CN103156888A - Application of bacteroides fragilis in preparation of composition for treating inflammatory bowel diseases - Google Patents
Application of bacteroides fragilis in preparation of composition for treating inflammatory bowel diseases Download PDFInfo
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- CN103156888A CN103156888A CN2013100857161A CN201310085716A CN103156888A CN 103156888 A CN103156888 A CN 103156888A CN 2013100857161 A CN2013100857161 A CN 2013100857161A CN 201310085716 A CN201310085716 A CN 201310085716A CN 103156888 A CN103156888 A CN 103156888A
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Abstract
The invention relates to the technical field of application of bacteroides fragilis, and in particular relates to application of bacteroides fragilis in preparation of a composition for treating inflammatory bowel diseases. The experiments show that bacteroides fragilis is safe and nontoxic and strong in pharmacologic action, and has good treating effect of treating inflammatory bowel diseases, thereby indicating that the bacteroides fragilis has good edible and medicinal prospects. According to the invention, a novel use of bacteroides fragilis is explored and a novel application field is developed. Bacteroides fragilis as a probiotic can be used for preparing foods or medical compositions for treating inflammatory bowel diseases so as to provide health-cared foods or treating medicines suitable for human body to take.
Description
Technical field
The present invention relates to the applied technical field of bacteroides fragilis, be specifically related to the application of bacteroides fragilis in preparation treatment inflammatory bowel compositions.
Background technology
Inflammatory bowel is a kind of agnogenic chronic nonspecific inflammatory bowel disease, comprises Crohn disease and ulcerative colitis.The clinical manifestation of ulcerative colitis is the diarrhoea continued or repeatedly show effect, mucus bloody purulent stool companion stomachache, tenesmus and General Symptoms in various degree, and the course of disease is many more than 4～6 weeks.Can there is the intestinal of skin, mucosa, joint, eye and liver and gall etc. to show outward.The clinical manifestation of Crohn disease is variation, comprises that digestive tract performance, general performance, intestinal show and complication outward.The digestive tract performance mainly contains diarrhoea and stomachache, and hemafecia can be arranged; General performance mainly contains that weight alleviates, heating, inappetence, fatigue, anemia etc., adolescent patient visible growth hypoevolutism; The outer performance of intestinal is similar to UC.Concrete coincident with severity degree of condition judgement all should be in conjunction with lab testing, iconography, endoscopic manifestations and pathology.All without the specific medicine that can cure inflammatory bowel fully, main medicine is aminosallcylic acid preparation, hormone, azathioprine and Ying Fulixi clinically at present.
In probiotic bacteria, humans and animals there is the bacterial strain of fine effect after absorption by the bacillus bifidus of extensive concern and lactobacillus, their specific bacterial strain can be settled down on intestinal mucosa, the built-in group of intestinal and prevent that harmful microorganism from adhering to thereon, be to promote the microorganism formulation of growing of bion health by keeping enteral natural microorganisms fauna, help maintains the health of humans and animals.In a large amount of laboratory researches, show, bacillus bifidus and lactobacillus change good prevention and therapeutical effect are arranged for enteritis.In a lot of clinical experiments, although probiotic bacteria for the Endoscopic Features of inflammatory bowel without obvious change, helpful for the alleviation of clinical symptoms.
Bacteroides fragilis (
bacteroides fragilis) as a kind of Gram-negative, shaft-like, blunt round and dense the dying in two ends, pod membrane is arranged, without brood cell, motorless obligate anaerobes, minute produce enterotoxin type and non-product enterotoxin type.Bacteroides fragilis, as the part of people and animal intestinal normal flora, mainly is stored in colon, in addition, but the also field planting growth of respiratory tract, gastrointestinal tract and urogenital tract mucosa.Studying more is that bacteroides fragilis is as a kind of conditioned pathogen, when host's mucosa is impaired, can invade tela submucosa, cause infection, also can be through blood flow, cause that other organ of health is as intestinal, abdominal cavity, liver, lung, cerebral tissue pyogenic infection the abscess and cause acute and chronic diarrhea etc. of occurring together.In addition, its generation to colorectal cancer also has facilitation.
Yet bacteroides fragilis is not only a kind of conditioned pathogen.Within 1991, Zhang Jijie has delivered " isolation identification of a kind of nontoxic bacteroides fragilis ", reported about nineteen eighty-three JIUYUE separate the large and purer antibacterial of output from a well-developed infant faeces, warp is with serial evaluation, basic test and observation on Clinical Application, belong to a strain in the bacteroides fragilis group without toadstool, be the probiotic bacteria of human body, name the bacterial strain into BF839.Thus, study and found, bacteroides fragilis is also a kind of probiotic bacteria.But bacteroides fragilis is as a kind of probiotic bacteria, and, in the related application of preparation treatment inflammatory bowel compositions, report is not yet arranged at present.
Summary of the invention
The object of the present invention is to provide the application of bacteroides fragilis in preparation treatment inflammatory bowel compositions.
In order to address the above problem, the technical solution adopted in the present invention is:
The application of bacteroides fragilis in preparation treatment inflammatory bowel compositions is provided, described bacteroides fragilis depositary institution name is called China Committee for Culture Collection of Microorganisms's common micro-organisms center, the depositary institution address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date is on March 12nd, 2013, deposit number is CGMCC NO.7280, and Classification And Nomenclature is
Bacteroides fragilis.
Wherein, food or the pharmaceutical composition of described compositions at least containing the bacteroides fragilis that can treat inflammatory bowel.
Wherein, described compositions is selected from any one in milk powder, cheese, curdled milk, Yoghourt, ice cream, milk base fermented food, fermented cereal food, baby food or pet food.
Wherein, described compositions is pill, tablet, granular preparation, capsule, oral liquid or tube feed preparation.
Wherein, described compositions is the liquid bacterial suspension that contains described bacteroides fragilis.
Wherein, the content of described bacteroides fragilis in described compositions is 10
8~ 10
10cfu/ml.
the invention has the beneficial effects as follows:
The present invention has excavated the new purposes of bacteroides fragilis, has opened up a new application.The present invention proves by experiment, the bacteroides fragilis safety non-toxic, and pharmacological action is strong, for inflammatory bowel, good therapeutical effect is arranged, thereby has indicated that bacteroides fragilis has well edible and prospect in medicine.Bacteroides fragilis, as a kind of probiotic bacteria, can be used for food or the pharmaceutical composition of preparation treatment inflammatory bowel, and then is clinical health food or the medicine that provides applicable human body to take.
the specific embodiment:
Below by embodiment, the present invention is described in further details, these embodiment only are used for illustrating the present invention, do not limit the scope of the invention.
the preparation of embodiment 1 bacteroides fragilis bacterium liquid
(1) separate bacteroides fragilis in infant faeces
Take 0.5 gram stool by " normal flora inspection technique ", be placed in the test tube that the 4.5ml normal saline first is housed, with this serial dilution, become 10
-1~ 10
-6, by the rare long-pending degree of height, to low rare long-pending degree, dripped on the Bd culture medium flat plate, be placed in the anaerobic gas generation box and add the anaerobic gas generation bag and carry out the anaerobism cultivation.Put 37 ℃, can opening inspection after 2 ~ 7 days.Choose single bacterium colony and carry out pure culture, dyeing microscopic examination, cultural character, Physiology and biochemistry detection.
(2) bacterium that increases of bacteroides fragilis is cultivated and bacterium solution preparation
Choose single colony inoculation after purification is increased bacterium and is cultivated in the tryptone meat soup of improvement, the centrifugation of gained bacterium liquid, rotating speed 3000r/min, centrifugal 15min, remove supernatant, after precipitate washs with normal saline, the precipitate normal saline dilution, do the bacterium number with the Maxwell opacity tube and measure, be diluted to 10
8cfu/ml, 10
9cfu/ml, 10
10cfu/ml, save backup.
embodiment 2 bacteroides fragilis virulence experiments
Adopt the bacteroides fragilis bacterium liquid of embodiment 1 preparation to carry out following experiment:
(1) chmice acute virulence experiment
Get 60 of body weight 22 ± 2 gram C57BR/C mices (male and female half and half), be divided into high dose group, low dose group and matched group, each organizes 20.Every mice gavage every day 10 of high dose group
12cfu/ml bacterium liquid 0.5ml, low dose group gavage every day 10
9cfu/ml bacterium liquid 0.5ml, matched group gavage every day 0.5ml normal saline.Continuous Observation 14 days.
Experimental result: the mice normal growth, have no dead, LD50 does not measure.
(2) rat virulence experiment
Get 30 of 150 ± 20 gram SD rats (male and female half and half), be divided into 3 groups: high low dose group and matched group.Every rat gavage every day 10 of high dose group
12cfu/ml bacterium liquid 2ml, low dose group gavage every day 10
9cfu/ml bacterium liquid 2ml, matched group gavage every day 2ml normal saline.Continuous Observation 14 days, phlebotomize after 14 days and survey hemogram, Liver and kidney merit.Get gastrointestinal liver spleen tissue and do the pathological section electron microscopic observation.
Experimental result: be showed no obvious abnormalities.
(3) Rhesus Macacus virulence experiment
Get 10 of Rhesus Macacus (male and female half and half), be divided into 2 groups: high low dose group and matched group.Every of high dose group Rhesus Macacus every day oral 10
12cfu/ml bacterium liquid 100ml, low dose group every day oral 10
9cfu/ml bacterium liquid 100ml.Continuous Observation 60 days.Phlebotomize respectively after 14 days, 30 days, 45 days, 60 days and survey hemogram, Liver and kidney merit, thought-read electrograph.Get gastrointestinal liver spleen tissue and do the pathological section electron microscopic observation.
Experimental result: be showed no obvious abnormalities.
embodiment 3 bacteroides fragiliss are for the short term therapy effect of inflammatory bowel
Adopt the bacteroides fragilis bacterium liquid of embodiment 1 preparation to carry out following experiment:
Laboratory animal: BALB/c mouse
Animal model builds: chemicals is induced inflammatory bowel (trinitro-benzene-sulfonic acid TNBS and alcohol inducedly go out to clone the grace disease, dextran sulfate sodium DSS induces ulcerative colitis)
Get 50 mices and be divided at random 5 groups: TNBS group, DSS group, TNBS+Bd group, DSS+Bd group, matched group.
The mice fasting (can't help water) of TNBS group, TNBS+Bd group, matched group, first with 50/L chloral hydrate 0.15ml lumbar injection, mice is slightly anaesthetized, about 25min is later with the 1ml syringe pump medicinal liquid that has been socketed small size catheter, from anus, catheter is slowly inserted to about 3.5cm, then medicinal liquid is slowly injected, stop extracting catheter out after 20s, then put into box after hanging mice by the feet about 30s; The alcoholic solution 0.1ml of TNBS group and TNBS+Bd group perfusion 20g/L TNBS 500ml/L; Matched group perfusion 0.15ml ethanol; TNBS+Bd group modeling success rear every day of gavage 0.5ml 10
9the Bd suspension of cfu/ml, 7d continuously.DSS group mice drinkable liquid is 5g/L DSS distilled water solution, continuous 7 days; DSS+Bd group modeling success rear every day of gavage 0.5ml 10
9the Bd suspension of cfu/ml, 7d continuously.
Measurement index: within the 8th day, put to death, observation index is disease activity index (DAI), histopathology, blood drawing detection cytokine IL-2, IL-6, IL-10, TNF-α.
Experimental result: the disease activity index of bacteroides fragilis gavage group obviously reduces than the modeling group, and IL-10 obviously raises, and TNF-α obviously reduces, but pathology are without obvious change.
embodiment 4 bacteroides fragiliss are for the long-term treatment effect of inflammatory bowel
Laboratory animal: BALB/c mouse
Animal model builds: get 60 mices, be divided at random 4 groups, 10 of Bd1 groups, 10 of Bd2 groups, 20 of Bd+DSS groups, 20 of blank groups.
The Bd+DSS group: (1) assurance mice is drunk the DSS of 5ml 2g/L every day; Within (2) the 3rd days, play drinking water and replace with the distilled water containing 2%DSS; (3) from (2), count, the DSS that stops using afterwards in 5 days reaches 2 weeks; (4) repeat (1) to (3) 22-26 days the time; Within the 29th day, play inactive DSS full 2 weeks; (5) 43-47 repeats (1) to (3); (6) the 50th days start DSS is changed to distilled water.
The Bd1 group: from first day every day gavage 0.5ml 10
9the bacteroides fragilis bacterium liquid of cfu/ml;
Bd2 group: guaranteed to drink every day 2ml 10 since the 50th day
9the bacterium liquid of cfu/ml.
Measurement index: Bd1 group, 10 of Bd+DSS groups, 10 mices of blank group are put to death on the 51st day, and observation index is that histopathology, blood drawing detect cytokine IL-2, IL-6, IL-10, TNF-α; Remaining mice is put to death on the 80th day, and observation index is that histopathology, blood drawing detect cytokine IL-2, IL-6, IL-10, TNF-α.
Experimental result: Bd1 group, Bd2 group mice pathology obviously approach normal than the Bd+DSS group, intestinal mucosa is complete, and inflammatory cell infiltration obviously reduces; IL-10 obviously raises, and TNF-α obviously reduces.
The presentation of results of embodiment 3 and embodiment 4, bacteroides fragilis has good therapeutical effect for inflammatory bowel.
Claims (6)
1. the application of bacteroides fragilis in preparation treatment inflammatory bowel compositions.
2. the application of bacteroides fragilis according to claim 1 in preparation treatment inflammatory bowel compositions, is characterized in that: food or the pharmaceutical composition of described compositions at least containing the bacteroides fragilis that can treat inflammatory bowel.
3. the application of bacteroides fragilis according to claim 2 in preparation treatment inflammatory bowel compositions, it is characterized in that: described compositions is selected from any one in milk powder, cheese, curdled milk, Yoghourt, ice cream, milk base fermented food, fermented cereal food, baby food or pet food.
4. the application of bacteroides fragilis according to claim 2 in preparation treatment inflammatory bowel compositions, it is characterized in that: described compositions is pill, tablet, granular preparation, capsule, oral liquid or tube feed preparation.
5. the application of bacteroides fragilis according to claim 1 in preparation treatment inflammatory bowel compositions, it is characterized in that: described compositions is the liquid bacterial suspension that contains described bacteroides fragilis.
6. the application of bacteroides fragilis according to claim 1 in preparation treatment inflammatory bowel compositions, it is characterized in that: the content of described bacteroides fragilis in described compositions is 10
8~ 10
10cfu/ml.
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