin and NO, to regulate gastrointestinal motility, and act on Cajal interstitial cells (ICC) to control gastrointestinal rhythm, ultimately achieving multi-dimensional effective prevention or improvement of constipation.

[0114] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0115] It should be noted that, in this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the term "comprises" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0116] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A Lactobacillus paracasei, characterized in that It comprises the 16S rDNA sequence shown in SEQ ID NO:

1.

2. Lactobacillus paracasei according to claim 1, characterized in that The strain is Lactobacillus paracasei BGI-N2 with a deposit number of CCTCC NO: M20221486.

3. A composition for preventing and / or treating digestive system diseases, characterized in that: The invention comprises an effective amount of the Lactobacillus paracasei as claimed in claim 1 or 2 and optional pharmaceutically or food acceptable auxiliary materials.

4. The composition according to claim 3, characterized in that It also contains other drugs for preventing and / or treating digestive disorders; Optionally, the composition further comprises prebiotics and / or probiotics.

5. The composition according to claim 3 or 4, characterized in that The composition is administered orally or rectally.

6. The composition according to any one of claims 3 to 5, characterized in that The composition is in the form of powder, granules, capsules, tablets, solutions, suspensions, syrups, ointments, plasters or suppositories.

7. The composition according to any one of claims 3 to 6, characterized in that Based on the total volume or total weight of the composition, the composition comprises 1.0×10 7 to 1.0×10 12 CFU / mL or CFU / g of Lactobacillus paracasei BGI-N2, preferably containing 5.0×10 7 Up to 5.0×10 11 CFU / mL Lactobacillus paracaseiBGI-N2.

8. Use of the composition according to claim 1 or 2 or any one of claims 3 to 7 in the preparation of a medicament for preventing and / or treating digestive system diseases.

9. The use according to claim 8, characterized in that: The digestive system disease is selected from the group consisting of constipation, dyspepsia, hemorrhoids and colon cancer.

10. The use according to claim 9, characterized in that: The composition regulates gastrointestinal motility by increasing the secretion of motilin, regulating the synthesis and transport of serotonin, and reducing the level of nitric oxide.

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