Lactobacillus rhamnosus MBP01 capable of relieving constipation and application thereof

Abstract

The present invention belongs to the field of microbial technology, and specifically relates to a Lactobacillus rhamnosus MBP01 capable of relieving constipation and its application. The present invention discloses the application of Lactobacillus rhamnosus in preparing products for preventing, treating and / or assisting in the treatment of constipation, and the deposit number of the Lactobacillus rhamnosus is GDMCC NO:64725. The Lactobacillus rhamnosus can relieve constipation in mice caused by ice water activated carbon by regulating the cytokine content and the mRNA expression level of constipation-related genes, accelerating the secretion of enzymes in gastric acid and pancreas, promoting gastric peristalsis and gastric acid secretion, accelerating gastrointestinal motility, accelerating intestinal smooth muscle contraction and inhibiting the activity of inflammatory cells. The Lactobacillus rhamnosus has good prospects in the preparation of health food production with the function of relieving constipation and promoting digestion and in the clinical treatment of constipation.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a Lactobacillus rhamnosus MBP01 capable of relieving constipation and an application thereof. Background Art

[0002] Probiotics refer to dietary supplements containing live microbial strains that amplify the beneficial effects of normal intestinal flora through exogenous microorganisms in food to improve the health of the host (SHARIFI-RAD J, RODRIGUES CF, STOJANOVIĆ-RADIĆ Z, et al. Probiotics: Versatile Bioactive Components in Promoting Human Health [J]. Medicina (Kaunas, Lithuania) , 2020, 56(9)). Probiotics have the functions of improving lactose intolerance, promoting gastrointestinal motility, controlling infections caused by disordered intestinal flora, improving human immunity, lowering serum cholesterol, and preventing cancer (Li Lin, Zhao Mouming. Safety and genetic modification of probiotics [J]. China Dairy Industry, 2005(06): 43-46).

[0003] Constipation can seriously affect the quality of life of patients. Constipation can be diagnosed in people of all ages, with women and the elderly being the most susceptible. Severe constipation can cause patients to experience symptoms such as prolonged defecation time, difficulty in defecation, incomplete defecation, scanty defecation, and painful defecation. In addition, patients may experience symptoms such as anal obstruction and lower abdominal distension and pain (Liu Z, Yan S, Wu J, et al. Acupuncture for Chronic Severe Functional Constipation: A Randomized Trial [J]. Annals of Internal Medicine, 2016, 165(11):761-769). The Rome III criteria found that patients with constipation were accompanied by multiple symptoms. Therefore, the Rome III criteria believed that the presence of two or more of the following six symptoms could be defined as functional constipation (Bharucha A E. Constipation[J]. Best Practice & Research Clinical Gastroenterology. 2007, 21(4): 709-731): (1) The frequency of bowel movements is significantly reduced (less than 3 times per week); (2) The stool becomes dry and hard; (3) Excessive force is required during bowel movements; (4) The anorectal area is blocked; (5) Manual operation is required to assist in bowel movements; (6) The feeling of incomplete bowel movements after bowel movements. Long-term constipation can cause feces to remain in the intestines, and then harmful substances such as hydrogen sulfide, ammonia, amines and endotoxins produced by harmful bacteria metabolism will enter the blood and spread throughout the body. In severe cases, headaches, nausea, hypertension, coronary heart disease and colon cancer may also occur (Li Cunliang. The hazards of constipation in the elderly and common treatment methods [J]. Inner Mongolia Traditional Chinese Medicine. 2011, 30(16): 31-32).

[0004] Chinese patent application CN117535176A discloses a strain of Bifidobacterium animalis subspecies lactis TA-2688 and its application in the preparation of products for improving constipation, repairing intestinal mucosal barriers and regulating immunity. The strain can promote the production of 5-hydroxytryptamine precursor - 5-hydroxytryptamine, regulate the secretion of gastrointestinal hormones, and gently promote intestinal peristalsis, thereby relieving constipation symptoms of the elderly in the third grade of high school, and also has the function of improving intestinal barriers and regulating immunity.

[0005] Chinese patent application CN117305190A discloses a strain of Lactobacillus casei HYZQF and its application in the preparation of products that assist postoperative recovery. Postoperative recovery includes wound recovery and the construction of an intestinal microbial barrier, especially gastrointestinal surgery is prone to cause postoperative constipation, increasing patient pain. The technical solution provides a strain of Lactobacillus casei HYZQF, the fermentation product of which includes orotic acid and methionine, which helps to improve the patient's blood circulation, promote wound healing, and effectively improve postoperative constipation, helping patients rebuild their intestinal microbial barriers, and is used to prepare special medical foods or probiotic preparations that assist postoperative recovery, and has important application prospects.

[0006] There is a need in the art for more probiotics that can relieve constipation. Summary of the invention

[0007] Lactobacillus rhamnosus (LGG) belongs to the genus Lactobacillus, subspecies rhamnosus in the bacteriological classification (Wu Guangwei, Han Jianchun. Research on bacteriocin production of Lactobacillus rhamnosus (ATTC 53103) [J]. Journal of Northeast Agricultural University, 2009, 40(08): 81-85). Lactobacillus rhamnosus is a Gram-positive bacterium without plasmids and cannot utilize lactose, but can metabolize monosaccharides (Ma Pengfei, Chen Youliang, Jin Niaojun. Research progress on functional characteristics of Lactobacillus rhamnosus [J]. Bulletin of Science and Technology, 2009, 25(02): 202-206). The colony is generally off-white or slightly yellow, with a rough and rounded morphology (Wang Yuhua, Gao Jing, Feng Yin et al. Research on acid and bile salt resistance of Lactobacillus rhamnosus [J]. Food Science, 2008, 29(12): 449-451). It has strong tolerance to extreme environments, can adhere to and colonize in human epithelial cells, and has multiple probiotic functional properties (Lu Xiao. Analysis of biochemical characteristics of three strains of Lactobacillus rhamnosus and research on their application in salad dressing [D]. South China University of Technology, 2021).

[0008] Lactobacillus rhamnosus (LGG) can tolerate the animal digestive tract environment and has the ability to colonize in the intestines of humans and animals, regulating intestinal flora, preventing and treating diarrhea, eliminating toxins, preventing dental caries and improving the body's immunity. Therefore, the use of this probiotic has broad application prospects (Cui Zhiwen. Research on the effect of Lactobacillus rhamnosus on the intestinal barrier function of piglets [D]. Zhejiang University, 2013).

[0009] The present invention uses the separated Lactobacillus rhamnosus MBP01 for treating, assisting in treating or alleviating constipation.

[0010] In order to achieve the above technical objectives, the present invention proposes the following technical solutions:

[0011] In a first aspect, the present invention provides the use of Lactobacillus rhamnosus in preparing a product for preventing, treating and / or assisting in treating constipation, wherein the Lactobacillus rhamnosus has a deposit number of GDMCC NO:64725.

[0012] Specifically, the Lactobacillus rhamnosus is Lactobacillus rhamnosus MBP01.

[0013] Specifically, the product includes medicine or food.

[0014] Furthermore, each unit of the product contains at least 1×10 8 CFU of Lactobacillus rhamnosus.

[0015] Specifically, the product further comprises at least one physiologically acceptable auxiliary material or pharmaceutically acceptable excipient.

[0016] Specifically, the food is a health food, including a nutrient supplement or a functional health food.

[0017] Furthermore, the functional health food is a functional health food that helps to moisten the intestines and promote bowel movements.

[0018] Furthermore, the dosage form of the health food includes tablets, capsules, soft capsules, granules, pills, gel candies, powders, oral liquids or drops.

[0019] Furthermore, the health food also includes a second probiotic for relieving constipation and / or promoting digestion.

[0020] Specifically, the medicament further comprises a second therapeutic agent for promoting digestion, and / or preventing, treating or assisting in treating constipation.

[0021] In some embodiments, the Lactobacillus rhamnosus is used to relieve constipation.

[0022] In some preferred embodiments, the Lactobacillus rhamnosus is used to promote digestion.

[0023] According to some embodiments of the present invention, the present invention provides a culture, wherein the culture is obtained by culturing any of the aforementioned Lactobacillus rhamnosus in a culture medium.

[0024] The culture medium of the present invention includes carbon sources, nitrogen sources and / or phosphorus sources necessary for the proliferation, survival or growth of microorganisms, and optionally, growth additives such as trace elements that are beneficial to the proliferation, survival or growth of microorganisms.

[0025] Those skilled in the art can prepare the "culture" or "microorganism culture" of the present invention for use in the compositions or products herein using any standard or known static drying or liquid fermentation techniques known in the art. The optimal conditions for microorganism cultivation may depend on the specific strain. Those skilled in the art will be able to determine the appropriate nutrients and conditions. Microorganisms can be grown in aerobic, anaerobic or facultative anaerobic liquid cultures on a culture medium.

[0026] In some embodiments, the culture medium is a culture medium for culturing lactic acid bacteria.

[0027] In some preferred embodiments, the culture medium is a culture medium for culturing lactobacilli.

[0028] In some preferred embodiments, the culture medium is MRS medium or MRS modified medium.

[0029] In some embodiments, the culture includes, but is not limited to: live bacteria, inactivated bacteria, secretions or metabolites of any of the aforementioned Lactobacillus rhamnosus.

[0030] According to some embodiments of the present invention, the present invention provides a bacterial agent, wherein the bacterial agent comprises any of the aforementioned Lactobacillus rhamnosus or cultures.

[0031] In some embodiments, the bacterial agent also includes an auxiliary agent that enhances the stability of the rhamnosus Lactobacillus or culture, or helps the rhamnosus Lactobacillus or culture to perform biological functions, for example, preservatives specified in the production standards of food, health products (health food) or drugs are allowed to be added in safe doses.

[0032] In yet another aspect, the present invention provides a product for preventing, treating and / or assisting in the treatment of constipation, wherein the product comprises any one of the aforementioned Lactobacillus rhamnosus.

[0033] In some embodiments, each unit of the product contains at least 1×10 8 CFU of Lactobacillus rhamnosus.

[0034] In some embodiments, each unit of the product contains at least 1×10 8 CFU, 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 The Lactobacillus rhamnosus.

[0035] In some preferred embodiments, each unit of the product contains at least 1×10 9 CFU of Lactobacillus rhamnosus.

[0036] The unit depends on the dosage form or packaging form of the product, such as each tablet, each can, each pill, each grain, each package, etc., which shall be subject to the specific production practice or product packaging.

[0037] In some embodiments, the product comprises a pharmaceutical or a food.

[0038] In some preferred embodiments, the food is a health food.

[0039] In some embodiments, the product further comprises at least one physiologically acceptable auxiliary material, or pharmaceutically acceptable excipient.

[0040] In some embodiments, the physiologically acceptable excipients include, but are not limited to: erythritol, D-mannitol, fumaric acid, glycerin, pectin, potassium alginate, sodium alginate, talc, sodium pyrophosphate, polydextrose, carrageenan, sodium ascorbate, ascorbyl palmitate, L-malic acid, L(+)-tartaric acid, maltitol, gelatin, xylitol, citric acid, potassium citrate, sodium citrate, citric acid fatty acid glycerides, agar, lactic acid, sodium lactate, sorbic acid and its potassium salt, sorbitol, acid red, calcium carbonate, sodium carbonate, sodium bicarbonate, beet red, vitamin C, vitamin E, oxidized starch, ethanol, sodium acetate, stearic acid, calcium stearate, magnesium stearate and / or dextrin.

[0041] In some embodiments, the pharmaceutically acceptable excipients include, but are not limited to: solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavors, sweeteners, ion exchangers, release agents, coating agents, flavoring agents and / or antioxidants.

[0042] In some embodiments, the product is a solid formulation, a semisolid formulation, a liquid formulation, or a gaseous formulation.

[0043] In some embodiments, the product is a food, including, but not limited to, confectionery products, fermented beverages, chocolate, fermented milk, ice cream, energy bars, baked goods, biscuits or canned goods, etc.

[0044] In some embodiments, the product is a health food, including a nutrient supplement or a functional health food.

[0045] In some embodiments, the health food further comprises a second probiotic for relieving constipation and / or improving digestion.

[0046] In some embodiments, the second probiotic is an acid-producing probiotic.

[0047] In some preferred embodiments, the acid-producing probiotics include, but are not limited to: Lactobacillus plantarum, Lactobacillus plantarum, Lactobacillus reuteri, Bifidobacterium animalis, Bifidobacterium longum, Bifidobacterium breve, Lactobacillus crispatus or Pediococcus acidilactici.

[0048] In some embodiments, the dosage form of the health food includes, but is not limited to: tablets, capsules, soft capsules, granules, pills, gel candies, powders, oral liquids or drops.

[0049] In some preferred embodiments, the nutrient supplement includes a single nutrient supplement and a complex nutrient supplement.

[0050] In some preferred embodiments, the functional health food is a functional health food that helps to moisten the intestines and promote bowel movements.

[0051] In some preferred embodiments, the functional health food is a functional health food that aids digestion.

[0052] In some embodiments, the product is a pharmaceutical.

[0053] In some embodiments, the medicament further comprises a second therapeutic agent for improving digestion, and / or preventing, treating or assisting in the treatment of constipation.

[0054] In some preferred embodiments, the second therapeutic agent includes, but is not limited to: lactulose, Maren Runchang Pills, Tongbianling Capsules, bisacodyl, enema, compound aloe capsules, Maren Pills, magnesium sulfate, Danggui Longhui Pills, Maren Zipi Pills or polyethylene glycol 4000 powder.

[0055] Compared with the prior art, the Lactobacillus rhamnosus MBP01 provided by the present invention has at least the following beneficial effects:

[0056] (1) Significantly shortened the defecation time of constipated mice and significantly increased the water content of feces of constipated mice, thereby alleviating constipation symptoms;

[0057] (2) Promote intestinal peristalsis and significantly alleviate the decrease in small intestinal propulsion rate caused by constipation;

[0058] (3) Significantly improve the serum cytokine levels related to gastrointestinal digestion;

[0059] (4) Significantly improve intestinal function, significantly relieve inflammation of the colon mucosa, and significantly restore intestinal villi damage;

[0060] (5) It has a significant positive regulatory effect on the mRNA expression level of constipation-related genes.

[0061] Collection Instructions

[0062] Strain name: MBP01;

[0063] Deposit number: GDMCC No: 64725;

[0064] Category naming: Lacticaseibacillus rhamnosus ;

[0065] Deposit date: June 5, 2024;

[0066] Depository: Guangdong Microbiological Culture Collection Center;

[0067] Abbreviation of depository unit: GDMCC;

[0068] Address of the preservation unit: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 The statistical chart shows the time of the first red stool appearance of mice in different experimental groups. **** indicates that each group has significant difference with the model group in Dunnett's multiple comparison test, * p <0.0001.

[0070] Figure 2 The curves of the water content of feces of mice in different experimental groups are shown. **** indicates that each group has significant difference with the model group in Dunnett's multiple comparison test, * p <0.0001.

[0071] Figure 3 A schematic diagram showing the small intestinal propulsion rate of mice in different experimental groups.

[0072] Figure 4 The following table shows the statistical graphs of the small intestinal propulsion rate of mice in different experimental groups. **** indicates that each group has significant differences from the model group in Dunnett's multiple comparison test, * p <0.0001.

[0073] Figure 5 The Gas content in the serum of mice in different experimental groups is shown. Among them, *, *, **, **** respectively indicate that each group has significant differences compared with the model group in Dunnett's multiple comparison test, p The values ​​are* p <0.05,** p <0.01,*** p <0.001, **** p <0.0001.

[0074] Figure 6 The SP content in the serum of mice in different experimental groups is shown. Among them, *, *, **, **** respectively indicate that each group has significant difference with the model group in Dunnett's multiple comparison test, p The values ​​are* p <0.05,** p <0.01,*** p <0.001, **** p <0.0001.

[0075] Figure 7 The figure shows the MTL content in the serum of mice in different experimental groups. Among them, *, *, **, **** respectively indicate that each group has significant difference with the model group in Dunnett's multiple comparison test,p The values ​​are* p <0.05,** p <0.01,*** p <0.001, **** p <0.0001.

[0076] Figure 8 The VIP content in the serum of mice in different experimental groups is shown. Among them, *, *, **, **** respectively indicate that each group has significant difference with the model group in Dunnett's multiple comparison test, p The values ​​are* p <0.05,** p <0.01,*** p <0.001, **** p <0.0001.

[0077] Fig. 9 The 5-HT content in the serum of mice in different experimental groups is shown. Among them, *, *, **, **** respectively indicate that each group has significant difference with the model group in Dunnett's multiple comparison test, p The values ​​are* p <0.05,** p <0.01,*** p <0.001, **** p <0.0001.

[0078] Fig.10 The IL-10 levels in the serum of mice in different experimental groups are shown. Among them, *, *, **, **** respectively indicate that each group has significant differences compared with the model group in Dunnett's multiple comparison test, p The values ​​are* p <0.05,** p <0.01,*** p <0.001, **** p <0.0001.

[0079] Fig.11 The IL-4 levels in the serum of mice in different experimental groups are shown. Among them, *, *, **, **** respectively indicate that each group has significant differences compared with the model group in Dunnett's multiple comparison test, p The values ​​are* p <0.05,** p <0.01,*** p <0.001, **** p <0.0001.

[0080] Fig.12The figure shows the IL-1β content in the serum of mice in different experimental groups. Among them, *, *, **, **** respectively indicate that each group has significant difference with the model group in Dunnett's multiple comparison test, p The values ​​are* p <0.05,** p <0.01,*** p <0.001, **** p <0.0001.

[0081] Fig.13 The IL-6 content in the serum of mice in different experimental groups is shown. Among them, *, *, **, **** respectively indicate that each group has significant difference with the model group in Dunnett's multiple comparison test, p The values ​​are* p <0.05,** p <0.01,*** p <0.001, **** p <0.0001.

[0082] Fig.14 Shown are the results of H&E staining of the colon of mice in different experimental groups.

[0083] Fig.15 Shown are the results of H&E staining of the small intestine of mice in different experimental groups.

[0084] Fig.16 The relative expression levels of SCF mRNA in the small intestine of mice in different experimental groups are shown. Among them, *, *, **, **** respectively indicate that each group has significant differences compared with the model group in Dunnett's multiple comparison test, p The values ​​are* p <0.05,** p <0.01,*** p <0.001, **** p <0.0001.

[0085] Fig.17 The relative expression levels of eNOS mRNA in the small intestine of mice in different experimental groups are shown. Among them, *, *, **, **** respectively indicate that each group has significant differences compared with the model group in Dunnett's multiple comparison test, p The values ​​are* p <0.05,** p <0.01,*** p <0.001, **** p <0.0001.

[0086] Fig.18The relative expression levels of c-Kit mRNA in the small intestine of mice in different experimental groups are shown. Among them, *, *, **, **** respectively indicate that each group has significant differences compared with the model group in Dunnett's multiple comparison test, p The values ​​are* p <0.05,** p <0.01,*** p <0.001, **** p <0.0001.

[0087] Fig.19 The relative expression levels of SCF mRNA in the colon of mice in different experimental groups are shown. Among them, *, *, **, **** respectively indicate that each group has significant differences from the model group in Dunnett's multiple comparison test, p The values ​​are* p <0.05,** p <0.01,*** p <0.001, **** p <0.0001.

[0088] Fig. 20 The relative expression levels of eNOS mRNA in the colon of mice in different experimental groups are shown. Among them, *, *, **, **** respectively indicate that each group has significant differences from the model group in Dunnett's multiple comparison test, p The values ​​are* p <0.05,** p <0.01,*** p <0.001, **** p <0.0001.

[0089] Fig.21 The relative expression levels of c-Kit mRNA in the colon of mice in different experimental groups are shown. Among them, *, *, **, **** respectively indicate that each group has significant differences from the model group in Dunnett's multiple comparison test, p The values ​​are* p <0.05,** p <0.01,*** p <0.001, **** p <0.0001.

[0090] Fig. 22 The relative expression levels of iNOS mRNA in the small intestine of mice in different experimental groups are shown. Among them, *, *, **, **** respectively indicate that each group has significant differences compared with the model group in Dunnett's multiple comparison test, p The values ​​are* p<0.05,** p <0.01,*** p <0.001, **** p <0.0001.

[0091] Fig.23 The relative expression levels of iNOS mRNA in the colon of mice in different experimental groups are shown. Among them, *, *, **, **** respectively indicate that each group has significant differences with the model group in Dunnett's multiple comparison test, p The values ​​are* p <0.05,** p <0.01,*** p <0.001, **** p <0.0001. DETAILED DESCRIPTION

[0092] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0093] Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0094] The term "about" as used herein refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0095] Numerical ranges used herein should be understood to include all numbers within the range. For example, a range of 1 to 20 should be understood to include any number, combination of numbers, or subrange from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0096] As used herein, the term "comprises" or "comprising" means "including but not limited to". The term is intended to be open-ended to specify the presence of any stated features, elements, integers, steps, or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Thus, the term "comprising" encompasses the more restrictive terms "consisting of" and "consisting essentially of". In one embodiment, the term "comprising" used throughout the application, especially in the claims, may be replaced by the term "consisting of".

[0097] The terms "optional", "either", "any", or "any" as used herein mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur. As used herein, "a" and "an" are used herein to refer to one or more than one of the grammatical objects.

[0098] The term "and / or" used herein should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

[0099] The term "culture" as used herein refers to a living cell group or living growth of a microorganism in a certain time and space, such as a slant culture of a microorganism, a shake flask culture, etc. In some examples, it refers to a living microbial colony cultured and propagated under artificially specified conditions.

[0100] The term "medium" as used herein refers to a carbon source, a nitrogen source and an inorganic salt source that can be absorbed by microorganisms and support effective cell growth. The carbon source can include hexoses, such as glucose, and other easily absorbed sources, such as amino acids, can be used. Many inorganic and protein materials can be used as nitrogen sources during growth. Nitrogen sources can include amino acids and urea, as well as ammonia, inorganic nitrates and ammonium, vitamins, purines, pyrimidines, yeast extracts, beef extracts, proteose peptones, soy flour, casein hydrolyzates, smelt lees residues, etc. The inorganic minerals that can be incorporated into the nutrient medium are salts that can produce calcium, zinc, iron, manganese, magnesium, copper, cobalt, potassium, sodium, molybdate, phosphate, sulfate, chloride, borate and similar ions.

[0101] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiment. If the specific conditions are not specified in the embodiment, it is carried out according to the conditions of normal conditions or manufacturer's suggestion. If the manufacturer is not specified in all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention. Such structures and technologies are also described in the publication of many publications.

[0102] The main reagents, instruments or materials involved in the present invention are shown in Table 1.

[0103] Table 1

[0104]

[0105] Table 1 (continued)

[0106]

[0107] Example 1 Fermentation culture of Lactobacillus rhamnosus MBP01

[0108] The frozen suspension of Lactobacillus rhamnosus MBP01 was inoculated into the fermentation medium at an inoculum rate of 4.5% and fermented at 37°C for 24 h to obtain Lactobacillus rhamnosus fermentation liquid. The bacterial liquid was resuspended after centrifugation for subsequent use.

[0109] The fermentation medium comprises the following components in parts by weight: 10 parts of peptone, 8 parts of yeast extract powder, 20 parts of glucose, 3 parts of dipotassium hydrogen phosphate, 0.2 parts of magnesium sulfate, 3 parts of sodium acetate, 0.01 parts of manganese sulfate and 1000 parts of water.

[0110] Example 2 Effect of Lactobacillus rhamnosus MBP01 on defecation time and feces water content

[0111] 2.1 Animal model construction

[0112] Female Kunming mice (SCXK (Xiang) 2021-0002, Hunan Slake Jingda Animal Co., Ltd.) were randomly divided into 5 groups according to their body weight of about 25.5±2.5 g after one week of adaptive feeding, and named them as normal group, model (constipation) group, high-dose (Lactobacillus rhamnosus MBP01) group, low-dose (Lactobacillus rhamnosus MBP01) group and bisacodyl (positive drug) group, with 10 mice in each group, and the wet weight of each single feces was recorded.

[0113] The experimental period was 14 days. During the experimental period, all mice except the normal group were gavaged with ice water activated carbon every day to cause constipation in mice. From the first day, mice in other groups except the normal group were gavaged with 0.2 mL of ice water activated carbon (mass concentration of 100 g / L) every day. After the gavage on the 13th day, each group of mice fasted for 12 hours. At the same time, the high-dose group was gavaged with 1×10 9 CFU / kg bacterial suspension, low-dose group gavage 1×10 8 CFU / kg bacterial suspension, and the bisacodyl group was gavaged with 100 mg / kg bisacodyl every day. After daily gavage, fresh feces of each mouse were taken and weighed on an electronic balance, then placed in a drying oven set at 105°C for one day, taken out and cooled, and weighed repeatedly until constant weight was reached. The final dry weight of feces was recorded and the moisture content of feces (%) was calculated = (feces wet weight - feces dry weight) / feces wet weight × 100.

[0114] 2.2 Lactobacillus rhamnosus MBP01 can shorten defecation time and increase fecal water content

[0115] After intragastric administration on the 13th day, mice in each group were fasted for 12 h and intragastric administration was performed with 0.2 mL of red ink. The time of the first red stool of the mice was observed and recorded. On the 14th day, mice were intragastrically administered with 0.2 mL of red ink and then killed.

[0116] The results are as follows Figure 1 and Figure 2 As shown, compared with the normal group, the time for the first red stool to appear in the model group was significantly increased and the fecal water content continued to decrease ( p <0.0001). After intervention with Lactobacillus rhamnosus MBP01, the time for the first red stool to appear was significantly shortened ( p <0.0001), which was equivalent to the normal group; the feces water content increased significantly ( p <0.0001, but still lower than the normal group. The bisacodyl group maintained the same trend as the high-dose group, and the difference was statistically significant ( p >0.05). The above results show that Lactobacillus rhamnosus MBP01 can significantly shorten the defecation time of constipated mice and significantly increase the water content of feces of constipated mice, thereby alleviating the constipation reaction of mice.

[0117] Example 3 Effect of Lactobacillus rhamnosus MBP01 on small intestinal propulsion rate

[0118] When mice show symptoms of constipation, intestinal motility slows down, so the rate of small intestinal propulsion decreases.

[0119] The experimental grouping and animal model construction of this example are the same as those of Example 2.

[0120] After gavage on the 13th day, each group of mice fasted for 12 h and were gavaged with 0.2 mL of red ink. On the 14th day, the mice were gavaged with 0.2 mL of red ink again. After the mice were killed, they were dissected and the small intestine was removed. The total length of the small intestine and the length of the red ink in the small intestine were measured, and then the propulsion rate of the red ink in the small intestine (%) was calculated = (the distance of the red ink in the small intestine / the total length of the small intestine) × 100%.

[0121] The results showed that the average advancement rate of mice in the normal group was 89.13%, the advancement rate of mice in the model group was 29.69%, the advancement rate of mice in the bisacodyl group was 58.68%, the high-dose group was 80.87%, and the low-dose group was 43.47% ( Figure 3 and Figure 4 ). It was concluded that activated carbon would reduce the propulsion rate of the small intestine, causing constipation in mice, indicating that the animal model was successfully established. Bisacodyl and Lactobacillus rhamnosus MBP01 had a relieving effect on constipation, and the high-dose Lactobacillus rhamnosus MBP01 had a stronger effect than the bisacodyl group.

[0122] Example 4 Effect of Lactobacillus rhamnosus MBP01 on cytokine content

[0123] The experimental grouping and animal model construction of this example are the same as those of Example 2.

[0124] After killing the mice, orbital venous blood was collected, and the levels of motilin (MTL), gastrin (Gas), vasoactive intestinal peptide (VIP), substance P (SP), IL-4, IL-6, IL-10, 5-HT and IL-1β in mouse serum were measured using the Elisa kits in Table 1.

[0125] Gastrin (Gas) can promote the secretion of gastric acid and enzymes in pancreatic juice, reduce the tension of the pyloric sphincter and gastric antrum, and has a similar effect on promoting gastric emptying as MTL. It can also act on the specific receptors of smooth muscle cells to induce the contraction reaction of gastric antral smooth muscle. Gas secretion disorders may lead to gastrointestinal motility disorders (Xu L, Gao S, Guo F, et al. Effect of motilin on gastric distension sensitive neurons in arcuate nucleus and gastric motility in rat[J]. Neurogastroenterology&Motility the Official Journal of the European Gastrointestinal Motility Society , 2011, 23(3):265-e121). The results showed that the average Gas content in the serum of mice in the high-dose group was 67.36 pg / mL, which was significantly higher than that in the model group with the lowest Gas content in serum ( p<0.05), indicating that Lactobacillus rhamnosus MBP01 increased the content of Gas in the serum of constipated mice ( Figure 5 ).

[0126] Substance P (SP) belongs to the tachykinin family and is mainly distributed in the intestinal wall muscle layer (Li Lin, Ru Liqiang. Distribution pattern and relationship of ACh-ergic, SP, VIP-peptide-ergic and NO-ergic nerves in rat intestine [J]. Journal of Huazhong University of Science and Technology (Medical Edition). 2009, 38(5)). SP can accelerate gastrointestinal motility and gastrointestinal smooth muscle contraction, speed up gastrointestinal emptying, make the intestine more lubricated, and significantly increase the frequency of bowel movements (Bouchoucha M, Devroede G, Mary F, et al. Painful or Mild-Pain Constipation A Clinically Useful Alternative to Classification as Irritable Bowel Syndrome with Constipation Versus Functional Constipation [J]. Digestive Diseases and Sciences . 2018, 63(7):1763-1773). The results showed that compared with the normal group, the content of SP in the serum of the model group mice was significantly reduced ( p <0.05); the expression levels of serum SP in the high-dose group and the low-dose group were significantly higher than those in the model group ( p <0.05) ( Figure 6 ). It can be seen that Lactobacillus rhamnosus MBP01 increased the content of SP in the serum of constipated mice.

[0127] Motilin (MTL) is an active linear polypeptide composed of 22 amino acids. In the intestine, motilin is mainly secreted by Mo cells. When gastrointestinal motility increases, under pressure stimulation, Mo cells secrete motilin, and the plasma motilin content increases, leading to the production of MMC (Fan Yufeng. Effect of Xuanfei Bushen Yangyin Decoction on MTL and iNOS in rats with slow-transmission constipation [D]. North Sichuan Medical College, 2016). Insufficient secretion of motilin can lead to slow intestinal transit, which may be an important cause of constipation. The results showed that the MTL content in the serum of mice in the model group was the lowest, and was significantly lower than that in the normal group ( p <0.05); compared with the model group mice, the MTL content in the serum of the high-dose group and the low-dose group was significantly increased ( p <0.05) ( Figure 7 ). It can be seen that Lactobacillus rhamnosus MBP01 increased the content of MTL in the serum of constipated mice.

[0128] Vasoactive intestinal peptide (VIP) can inhibit gastric motility and gastric acid secretion, slowing down gastric emptying (Evangelista S, Publisher B S. Involvement of Tachykinins in Intestinal Inflammation[J]. Current Pharmaceutical Design , 2001, 7(1):19-30). Related studies have shown that the increased VIP content in the intestinal mucosa of constipated patients causes an inhibitory background of intestinal gastric motility, which causes constipation because intestinal peristalsis is not easy to occur (Ma Xiaosong, Fan Xueping, Chen Zhong, et al. Effects of Atractylodes macrocephala on gastrointestinal motility in animals and its mechanism [J]. Chinese Journal of Digestion. 1996(5): 261-264; Wang Yuan, Liu Zhibin, Niu Wenmin, et al. Effects of electroacupuncture at different acupoints on GAS and VIP in serum and tissues of rats with functional constipation [J]. Lishizhen Traditional Chinese Medicine. 2013(01): 242-244). The results showed that the average VIP content in the serum of mice in the model group was 252.15 pg / mL, which was significantly higher than that in the other four groups ( p <0.05); the levels of VIP in serum of the high-dose group and the low-dose group were 162.30 pg / mL and 198.73 pg / mL, respectively, and were significantly lower than those of the model group ( p <0.05) ( Figure 8 ). It can be seen that Lactobacillus rhamnosus MBP01 reduces the content of VIP in the serum of mice.

[0129] 5-HT, also known as serotonin, is a heterocyclic amine. The 5-HT in the human body is mainly present in the chromaffin cells of the gastrointestinal mucosa, accounting for about 80% of the total, and the rest is mainly distributed in platelets, pineal glands and 5-HT neurons in the brain (Li Guodong, Qiu Jianfeng, Xiao Cheng, et al. Effects of Yangxue Runchang Recipe on 5-HT, AchE, SP, and VIP in the colonic myenteric plexus of experimental mice with blood deficiency type chronic functional constipation [C]. Selected Outstanding Papers on Traditional Chinese Medicine of the Chinese Association of Traditional Chinese Medicine (Volume 1). 2009: 6). 5-HT can promote the contraction, proliferation and migration of vascular smooth muscle cells, participate in the regulation of blood pressure, activate platelets, induce platelet aggregation, stimulate the occurrence of mitosis, activate T, B, NK and phagocytes, participate in the neurotransmission of the central nervous system, and participate in the regulation of behavioral activities, emotions, appetite and body temperature together with other neurotransmitters (Wang Qiang. Detection and clinical significance of 5-hydroxytryptamine [J]. Foreign Medicine. Clinical Biochemistry and Laboratory Science, 2004 (02): 149-151). The results showed that the average 5-HT content in the serum of mice in the high-dose group was 197.97 pg / mL, which was significantly lower than that in the model group ( p <0.05) ( Fig. 9 ). It can be seen that Lactobacillus rhamnosus MBP01 reduces the content of 5-HT in the serum of mice.

[0130] Mouse interleukin 10 (IL-10) has an important anti-inflammatory effect. It can reduce the activity of myeloperoxidase (MPO), inhibit the activation of inflammatory cell NF-κB, and inhibit the production of other pro-inflammatory cytokines (Li Juan. TNF-α and IFN-γ regulate the inflammatory response of DSS-induced mouse enteritis by activating the NF-κB / STAT3 pathway of bone marrow mesenchymal stem cells [D]. Xiamen University, 2014). The results showed that compared with the model group mice, the high-dose group significantly increased the level of IL-10 expression in the serum of constipated mice ( p <0.05); the bisacodyl group had the same trend as the low-dose group, but the high-dose group was significantly better than the bisacodyl group ( Fig.10 ).

[0131] IL-4 is released by immune effector cells such as T cells, B cells, and eosinophils after binding to allergens. It can promote the secretion of IgE by activated B cells, promote the occurrence of inflammation and allergic reactions (Chen Wei, Ma Lei, Yang Lishan. Effects of glycyrrhetinic acid on IgE, IL-4 and TNF-α in rats with bronchial asthma [J]. Chinese Pharmacology and Clinical Medicine, 2015, 31(03): 52-55). The results showed that the average IL-4 content in the serum of mice in the model group was 221.15 pg / mL, which was significantly different from the other four groups ( p <0.05), indicating that ice water activated carbon can increase the content of IL-4 in the serum of mice, and bisacodyl and Lactobacillus rhamnosus can reduce the content of IL-4 in the serum of constipated mice ( Fig.11 ).

[0132] IL-1β is a pro-inflammatory factor that can promote the entry of inflammatory cells such as chemotactic neutrophils into the relevant lesions of the intestine through autocrine and paracrine pathways, promote the expression of leukocyte adhesion molecules in the vascular endothelium, and then cause intestinal tissue destruction and inflammatory response (Mulay SR, Kulkarni OP, Rupanagudi KV, et al. Calciumoxalate crystals induce renal inflammation by NLRP3-mediated IL-1β secretion[J]. Journal of Clinical Investigation . 2013, 123(1): 236-246). The results showed that compared with the normal group, the IL-1β content in the serum of the model group mice increased significantly ( p<0.05, while the expression levels of serum IL-1β in the bisacodyl group, high-dose group and low-dose group were significantly lower than that in the constipation group ( p <0.05) ( Fig.12 ). It can be seen that Lactobacillus rhamnosus MBP01 reduces the content of IL-1β in the serum of mice.

[0133] There are many target cells for mouse interleukin 6 (IL-6), mainly including resting T cells, macrophages, hepatocytes, activated B cells and plasma cells. Its main functions include stimulating the proliferation of activated B cells to secrete antibodies; stimulating T cell proliferation and CTL activation; stimulating hepatocytes to synthesize acute phase proteins and participate in inflammatory response; promoting the development of blood cells; promoting the osteoblastic differentiation and calcium formation of human aortic valve interstitial cells (Ban Jinbao, Liu Donghong, Tao Yu'an, Sun Fuqiang. Study on interleukin-6-induced osteoblastic differentiation of human aortic valve interstitial cells [J]. Chinese Journal of Experimental Surgery. 2020). The results showed that the average IL-6 content in the serum of mice in the model group was 312.46 pg / mL, and was significantly higher than that in the other four groups ( p <0.05), the average IL-6 content in the serum of mice in the high-dose group was 173.95 pg / mL, and was significantly lower than that in the other three groups except the normal group ( p <0.05) ( Fig.13 ). This indicates that Lactobacillus rhamnosus MBP01 reduces the content of IL-6 in the serum of mice.

[0134] Example 5 Effects of Lactobacillus rhamnosus MBP01 on intestinal function

[0135] The experimental grouping and animal model construction of this example are the same as those of Example 2.

[0136] After the experiment, the mice were killed and all the materials in the colon and small intestine were carefully removed. After washing them with saline, 0.5 cm of the colon and small intestine were cut off and fixed in 10% formalin and H&E-stained sections were made. The remaining parts were stored in a -80℃ refrigerator for backup.

[0137] 5.1 Effect of Lactobacillus rhamnosus MBP01 on colonic inflammation

[0138] The results of H&E staining of the colon showed that the colon mucosa of the normal group and the bisacodyl group was smooth and neatly arranged, with no obvious pathological changes. However, the colon mucosa of the constipation group, which was only gavaged with ice water activated carbon, showed obvious inflammation ( Fig.14 The colon mucosa of the high-dose group and the low-dose group showed mild inflammation ( Fig.14 ). This shows that Lactobacillus rhamnosus MBP01 has the effect of alleviating inflammation in mice.

[0139] 5.2 Effects of Lactobacillus rhamnosus MBP01 on small intestinal villi

[0140] When the small intestinal villi are damaged, the intestinal motility will be affected to varying degrees, and slow intestinal motility is one of the factors that cause constipation, so the integrity of the small intestinal villi is also important for evaluating constipation (Qian Yu. Effects of resistant starch on the physiological environment of the intestine and gastrointestinal function of experimental mice [D]. Southwest University, 2013). The results showed that the intestinal villi of the normal group were distributed more evenly, with almost no breakage, but obvious breakage occurred in the model group ( Fig.15 Although the intestinal villi in both the high-dose and low-dose groups were damaged to varying degrees, they were more intact than those in the model group, and the villi in the high-model group were almost the same as those in the normal group and the bisacodyl drug group ( Fig.15 ).

[0141] Example 6 Effect of Lactobacillus rhamnosus MBP01 on mRNA expression levels of constipation-related genes

[0142] The experimental grouping and animal model construction of this example are the same as those of Example 2.

[0143] 6.1 Extraction of RNA from mouse colon and small intestine tissues

[0144] Weigh 50 mg of small intestine or colon tissue and place it in a sterile homogenization tube containing homogenization beads. Add 1 mL of TRizol to the homogenization tube containing the sample in a fume hood and homogenize it with a homogenizer for 8 minutes. After the homogenization is completed, observe whether it is completely homogenized. If it is not completely homogenized, homogenize it again. After the homogenization is complete, add 200 μL of chloroform to the homogenization tube in the fume hood, tighten the homogenization tube, shake it up and down vigorously, and the color will fade from purple to pink. Place the sample on ice for 15-30 minutes. After standing, the homogenate was pipetted into a 1.5 mL centrifuge tube, placed symmetrically in a centrifuge, and centrifuged at centrifugation parameters (14000 r / min, 15 min, 4°C). After the centrifugation, the supernatant was transferred to a sterilized 1.5 mL centrifuge tube with a pipette and the same volume of isopropanol as the supernatant was added to the centrifuge tube. The tube was gently shaken up and down. After the sample was placed on ice for 15 min, it was placed symmetrically in a centrifuge and centrifuged again at centrifugation parameters (14000 r / min, 20 min, 4°C). After the supernatant was poured out, the yellow-white precipitate at the bottom of the centrifuge tube was RNA. Add 500 μL of 75% ethanol to the centrifuge tube containing RNA precipitate, tap the RNA precipitate on the tube wall with a pen to loosen it, and then place it in a centrifuge and centrifuge at centrifugation parameters (14000 r / min, 15 min, 4°C). After centrifugation, use a pipette to aspirate the liquid, and gently wipe the inner wall of the second batch of centrifuge tubes with sterilized dust-free paper. Be careful not to touch the RNA precipitate. Leave it open. After the alcohol evaporates, add 20 μL of sterile enzyme-free water (DEPC) to the centrifuge tube and blow the RNA precipitate (do not touch the precipitate) until there is no obvious precipitate at the bottom of the centrifuge tube and there is no liquid residue in the tip of the gun. Add 49 μL of DEPC and 1 μL of RNA to a 200 μL centrifuge tube and mix well. Use an ultra-micro spectrophotometer to measure and record its content and purity in parallel, and then use the total RNA as a template to reverse transcribe and synthesize cDNA.

[0145] 6.2 Detection of mRNA expression of related indicators in mouse colon and small intestine tissues

[0146] Centrifuge at 14000 r / min, 4℃ for 10 min, dilute the forward and reverse primers with filtered enzyme-free water, add equal proportions of SYBR, forward and reverse primers and sterile ultrapure water according to the volume size as required by the instructions and mix evenly, take 18μL of the mixed solution into eight tubes. Add 2 μL of the corresponding cDNA according to the set layout, cover the lid and centrifuge the eight tubes until there are no bubbles. Fluorescence PCR was used to detect the relative expression levels of mRNA of related genes (iNOS, SCF, C-Kit, eNOS and GAPDH). The primers for detecting related genes are shown in Table 2.

[0147] Table 2

[0148]

[0149] c-kit is a specific marker of gastrointestinal pacemaker cells ICC. The signaling pathway initiated by its binding to its ligand stem cell factor (SCF) is of great significance to the development, differentiation and phenotypic maintenance of ICC (Chen Lijun, Zhang Tingmo, Peng Cheng. Effects of different preparations of rhubarb on the expression of c-kit mRNA in the colon of rats with heat-constipation model [J]. Chinese Pharmacology and Clinical Medicine, 2009, 25(04): 37-40). SCF, also known as mast cell growth factor (MGF), is the natural ligand of c-Kit. The SCF / c-Kit signaling pathway plays a vital role in the proliferation, differentiation and phenotypic maintenance of ICC (Davis B H. Roles of stem cell factor on the depletion of interstitial cells of Cajal in the colon of diabetic mice. [J]. Am J Physiol Gastrointest Liver Physiol , 2010, 298(1): 241-247). iNOS is expressed only when cells are stimulated and activated. iNOS catalyzes the synthesis of large amounts of non-physiological NO, producing a series of pathological effects (Zhang Ning, Ma Tengfei, Wang Yeqiu et al. Study on the intervention effect of Smilax glabra on constipation-type irritable bowel syndrome in rats [J]. Chinese Pharmacology and Clinical Medicine, 2012, 28(04): 63-65). Endothelial nitric oxide synthase (eNOS) is expressed in the normal physiological state of the body and is used to maintain the normal physiological function of the body (Zhang J, Zhou X, Chen B, et al. Preventive Effect of Lactobacillus Plantarum CQPC10 on Activated CarbonInduced Constipation in Institute of Cancer Research (ICR) Mice [J]. Applied Sciences . 2018, 8(9): 1498).

[0150] The results showed that the expression levels of c-Kit, SCF, and eNOS mRNA in the model group were the lowest, and those in the normal group, bisacodyl group, high-dose group, and low-dose group were significantly higher than those in the model group ( p<0.05, and the high-dose group was higher than the low-dose group ( Figure 16-Figure 22 ). Lactobacillus rhamnosus MBP01 can increase the mRNA expression of c-Kit, SCF, and eNOS in the colon and small intestine tissues of constipated mice.

[0151] However, in the expression of iNOS mRNA, the expression level of the model group mice was the highest and significantly higher than that of the normal group, bisacodyl group, high-dose group, and low-dose group ( p <0.05), the order of iNOS mRNA expression in the five groups from high to low was model group > low-dose group > bisacodyl group > high-dose group > normal group ( Fig.23 ). It can be seen that constipation can cause a significant increase in the expression of iNOS mRNA in mice, and bisacodyl and Lactobacillus rhamnosus MBP01 can reduce the expression of iNOS mRNA in constipated mice.

[0152] In summary, Lactobacillus rhamnosus MBP01 relieves constipation caused by ice water activated carbon in mice by regulating cytokine content and mRNA expression levels of constipation-related genes, accelerating the secretion of gastric acid and enzymes in the pancreas, promoting gastric peristalsis and gastric acid secretion, accelerating gastrointestinal motility, accelerating intestinal smooth muscle contraction, and inhibiting the activity of inflammatory cells.

[0153] Comparative Example

[0154] The strain used to study the effect of constipation in Comparative Example 1 was Lactobacillus rhamnosus 4F225 with a preservation number of CGMCC No. 26437. The constipation modeling process and the dosage (1×10 9 The CFU / kg) were the same as those in Example 1, except for the specific strains.

[0155] Experimental results: The average IL-6 content in the serum of mice in the model group was 312.46 pg / mL, the average IL-6 content in the serum of mice in the high-dose group of Lactobacillus rhamnosus with a preservation number of GDMCCNO:64725 was 173.95 pg / mL, and the average IL-6 content in the serum of mice in the Lactobacillus rhamnosus 4F225 group with a preservation number of CGMCC No. 26437 was 283.30 pg / mL, which was not significantly different from the model group.

[0156] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. Use of Lactobacillus rhamnosus in the preparation of a product for preventing, treating and / or assisting in the treatment of constipation, characterized in that: The deposit number of the Lactobacillus rhamnosus is GDMCC NO:64725.

2. The use according to claim 1, characterized in that: The product includes a medicine or a food.

3. The use according to claim 1, characterized in that: Each unit of the product contains at least 1×10 8 CFU of the Lactobacillus rhamnosus described in claim 1.

4. The use according to claim 1, characterized in that: The product further comprises at least one physiologically acceptable auxiliary material or pharmaceutically acceptable excipient.

5. The use according to claim 2, characterized in that: The food is a health food.

6. The use according to claim 5, characterized in that: The health food includes nutrient supplements or functional health food.

7. The use according to claim 5, characterized in that: The functional health food is a functional health food that helps to moisten the intestines and promote bowel movements.

8. The use according to claim 5, characterized in that: The dosage forms of the health food include tablets, capsules, soft capsules, granules, pills, gel candies, powders, oral liquids or drops.

9. The use according to claim 5, characterized in that: The health food further comprises a second probiotic for relieving constipation and / or promoting digestion.

10. The use according to claim 2, characterized in that: The medicament may also include a second therapeutic agent for promoting digestion, and / or preventing, treating or assisting in the treatment of constipation.

11. A product for preventing, treating and / or assisting in the treatment of constipation, characterized in that: The product contains Lactobacillus rhamnosus, and the preservation number of the Lactobacillus rhamnosus is GDMCC NO:64725.

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