CN110023484A - A kind of small bifidobacterium catenulatum of vacation and its cultural method and application - Google Patents
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Abstract
Provide a kind of small bifidobacterium catenulatum of vacation and its cultural method and application, the small bifidobacterium catenulatum of the vacation (Bifidobacterium pseudocatenulatum) TM12-14, it is preserved in Guangdong Province's Culture Collection, deposit number is GDMCC No:60089.The bacterial strain can significantly improve the performance state of ulcerative colitis mouse, reduce mouse disease activity index and inflammatory reaction, while have the ability for reducing cholesterol.
Description
The present invention relates to microorganisms technical field more particularly to a kind of small bifidobacterium catenulatum of vacation (Bifidobacterium pseudocatenulatum) and its cultural method and applications.
Human body intestinal canal is by a large amount of microorganism group at these microbe species are various, substantial amounts, form a complicated ecosystem in people's enteron aisle, and the composition and human health of microorganism therein are closely bound up.A large number of studies show that enteric microorganism and many diseases have it is close be associated with, such as diseases associated with inflammation such as the metabolic diseases such as diabetes, obesity, hyperlipidemia and colitis, rheumatoid arthritis.There is most of microorganism to belong to beneficial bacterium in the enteron aisle of healthy human body, it is the indispensable type of human health, wherein Bifidobacterium is that a kind of important bacterium, Bifidobacterium colonize in baby intestinal since birth in people's enteron aisle, with advancing age, the content of Bifidobacterium is gradually reduced.Bifidobacterium has effects that anti-inflammatory, antitumor, anti-aging and inhibition harmful bacteria etc. are important, while Bifidobacterium can also stimulate intestines peristalsis by generating short chain fatty acids (SCFA) and organic acid, prevent constipation and other diseases.
Ulcerative enteritis (Ulcerative colitis, UC) is one kind of inflammatory bowel disease (Inflammatory bowel disease, IBD), is a kind of chronic gut inflammation disease that pathogenesis is unknown.Clinical pathological study thinks that the morbidity of UC is mainly related with the intestinal mucosal immune reaction that individual tumor susceptibility gene and enteric microorganism cause, and symptom is mainly shown as lasting abdominal pain, diarrhea and mucus bloody stool, and Relapse rate, visible any age level of falling ill.
Currently, clinically mainly having salicylic acid, glucocorticoid, immune formulation for the medication of UC.Salicylates can inhibit prostaglandin to synthesize better, scavenging activated oxygen is to achieve the purpose that alleviate inflammatory reaction, clinical treatment UC common salicylic acid Western medicine is mainly salicylazosulfapyridine (SASP), mainly for slight, moderate and chronic UC patient；Glucocorticoid is severe or explosive UC patient
Preferred medication, such as betamethasone；Immunosuppressor such as cyclosporine can influence the progress of immune response, to inhibit to UC by the generation of inhibition T cell IL-2.
The existing three classes drug for UC can to a certain extent alleviate UC, but also all there is certain side effect, the side effect of salicylic acid is fash, hepatotoxicity wind agitation, oligoleukocythemia, anaemia caused by causing digestive tract reaction, headache, ceticulocytosis, oligozoospermia and allergic reaction etc..Glucocorticoid will lead to organism metabolic disorder, and the side effects such as water retention only can be used as emergency medication, cannot take for a long time.Immunosuppressant treatment is larger to drug dependence, and treatment cycle is long, easily causes renal toxicity and superinfection, can only be as a kind of means of adjuvant treatment.
Summary of the invention
The present invention provides a kind of small bifidobacterium catenulatum novel species of vacation, has the function of preventing and/or treating ulcerative enteritis.The present invention further provides the cultural method of the enteric bacteria novel species and products made of it and its application.
The present invention includes following technical solution:
According to the first aspect of the invention, the present invention provides a kind of small bifidobacterium catenulatum of vacation (Bifidobacterium pseudocatenulatum) TM12-14, is preserved in Guangdong Province's Culture Collection, and deposit number is GDMCC 60089.
According to the second aspect of the invention, the present invention provides the cultural method of the small bifidobacterium catenulatum TM12-14 of vacation of first aspect a kind of, and the small bifidobacterium catenulatum TM12-14 of the vacation is inoculated in PYG culture medium and carries out Anaerobic culturel.
According to the third aspect of the invention we, the present invention provides the probiotics of small bifidobacterium catenulatum TM12-14 of vacation and/or its metabolite containing first aspect a kind of.
According to this field it is generally understood that all can promote normal microflora growth and breeding and the preparation of pathogenic bacteria growth and breeding is inhibited to be referred to as " probiotics ".It is so-called " probiotics " in the present invention, refer to using preparation made of the small bifidobacterium catenulatum TM12-14 of vacation and/or its metabolite, have adjusting enteron aisle it
Effect, rapid build intestinal microecology balance.Typical probiotics can be probiotics preparation, be used for preventing/treating ulcerative enteritis.As probiotics of the invention, false small bifidobacterium catenulatum TM12-14 has the effect for the treatment of ulcerative enteritis, it can be by further changing probiotics preparation type, using Different Package and processing method, for example bacterial activity is kept to reach corresponding therapeutic effect using embedding techniques, or can all realize same therapeutic effect by additionally adding the false small bifidobacterium catenulatum TM12-14 of prebiotics (bacterium powder, oligosaccharide etc.) combination to treat ulcerative enteritis.In addition the false small bifidobacterium catenulatum TM12-14 of probiotics of the invention can alleviate ulcerative enteritis, it is also possible to can play its therapeutic effect in the relevant disease (common enteritis, gastritis etc.) of some inflammation of others.
According to the fourth aspect of the invention, the present invention provides food compositions, health care product or the auxiliary material additive of a kind of small bifidobacterium catenulatum TM12-14 of vacation and/or its metabolite containing first aspect.
Food compositions in the present invention can also contain various raw-food materials or food additives etc., such as milk, white sugar and vitamin etc. in addition to containing the small bifidobacterium catenulatum TM12-14 of vacation and/or its metabolite.Auxiliary material additive in the present invention, such as various consumption additives.
According to the fifth aspect of the invention, the present invention provides the pharmaceutical composition of small bifidobacterium catenulatum TM12-14 of vacation and/or its metabolite containing first aspect a kind of.
Pharmaceutical composition in the present invention, in addition to containing the small bifidobacterium catenulatum TM12-14 of vacation and/or its metabolite, various pharmaceutically acceptable carriers and/or auxiliary material can also be contained, including but not limited to: lactose, yeast powder, peptone, pure water, starch and vitamin etc., various excipient can also be contained, tablet or capsule preparations etc. can be made.In addition, the pharmaceutical composition in the present invention can also help to maintain the substance of false small bifidobacterium catenulatum TM12-14 vigor, such as protective agent, typical but non-limiting protective agent is vitamin C.
In pharmaceutical composition of the invention, the content of false small bifidobacterium catenulatum TM12-14 can be by the total volume or total weight of pharmaceutical composition, for example, typical but contain 1 × 10 in non-limiting manner-1To 1 × 1020The small bifidobacterium catenulatum TM12-14 of vacation of cfu/mL or cfu/g, preferably contains 1 × 104To 1 × 1015The small bifidobacterium catenulatum TM12-14 of vacation of cfu/mL or cfu/g.
According to the sixth aspect of the invention, the present invention provides application of the small bifidobacterium catenulatum TM12-14 of vacation of first aspect in the drug of preparation prevention and/or treatment ulcerative enteritis.
According to the seventh aspect of the invention, the small bifidobacterium catenulatum TM12-14 of vacation that the present invention provides first aspect is preparing the application in probiotics.
According to the eighth aspect of the invention, the small bifidobacterium catenulatum TM12-14 of vacation that the present invention provides first aspect is preparing the application in food compositions, health care product or auxiliary material additive.
According to the ninth aspect of the invention, the present invention provides application of the small bifidobacterium catenulatum TM12-14 of vacation of first aspect in production organic acid.
Organic acid in the present invention, refer in particular to short chain fatty acids (SCFA), such as formic acid, acetic acid, butyric acid etc., organic acid can also include one of 3 Methylbutanoic acid, valeric acid, quininic acid, lactic acid, oxalic acid, malonic acid, benzoic acid, maleic acid, succinic acid, anti-fumaric acid, malic acid, adipic acid, tartaric acid, shikimic acid, citric acid, isocitric acid and L-AA or a variety of.
According to the tenth aspect of the invention, the present invention provides application of the small bifidobacterium catenulatum TM12-14 of vacation of first aspect in production short chain fatty acids.
According to the eleventh aspect of the invention, the present invention provides a kind of method prevented and/or treat ulcerative enteritis, the pharmaceutical composition including the vacation aspect of small bifidobacterium catenulatum TM12-14 or the 5th to study subject application first aspect.
According to the twelfth aspect of the invention, the present invention provides a kind of method of decline for reducing blood lipid, controlling weight of mammal, and/or the disease activity index (DAI) for reducing mammal, including the pharmaceutical composition in terms of the vacation small bifidobacterium catenulatum TM12-14 or the 5th to study subject application first aspect.
In the present invention, study subject can be people or other mammals.
It finds after study, the small bifidobacterium catenulatum TM12-14 of vacation of the invention, there is apparent relaxation effect to ulcerative enteritis, it is in particular in the apparent state that can significantly improve ulcerative colitis mouse, reduce mouse disease activity index, and mouse inflammatory reaction is reduced, while there is the ability of norcholesterol.Therefore, probiotics, food compositions, pharmaceutical composition can be made in the small bifidobacterium catenulatum TM12-14 of the vacation
Equal products, among the prevention and/or treatment of ulcerative enteritis.
Preservation information
Strain name: Bifidobacterium pseudocatenulatum TM12-14
Preservation date: on October 13rd, 2016
Depositary institution: Guangdong Province's Culture Collection (GDMCC)
Preservation address: 5 building, the building of compound the 59th of XianLie Middle Road, GuangZhou City, GuangDong Province 100
Deposit number: GDMCC 60089
Fig. 1 shows that the picture of false small bifidobacterium catenulatum TM12-14 culture 48h bacterium colony, bacterium colony are white, protrusion, round, neat in edge, colony diameter about 1-2mm.
Fig. 2 shows the Gram's staining picture (1000 times) of false small bifidobacterium catenulatum TM12-14 under the microscope, and thallus presentation disagreement is rod-shaped, and Gram's staining is the positive, generates without gemma and flagellum.
Fig. 3 shows cholesterol standard curve, the detection of cholesterol is carried out using o-phthalaldehyde colorimetric method (OPA method), by using various concentration (20ug/mL, 40ug/mL, 60ug/mL, cholesterol and OPA 80ug/mL) carries out reaction solution, obtains standard curve, the equation of linear regression are as follows: y=0.0085x+0.0072；Coefficient R2It is 0.9992.
Fig. 4 shows control group, model group, VSL#The variation of the weight of 3 and TM12-14 treatment group mouse.
Fig. 5 shows control group, model group, VSL#The variation of the DAI index of 3 and TM12-14 treatment group mouse.
The present invention will be further explained with reference to the examples below.
Embodiment 1: the separation identification of false small bifidobacterium catenulatum TM12-14
1, sample collection
Sample is separated from 14 years old healthy male fecal, laboratory is taken back by feces collection into sterile sample cell, in 1h and is sorted.
2, Bifidobacterium isolates and purifies
The fresh sample of collection is transferred at once in anaerobic operation case, take 0.2g sample in 1ml sterile PBS (phosphate buffer), mixing fullys shake, then carries out gradient dilution coating, culture medium is using PYG culture medium (being purchased from Huan Kai microorganism scientific & technical corporation), specific formula is (1L): peptone 5g, pancreas casein 5g, yeast powder 10g, beef extract 5g, glucose 5g, K2HPO42g, Tween 80 1mL, Cysteine-HClH2O 0.5g, ferroheme 5mg, vitamin K11uL, (every L inorganic salt solution contains CaCl to inorganic salt solution2·2H2O 0.25g, MgSO4·7H2O 0.5g, K2HPO41g, KH2PO41g, NaHCO310g, NaCl 2g) 40mL, resazurin 1mg, distilled water 950mL, adjust pH to 6.8~7.0,115 DEG C of sterilizing 25min.1.5% agar is added in solid medium, topples in anaerobic operation case.The plate of coating is placed in 37 DEG C of Anaerobic culturels, and the gas component of anaerobism is N2: CO2: H2=90:5:5.After culture 3 days, picking single colonie carries out scribing line and divides pure, the pure culture of every plant of single bacterium of acquisition.
3, culture presevation
The pure culture bacterial strain of acquisition is cultivated, until concentration is about 109Cfu/ml takes 400ul bacterium solution to add 40% glycerol 400ul, its glycerol concentration is made to reach 20%, then carries out -80 DEG C of ultralow temperature preservations.
4,16S rDNA is identified
The isolated strains of acquisition are cultivated for 24 hours in liquid PYG culture medium, takes 1ml bacterium solution to carry out 10000r/min centrifugation 5min, collects thallus, extract genomic DNA.Using genomic DNA as template, PCR amplification is carried out using 16S rDNA universal primer (8F-AGAGTTTGATCATGGCTCAG (SEQ ID NO:1) and 1492R-TAGGGTTACCTTGTTACGACTT (SEQ ID NO:2)), amplification system are as follows: 10 × PCR buffer, 3uL；DNTP, 2.5uL；27F, 0.5uL；1492R, 0.5uL；Taq enzyme, 0.3uL；Template, 1uL；ddH2O, 18.2uL.PCR amplification condition are as follows: 95 DEG C of initial denaturation 4min,
Then 95 DEG C of denaturation 30s, 57 DEG C of annealing 40s, 72 DEG C of extension 1min 30s, 30 recycle.The 16S rDNA amplified production of acquisition is subjected to electrophoresis detection, purifying, 3730 sequencings, obtains the 16S rDNA sequence that length is 1400bp (see SEQ ID NO:3 in sequence table).This section of sequence is carried out to blast in Genebank and compares analysis, the qualification result for obtaining TM12-14 is small bifidobacterium catenulatum Bifidobacterium pseudocatenulatum.
5, the physiological and biochemical property of TM12-14
It is cultivated 48 hours on PYG culture medium, the bacterium colony of TM12-14 is white, protrusion, round, neat in edge, colony diameter about 1-2mm (Fig. 1).At 1000 times of microscopically observations (Fig. 2), thallus presentation disagreement is rod-shaped, and Gram's staining is the positive, generates without gemma and flagellum.The catalase reaction of TM12-14 is negative, oxidase negative, strictly anaerobic, and utilization of carbon source situation is detected using API 20A kit (French Mei Liai).As a result as table 1 (+, indicate positive reaction；, indicate negative reaction；W indicates weakly positive reaction).
Table 1
Embodiment 2: the bioactive substance of false small bifidobacterium catenulatum TM12-14
The bioactive substance of TM12-14 mainly investigates short chain fatty acids (SCFA) and organic acid production in metabolite.
1, sample pretreatment
TM12-14 is cultivated into 48h, takes 1ml bacterium solution to carry out 10000r/min centrifugation 5min, takes supernatant, be ready for the detection of short chain fatty acids (SCFA) and organic acid.
2, the measurement of SCFA
The detection of SCFA predominantly detects the content of this 4 kinds of substances of acetic acid, propionic acid, butyric acid, valeric acid.Using Agilent gas chromatograph (GC-7890B, Agilent), HP-INNOWax (Cross-Linked PEG) is selected, 30m × 0.25mm × 0.25um capillary column is analyzed, detector is hydrogen flame ionization detector, and GC parameter is set as column temperature: 180~200 DEG C；Gasify room temperature: 240 DEG C；Detection temperature: 210 DEG C；Sample volume: 2 μ L；Carrier gas flux: N2, 50mL/min；Hydrogen flowing quantity: 50mL/min；Air mass flow: 600~700ml/min.
3, the measurement of organic acid
The examination criteria product of organic acid are selected: 3 Methylbutanoic acid, valeric acid, quininic acid, lactic acid, oxalic acid, malonic acid, benzoic acid, maleic acid, succinic acid, anti-fumaric acid, malic acid, adipic acid, tartaric acid, shikimic acid, citric acid, isocitric acid and L-AA.Still Agilent gas chromatograph (GC-7890B, Agilent) is used, chromatographic column selects 122-5532G DB-5ms (40m × 0.25mm × 0.25um), column temperature: 270~290 DEG C；Injector temperature: 250 DEG C；Gas flow: 0.86ml/min.
4, experimental result
The results are shown in Table 2.
Table 2
Embodiment 3: the antibiotic sensitive situation of false small bifidobacterium catenulatum TM12-14
TM12-14 is investigated to the sensitive situations of common 20 kinds of antibiotic, is tested using quick paper disk method, the bacterium solution 100ul for taking culture to the TM12-14 of logarithmic phase carries out plate coating, antibiotic drug sensitive piece is attached to planar surface, 37 DEG C of culture 48h measure inhibition zone size, result such as table 3.
Table 3
The results show that TM12-14 is resistant to oxacillin, kanamycins and neomycin, it is more sensitive to other 17 kinds of antibiotic.
Embodiment 4: tolerance of the false small bifidobacterium catenulatum TM12-14 to acid and cholate
It needs because people probiotics reaches enteron aisle by stomach and small intestine, it is therefore desirable to undergo the gastric acid of pH2.5 or so and the cholate of 0.3% concentration.Only there is the bacterium of acid and cholate tolerance to get to enteron aisle and play prebiotic effect.So the present embodiment investigates acid and cholate the tolerance situation of TM12-14.
1, the acid tolerance situation of TM12-14
Prepare the PYG culture medium of pH 2.5, in the PYG culture medium that the TM12-14 of culture to logarithmic phase is seeded to pH2.5 according to 10% inoculum concentration, takes the TM12-14 bacterium solution of the normal PYG culture medium culture of equivalent and the TM12-14 bacterium solution of the PYG culture medium culture of pH2.5 to carry out plate coating after 37 DEG C of culture 2h and count
Number, is calculated according to the following formula the survival rate of the TM12-14 bacterium handled under conditions of pH2.5:
PH2.5 handles survival rate=(the bacterium solution plate plate colonies number of the PYG culture medium culture of pH2.5/normal PYG culture medium culture bacterium solution plate plate colonies number) × 100%
It is 78% that TM12-14 handles the survival rate of 2h under conditions of pH2.5 as the result is shown.
2, the cholate of TM12-14 is resistant to situation
Prepare 0.3% bile salt culture-medium, by the cholate for adding 0.3% in PYG, the TM12-14 of culture to logarithmic phase is seeded to according to 10% inoculum concentration to 0.3% PYG bile salt culture-medium, it takes the TM12-14 bacterium solution of the normal PYG culture medium culture of equivalent and the TM12-14 bacterium solution of 0.3% PYG bile salt culture-medium culture to carry out plate coating after 37 DEG C of culture 2h to count, the survival rate of the TM12-14 bacterium handled under conditions of 0.3% cholate is calculated according to the following formula:
0.3% cholate handles survival rate=(the bacterium solution plate plate colonies number of 0.3% PYG bile salt culture-medium culture/normal PYG culture medium culture bacterium solution plate plate colonies number) × 100%
It is 65% that TM12-14 handles the survival rate of 2h under conditions of 0.3% cholate as the result is shown.
By the above tolerance test, TM12-14 has certain tolerance in the condition of pH2.5 and the condition of 0.3% cholate respectively.
Embodiment 5: the norcholesterol characteristic of false small bifidobacterium catenulatum TM12-14
1, the bile salt hydrolase activity of TM12-14
Bile salt hydrolase is detected using TDCA method, prepares TDAC plate, the CaCl of TDAC (Taurodeoxycholate sodium) and 0.37g/L of addition 4% in PYG solid medium2, TM12-14 is cultivated to concentration about 108Cfu/ml takes 10ul bacterium solution drop on the filter paper that diameter is 0.6mm, and filter paper is placed in TDAC planar surface, and 37 DEG C are cultivated 2 days, and the white precipitate situation that observation filter paper periphery generates, the diameter of white precipitate represents the activity of bile salt hydrolase.
By measurement, the diameter of the white precipitate of TM12-14 is 8mm, shows that TM12-14 has the activity of bile salt hydrolase.
2, the external norcholesterol situation of TM12-14
The content assaying method of cholesterol uses o-phthalaldehyde colorimetric method (OPA method), is investigating the degradation capability to cholesterol containing the variation before and after the cholesterol level for cultivating a period of time in certain density cholesterol culture medium by bacterial strain.The specific method is as follows:
(1) culture of the preparation of cholesterol culture medium and experimental strain
The cholesterol for weighing certain mass is dissolved in ethyl alcohol, concentration 10mg/mL, filtration sterilization.Configured PYG culture medium is separately added into the cholate (high pressure sterilization) of 10mg/mL, the sodium thioglycolate (filtration sterilization) and cholesterol of 10% mass concentration, it mixes well, then strain to be tested is seeded in the culture medium according to 3% inoculum concentration, strain to be tested is in addition to TM12-14, other one plant of business norcholesterol probiotics lactobacillus plantarum Lp299v (purchased from Sweden Probi company) is also selected to make comparisons, two kinds of bacterium all cultivate 72h under 37 DEG C of anaerobic conditions.
(2) production of standard curve
The cholesterol standard solution 40uL of accurate measuring 0.5mg/mL, 80uL, 120uL, 160uL, 200uL is in clean tube, dehydrated alcohol is added and is settled to 1mL, OPA4mL (0.5mg o-phthalaldehyde is added to 1mL glacial acetic acid) is added in each test tube, concussion mixes, it is stored at room temperature 10min, then the concentrated sulfuric acid that 2mL is added mixes, and stands reaction 10min, absorbance is measured at 550nm.Using concentration as abscissa, absorbance draws standard curve (Fig. 3) as ordinate, by calculating, the equation of linear regression are as follows: y=0.0085x+0.0072；Coefficient R2It is 0.9992.
(3) in culture medium cholesterol measurement
The centrifugation that the cultured bacterium solution of PYG culture medium containing cholesterol is carried out to 10000r/min, collects supernatant, carries out cholesterol detection, while using nonvaccinated cholesterol PYG culture medium as blank control group.Take 1ml sample to be tested in clean test tube, the KOH 4ml of 95% ethyl alcohol 6ml and 50% is added, concussion mixes, and saponification 10min is then carried out in 60 DEG C of water-baths, is cooled down rapidly, 10ml n-hexane is added to be extracted, it mixes well, is stored at room temperature 20min, measure 8ml organic phase (n-hexane layer) into another clean tube, then it is dried with nitrogen in 60 DEG C of water-baths, 4ml 0.5g/L o-phthalaldehyde acetic acid is added
Solution is stored at room temperature 10min, adds the dense H of 2ml2SO410min is reacted, the light absorption value at 550nm is finally measured.
(4) calculating of degrading rate of cholesterol
The content of cholesterol in the culture medium of culture front and back is calculated according to standard curve, the degradation rate of cholesterol is calculated as follows:
L=(A-B)/A × 100%
L: degrading rate of cholesterol；A: it is not inoculated with the content of cholesterol in the cholesterol culture medium of bacterium；B: the content of cholesterol in strain to be tested culture 48h culture solution.
(5) cholesterol degradation result
By calculating, the degrading rate of cholesterol for obtaining TM12-14 is 78%, and Lp299v degradation rate is 70%, thus illustrates that TM12-14 ratio Lp299v has stronger cholesterol degradation ability.
Embodiment 6: treatment of the false small bifidobacterium catenulatum TM12-14 to UC mouse
Mouse model selected by the present embodiment are as follows: the ulcerative enteritis mouse model of DSS induction, using C57bl/6 mouse (being purchased from Hubei medical experiment animal center), 8 week old, weight 20g ± 2g, mouse feeding environment is SPF grades, DSS (dextran sulfate sodium, molecular weight 36000-50000) 7 days of 0.2% are continuously drunk in adaptable fed progress DSS induction in 1 week to mouse.It is used as positive control by using VSL#3 (being purchased from U.S. Sigma Tau), compares the therapeutic effect of TM12-14 and VSL#3.
Test mice amounts to 48, is randomly divided into 4 groups, and every group 12, including control group (control group), the model group (every daily stomach-filling 0.2ml PBS) of DSS induction, TM12-14 treatment group and VSL#3 treatment group.TM12-14 treatment process are as follows: TM12-14 bacterium solution for 24 hours is cultivated, thalline were collected by centrifugation, it is suspended with PBS, adjustment bacteria concentration to 109Cfu/ml, the TM12-14 of the daily stomach-filling 200ul of every mouse.VSL#3 also uses PBS to suspend, and the same concentration that adjusts is to 109Cfu/ml, the daily stomach-filling 200ul of every mouse.Treatment method in modeling by the way of being administered, record mouse weight, diet and drinking-water situation daily, the fecal character and fecal occult blood situation of mouse are observed simultaneously, respectively on day 1, the 3rd day, the 5th day and the 7th day calculate the disease activity index (DAI) of mouse, see Table 4 for details for DAI scoring.Therapy lasted 7
It, the day stomach-filling amount of probiotics and PBS are 200ul.Mouse is put to death after experiment, all mouse take blood, de- neck, take colon, take pictures, weighing, measuring colon lengths.Colonic tissue is stored in -80 DEG C of refrigerators and paraformaldehyde.
Table 4.DAI index score table
Stool in table: normal stool-forming stool；It loosely defecates-is not adhere to the paste of anus, half-formed stool；Loose stools-adheres to dilute sample water of anus just.Wherein hematochezia situation: normal mouse hematochezia is the positive；Naked eyes bloody stool is red or brown；Occulting blood positive is unconspicuous naked eyes bloody stool, is detected using tetramethyl benzidine.DAI index is equal to the sum of weight, stool and stool blood/integral of weak eye bloody stool three.
The changes of weight of pretherapy and post-treatment mouse is as shown in the following table 5 and Fig. 4:
Table 5
It can be learnt from the result in table 5 and Fig. 4, the weight of control group mice maintains slow raised trend substantially, the weight of the model group subordinate of DSS induction is gradually reduced, the ratio for starting weight loss on the 3rd day is more significant (P < 0.05 *), start within 5th day, significant difference degree between the two is more obvious (P < 0.01 * *).And TM12-14 and VSL#3 treatment can slow down the decline of UC mouse weight, at the 7th day, TM12-14 and VSL#The control of the weight loss of 3 mouse relative to model group than it is more significant (▲P<0.05).Illustrate that this two groups of probiotics can control weight loss situation caused by UC.It can be found that the weight of TM12-14 group mouse is slightly above VSL by comparing the weight numerical value of the 7th day each group#3, illustrate that TM12-14 is better than VSL in the ability that control UC mouse weight reduces#3。
The mouse of the ulcerative enteritis of DSS induction is since the variation of weight loss, stool and hematochezia situation causes the variation of DAI index, mouse DAI index variation such as table 6 and Fig. 5 before and after treatment:
Table 6
Table 6 and Fig. 5 statistics indicate that, the DAI of control group mice maintains a just constant low-level, and the mouse of DSS induction is due to there are a series of pathology, DAI is caused to gradually rise, beginning model group mouse DAI relative comparison group becomes extremely significant (P < 0.01 * *) within 3rd day, and model group mouse DAI reaches highest level within the 7th day.The intervention of probiotics can control the raising of DAI, TM12-14 and VSL#3 groups of mouse the 7th day DAI relative to model group obtained a degree of control (▲P<0.05).It can be with by the 7th day DAI numerical value
It was found that the DAI of TM2-14 group mouse is slightly below VSL#3, it may be said that bright TM12-14 is better than VSL in the control raised effect of UC mouse DAI#3。
The colonic tissue of UC model mice can change, and being primarily due to ulcer and inflammation causes colonic tissue to shorten, and after treatment end, be shown in Table 7 by the mouse Colon length of anatomic measurement.
Table 7
For table 7 the results show that the colonic tissue of the mouse (model group) after carrying out DSS and inducing 7 days shortens situation than more serious, compared with the control group difference is extremely significant (P < 0.01 * *).And probiotics TM12-14 and VSL#3 intervention can significantly control the shortening of mouse Colon (relative to model group▲P<0.05).The colon lengths ratio VSL of TM12-14 group mouse is can be found that by data in table#3 groups of mouse Colon length are long, it may be said that bright TM12-14 is better than VSL in the ability that control UC mouse Colon shortens#3。
Embodiment 7: the food compositions containing false small bifidobacterium catenulatum TM12-14
Raw material proportioning such as table 8.
Table 8
| Raw material | Mass percent (%) |
| False small bifidobacterium catenulatum TM12-14 | 0.5 |
| Milk | 90.0 |
| White sugar | 9.0 |
| Vitamin C | 0.5 |
According to above-mentioned formula rate mixing milk, white sugar, stirring preheats, 20Mpa pressure homogeneous to being thoroughly mixed, 90 DEG C or so sterilization 5-10 minutes, be cooled to 40-43 DEG C, be mixed into protective agent vitamin C, inoculation 1-100 × 106The small bifidobacterium catenulatum TM12-14 bacterium of vacation of cfu/g, that is, be made the food compositions containing false small bifidobacterium catenulatum TM12-14 bacterium.
Embodiment 8: the pharmaceutical composition containing false small bifidobacterium catenulatum TM12-14
Raw material proportioning is shown in Table 9.
Table 9
| Raw material | Mass percent (%) |
| False small bifidobacterium catenulatum TM12-14 | 1.0 |
| Lactose | 2.0 |
| Yeast powder | 2.0 |
| Peptone | 1.0 |
| Pure water | 93.5 |
| Vitamin C | 0.5 |
Lactose, yeast powder, peptone are uniformly mixed with pure water proportionally, are preheating to 60-65 DEG C, 20Mpa pressure homogeneous; 90 DEG C or so sterilization 20-30 minutes; it is cooled to 36-38 DEG C, is mixed into protective agent vitamin C, false small bifidobacterium catenulatum TM12-14 viable bacteria (1-500 × 10 of access6Cfu/mL), 36-38 DEG C of fermentation to pH value is 6.0, centrifugation, and freeze-drying less than 3%, that is, prepares false small bifidobacterium catenulatum TM12-14 bacterium freeze-drying object to water content.It is fitted into capsule after weighing the small bifidobacterium catenulatum TM12-14 freeze-drying object of 0.5 gram of vacation and maltodextrin mixed in equal amounts, that is, the pharmaceutical composition containing false small bifidobacterium catenulatum TM12-14 bacterium is made.
Embodiment 9: for treating the preparation method of the drug of ulcerative enteritis (UC)
1, bacterium solution prepares: by false small bifidobacterium catenulatum TM12-14 (1 × 109Cfu/ml) carry out Anaerobic culturel, anaerobic culture medium use PYG culture medium, by 37 DEG C anaerobic fermentation 2-3 days.
2, prepared by growth factor: skim milk, casein being mixed, are centrifuged, ultrafiltration obtains milk growth factor crude extract (containing ingredients such as vitamin substances, purine substance, pyrimidine substances).
3, pharmaceutical dosage form makes: the protective agent vitamin C of the 5 volume growth factors and 1 volume being added in the bacterium solution of TM12-14 fermentation of 100 volumes, mixing is sufficiently stirred, starch supplementary material (such as maltodextrin) is then added and prepares pharmaceutical dosage form.
The above content is specific embodiment is combined, further detailed description of the invention, and it cannot be said that specific implementation of the invention is only limited to these instructions.For those of ordinary skill in the art to which the present invention belongs, without departing from the inventive concept of the premise, a number of simple deductions or replacements can also be made, all shall be regarded as belonging to protection scope of the present invention.
Claims (15)
- A kind of small bifidobacterium catenulatum of vacation (Bifidobacterium pseudocatenulatum) TM12-14, is preserved in Guangdong Province's Culture Collection, and deposit number is GDMCC 60089.
- A kind of cultural method of the small bifidobacterium catenulatum TM12-14 of vacation described in claim 1, which is characterized in that the small bifidobacterium catenulatum TM12-14 of the vacation is inoculated in PYG culture medium and carries out Anaerobic culturel.
- A kind of probiotics containing the small bifidobacterium catenulatum TM12-14 of vacation described in claim 1 and/or its metabolite.
- A kind of food compositions containing the small bifidobacterium catenulatum TM12-14 of vacation described in claim 1 and/or its metabolite, health care product or auxiliary material additive.
- A kind of pharmaceutical composition containing the small bifidobacterium catenulatum TM12-14 of vacation described in claim 1 and/or its metabolite.
- Pharmaceutical composition as claimed in claim 5, which is characterized in that by the total volume or total weight of described pharmaceutical composition, described pharmaceutical composition contains 1 × 10-1To 1 × 1020The small bifidobacterium catenulatum TM12-14 of vacation of cfu/mL or cfu/g, preferably contains 1 × 104To 1 × 1015The small bifidobacterium catenulatum TM12-14 of vacation of cfu/mL or cfu/g.
- Pharmaceutical composition as described in claim 5, which is characterized in that described pharmaceutical composition also contains pharmaceutically acceptable carrier and/or auxiliary material.
- Pharmaceutical composition as described in claim 5, which is characterized in that described pharmaceutical composition also contains the substance for helping to maintain false small bifidobacterium catenulatum TM12-14 vigor.
- Application of the small bifidobacterium catenulatum TM12-14 of vacation described in claim 1 in the drug of preparation prevention and/or treatment ulcerative enteritis.
- The small bifidobacterium catenulatum TM12-14 of vacation described in claim 1 is preparing the application in probiotics.
- The small bifidobacterium catenulatum TM12-14 of vacation described in claim 1 is preparing the application in food compositions, health care product or auxiliary material additive.
- Application of the small bifidobacterium catenulatum TM12-14 of vacation described in claim 1 in production organic acid.
- Application of the small bifidobacterium catenulatum TM12-14 of vacation described in claim 1 in production short chain fatty acids.
- A method of prevention and/or treatment ulcerative enteritis, which is characterized in that apply pharmaceutical composition described in the small bifidobacterium catenulatum TM12-14 of vacation described in claim 1 or claim 5 to study subject.
- A method of blood lipid is reduced, the decline of weight of mammal is controlled, and/or reduces the disease activity index of mammal, it is characterized in that, applying pharmaceutical composition described in the small bifidobacterium catenulatum TM12-14 of vacation described in claim 1 or claim 5 to study subject.
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| WO2024066079A1 (en) * | 2022-09-28 | 2024-04-04 | 中国科学院深圳先进技术研究院 | Bile acid metabolizing bacterium for preventing and treating inflammatory bowel disease and use thereof |
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| CN113897302B (en) * | 2021-08-06 | 2022-08-09 | 东北农业大学 | Bifidobacterium capable of relieving colitis and application thereof |
| WO2023118868A1 (en) * | 2021-12-21 | 2023-06-29 | Quadram Institute Bioscience | Bifidobacterium and compositions thereof for breast cancer treatment |
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Non-Patent Citations (1)
| Title |
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| WO2024066079A1 (en) * | 2022-09-28 | 2024-04-04 | 中国科学院深圳先进技术研究院 | Bile acid metabolizing bacterium for preventing and treating inflammatory bowel disease and use thereof |
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