CN105586299B - Pro-Bionate and its application - Google Patents
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- CN105586299B CN105586299B CN201610143741.4A CN201610143741A CN105586299B CN 105586299 B CN105586299 B CN 105586299B CN 201610143741 A CN201610143741 A CN 201610143741A CN 105586299 B CN105586299 B CN 105586299B
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- lactobacillus acidophilus
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Classifications
-
- 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
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/113—Acidophilus
-
- 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
- A61K2035/11—Medicinal preparations comprising living procariotic cells
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention relates to Pro-Bionate and its applications, probiotics are made as main active ingredient more particularly to lactobacillus acidophilus, and application of the probiotics in the related diseases such as treatment allergy, the lactobacillus acidophilus is lactobacillus acidophilus DH213, deposit number CGMCC No.11228, belongs to biological medicament field.
Description
Technical field
The present invention relates to Pro-Bionate and its applications, and in particular to lactobacillus acidophilus is as main active ingredient system
At the application of probiotics and the probiotics in treatment related disease, belong to biological medicament field.
Background technique
Lactobacillus acidophilus plays an increasingly important role in terms of preventing and treating disease, but what is listed at present contains
There is the product effect of lactobacillus acidophilus undesirable, therefore screen the problem of efficient Lactobacillus acidophilus species are current urgent need to resolve,
Especially screen the Lactobacillus acidophilus species with significant anti-allergic effects.
In recent years, the incidence of anaphylactic disease is in rising trend, and according to the data of the World Health Organization, the current whole world has
22% people suffers from anaphylactia, and disease incidence rapidly increases, and by the end of 2015, the people of the existing 40-50% in the whole world was suffered from
Anaphylactia is classified as one of the three big diseases that 21 century needs primary study and prevention and treatment by allergy, WHO.China according to statistics
The disease incidence of anaphylactia is up to 37.3%, main to influence crowd for infant.
It is extremely urgent to find effective allergy control measure.It is for the commonplace method for the treatment of anaphylactia at present
Use drug therapy.Such as histamine antagonist, histamine can be inhibited to react and steroids and inhibition by blocking histamine receptor
Drug, the trachea expanding agent drug etc. of mast cell release inflammation substance.But this kind of drug only provides temporary remission,
Allergic immune response can not really be changed, it is palliative.
However not all probiotic strain has antianaphylactic effect.Taylor etc. will be pregnant with anaphylactia
The newborn of woman is divided into two groups, after birth in 6 months, takes lactobacillus acidophilus or placebo daily, at as a result 6 months
The atopic dermatitis disease incidence of two groups of babies does not have difference, and the lactobacillus acidophilus for prompting the test to use can't reduce spy
Anisotropic dermatitis disease incidence (Taylor AL, Dunstan JA, Prescott SL, et a1.Probiotic
supplementationfor the first 6months of life falls to reduce the risk of
atopic dermatitis and increases the risk of allergen sensitization in high-
Risk children.a randomized controlled trial.J Allergy Clin lnlmunol, 2007,119
(1): 184-191.).One randomized control study give through suck allergen-induced asthma teenager and adult take and contain
There is the dairy produce of streptococcus thermophilus and lactobacillus acidophilus, as the result is shown the laboratory of the clinical symptoms of asthma, lung function and inflammation
Marker is relative to control group no change has taken place (Wheeler JG, Shema SJ, Bogle ML, et a1.Immune
and clinical impact of Lactobacillus acidophilus on asthma.Ann Allergy Asthma
Immunol, 1997,79 (3): 229-233.).Illustrate that only certain lactic acid bacteria strains have antianaphylactic immunization.The present invention
It is significant that lactobacillus acidophilus probiotics prevent and treat the curative effect of disease such as allergy, and can be used as drug, health care product, food, veterinary drug and feeding
The application such as feed additives, social effect is huge, and has no that correlative study is reported, spy's application the invention patent.
Summary of the invention
The object of the present invention is to provide a kind of probiotics, the active constituent of the probiotics is lactobacillus acidophilus
DH213, deposit number CGMCC No.11228, the probiotics have the effects that prevent or treat body allergic reaction.
According to the bacterium colony of bacterial strain, morphological features, the analysis of 16SrDNA sequence and API Bacteria Identification network analysis result
To confirm that bacterial strain in taxonomy, it is found that the bacterial strain of number DH213 is lactobacillus acidophilus；This plant of bacterium is in morphology and generality
Feature in matter is as follows:
1, thallus or colonial morphology
Thalli morphology: thallus is in rod-short under the microscope, and both ends are circle, 1.5-6 microns long, 0.6-0.9 microns wide,
With the arrangement of single, in pairs or short chain.It does not move, atrichia, no gemma, Gram-positive.
Colonial morphology: colonial morphology is usually rougher on solid medium, is shown as winding or microtriche with micro- sem observation
Filiform radiates to come from bacterium heap center, and deep colony is in radiation or the irregular shape of branch, non-pigment.
2, physiological and biochemical property:
Optimum growth temperature is 35-38 DEG C, and 20 DEG C or less do not grow, amphimicrobian.The most suitable growth pH value 5.5- of bacterial strain
6.0, acid resistance is strong.Without pathogenic, not toxin producing.
It is amarogentin, cellobiose, aesculin, fructose, galactolipin, glucose, lactose, maltose, sweet in sugar fermentation
Dew sugar, salicin, sucrose can be fermented；Unfermentable arabinose, gluconate, mannitol, melezitose, rhamnose, core
Sugar, sorbierite, xylose；Arginine hydrolysis does not produce NH3, glucose fermentation do not produce gas, and catalase is negative.
This probiotics is made total viable count that solid composite includes and is not less than 1 × 106CFU/g, generally 1 ×
109CFU/g or more can reach 1 × 1011CFU/g or 1 × 1011CFU/g or more；Total work that liquid composition includes is made
Bacterium number cannot be below 1 × 106CFU/mL, generally 1 × 109CFU/mL or more can reach 1 × 1011CFU/mL or 1 ×
1011CFU/mL or more.
This probiotics can be used for preventing or treating a variety of human body diseases such as body allergic reaction, further treat wet
The diseases such as rash, allergic rhinitis, atopic dermatitis, allergic asthma or food hypersenstivity.
This probiotics can be made into composite medicine, health care product, food, veterinary drug or feed addictive etc..
Lactobacillus acidophilus described in this probiotics refers to bion living.
Specific embodiment
Preparation example explanation: here by being carried out for lactobacillus acidophilus DH213 deposit number CGMCC No.11228 powder
It illustrates, preparation method those skilled in the art of other Lactobacillus acidophilus species' preparations are easy to slap through this embodiment
It holds, preparation method those skilled in the art of other dosage forms are easy to grasp by this implementation, no longer describe explanation one by one herein.
Preparation method is not limited to described in the embodiment of the present invention, and the well known method that can reach preparation purpose can be with embodiment
Preparation explanation be the description of the invention, be not limiting the scope of the invention.
Prepare the preparation of embodiment lactobacillus acidophilus powder
The preparation of 1 bacterium powder and the identification of strain
The fresh excreta 5-10g naturally drained is collected with sterile chamber, is put into anaerobic jar rapidly, and laboratory progress is taken back
Strain separating.Fecal specimens 1.5g is taken, is suspended and is mixed with 15mlPBS buffer, pure and turbid liquid in 400g centrifugation 5min collection, then plus
Enter appropriate PBS buffer solution 8000g centrifugation 10min, pure and turbid liquid is in collections to get to test sample.It draws turbid on 1ml and is added one
Branch is mixed equipped with the test tube mesoscale eddies of 9mlPBS dilution (containing 5 ‰ L-cysteines), as 10-2Dilution, with same sample prescription
Method is diluted to 10-3, excrement stoste, 10 are taken respectively-2Dilution, 10-3Each 0.1ml of dilution is coated on MRS plate, is placed in 37
After cultivating 24 hours in DEG C constant incubator, the more coarse bacterium colony of colonial morphology on Selective agar medium is inoculated in the training of MRS liquid
It supports in base, after 37 DEG C of cultures for 24 hours, after gained medium centrifugal (12000rpm) is isolated thallus, thallus freezing vacuum is done
It is dry, dry bacterium powder is modulated, viable count is 1 × 109CFU/g or more.Simultaneously to bacterial strain expansion physiological and biochemical property identification.It is logical
It crosses and catalase experiment and curdled milk experiment, removal contact enzyme positive and the bacterium without curdled milk phenomenon is carried out respectively to the bacterial strain that separation obtains
Strain after excluding non-purpose bacterial strain, continues through BIOLOG and API 50CH to remaining bacterial strain and carries out physiological and biochemical property identification,
Finally by DNA sequencing is extracted, the classification position of bacterial strain is further clarified.It is identified as lactobacillus acidophilus DH213, acidophilus cream bar
DH429, lactobacillus acidophilus S3, lactobacillus acidophilus S1.
2 toxicity tests
2.1 animals and grouping take 50 SPF rank mouse, 6~8 week old, and 15~19g of weight is randomized into 4 acidophilus creams
Bacillus group and non-administered group, every group 10.
Above-mentioned lactobacillus acidophilus bacterium powder is modulated to containing bacterium number be 1 × 10 by 2.2 preparation bacterium solutions with purified water respectively9CFU/
The bacterium solution of mL.
2.3 method lactobacillus acidophilus groups and non-administered group give identical basal feed, and rearing conditions are consistent, thermophilic
Sour bacterium group gavages each lactobacillus acidophilus bacterium solution 0.5mL daily, and non-administered group gavages purified water 0.5mL daily, feeds 6 months, sees
Examine toxic reaction.
2.4 result
Each group mouse does not occur abnormal conditions, and chatter, spasm, ataxia, posture exception do not occur, and no eyeball is prominent
Out, urination is normal, and skin, breathing are normal, no death condition, and showing lactobacillus acidophilus, there is no toxicity.
3 are prepared into the dosage forms such as powder
It is separated and identified according to above-mentioned steps and method, through experimental check nontoxicity, so that it may by Lactobacillus acidophilus species' system
It at bacterium powder, is then added as required relevant auxiliary materials and various dosage forms is made, preferably according to the viable count of lactobacillus acidophilus bacterium powder, system
At powder, make viable count not less than 1 × 107Then CFU/g is packed.
Application effect embodiment explanation: the present invention is said using lactobacillus acidophilus DH213 deposit number CGMCC11228 as representative
The application effect of bright lactobacillus acidophilus, but be not limitation of the present invention.
Application effect embodiment 1: lactobacillus acidophilus Reduce allergy experimental study
1 materials and methods
1.1 material
1.1.1 test products
Live lactobacillus acidophilus DH213, DH429, S3, S1 powder, viable count are 1 × 107CFU/g or more.
1.1.2 experimental animal
3 week old of cleaning grade is weaned male BALB/c inbred mouse 42.
1.2 method
42 mouse are randomly divided into 6 groups, every group each 7.The probiotics of each group is dissolved in mix in physiological saline and is made
Dirty solution makes final concentration of 1 × 108cfu/ml.20ml/kg, daily dosage stomach-filling in two times are given in stomach-filling.Negative control group:
Freeze drying protectant is dissolved in physiological saline, turbid solution is made, the daily stomach-filling 20ml/kg of mouse, daily dosage stomach-filling in two times.
Positive controls: the lactobacillus acidophilus for separating acquisition in similar probiotic products from the market is selected, which is dissolved in
1 × 10 is made in physiological saline8Cfu/ml dirty solution, the daily stomach-filling 20ml/kg of mouse, daily dosage stomach-filling in two times.
1.3 establish delayed allergy animal model
Stomach-filling second day mouse web portion shaving for the first time, 3cm × 3cm range, so that sensitization position sufficiently exposes.Even spread
20 μ L 0.5%DNFB solution (being dissolved in 4:1 acetone: in olive oil) sensitization.To each group mouse continuous gavage 14 according to the above method
It, 1 hour after the last administration, is attacked in the left 10 μ L of ear two sides even spread 0.25%DNFB solution of mouse, induces mouse
Delayed allergy model.Ears weighing is cut after 60min.
1.4 observation index
Ear weight: tissue block in the middle part of ear is taken with punch, assay balance weighing calculates the weight difference of left and right ear tissue block.
Compare each group numerical value, the processing that takes statistics is examined with sample average significance of difference t.
1.5 calculation formula
Swelling (mg)=auris dextra sheet weight-left auricle weight
Inhibiting rate (%)=(blank control group be averaged each administration group of swelling-be averaged swelling)/blank control group is average
Swelling × 100%
2 test results and analysis
2.1 test result
Influence (n=7) of the different probiotics strains of table 1 to mouse delayed allergy
2.2 analysis
The other bacterial strains for separating the lactobacillus acidophilus of acquisition with well-known similar product in the market and being separated simultaneously from sample
It compares, lactobacillus acidophilus DH213 can be significantly inhibited by the ear thickness of the DNFB mouse delayed allergy induced, be inhibited
Rate is 65.60%, and difference has statistical significance (P < 0.05).Therefore the probiotics have good compared to other bacterial strains
Inhibition body allergic reaction function.
Microorganism fungus kind of the present invention used in implementation process is in 11 days 08 year 2015 in China Microbiological bacterium
Kind preservation administration committee common micro-organisms center (Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences microorganism
Research institute's postcode 100101) preservation, microorganism fungus kind information is as follows.
Classification naming: lactobacillus acidophilus Lactobacillus acidophilus saves number CGMCC No.11228.
For mentioned microorganism strain through the microorganism Spot detection, testing result is survival.
Claims (9)
1. a kind of probiotics, it is characterised in that the active constituent of the probiotics is lactobacillus acidophilus, the acidophilus cream
Bacillus is lactobacillus acidophilus DH213, deposit number CGMCC No.11228.
2. probiotics according to claim 1, it is characterised in that lactobacillus acidophilus refers to bion living.
3. the application of probiotics described in claim 1, it is characterised in that the lactobacillus acidophilus of effective dose is as activity
Composite medicine, health care product, food or feed addictive is made in composition.
4. composite medicine as claimed in claim 3 includes veterinary drug.
5. purposes of the probiotics described in claim 1 in preparation prevention or treatment body allergic reaction composition.
6. probiotics described in claim 1 are roared in preparation treatment eczema, allergic rhinitis, atopic dermatitis, anaphylaxis
Purposes in asthma or food hypersenstivity composition.
7. probiotics according to claim 1, it is characterised in that total viable count that solid composite includes not less than 1 ×
106Total viable count that CFU/g or liquid composition include cannot be below 1 × 106CFU/mL。
8. probiotics according to claim 1, it is characterised in that total viable count preferably 1 that solid composite includes ×
109Total viable count preferably 1 × 10 that CFU/g or more or liquid composition include9CFU/mL or more.
9. probiotics according to claim 1, it is characterised in that total viable count preferably 1 that solid composite includes ×
1011Total viable count preferably 1 × 10 that CFU/g or more or liquid composition include11CFU/mL or more.
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| CN106011006A (en) * | 2016-06-01 | 2016-10-12 | 河北然生物科技有限公司 | Lactobacillus acidophilus La28 with immunoregulation function and allergy-resisting function and applications of lactobacillus acidophilus La28 |
| CN106350468B (en) * | 2016-09-08 | 2019-05-10 | 济南康多宝生物技术有限公司 | A kind of acidophilic lactobacillus |
| WO2018112741A1 (en) * | 2016-12-20 | 2018-06-28 | 深圳华大基因研究院 | Lactobacillus acidophilus, culture method therefor and application thereof |
| CN107616985A (en) * | 2017-10-01 | 2018-01-23 | 青岛东海药业有限公司 | Bacillus coagulans preparation and its application |
| CN110643542B (en) * | 2019-10-25 | 2021-01-29 | 江南大学 | Lactobacillus reuteri capable of relieving Th2 reaction of allergic asthma and application thereof |
| CN110787190B (en) * | 2019-12-17 | 2020-06-16 | 杨波 | Compound probiotic composition with anti-aging effect and preparation method and application thereof |
| CN117085046A (en) * | 2023-10-20 | 2023-11-21 | 潍坊君薇生物科技有限责任公司 | Lactobacillus acidophilus LS001 metaplasia and application thereof |
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