CN105624069A - Lactobacillus rhamnosus preparation and application thereof - Google Patents
Lactobacillus rhamnosus preparation and application thereof Download PDFInfo
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- CN105624069A CN105624069A CN201610143184.6A CN201610143184A CN105624069A CN 105624069 A CN105624069 A CN 105624069A CN 201610143184 A CN201610143184 A CN 201610143184A CN 105624069 A CN105624069 A CN 105624069A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
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- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
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Abstract
The invention belongs to the field of biological medicine and relates to a lactobacillus rhamnosus preparation and application thereof, in particular to a method for preparing a microecological preparation from lactobacillus rhamnosus serving as a main active ingredient and application of the microecological preparation in treating allergy and other related diseases. The lactobacillus rhamnosus is lactobacillus rhamnosus DH099 with the preservation number of CGMCC No.11229.
Description
Technical field
The present invention relates to lactobacillus rhamnosus preparation and application thereof, it is specifically related to lactobacillus rhamnosus and makes probiotics as main active ingredient, and this probiotics is in the application treated in relative disease, belongs to biological medicament field.
Background technology
Lactobacillus rhamnosus plays an increasingly important role in prevention and therapy disease, but the product effect containing lactobacillus rhamnosus listed at present is undesirable, therefore screening efficient lactobacillus rhamnosus bacterial classification is the problem needing solution at present badly, especially screens the lactobacillus rhamnosus bacterial classification with remarkable anti-allergic effects.
In recent years, the incidence of anaphylactic disease is in rising trend, data according to the World Health Organization, the current whole world has the people of 22% to suffer from anaphylactic disease, and sickness rate rapidly increases, by the end of 2015, the people of the existing 40-50% in the whole world suffered from allergy, and WHO has been classified as anaphylactic disease needs one of the three of primary study and control big diseases 21st century. The sickness rate of China's anaphylactic disease is up to 37.3% according to statistics, and main to affect crowd be infant.
Find effective irritated prophylactico-therapeutic measures extremely urgent. The current method commonplace for treatment anaphylactic disease uses pharmacological agent. Such as histamine antagonist, histamine is suppressed to react by blocking histamine susceptor, and the medicine of steroid and suppression mastocyte release inflammation material, trachea expanding agent medicine etc. But this kind of medicine only provides temporary transient remission, cannot really change allergic immune response, cure the symptoms, not the disease.
Lactobacillus rhamnosus probiotics of the present invention control is irritated waits curative effect of disease remarkable, and can be used as the application such as medicine, healthcare products, food, veterinary drug and fodder additives, and social effect is huge, and has no correlative study report, special this patent of invention of application.
Summary of the invention
It is an object of the invention to provide a kind of probiotics, the activeconstituents of this probiotics is lactobacillus rhamnosus DH099, deposit number CGMCCNo.11229, and this probiotics has the effect such as prevention or treatment body anaphylaxis.
Bacterium colony according to bacterial strain, morphological features, 16SrDNA sequential analysis and API Bacteria Identification systems analysis result confirm that bacterial strain is in taxonomy status, it has been found that the bacterial strain of numbering DH099 is lactobacillus rhamnosus; The feature of this strain bacterium on morphology and general character is as follows:
1, thalline or colonial morphology
Thalli morphology: thalline is under the microscope in rod-short, and two ends are circular. Thalline Cheng Dan, paired or one-tenth chain, do not form gemma.
Colonial morphology: form obvious bacterium colony on solid medium, diameter is between 0.5-1.0mm, and circular, neat in edge, oyster white, opaque, surface wettability is smooth.
2, physiological and biochemical property:
Growth temperature scope 2-53 DEG C, optimum growth temperature is 35-38 DEG C, amphimicrobian. The most suitable growth pH value 5.5-6.0 of bacterial strain. Not there is pathogenic property, not toxin producing.
In sugar-fermenting, amygdaloside, pectinose, cellobiose, polychrom, fructose, semi-lactosi, glucose, gluconate, lactose, maltose, N.F,USP MANNITOL, seminose, melizitose, rhamnosyl, ribose, saligenin, sorbyl alcohol, sucrose, trehalose all can be fermented, and melibiose, silk floss sugar, wood sugar can not be fermented; Arginine produces NH3; Nitrate can be reduced when PH value is more than 6.0; Not liquefy gelatin; Do not decompose casein, a small amount of soluble nitrogen can be produced; Do not produce indoles and H2S; Catalase is negative; Acellular pigment; Benzidine reaction is negative.
This probiotics is made total viable count that solids composition comprises and is not less than 1 �� 106CFU/g, generally 1 �� 109More than CFU/g, can reach 1 �� 1011CFU/g or 1 �� 1011More than CFU/g; Making total viable count that liquid composition comprises can not lower than 1 �� 106CFU/mL, generally 1 �� 109More than CFU/mL, can reach 1 �� 1011CFU/mL or 1 �� 1011More than CFU/mL.
This probiotics can be used for the multiple human body diseases such as prevention or treatment body anaphylaxis, treats the diseases such as eczema, allergic rhinitis, atopic dermatitis, allergic asthma or food anaphylaxis further.
This probiotics can be made into composite medicine, healthcare products, food, veterinary drug or fodder additives etc.
Lactobacillus rhamnosus described in this probiotics refers to the biont lived.
Embodiment
Preparation example illustrates: be that example is specifically described by lactobacillus rhamnosus DH099 deposit number CGMCC11229 powder here, preparation method those skilled in the art of other lactobacillus rhamnosus bacterial classification preparations are easy to grasp by the present embodiment, preparation method those skilled in the art of other formulations are easy to grasp by this enforcement, describe explanation no longer one by one at this. Preparation method is not limited to described in the embodiment of the present invention, and known can to reach the method preparing object all passable, and the preparation of embodiment illustrates just explanation to the present invention, is not limiting the scope of the invention.
The preparation of preparation embodiment lactobacillus rhamnosus powder
The preparation of 1 bacterium powder and the qualification of bacterial classification
Collect the fresh excreta 5-10g of Natural excrement with sterile chamber, put into rapidly anaerobic jar, take back laboratory and carry out strain separating. Get faecal samples 1.5g, even with 15mlPBS damping fluid suspended mixture, pure and turbid liquid in the centrifugal 5min collection of 400g, then add the appropriate centrifugal 10min of PBS 8000g, pure and turbid liquid in collection, namely obtains test sample. Draw turbid liquid on 1ml to add a test tube mesoscale eddies that 9mlPBS diluent (containing 5 �� Cys) is housed and mix even, as 10-2Diluent, is diluted to 10 by same method-3, get ight soil stoste, 10 respectively-2Diluent, 10-3The each 0.1ml of diluent coats on MRS flat board, is placed in 37 DEG C of constant incubators cultivations after 24 hours, rounded on Selective agar medium, neat in edge, protuberance, oyster white is opaque, show moistening smooth, diameter, at the bacterium colony of 0.5-1.0mm, is inoculated in MRS liquid nutrient medium, after 37 DEG C of cultivation 24h, after gained medium centrifugal (12000rpm) is isolated thalline, by thalline vacuum freezedrying, modulating dry bacterium powder, viable count is 1 �� 109More than CFU/g. Bacterial strain is launched physiological and biochemical property qualification simultaneously. Catalase experiment and curdled milk experiment is carried out respectively by bacterial strain separation obtained, remove the catalase positive and the bacterial strain without curdled milk phenomenon, after getting rid of non-object bacterial strain, remaining bacterial strain is continued through BIOLOG and API50CH and carries out physiological and biochemical property qualification, finally by extraction DNA sequencing, the classification position of further clear and definite bacterial strain. Through being accredited as lactobacillus rhamnosus DH099, rhamnosyl breast bar DH105, rhamnosyl breast bar DH117, rhamnosyl breast bar DH033, rhamnosyl breast bar DH021.
2 toxicity tests
60 SPF rank mouse are got in 2.1 animals and grouping, 6��8 week age, and body weight 15��19g, is randomized into each lactobacillus rhamnosus group and non-administered group, often organize 10.
Above-mentioned lactobacillus rhamnosus bacterium powder is modulated to by purified water and is 1 �� 10 containing bacterium number by 2.2 preparation bacterium liquid respectively9The bacterium liquid of CFU/mL.
2.3 method lactobacillus rhamnosus groups and non-administered group all give identical basal feed, and rearing conditions is all consistent, rhamnosyl bacillus group gavages lactobacillus rhamnosus bacterium liquid 0.5mL every day, and non-administered group gavages purified water 0.5mL every day, feed 6 months, observe toxic reaction.
2.4 result
All there are not abnormal conditions in each group mouse, shake quiver, spasm, ataxia, attitude abnormal, without ophthalmoptosis, urinate normal, skin, breathe normally, without death condition, show that lactobacillus rhamnosus does not exist toxicity.
3 are prepared into the formulations such as powder
After above-mentioned steps and method isolation identification, through examination nontoxicity, so that it may lactobacillus rhamnosus bacterial classification is made bacterium powder, then add relevant auxiliary materials as required and make various formulation, preferably according to the viable count of lactobacillus rhamnosus bacterium powder, make powder, make viable count be not less than 1 �� 107CFU/g, then packs.
Effect embodiment illustrates: the present invention illustrates the effect of lactobacillus rhamnosus by representative of lactobacillus rhamnosus DH099 deposit number CGMCC11229, but is not limitation of the present invention.
Effect embodiment 1: lactobacillus rhamnosus alleviates hypersensitive test research
1 materials and methods
1.1 material
1.1.1 test products
Lactobacillus rhamnosus bacterium DH099, DH105, DH117, DH033, DH021 powder alive, viable count is 1 �� 107More than CFU/g.
1.1.2 experimental animal
Cleaning grade is weaned male BALB/c inbred mouse 49 3 week age.
1.2 method
49 mouse are divided into 7 groups at random, often organize each 7. To be dissolved in physiological saline by the probiotic bacterium of each group mixed even makes muddy liquid, makes final concentration be 1 �� 108Cfu/ml. Gavage gives 20ml/kg, and every per daily dose divides twice gavage. Negative control group: be dissolved in physiological saline by lyophilized vaccine and make turbid solution, mouse gavage 20ml/kg every day, every per daily dose divides twice gavage. Positive controls: select the lactobacillus rhamnosus being separated acquisition in similar probiotic products from the market, be dissolved in physiological saline to make 1 �� 10 by this bacterial strain thalline8The muddy liquid of cfu/ml, mouse gavage 20ml/kg every day, every per daily dose divides twice gavage.
1.3 set up delayed type hypersensitivity animal model
Gavage second day mouse web portion shaves hair first, 3cm �� 3cm scope, so that sensitization position fully exposes. Evenly coating 20 �� L0.5%DNFB solution (being dissolved in 4:1 acetone: in sweet oil) sensitization. To each group of mouse continuous gavage 14 days as stated above, after last administration 1 hour, evenly it is coated with 0.25%DNFB solution 10 �� L on the left ear two sides of mouse and attacks, bring out mouse delayed type hypersensitivity model. Cut ears after 60min to weigh.
1.4 observation index
Ear weight: getting tissue block in the middle part of ear with punch tool, analytical balance is weighed, calculates the weight difference of left and right ear tissue block. Respectively organize numerical value, check with sample mean significance of difference t and make statistical procedures.
1.5 calculation formula
Swelling (mg)=right auricle weight-left auricle weight
Inhibiting rate (%)=(the average swelling of blank group-each average swelling of administration group)/average swelling �� 100% of blank group
2 test-results and analysis
2.1 test-results
The different probiotic strain of table 1 is on the impact (n=7) of mouse delayed type hypersensitivity
2.2 analyze
Compared with the lactobacillus rhamnosus that like product well-known on market is separated acquisition and other bacterial strain of being simultaneously separated from sample, lactobacillus rhamnosus DH099 can significantly suppress the ear thickness of the mouse delayed type hypersensitivity brought out by DNFB, inhibiting rate is 67.35%, and difference has statistical significance (P < 0.05). Therefore this probiotics has the anaphylaxis function of good suppression body compared to other bacterial strain.
The microbial strains that the present invention uses in implementation process in 2015 08 year 11 days in (No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center, Institute of Microorganism, Academia Sinica's postcode 100101) preservation, microbial strains information is as follows.
Classification name: lactobacillus rhamnosus Lactobacillusrhamnosus, preserves numbering CGMCCNo.11229.
Mentioned microorganism bacterial classification is through this microorganism Spot detection, and detected result is survival.
Claims (6)
1. a probiotics, it is characterised in that the activeconstituents of this probiotics is lactobacillus rhamnosus, described lactobacillus rhamnosus is lactobacillus rhamnosus DH099, deposit number CGMCCNo.11229.
2. by probiotics according to claim 1, it is characterised in that lactobacillus rhamnosus refers to the biont lived.
3. the application of probiotics according to claim 1, it is characterised in that the lactobacillus rhamnosus of effective dose makes composite medicine, healthcare products, food, veterinary drug or fodder additives as active ingredient.
4. the purposes that probiotics according to claim 1 prevents in preparation or treats in body anaphylaxis composition.
5. probiotics according to claim 1 treats the purposes in eczema, allergic rhinitis, atopic dermatitis, allergic asthma or food anaphylaxis composition in preparation.
6. by claim 1 probiotics, it is characterised in that total viable count that solids composition comprises is not less than 1 �� 106CFU/g, generally 1 �� 109More than CFU/g, can reach 1 �� 1011CFU/g or 1 �� 1011More than CFU/g; Or total viable count that liquid composition comprises can not lower than 1 �� 106CFU/mL, generally 1 �� 109More than CFU/mL, can reach 1 �� 1011CFU/mL or 1 �� 1011More than CFU/mL.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106520598A (en) * | 2016-10-25 | 2017-03-22 | 天益健康科学研究院（镇江）有限公司 | Lactobacillus rhamnosus and application |
| CN107217022A (en) * | 2017-07-18 | 2017-09-29 | 邓禹 | One plant of Lactobacillus rhamnosus and its application |
| CN109666614A (en) * | 2019-02-14 | 2019-04-23 | 西南大学 | A kind of Lactobacillus rhamnosus and its application in the health food and drug of preparation prevention allergic asthma |
| CN111991428A (en) * | 2020-06-12 | 2020-11-27 | 郑州和合生物工程技术有限公司 | Probiotic composition with asthma improving effect and preparation method thereof |
| CN114774315A (en) * | 2022-04-13 | 2022-07-22 | 微康益生菌(苏州)股份有限公司 | Application of lactobacillus rhamnosus strain LRa05 in preparing immunity enhancing and/or eczema relieving product |
-
2016
- 2016-03-14 CN CN201610143184.6A patent/CN105624069B/en active Active
Non-Patent Citations (2)
| Title |
|---|
| MARTIN L. CROSS ET AL.: "Dietary intake of Lactobacillus rhamnosus HN001 enhances production of both Th1 and Th2 cytokines in antigen-primed mice", 《MED MICROBIOL IMMUNOL》 * |
| 贺璟等: "鼠李糖乳杆菌功能特性的研究进展", 《农产品加工·学刊》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106520598A (en) * | 2016-10-25 | 2017-03-22 | 天益健康科学研究院（镇江）有限公司 | Lactobacillus rhamnosus and application |
| CN107217022A (en) * | 2017-07-18 | 2017-09-29 | 邓禹 | One plant of Lactobacillus rhamnosus and its application |
| CN109666614A (en) * | 2019-02-14 | 2019-04-23 | 西南大学 | A kind of Lactobacillus rhamnosus and its application in the health food and drug of preparation prevention allergic asthma |
| CN111991428A (en) * | 2020-06-12 | 2020-11-27 | 郑州和合生物工程技术有限公司 | Probiotic composition with asthma improving effect and preparation method thereof |
| CN114774315A (en) * | 2022-04-13 | 2022-07-22 | 微康益生菌(苏州)股份有限公司 | Application of lactobacillus rhamnosus strain LRa05 in preparing immunity enhancing and/or eczema relieving product |
| CN114774315B (en) * | 2022-04-13 | 2023-12-19 | 微康益生菌(苏州)股份有限公司 | Application of lactobacillus rhamnosus strain LRa05 in preparation of immunity enhancing product and/or eczema relieving product |
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