LU502579B1 - Bifidobacterium lactis for relieving constipation and application thereof - Google Patents
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- LU502579B1 LU502579B1 LU502579A LU502579A LU502579B1 LU 502579 B1 LU502579 B1 LU 502579B1 LU 502579 A LU502579 A LU 502579A LU 502579 A LU502579 A LU 502579A LU 502579 B1 LU502579 B1 LU 502579B1
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- constipation
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- bifidobacterium lactis
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- 206010010774 Constipation Diseases 0.000 title claims abstract description 74
- 241000901050 Bifidobacterium animalis subsp. lactis Species 0.000 title claims abstract description 27
- 229940009289 bifidobacterium lactis Drugs 0.000 title claims abstract description 27
- 230000000968 intestinal effect Effects 0.000 claims abstract description 37
- 210000003608 fece Anatomy 0.000 claims abstract description 30
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 18
- 230000013872 defecation Effects 0.000 claims abstract description 13
- 210000001035 gastrointestinal tract Anatomy 0.000 claims abstract description 13
- 230000008855 peristalsis Effects 0.000 claims abstract description 11
- 229960002983 loperamide hydrochloride Drugs 0.000 claims abstract description 10
- PGYPOBZJRVSMDS-UHFFFAOYSA-N loperamide hydrochloride Chemical compound Cl.C=1C=CC=CC=1C(C=1C=CC=CC=1)(C(=O)N(C)C)CCN(CC1)CCC1(O)C1=CC=C(Cl)C=C1 PGYPOBZJRVSMDS-UHFFFAOYSA-N 0.000 claims abstract description 10
- 210000001809 melena Anatomy 0.000 claims abstract description 10
- 150000004666 short chain fatty acids Chemical class 0.000 claims abstract description 4
- 238000009629 microbiological culture Methods 0.000 claims abstract description 3
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/745—Bifidobacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/10—Laxatives
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/51—Bifidobacterium
- A23V2400/531—Lactis
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
Disclosed is Bifidobacterium lactis TY-S01 for relieving constipation. The strain is deposited with the China General Microbiological Culture Collection Center (CGMCC) on November 27, 2020, and assigned the accession number CGMCC No.21255. Through an animal validation experiment, it is confirmed that TY-S01 can relieve loperamide hydrochloride-induced constipation in mice, shorten the first defecation time of melena, increase water content in feces, accelerate intestinal peristalsis, and effectively inhibit damage of small intestinal villi. In addition, the TY-S01 significantly elevates levels of acetic acid, propionic acid, butyric acid, isobutyric acid, and valeric acid in feces of mice. Finally, the TY-S01 may relieve constipation by elevating SCFAs levels of the intestinal tract. The Bifidobacterium lactis TY-S01 provided in the present invention may increase the content of short-chain fatty acid in the intestinal tract of the mice with constipation, so that the constipation can be relieved. As the Bifidobacterium lactis TY-S01 has no side effects and desirable effects, a new treatment idea can be provided for patients with constipation.
Description
BIFIDOBACTERIUM LACTIS FOR RELIEVING CONSTIPATION AND
APPLICATION THEREOF “ >%20502579
The present invention relates to the technical field of microorganisms, and in particular, to
Bifidobacterium lactis for relieving constipation and an application thereof.
Constipation is a common chronic disease and a common clinical digestive system disease, which is mainly manifested as decreased defecation, dry feces, difficulty defecation, and decreased frequency of defecation. Due to the long course of the disease and persistent and repeated symptoms, it brings great pain to patients. The incidence of constipation in the population is as high as 27%, and people in different regions, genders and ages may be affected by constipation. Long-term defecation difficulties may cause hemorrhoids, flatulence, insomnia, endocrine disorders, obesity and blood loss anemia, and the like. In severe cases, it may induce or exacerbate other diseases, such as cardiovascular disease, kidney disease, Parkinson's disease and colorectal cancer.
For constipation symptoms, clinically, drugs or surgery are usually used to treat constipation.
Although drug therapy has a certain effect, long-term medication may cause drug resistance, dependence and other complications in the body, for example, drug dependence, nausea and severe diarrhea. Surgical treatment can get good results temporarily, but the treatment only treats the symptoms and not the cause, so that it is very easy to relapse if people do not pay attention at ordinary times.
Therefore, in the process of treating constipation, the above deficiencies still exist in the prior art, so that it is particularly important to find a constipation treatment concept with high safety, desirable curative effect and less toxic and side effects.
The present invention is intended to provide Bifidobacterium lactis TY-SO1 for relieving constipation. 16S rDNA homology analysis results show that TY-S01 is bifidobacterium lactis.
Through a validation experiment, it is confirmed that TY-S01 can relieve loperamide hydrochloride-induced constipation, shorten the first defecation time of melena, increase water content in feces, accelerate intestinal peristalsis, and effectively inhibit damage of small intestinal villi. In addition, the TY-S01 significantly elevates levels of acetic acid, propionic acid, butyric acid, isobutyric acid, and valeric acid in feces. Finally, the TY-SO1 may relieve constipation by elevating short-chain fatty acid levels of the intestinal tract.
Separation and purification of bifidobacterium lactis: a feces sample from the PPÉ0502579 longevous in Bama, Guangxi without urine contamination is collected. The sample is diluted with sterile saline, and then spread on an MRS solid medium added with cysteine. After anaerobic culture, single colonies with different morphology are selected, and a streak plate method is adopted to isolate strains. Then, a single colony is selected from the streak-purified plate to continue perform streaking purification, so as to obtain purified strains.
Colonial morphology and cell morphology of the isolated strain: The strains form a single colony in solid medium after purification, and the colony is consistent in morphology, hemispherical in shape, white, smooth and moist in surface, and flat in edge. After gram staining, purple cell morphology with a rod-like shape is observed under a microscope, which is determined as gram-positive bacteria (G").
PCR amplification of a 16S rDNA sequence: PCR amplification is performed on the purified strains by using a 25 pL of a reaction system. After sequence amplification, Sangon
Biotech (Shanghai) Co., Ltd. is entrusted to sequence qualified PCR amplification products.
After the sequence is obtained, searching and similarity comparison are performed in GenBank by using BLAST (http://www.ncbi.nlm.nih.gov/BLAST).
Analysis of a 16S rDNA sequence of the strain: Sequence alignment is performed in a
GenBank database, and the 16S rDNA homology analysis results show that TY-SO1 is
Dbifidobacterium lactis. A sequence of a 16S rDNA gene amplification product of the
Bifidobacterium lactis TY-S01 is shown as SEQ ID No.1.
Preservation information:
The Bifidobacterium lactis TY-S01 for relieving constipation described in the present invention has been deposited, and detailed preservation information is shown as follows.
Depositary authority: China General Microbiological Culture Collection Center (CGMCC);
Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;
Preservation date: November 27, 2020;
Accession number: CGMCC No.21255; and
Classification name: Bifidobacterium lactis TY-SO1.
A principle of the present invention includes the following. Some microorganisms, such as bifidobacteria and lactobacilli, are less in side effect and effective, and may benefit the body in many aspects, so the microorganisms have become a new treatment concept in the treatment of constipation. Bifidobacterium is an important part of colonic flora, and its mechanism of regulating constipation is related to own metabolites regulating intestinal peristalsis, regulating intestinal flora and other factors affecting intestinal environments. Short-Chain Fatty Acids
(SCFAs) are the metabolites of probiotics such as bifidobacterium. The SCFAs are not only related to some physiological metabolic processes, but also play an irreplaceable role Le PPÉ0502579 maintaining intestinal health. In an aspect, the SCFAs are acidic metabolites that can reduce the pH value in the intestinal lumen, thereby regulating normal intestinal peristalsis. In another aspect, different SCFAs have different functions in relieving constipation. Acetic acid is the main energy source of colonic mucosal cells and may promote intestinal peristalsis by promoting the absorption of water and electrolytes. Propionic acid, butyric acid and valeric acid may induce the contraction of the mid- and distal colon and may directly act on smooth muscle, thereby relieving constipation.
Beneficial effects of the present invention are as follows. The Bifidobacterium lactis
TY-S01 for relieving constipation with the accession number CGMCC No.21255 provided in the present invention may increase the content of SCFAs in the intestinal tract, so that the constipation can be relieved. As the Bifidobacterium lactis TY-SO01 has no side effects and desirable effects, a new treatment idea can be provided for patients with constipation.
In order to more clearly illustrate the technical solutions in the embodiments of the present invention or prior art, the drawings used in the technical description of the embodiments will be briefly described below. It is apparent that the drawings in the following descriptions are merely some embodiments of the present invention. Other drawings can be obtained from those skilled in the art according to these drawings without any creative work.
Fig. 1 is a colonial morphology diagram of isolated strains.
Fig. 2 is a result diagram of gram staining.
Fig. 3 is a standard chromatogram when SCFAs content in mouse feces is detected by using gas chromatography.
Fig. 4 is a statistical diagram showing changes in weight, food ration, and water intake of the mice during experiment.
Fig. 5 is a defecation situation diagram of mice. (A) in Fig. 5 is a comparison diagram of time of first melena in each group. (B) in Fig. 5 is a comparison diagram of water content in feces in each group. (C) in Fig. 5 is a comparison diagram of an activated carbon propulsion distance of mice in each group.
Fig. 6 is a complete comparison diagram of small intestinal villi of mice in each group.
Fig. 7 is a situation diagram of changes in levels of acetic acid, propionic acid, butyric acid, — isobutyric acid, and valeric acid in the intestinal tract during experiment.
(A), (D), (G), (J), and (M) in Fig. 7 are respectively situation diagrams of changes in levels of acetic acid, propionic acid, butyric acid, isobutyric acid, and valeric acid in a normal group PPÉ0502579 and a TY-S01 group during 1 to 14d of an experiment. (B), (E), (H), (K), and (N) in Fig. 7 are respectively situation diagrams of changes in levels of acetic acid, propionic acid, butyric acid, isobutyric acid, and valeric acid in a constipation group and a TY-S01 group during 14 to 17d of the experiment. ©), (F), (D, (L), and (0) in Fig. 7 are respectively level diagrams of acetic acid, propionic acid, butyric acid, isobutyric acid, and valeric acid in mice in each group at the last day (17d) of a modeling phase.
The technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the drawings in the embodiments of the present invention. It is apparent that the described embodiments are only part of the embodiments of the present invention, not all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skilled in the art without creative work shall fall within the protection scope of the present invention.
Embodiment 1
Separation, purification and identification of TY-S01 1. Experimental material
A feces sample from the longevous in Bama, Guangxi is collected. Feces without urine contamination is collected, a spoon of a feces sampler is used to collect 4 to 5 spoons (about 20 grams) of feces to put into the 20 mL feces sampler containing 30% glycerol, and a cap is tightened up. 2. Separation and purification of TY-S01 1 mL of sample is pipetted to 9 mL of sterile saline to obtain 10! diluent. Then, 1 mL of 10"! diluent is pipetted to 9 mL of the sterile saline, to obtain 10? diluent. According to the operation, 107 diluent, 10“ diluent, 10-3 diluent, and 10° diluent are successively obtained. 200 pL of the diluent with different dilutions (107, 104, 10-5, and 10°) is separately spread on an MRS solid medium added with 0.05% cysteine for anaerobic culture at 37°C for 48-72h.
Colonial morphology on the plate is observed. Single colonies with different morphology are selected, and a streak plate method is adopted to isolate strains. Then, a single colony is selected from the streak-purified plate to continue perform streaking purification, so as to obtain purified strains. The streaking operation is repeated until purified strains are obtained. Then, morphological observation is performed by means of gram staining.
3. PCR amplification of a 16S rDNA sequence
PCR amplification is performed by using a 25 uL of a reaction system, including 1 pL ofa PPÉ0502579 template, 1 pL of an upstream primer (10 uM), 1 pL of a downstream primer (10 uM), and 12.5 uL of 2xTaq PCR Master Mix, and making up to 25 uL with sterile ultrapure water. 5 PCR amplification conditions include: performing pre-denaturation at 94°C for 5 min, performing denaturation at 94°C for 30 s, performing annealing at 55°C for 30 s, and performing extension at 72°C for 1 min, where there is a total of 35 cycles; and performing end extension at 72°C for 10 min. After sequence amplification, Sangon Biotech (Shanghai) Co., Ltd. is entrusted to sequence qualified PCR amplification products. After the sequence is obtained, searching and similarity comparison are performed in GenBank by using BLAST (http://www.ncbi.nlm.nih.gov/BLAST). 4. Experimental result and analysis 4.1. Colonial morphology and cell morphology of the isolated strain
The strains form a single colony in solid medium after purification, and the colony is consistent in morphology, hemispherical in shape, white, smooth and moist in surface, and flat in edge. After gram staining, purple cell morphology with a rod-like shape is observed under a microscope, which is determined as gram-positive bacteria (G”). The colonial morphology of the strain and a gram staining result are shown in Fig. 1 to Fig. 2. Fig. 1 is a colonial morphology diagram of isolated strains. Fig. 2 is a result diagram of gram staining. 4.2. Analysis of a 16S rDNA sequence of the strain
Sequence alignment is performed in a GenBank database, and the 16S rDNA homology analysis results show that TY-SO1 is bifidobacterium lactis. A sequence of a 16S rDNA gene amplification product of the Bifidobacterium lactis TY-S01 is shown as SEQ ID No.1.
Embodiment 2
A relieving effect of TY-S01 on constipation 1. Experimental material
The Bifidobacterium lactis TY-S01 for relieving constipation is isolated from the intestinal tract of the longevous in Bama, Guangxi, is deposited with the CGMCC, and is assigned the accession number CGMCC No.21255. 2. Animal validation experiment 2.1. Grouping and treatment of experimental animals 7-Week old BALB/c male mice (20 g to 25 g) are selected and fed in a standardization laboratory with at room temperature being 25+2°C, relative humidity being 50+5% and a condition of 12h light/12h dark. Experiments are started after a week of adaptive feeding. After an adaptive phase ends, the mice are randomly grouped into a normal group, a constipation group, and a TY-S01 group, and there are 10 mice in each group.
An experimental period is 18 days, and a TY-SO1 intervention duration is 1-14d. The PPÉ0502579 normal group and the constipation group are treated with 10mL/kg.BW of normal saline; and the
TY-S01 group is treated with 10°CFU/kg.BW of TY-S01 bacterial fluid, and an intragastric administration volume is the same as that of the normal group and the constipation group. A modeling experiment in constipation mice starts at Day 15. 10mg/kg-BW of a loperamide hydrochloride solution is given to the mice in the groups in addition to the normal group; 10mL/kg.BW of the normal saline is given to the mice in the normal group. After 1h interval, 10mL/kg.BW of the normal saline is given to the mice in the normal group and a model group; and 10°CFU/kg.BW of the TY-S01 bacterial fluid is given to the mice in the TY-S01 group. The experiment lasts 3d until Day 17. After intragastric administration is completed, all of the mice start to fast. After 16h of fasting (Day 18), 10mL/kg.BW of an activated carbon solution is given to all of the mice. 5 mice are taken from each group for measuring the time when melena is defecated for the first time (after the first melena is defecated, the mice are immediately dissected for blood and tissue). The remaining 5 mice are killed and dissected 30 min after the activated carbon solution is given, for the observation of an intestinal propulsion distance of the mice. 2.2. Growth performance monitoring of mice during experiment
The weight, food ration, and water intake of the mice are recorded every day within 17d of the experiment. 2.3. Collection of mouse feces and measurement of water content in feces
Feces of the mice in each group within a probiotic intervention duration (1d, 7d, and 14d) and a constipation modeling phase (15d, 16d, and 17d) is collected to measure the SCFAs in the feces. In addition, the water content in feces at Day 17 is measured according to the following formula.
Water content(%) = Weight of Fresh Weight ar feces after constant Weight 2.4. Histopathological section observation of small intestine tissue
HE staining is performed on the fixed small intestine tissue after dehydration, clearing, paraffin immersing, embedding, and slicing, and then histopathological changes are observed under a microscope (50x). 2.5. Detection of SCFAs content in mouse feces using gas chromatography 500 pL of a saturated NaCl solution is added in a 50 mg of mouse feces sample, and is shaken until there are no obvious lumps (a tissue crusher, 60HZ, 30s, repeating 5 times). 40 uL of 10% sulfuric acid is added to acidify, shaken and well mixed. Then, 1000 pL of ether is added for extraction, shaken and well mixed, and centrifugation is performed for 15 min at 4°C and
12000rpm. Supernatant is taken and added to an EP tube containing 0.25 g of anhydrous sodium sulfate to stand for 15 min, and centrifugation is also performed with same conditions. (0002579
Supernatant is taken and added to a gas-phase vial for analysis. Gas chromatographic detection conditions are shown in Table 1 below.
Table 1 Gas chromatographic detection condition table
Chromatographic 100°C for 5 min, a temperature is raised to 250°C at 10°C/min for 12 min program
Ee [mf 300°C Inlet temperature 250°C temperature 30mL/min Air flow rate 400mL/min rate
Acetic acid, propionic acid, butyric acid, isobutyric acid and valeric acid in mouse feces are detected by means of gas chromatography. A SCFAs concentration is calculated by means of an external standard method. The standard chromatogram is shown in Fig. 3. 3. Experimental result and analysis 3.1. Changes in weight, food ration, and water intake of the mice during experiment
Changes in the weight, food ration, and water intake of the mice can reflect their health conditions to a certain extent. À statistical diagram showing changes in weight, food ration, and water intake of the mice during experiment is specifically shown in Fig. 4.
It may be learned from Fig. 4 that, during the entire experiment, there is not obvious difference (P < 0.05) in the weight, food ration and water intake of the mice in each group, indicating that the mice had desirable growth performance during the construction of a mouse constipation model by loperamide hydrochloride and the prevention of TY-S01 on constipation in mice, and this experiment does not affect the normal life and growth of the mice. 3.2. Prevention and improvement effects of TY-S01 on loperamide hydrochloride-induced constipation in mice
The time when mice first excrete melena is an important indicator to measure the defecation situation of the mice, which plays an important role in the evaluation of constipation in mice.
The defecation situation of the mice is specifically shown in Fig. 5. In Fig. 5, "*" represents that PPÉ0502579 there is a statistical difference (p < 0.05) between two groups. (A) in Fig. 5 is a comparison diagram of time of first melena in each group. Dry feces are one of the main symptoms of constipation and an important indicator for evaluating the degree of constipation. It may be learned from (A) in Fig. 5 that, the defecation time of the first melena in the constipation group is significantly more than that in normal group, indicating that the loperamide hydrochloride-induced constipation mouse model is succeeded. Therefore, the
TY-S01 can significantly relieve constipation in mice and shorten the defecation time of the first melena (P < 0.05). (B) in Fig. 5 is a comparison diagram of water content in feces in each group. The propulsion of activated carbon in the small intestine may directly reflect the intestinal peristalsis function of a body. It may be learned from (B) in Fig. 5 that, TY-SO1 intervention can significantly increase the water content in feces (P < 0.05), thereby improving the bulkiness of feces. (C) in Fig. 5 is a comparison diagram of an activated carbon propulsion distance of mice in each group. The propulsion of activated carbon in the small intestine may directly reflect the intestinal peristalsis function of a body. It may be learned from (C) in Fig. 5 that, at the same time, compared with normal mice, mice with constipation have a shorter activated carbon propulsion distance, and this phenomenon can be reversed through intragastric administration of
TY-S01, so as to prolong the propulsion distance.
The results of the defecation of the mice in each group show that the loperamide hydrochloride can cause constipation in mice, and TY-S01 has the effect of preventing and relieving constipation. 3.3. Impact of TY-S01 on intestinal villi of mice with constipation
The function of intestinal peristalsis is closely related to the integrity of the small intestinal villi, and patients with constipation are often accompanied by different degrees of small intestinal villi damage. A complete comparison diagram of small intestinal villi of mice in each group is shown in Fig. 6.
It may be learned from Fig. 6 that, the small intestinal villi of the mice in the normal group are uniform and neat without damage, while the small intestinal villi in the model group are severely damaged, such as breakage and atrophy, and inflammatory infiltration occurred in the intestinal lumen. After TY-S01 intervention, the damage of small intestinal villi is uniform, neat and complete, indicating that TY-SO1 intervention may effectively inhibit the damage of small intestinal villi and protect the small intestinal villi.
3.4. Impact of TY-S01 on SCFAs levels of intestinal tract of mice with constipation
The SCFAs are the final metabolites produced by bacterial fermentation of indigestible PPÉ0502579 carbohydrates, including acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid, among which more than 90% of the SCFAs are acetic acid, propionic acid, and butyric acid. The SCFAs participate in the normal physiological activities of the human body, are related to blood glucose homeostasis, lipid metabolism, appetite regulation, immune regulation and neuromodulation, and can promote the normalization of intestinal peristalsis. Therefore, determination of intestinal SCFAs levels can evaluate the impact of TY-S01 on the intestinal microenvironment of mice with constipation. Different SCFAs have different effects in preventing constipation. The scetic acid is the SCFAs with the largest content. As the main product of intestinal fermentation, the acetic acid may increase the osmotic pressure in the intestinal tract, allowing water to enter the intestinal tract, thereby increasing the water content in the feces and stimulating the intestinal tract to excrete feces. The butyric acid may affect intestinal motility by increasing the concentration of regulatory T cells. Flevated levels of the isobutyric acid facilitate the acceleration of peristalsis and the excretion of feces. The propionic acid, the butyric acid and the valeric acid may relieve constipation by inducing contractions of the mid- and distal colon and directly acting on smooth muscle.
Fig. 7 is a situation diagram of changes in levels of acetic acid, propionic acid, butyric acid, isobutyric acid, and valeric acid in the intestinal tract during experiment. (A), (D), (G), (J), and (M) in Fig. 7 are respectively situation diagrams of changes in levels of acetic acid, propionic acid, butyric acid, isobutyric acid, and valeric acid in a normal group and a TY-S01 group during 1 to 14d of an experiment. It may be learned from (A), (D), (G), (I), and (M) in Fig. 7 that, during 1-14d of the experiment, the mice are not modeled for constipation, and the levels of acetic acid, propionic acid, butyric acid, isobutyric acid, and valeric acid remained basically unchanged in the mice in the normal group within 1-14d. During this period, the levels of SCFAs in a TY-S01 intervention group show an upward trend and are always higher than those in the normal group, indicating that continuous supplementation of TY-S01 in the mice under normal conditions may increase intestinal SCFA levels, thereby having beneficial effects on health.
At Day 15 to Day 17 of the experimental period, the mice in the constipation group and the
TY-S01 group are given constipation modeling drugs through intragastric administration. (B), (E), (H), (K), and (N) in Fig. 7 are respectively situation diagrams of changes in levels of acetic acid, propionic acid, butyric acid, isobutyric acid, and valeric acid in a constipation group and a TY-S01 group during 14 to 17d of the experiment. It may be learned from (B), (E), (H), (K), and (N) in Fig. 7 that, after the mice received the modeling drug loperamide hydrochloride, the intestinal SCFA levels of the mice in each group decrease sharply at D15, and the acetic acid levels of the mice in the constipation group at Day 16 and Day 17 show a Slight PPÉ0502579 upward trend. The levels of propionic acid, isobutyric acid, and valeric acid are basically maintained at the same level, but the butyric acid shows a downward trend. After a 1-day adaptive phase, the levels of SCFAs in the TY-S01 group increase significantly at Day 16 and
Day 17, and are always significantly higher than those in groups without TY-SO1 intervention. ©), (F), (D, (L), and (0) in Fig. 7 are respectively level diagrams of acetic acid, propionic acid, butyric acid, isobutyric acid, and valeric acid in mice in each group at the last day (17d) of a modeling phase. It may be learned from (C), (F), (I), (L), and (O) in Fig. 7 that, the levels of acetic acid, butyric acid and valeric acid in the constipation group are significantly lower than those in the normal group, but there is no statistical difference in the levels of propionic acid and isobutyric acid (P < 0.05). After TY-S01 intervention, the intestinal SCFA levels are significantly increased (P < 0.05). The above results show that the TY-S01 can not only significantly increase the intestinal SCFA levels in the mice with constipation, but also increase the intestinal SCFA levels in healthy normal mice to a certain extent, so that the TY-S01 has the effect of maintaining intestinal health.
It may be seen from the situation diagram of changes in levels of acetic acid, propionic acid, butyric acid, isobutyric acid, and valeric acid in the intestinal tract during experiment in Fig. 7 that, the Bifidobacterium lactis TY-SO1 for relieving constipation disclosed in the present invention can relieve loperamide hydrochloride-induced constipation in mice, and the mechanism of relief is to increase the intestinal SCFA levels in the mice with constipation.
Therefore, the present invention brings good news to patients suffering from constipation, and provides a treatment concept without side effects.
It is finally to be noted that, the above embodiments are merely used to describe the technical solutions of the present invention and not to limit the technical solutions. Although the present invention has been described with reference to the preferred embodiments of the present invention, it should be understood by those of ordinary skill in the art that, various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the appended claims.
SEQUENCE LISTING LUSO2BBh2579 <110> Chongqing Tianyou Dairy Co. Ltd. <120> Bifidobacterium lactis for relieving constipation and application thereof <160> 1 <170> SIPOSequencelisting 1.0 <210> 1 <211> 1188 <212> DNA <213> Bifidobacterium lactis TY-S01 <400> 1
GCGGCCTAGG GGGGGGTTCT TACACATGCA GTCGACGGGA TCCCTGGCAG CTTGCTGTCG 60
GGGTGAGAGT GGCGAACGGG TGAGTAATGC GTGACCAACC TGCCCTGTGC ACCGGAATAG 120
CTCCTGGAAA CGGGTGGTAA TACCGGATGC TCCGCTCCAT CGCATGGTGG GGTGGGAAAT 180
GCTTTTGCGG CATGGGATGG GGTCGCGTCC TATCAGCTTG TTGGCGGGGT GATGGCCCAC 240
CAAGGCGTTG ACGGGTAGCC GGCCTGAGAG GGTGACCGGC CACATTGGGA CTGAGATACG 300
GCCCAGACTC CTACGGGAGG CAGCAGTGGG GAATATTGCA CAATGGGCGC AAGCCTGATG 360
CAGCGACGCC GCGTGCGGGA TGGAGGCCTT CGGGTTGTAA ACCGCTTTTG TTCAAGGGCA 420
AGGCACGGTT TCGGCCGTGT TGAGTGGATT GTTCGAATAA GCACCGGCTA ACTACGTGCC 480
AGCAGCCGCG GTAATACGTA GGGTGCGAGC GTTATCCGGA TTTATTGGGC GTAAAGGGCT 540
CGTAGGCGGT TCGTCGCGTC CGGTGTGAAA GTCCATCGCC TAACGGTGGA TCTGCGCCGG 600
GTACGGGCGG GCTGGAGTGC GGTAGGGGAG ACTGGAATTC CCGGTGTAAC GGTGGAATGT 660
GTAGATATCG GGAAGAACAC CAATGGCGAA GGCAGGTCTC TGGGCCGTCA CTGACGCTGA 720
GGAGCGAAAG CGTGGGGAGC GAACAGGATT AGATACCCTG GTAGTCCACG CCGTAAACGG 780
TGGATGCTGG ATGTGGGGCC CTTTCCACGG GTCCCGTGTC GGAGCCAACG CGTTAAGCAT 840
LUSO2HE) 2579
CCCGCCTGGG GAGTACGGCC CGCAAGGCTA AAACTCAAAG AAATTGACGG GGGGCCTCGC 900
ACAAGCGGGC GGGAGCATGC GGGATTAATT CGATGCAACG CGAAGAACCT TTACCTTGGG 960
CTTGACATGT ~~ GCCCGGATCG ~~ TCCGTGGAGA CACGGTTTIC CCTTTCGGGG TCGGTCCACA 1020
GTTGTTGCCA TGGGTCGTTC GTCAGCCTCG TGTCGGTGAA GATGTTGGGG TTTAAGTCCC 1080
CGTCAATCGA GCGTCATCTC CTCGTCCTGC ATTGTTTGCC AGCCGGATGA TTGCCGGAAA 1140
CCTCATGTGA CCGCTGGGGA ATCACCTCGG AAGCAAAGTT TGTGGGAC 1188
Claims (7)
1. Bifidobacterium lactis TY-SO1 for relieving constipation, wherein the strain is deposited with the China General Microbiological Culture Collection Center (CGMCC) on November 27, 2020, and assigned the accession number CGMCC No.21255.
2. The Bifidobacterium lactis TY-SO1 for relieving constipation as claimed in claim 1, wherein a 16S rDNA sequence thereof is shown as SEQ ID No.1.
3. The Bifidobacterium lactis TY-SO1 for relieving constipation as claimed in claim 1 or 2, applicable to a drug or food for increasing the content of short-chain fatty acid in the intestinal tract.
4. The Bifidobacterium lactis TY-SO1 for relieving constipation as claimed in claim 1 or 2, applicable to an application for relieving loperamide hydrochloride-induced constipation.
5. The Bifidobacterium lactis TY-SO1 for relieving constipation as claimed in claim 1 or 2, able to shorten the first defecation time of melena, increase water content in feces, and accelerate intestinal peristalsis.
6. The Bifidobacterium lactis TY-SO1 for relieving constipation as claimed in claim 1 or 2, applicable to a drug or food for inhibit damage of small intestinal villi.
7. The Bifidobacterium lactis TY-SO1 for relieving constipation as claimed in claim 1 or 2, applicable to a drug or food for preventing or relieving constipation.
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