CN111996153B - Bifidobacterium breve and application thereof - Google Patents

Bifidobacterium breve and application thereof Download PDF

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CN111996153B
CN111996153B CN202010984015.1A CN202010984015A CN111996153B CN 111996153 B CN111996153 B CN 111996153B CN 202010984015 A CN202010984015 A CN 202010984015A CN 111996153 B CN111996153 B CN 111996153B
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bifidobacterium breve
mice
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liver
constipation
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李珮铷
贾长生
边高瑞
王信超
李苗
许登泉
王林
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Shenzhen Chuangxin Medical Technology Co., Ltd
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    • 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
    • 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
    • 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
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • C—CHEMISTRY; METALLURGY
    • 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
    • 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
    • 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
    • A23V2400/519—Breve
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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

Abstract

The invention also provides Bifidobacterium breve (Bifidobacterium breve) TR103 which is preserved in China center for type culture collection with the preservation number of CCTCC NO: m2018728, the preservation address is Wuhan university in Wuhan city of Hubei province of the people's republic of China, and the preservation date is 10 months and 31 days in 2018. The bifidobacterium breve TR103 provided by the invention is derived from the elderly with long life, and has the effects of protecting intestinal mucosa, protecting liver, avoiding liver injury, regulating defecation frequency and relieving obesity.

Description

Bifidobacterium breve and application thereof
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to bifidobacterium breve and application of the strain.
Background
The factors causing the damage of the intestinal mucosa are mainly serious trauma, serious infection, burn, shock, acute pancreatitis and the damage of the intestinal mucosa barrier of other critical patients, and the latter can aggravate the conditions of various primary diseases, and the two are mutually promoted and cause and effect. The damage of chemotherapy drugs, abdominal dialysis, bacterial peritonitis, inflammatory bowel disease and other diseases to the intestinal mucosa is the most common, and the damage of the intestinal mucosa barrier can cause the bacterial displacement, so that pathogenic bacteria parasitic on the large intestine can be displaced to the small intestine (transverse displacement) or cross the intestinal mucosa to enter mesenteric lymph nodes and portal vein and then enter the systemic circulation (longitudinal displacement). All cause severe systemic physiological dysfunction. Especially, the massive propagation of intestinal bacteria causes excessive toxin to enter portal vein and liver, once the capability of liver for removing toxin is reduced, endotoxemia occurs, and endotoxin can not only directly damage important organs, but also activate complement and coagulation mechanisms, so that respiratory distress syndrome and disseminated intravascular coagulation occur, and even multiple organ failure occurs. At present, the varieties of medicines for treating the intestinal mucosa injury disease are few, the curative effect of the existing western medicines for clinically treating the intestinal mucosa injury disease is limited, the western medicines have large side effect on liver and kidney, cannot be used for a long time, and are unfavorable for or have injury to the liver and kidney of a patient. The Chinese medicine has relatively few varieties, single treatment function and unobvious short-term curative effect.
The hazards of alcohol to the human body are well known. Excessive drinking can cause damage to the digestive system, circulatory system, etc., manifested as loss of control of mood, disturbance of consciousness, discomfort of digestive tract, and even death. Liver diseases such as alcoholic fatty liver, alcoholic hepatitis, alcoholic cirrhosis and the like caused by long-term drinking. The number of alcoholic liver patients in China is nearly hundred million, and the incidence rate is in an increasing trend every year. The long-term drinking can cause the myocardial cell membrane damage, the organelle dysfunction, the internal environment calcium ion unbalance and the protein contraction dysfunction to cause alcoholic heart disease. How to maintain the physical health of the drinkers is always a subject of high attention in the medical and social circles.
Constipation is a common symptom, which refers to decreased stool frequency and/or difficulty in stool drying, and generally no defecation occurs for more than two days, indicating that constipation exists. According to statistics, the number of constipation patients in China is more than 30%, women are more than 4 times of men, and the incidence rate of constipation patients increases with the increase of age. Modern scientific research shows that the excrement contains up to 796 toxic substances such as indole, hydrogen sulfide, methane, phenol, ammonia and the like, and the bacteria, the toxic substances and metabolic wastes can be discharged out of the body in time by people who defecate once every day. If defecation time exceeds 48 hours, virulence is multiplied. Patients with long-term constipation are easy to cause diseases such as hemorrhoids, anal fissure, rectocele and the like; the defecation force of patients suffering from heart disease and hypertension can induce stroke, myocardial infarction and cerebral hemorrhage, and even can cause life accidents; constipation also plays an important role in the development of diseases such as colon cancer (data indicate that about 10% of people with severe constipation suffer from colon cancer), hepatic encephalopathy, breast diseases, and alzheimer's disease. Therefore, the constipation can be prevented, treated reasonably in time, serious consequences and social burden caused by the constipation can be greatly reduced, and huge consumer groups of patients can strongly stimulate the constipation market to continuously expand. At present, many drugs can be used for treating constipation, but most drugs are not suitable for patients with chronic constipation and are not suitable for long-term use.
The increasing awareness of the relationship between diet and health has led to an increasing demand for products that enhance health in addition to providing basic nutrition. There are studies that show that ingestion of probiotics is beneficial to maintain a delicate microbial balance in the body. It is well known that this balance promotes the health of the intestine and enhances the immune system and other physiological functions, thereby becoming a key factor in overall improvement of human health.
Disclosure of Invention
The purpose of the invention is as follows: in order to ensure the health of people, the invention provides bifidobacterium breve.
The technical scheme is as follows: the technical scheme adopted by the invention is as follows:
bifidobacterium breve (Bifidobacterium breve) TR103, which is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2018728, the preservation address is Wuhan university in Wuhan city of Hubei province of the people's republic of China, and the preservation date is 10 months and 31 days in 2018.
The large-scale fermentation method of the Bifidobacterium breve (Bifidobacterium breve) TR103 is characterized by comprising the following steps of:
the components are as follows: aqueous solution, wherein, 2 percent of yeast extract, 1 percent of soybean peptone, 0.5 percent of anhydrous sodium acetate, 0.2 percent of dipotassium phosphate, 0.2 percent of diammonium hydrogen citrate and 800.1 percent of tween-800 are sterilized for 20 minutes at 121 ℃;
and (2) component: an aqueous solution, wherein the glucose anhydrous is 2.3%, the galacto-oligosaccharide is 0.4%, the anhydrous magnesium sulfate is 0.025%, the manganese sulfate monohydrate is 0.02%, and the solution is sterilized at 115 ℃ for 20 minutes;
and (3) component: an aqueous solution, wherein, L-cysteine hydrochloride is 0.1 percent, and the sterilization kettle is sterilized for 20 minutes at the temperature of 121 ℃;
cooling to below 40 deg.C, mixing component 1 and component 2, and adjusting pH to 7.1; inoculating; then adding the component 3; culturing at constant temperature of 37 ℃.
The invention also provides application of Bifidobacterium breve TR103 in preparing a medicament for treating/preventing intestinal mucosal injury.
The invention also provides application of Bifidobacterium breve TR103 in preparing a medicament for preventing and/or treating liver injury caused by alcohol.
The invention also provides application of Bifidobacterium breve TR103 in preparing a medicament for preventing and/or treating constipation.
The invention also provides a pharmaceutical composition, which comprises bifidobacterium breve (bifidobacterium breve) TR103 and at least one pharmaceutically acceptable carrier, diluent, excipient or auxiliary agent.
Preferably, the pharmaceutical composition is used for treating/preventing intestinal mucosa damage of mice, preventing and/or treating liver damage caused by alcohol, and preventing and/or treating constipation; the Bifidobacterium breve TR103 is administered at a dose of (0.3-2.0) x 1010CFU/g。
Has the advantages that: the bifidobacterium breve TR103 provided by the invention is derived from the elderly with long life, and has the effects of protecting intestinal mucosa, protecting liver, avoiding liver injury, regulating defecation frequency and relieving obesity.
Drawings
FIG. 1 is a gram-stained optical micrograph of Bifidobacterium breve TR 103.
Fig. 2 is a biological phylogenetic tree diagram of bifidobacterium breve TR 103.
FIG. 3 is a photograph of the colon of each group of mice taken out after the groups of mice were dissected.
FIG. 4 is a graph showing the daily body weight of each group of mice after molding.
FIG. 5 is a graph showing the body weight change of mice after drinking alcohol and feeding stomach with Lactobacillus brevis TR 103.
FIG. 6 is a graph of the effect of Lactobacillus brevis TR103 on serum ALT in mice.
FIG. 7 is a graph showing the effect of Lactobacillus brevis TR103 on mouse serum AST.
Detailed Description
The invention is further described below with reference to the accompanying drawings.
Example 1 isolation, purification and characterization of the strains
1. Bacterial species and culture medium and culture conditions
(1) Bacterial source
New bifidobacterium breve is obtained by separating and screening strains in the body of one hundred years old.
Host health and dietary characteristics: the centenarian was able to move freely, had a good mental status and a normal body mass index. The daily diet mainly comprises coarse cereals and white meat, and the amount of Chinese liquor is about 50ml per day.
(2) Culture medium
Enrichment culture medium: 5g/L yeast powder, 10g/L peptone and 2.5g/L NaCl;
plate culture medium: 20g/L glucose, 2g/L (NH)4)2SO4，13.3g/L KH2PO4，1.2g/L MgSO4·7H2O, 1.7g/L citric acid, 1.7g/L microelement solution, 15g/L agar and 0.5 mu g/mL Nile blue A.
2. Isolation and characterization of strains
(1) Isolation of the Strain
Taking about 0.2g of sample, adding 1ml of MRS culture medium, vortexing and shaking for 1min, and sequentially10 times diluted to 10-6, 200. mu.L of each diluted concentration was applied to a coating containing 1% CaCO3Culturing in 37 deg.C anaerobic jar for 48h on MRS culture medium, selecting single colony on the plate, streaking and purifying on MRS solid culture medium, and culturing in 37 deg.C anaerobic jar for 48h to obtain pure colony.
(2) Identification of strains
Cell morphology:
short, fine or coarse cells, usually corynebacteria, with or without bifurcations. Particles are sometimes visible with gram staining. Cells self-agglutinate in saline. The colony is convex to pad, the surface is smooth or wave-curved, the whole edge is 2-3mm in diameter, and the quality is soft. Strains in the presence of CaCO3The diameter of the colony on the MRS culture medium is 2-3mm, the colony is white and convex, a calcium dissolving ring is arranged, and the surface is smooth or curvelling.
Biolog gram negative test:
gram-positive bacteria, cells are rod-shaped, and one end is sometimes bifurcated; gram staining characteristics of the cells are shown in FIG. 1.
Determination of 16S rRNA sequence:
use of published 16S Universal primers
The primer sequence is as follows:
S4-1：5’-ATAATGCGGCCGCACGGGCGGTGTGTRC-3’；
S4-2：5’-TAATAGCGGCCGCAGCMGCCGCGGTAATWC-3’。
amplifying and sequencing the 16SrDNA gene sequence of the strain TR103, sending a PCR amplification product to Huada Gene Co., Ltd for sequencing, and setting the nucleotide sequence of the 16SrDNA of the strain TR103 as a sequence 1 in a sequence table; through 16SrDNA gene comparison, the similarity rate of the strain to the Bifidobacterium breve strain in Genebank reaches 100 percent; combining with the identification of a microbial system, wherein TR103 is a strain of Bifidobacterium breve and is named as Bifidobacterium breve TR 103; the 16SrDNA of the Bifidobacterium breve TR103 is shown in SEQ ID NO.1, and the biological evolutionary tree is shown in FIG. 2.
Example 2 Effect of Bifidobacterium breve TR103 on relieving intestinal mucosal injury in mice and mechanism research thereof
1. Establishment and grouping of acute ulcerative colitis mouse model
60 male BALB/c mice were randomly divided into 5 groups: normal, model and TR103 low, medium and high dose groups, 12 per group.
From the first day of experiment, the normal group and the model group are administered with sterile physiological saline for intragastric administration, and TR103 low, medium and high dose groups are respectively administered with the concentration of 1 × 106、1×108、1×1010And (3) gavage of the bacterial solution of the TR103 cfu/mL, wherein each mouse is gavaged once a day, 0.2mL of the liquid is taken each time, and the gavage lasts for 14 days.
From the 8 th day of the test, according to the method of Murthy and the like, the distilled water drunk by the mice of the model group and the TR103 low, medium and high dose group is changed into 2.5% DSS solution, the experimental colitis model is caused by freely drinking for 7 days, the weight, the stool character and the occult blood condition of the mice are observed and recorded every day during the molding period, and the normal group drinks the distilled water.
2. Observation and determination of growth status of colitis mice
The body weights of the mice were measured and recorded every day after the model was made, and the average body weight of each group of mice after the model was made was calculated and the change was recorded.
3. Gross condition and length change of mouse colon
On the 15 th day of the experiment, the mouse was sacrificed by dislocation of cervical vertebrae, the colon was separated, and the general change of the colon was observed in each group of mice, and the length of the colon was measured from the junction of the small intestine and the cecum to the anus.
The pathological changes of the colons of all groups of mice are observed and recorded on the same day when the mice are sacrificed, the colons of all groups of mice taken out after dissection are shown in figure 3, and the colons of the normal group of mice are found to be 10.48 +/-0.74 cm in length, the intestinal tissues are intact and the excrement in the intestinal cavity is formed; the colon length of the model group and the colon length of the mouse with the low dose of the Bifidobacterium breve TR103 are respectively 7.33 +/-1.32 cm and 7.99 +/-1.29 cm, the intestinal wall is thinned, unformed stool can be seen in the intestinal cavity, and dark red bloody stool is formed in part of the intestinal cavity of the mouse; the colon lengths of the mice in the high dose group and the middle dose group of the Bifidobacterium breve TR103 are 8.76 +/-0.63 cm and 8.06 +/-0.94 cm respectively, the intestinal intracavity bleeding of the two groups is rare, and the feces are basically formed, wherein the colon state of the mice in the high dose group of the Bifidobacterium breve TR103 is closest to that of the normal group. Each group of bifidobacterium breve TR103 was significantly different from the model group (P < 0.05).
The daily body weights of the mice in each group after molding are shown in fig. 4, and the results show that: starting from mice freely drinking 2.5% DSS aqueous solution, the model group mice showed decreased food intake and weight loss with time, and gradually increased. The growth condition of the mice of the Bifidobacterium breve TR103 group is gradually improved along with the increase of the concentration of the gavage liquid, the weight is reduced slowly compared with the weight of the mice of the model group, and the symptoms of diarrhea and hematochezia are also reduced to different degrees, wherein the group of the Bifidobacterium breve TR103 high dose shows significant difference. The weight of the mice in the normal group is in a continuous rising trend, and the activity is normal.
Example 3 study of Lactobacillus brevis TR103 on reduction of chronic alcoholic liver injury in mice
1. Grouping and handling of test animals
Feeding 5 ICR mice in one cage, freely drinking water and eating, regularly replacing padding, keeping clean and dry in the mouse cage, adaptively feeding for 1 week, randomly dividing the mice into 4 groups, and feeding 15 mice in each group; respectively blank control group, alcohol injury group, and Bifidobacterium breve TR103 low dose test group (1.0 × 10)8CFU/mL), Bifidobacterium breve TR103 high dose test group (1.0X 10)10CFU/mL)。
The test is started for 0-2 weeks, and the control group and the alcohol-damaged group of mice are correspondingly given with 0.2 mL/(d) of intragastric normal saline, and the Bifidobacterium breve TR103 groups are respectively given with 0.2 mL/(d) of intragastric lactobacillus. On the basis of the intragastric administration mode of the first two weeks from the 3 rd week, after half an hour, the intragastric administration of 0.15 mL/(d) of physiological saline is added once for the control group, and the intragastric administration of 0.15 mL/(d) of 50% ethanol solution is added once for the alcohol injury group and the test group, and the duration is 1 week. The stomach is irrigated after half an hour for 4-6 weeks, and the volume of the physiological saline and the alcohol solution is increased to 0.20 mL/(d). And (3) after the stomach is perfused for 7-12 weeks and half an hour later, the volume of the physiological saline and the alcohol solution is increased to 0.25 mL/(d only).
2. Detection index of test animal
2.1 mouse body weight and apparent characteristics
During the test, the padding is replaced at regular time, and the padding is kept dry and the drinking water is sufficient. The food intake and water intake were recorded daily, the apparent characteristic changes of the mice were closely observed, and the body weights of the mice in each group were regularly weighed every week.
2.2 sample processing method
Serum: after fasting for 12h in mice at the end of 12 weeks of the experiment, the final body weight was weighed, blood was collected, and the mice were euthanized. Standing the blood at room temperature for 1h, centrifuging (3500r/min,15min), collecting supernatant, and storing at-80 deg.C. The Total Cholesterol (TC), total Triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) contents of mouse serum were measured using a fully automated biochemical analyzer. The kit method is used for measuring the content of glutamic acid Aminotransferase (ALT) and aspartic acid Aminotransferase (AST) in the serum of the mouse.
Organ tissues: the visceral tissues were harvested quickly after sacrifice. The organ tissues include brain, liver, kidney, spleen and small intestine. Weighing each tissue, placing in liquid nitrogen for quick freezing, and storing at-80 deg.C to be tested. Organ index (liver, spleen, kidney) (%) -visceral weight/rat body weight × 100%.
2.3 preparation of mouse liver homogenate
After dissecting the mouse, the right lobe tissue of the mouse liver was rapidly picked up, weighed, cut into pieces, mixed with 9 times volume of pre-cooled physiological saline, and fully ground into 10% liver tissue homogenate. After the 10% liver homogenate is prepared, centrifuging for 15min at 3000r/min, discarding the precipitate and leaving the supernatant for later use, and freezing the liver homogenate at-80 ℃.
2.4 measurement of liver homogenate index in mouse
The concentration of the contents of glutamic acid Aminotransferase (ALT) and aspartic acid Aminotransferase (AST) in the liver homogenate of the mouse is determined strictly according to the specific operation steps of the corresponding kit instructions.
3. Results of the experiment
3.1 Effect of Lactobacillus brevis TR103 on mouse body weight
FIG. 5 shows the trend of body weight change after intragastric administration of Bifidobacterium breve TR103 to mice receiving alcohol. In the adaptation period of 0-2 weeks, the body weight of each group of mice shows a trend of obvious increase; the test substance was gavaged in each group from week 3, and the body weight of the mice in the control group was continuously increased and the body weight of the mice in the alcohol-damaged group was decreased (P < 0.05). The weight of the mice perfused with Lactobacillus brevis TR103 was higher than that of the alcohol-injured group, and there was no significant difference between the high-dose group and the low-dose group (P > 0.05).
3.2 Effect of Lactobacillus brevis TR103 on mouse serum ALT and AST
As shown in FIG. 6, the serum AST level of the alcohol-damaged mice was increased by 11.8% (P >0.05) compared to the control group. After the administration of bifidobacterium breve TR103, the AST level of each group has no significant change compared with the alcohol-damaged group (P is more than 0.05). The AST level of mice in the low-dose group is obviously increased compared with that in the control group (P <0.05), and the Lactobacillus brevis TR103 high-dose group has no obvious difference with the control group (P > 0.05).
Figure 7 compares the serum ALT activity levels of the mice in each group, the alcohol-injured group and the lactobacillus brevis TR103 dose group were not significantly different from the control group (P > 0.05).
The test results show that the serum AST level of the alcohol injury group is increased due to the intragastric alcohol, which indicates that the alcohol already causes the liver cell injury. After the action of the bifidobacterium breve TR103, the difference between the high-dose group and the control group is not obvious (P is more than 0.05), which indicates that the bifidobacterium breve TR103 has certain capacity of relieving cell damage.
Example 4 study of the Effect of Lactobacillus brevis TR103 on relieving constipation in mice
1. Grouping and processing of laboratory animals
The experimental strains were assigned 3 dose groups, blank groups and model groups. The blank group and the model group mice are filled with skim milk with the mass fraction of 10 percent of intragastric administration of 0.1 mL/(kg. d) (by the weight of the physique); lactobacillus brevis TR103 low-dose, medium-dose and high-dose mice are respectively gavaged by 1 × 106、1×108、1×1010CFU/mL of bacterial suspension 0.1 mL/(kg. d) (based on body mass). Once a day, free access to food during the trial, and body mass changes were recorded for each group of mice.
2. Stool test of mice
After the test sample was given for 7d, each group of mice was fasted for 16h without water deprivation. The mice of the model group and 3 dose groups were gavaged with compound diphenoxylate (10mg/kg) and the blank group was given distilled water. 2X 106、2×108、2×1010The TR103 bacterial suspension of CFU/mL and the ink are mixed in equal volume, after the compound diphenoxylate is administered for 0.5h, the blank group and the model group are perfused with the ink of 0.1mL/kg (by a physique amount), the low, medium and high dose groups and the control group are administered with the ink containing the tested sample of 0.1mL/kg (by a physique amount), and the animals are all raised in a single cage and fed with normal drinking water. Starting from the gastric lavage ink, recording the first grain defecation time of each animal, the number of black grains in 5h and the quality of the black grains. The collected feces were dried at 105 ℃ until the mass was constant, and the moisture content of the feces was calculated according to the formula (1).
Figure GDA0003215132260000071
TABLE 1
Group of 1d 7d 15d
Blank group 19.9±0.5 20.2±0.6 21.0±0.9
Model set 20.4±1.1 20.9±0.8 21.4±1.0
TR103 Low dose group 21.0±1.1 21.4±1.3 22.0±1.1
TR103 Medium dose group 21.3±1.4 21.8±1.5 22.3±1.3
TR103 high dose group 20.5±1.0 21.0±1.2 21.7±1.4
The study was performed under normal physiological conditions in mice. As can be seen from table 1, the body weight of the mice during the test did not have significant difference between each dose group and the control group (P >0.05), indicating that the lactobacillus brevis TR103 did not have significant effect on the body weight of the mice during the test.
TABLE 2
Figure GDA0003215132260000081
The experimental mice orally administrate the Bifidobacterium breve TR103 strain once a day, and the influence on the first grain defecation time, the number of the defecation granules within 5h, the quality and the water content of the excrement of the constipation model mice is continuously measured after 7 days, and the result is shown in Table 2. Compared with the blank group, the mice in the model group have very significant differences (P <0.01) in the first defecation time, the number of defecation particles in 5h, the defecation quality and the water content of the excrement, which indicates that the constipation mouse model is established.
Through gastric lavage for 7d, the first defecation time, the defecation quality within 5h and the defecation grain number of mice in each dose group of the Lactobacillus brevis TR103 reach extremely significant levels (P is less than 0.01) compared with those in a model group; the water content of the defecate of the mice in the medium and high dose groups is also obviously improved compared with that in the model group (P < 0.01). And the dosage effect is shown, and with the increase of the gavage dosage, the first defecation time of the mouse, the number of defecation particles in 5h, the defecation quality, the water content of excrement and the like are gradually enhanced.
Sequence listing
<110> biological science and technology Limited of Yiruilan, Nanjing
<120> Bifidobacterium breve and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 896
<212> DNA
<213> Bifidobacterium breve (Bifidobacterium breve)
<400> 1
gaggtcggga acgcattcac cgcgacgttg ctgattcgcg attactagcg actccgcctt 60
cacgcagtcg agttgcagac tgcgatccga actgagaccg gttttcaggg atccgctcca 120
gctcgcactg tcgcatcccg ttgtaccggc cattgtagca tgcgtgaagc cctggacgta 180
aggggcatga tgatctgacg tcatccccac cttcctccga gttaaccccg gcggtccccc 240
gtgagttccc ggcacaatcc gctggcaaca cggggcgagg gttgcgctcg ttgcgggact 300
taacccaaca tctcacgaca cgagctgacg acgaccatgc accacctgtg aacccgcccc 360
gaagggaaac cccatctctg ggatcgtcgg gaacatgtca agcccaggta aggttcttcg 420
cgttgcatcg aattaatccg catgctccgc cgcttgtgcg ggcccccgtc aatttctttg 480
agttttagcc ttgcggccgt actccccagg cgggatgctt aacgcgttag ctccgacacg 540
gaacccgtgg aacgggcccc acatccagca tccaccgttt acggcgtgga ctaccagggt 600
atctaatcct gttcgctccc cacgctttcg ctcctcagcg tcagtaacgg cccagagacc 660
tgccttcgcc attggtgttc ttcccgatat ctacacattc caccgttaca ccgggaattc 720
cagtctcccc taccgcactc aagcccgccc gtacccggcg cggatccacc gttaagcgat 780
ggactttcac accggacgcg acgaaccgcc tacgagccct ttacgcccaa taattccgga 840
taacgcttgc accctacgaa ttaccgcggc tgctggcccg cgcggcttta taaaaa 896

Claims (6)

1. Bifidobacterium breve: (Bifidobacterium breve) TR103, preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2018728, the preservation address is Wuhan university in Wuhan city of Hubei province of the people's republic of China, and the preservation date is 10 months and 31 days in 2018.
2. Use of Bifidobacterium breve TR103 according to claim 1 for the preparation of a medicament for the prevention and/or treatment of mucosal lesions of the intestinal tract.
3. Use of Bifidobacterium breve TR103 according to claim 1 for the preparation of a medicament for the prevention and/or treatment of liver damage caused by alcohol.
4. Use of Bifidobacterium breve TR103 according to claim 1 for the preparation of a medicament for the prevention and/or treatment of constipation.
5. A pharmaceutical composition comprising Bifidobacterium breve (Bifidobacterium breve) TR103 as claimed in claim 1 together with at least one pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
6. The pharmaceutical composition of claim 5, wherein the pharmaceutical composition is used for treating/preventing intestinal mucosal injury in mice, preventing and/or treating alcohol-induced liver injury, preventing and/or treating constipation; the Bifidobacterium breve TR103 is administered at a dose of (0.3-2.0) x 1010CFU/g。
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CN112807328B (en) * 2021-02-26 2023-04-11 江苏德禧生物科技有限公司 Application of selenium-rich probiotics in preparation of medicine for treating and/or preventing radiation liver injury
CN114748514A (en) * 2022-05-13 2022-07-15 浙江大学医学院附属邵逸夫医院 Application of three kinds of bifidobacteria in preventing or treating acute pancreatitis
CN114774328B (en) * 2022-05-13 2023-07-18 江南大学 Bifidobacterium breve capable of down-regulating IL-17 and relieving constipation and application thereof
CN114949007A (en) * 2022-07-04 2022-08-30 南昌大学第一附属医院 Application of bifidobacterium pseudolongum in preparing medicine for preventing and treating pancreatitis
CN116970530B (en) * 2023-08-07 2024-02-06 江苏新申奥生物科技有限公司 Bifidobacterium breve FPHC4024 for preventing and treating infantile diarrhea and application thereof

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