CN105624069B - Lactobacillus rhamnosus preparation and its application - Google Patents
Lactobacillus rhamnosus preparation and its application Download PDFInfo
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Abstract
The present invention relates to Lactobacillus rhamnosus preparation and its applications, probiotics are made as main active ingredient more particularly to Lactobacillus rhamnosus, and application of the probiotics in the related diseases such as treatment allergy, the Lactobacillus rhamnosus is Lactobacillus rhamnosus DH099, deposit number CGMCC No.11229, belongs to biological medicament field.
Description
Technical field
The present invention relates to Lactobacillus rhamnosus preparation and its applications, and in particular to Lactobacillus rhamnosus as chief active at
The application of probiotics and the probiotics in treatment related disease is made in part, belongs to biological medicament field.
Background technique
Lactobacillus rhamnosus plays an increasingly important role in terms of preventing and treating disease, but listed at present
Product effect containing Lactobacillus rhamnosus is undesirable, therefore screening efficient Lactobacillus rhamnosus strain is current urgent need to resolve
Problem especially screens the Lactobacillus rhamnosus strain 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.
The curative effect of disease such as Lactobacillus rhamnosus probiotics prevention and treatment allergy of the present invention are significant, and can be used as drug, health care
The application such as product, food, veterinary drug and feed addictive, social effect is huge, and has no that correlative study is reported, spy's application the invention is special
Benefit.
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 rhamnosus
DH099, deposit number CGMCC No.11229, 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 DH099 is Lactobacillus rhamnosus；This plant of bacterium is in morphology and generally
Qualitative feature is as follows:
1, thallus or colonial morphology
Thalli morphology: thallus is in rod-short under the microscope, and both ends are round.Thallus Cheng Dan, pairs of or chaining, do not form bud
Spore.
Colonial morphology: forming obvious bacterium colony on solid medium, diameter is round between 0.5-1.0mm, neat in edge,
Milky, opaque, surface wettability is smooth.
2, physiological and biochemical property:
2-53 DEG C of growth temperature range, optimum growth temperature is 35-38 DEG C, amphimicrobian.The most suitable growth pH value of bacterial strain
5.5-6.0.Without pathogenic, not toxin producing.
In sugar fermentation, amarogentin, arabinose, cellobiose, aesculin, fructose, galactolipin, glucose, glucose
Hydrochlorate, lactose, maltose, mannitol, mannose, melezitose, rhamnose, ribose, salicin, sorbierite, sucrose, trehalose are equal
It can be fermented, melibiose, silk floss sugar, xylose cannot be fermented；Arginine produces NH3；PH value can go back orthonitric acid at 6.0 or more
Salt；Do not liquefy gelatin；Casein is not decomposed, a small amount of soluble nitrogen can be generated；Do not produce indoles and H2S；Catalase is negative；It is cell-free
Pigment；Benzidine reaction 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 rhamnosus described in this probiotics refers to bion living.
Specific embodiment
Preparation example explanation: here by being had for Lactobacillus rhamnosus DH099 deposit number CGMCC11229 powder
Body explanation, preparation method those skilled in the art of other Lactobacillus rhamnosus strain 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 rhamnosus 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, rounded on Selective agar medium, neat in edge, protuberance, milky is opaque, shows
Moisten smooth, diameter is inoculated in MRS fluid nutrient medium in the bacterium colony of 0.5-1.0mm, after 37 DEG C of cultures for 24 hours, by gained
After medium centrifugal (12000rpm) isolates thallus, by thallus vacuum freezedrying, 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 connect respectively by the bacterial strain obtained to separation
Catalase experiment and curdled milk experiment, removal contact enzyme positive and the bacterial strain without curdled milk phenomenon, after excluding non-purpose bacterial strain, to remaining
Bacterial strain continues through BIOLOG and API 50CH and carries out physiological and biochemical property identification, further bright finally by extraction DNA sequencing
The classification position of true bacterial strain.It is identified as Lactobacillus rhamnosus DH099, rhamnose cream bar DH105, rhamnose cream bar DH117, mouse
The newborn bar DH033 of Lee's sugar, rhamnose cream bar DH021.
2 toxicity tests
2.1 animals and grouping take 60 SPF rank mouse, 6~8 week old, and 15~19g of weight is randomized into each rhamnose
Lactobacillus group and non-administered group, every group 10.
2.2 preparation bacterium solutions by above-mentioned Lactobacillus rhamnosus bacterium powder respectively with purified water be modulated to containing bacterium number be 1 ×
109The bacterium solution of CFU/mL.
2.3 method Lactobacillus rhamnosus groups and non-administered group give identical basal feed, and rearing conditions are uniform
It causes, sandlwood saccharobacillus group gavages Lactobacillus rhamnosus bacterium solution 0.5mL daily, and non-administered group gavages purified water 0.5mL daily, feeding
6 months, observe 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 rhamnosus, 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 rhamnosus strain
Bacterium powder is made, is then added as required relevant auxiliary materials and various dosage forms is made, preferably according to the viable bacteria of Lactobacillus rhamnosus bacterium powder
Number, is made powder, makes viable count not less than 1 × 107Then CFU/g is packed.
Application effect embodiment explanation: the present invention is using Lactobacillus rhamnosus DH099 deposit number CGMCC11229 as representative
Illustrate the application effect of Lactobacillus rhamnosus, but is not limitation of the present invention.
Application effect embodiment 1: Lactobacillus rhamnosus Reduce allergy experimental study
1 materials and methods
1.1 material
1.1.1 test products
Lactobacillus rhamnosus viable bacteria DH099, DH105, DH117, DH033, DH021 powder, viable count is 1 ×
107CFU/g or more.
1.1.2 experimental animal
3 week old of cleaning grade is weaned male BALB/c inbred mouse 49.
1.2 method
49 mouse are randomly divided into 7 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: selecting the Lactobacillus rhamnosus that acquisition is separated in similar probiotic products from the market, and the bacterial strain thallus is molten
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 bacterium for separating the Lactobacillus rhamnosus of acquisition with well-known similar product in the market and being separated simultaneously from sample
Strain is compared, and Lactobacillus rhamnosus DH099 can be significantly inhibited by the ear thickness of the DNFB mouse delayed allergy induced,
Inhibiting rate is 67.35%, and difference has statistical significance (P < 0.05).Therefore the probiotics have compared to other bacterial strains
The good allergic reaction function of inhibiting body.
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 rhamnosus Lactobacillus rhamnosus saves number CGMCC No. 11229.
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 rhamnosus, the sandlwood
Sugared lactobacillus is Lactobacillus rhamnosus DH099, deposit number CGMCC No.11229.
2. probiotics according to claim 1, it is characterised in that Lactobacillus rhamnosus refers to bion living.
3. the application of probiotics described in claim 1, it is characterised in that the Lactobacillus rhamnosus of effective dose is as work
Composite medicine, health care product, food or feed addictive is made in sexual element.
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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| 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 |
| CN109666614B (en) * | 2019-02-14 | 2022-05-06 | 西南大学 | Lactobacillus rhamnosus and application thereof in preparing health food and medicine for preventing 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 |
| 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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- 2016-03-14 CN CN201610143184.6A patent/CN105624069B/en active Active
Non-Patent Citations (2)
| Title |
|---|
| Dietary intake of Lactobacillus rhamnosus HN001 enhances production of both Th1 and Th2 cytokines in antigen-primed mice;Martin L. Cross et al.;《Med Microbiol Immunol》;20020326;第191卷;摘要，第52页第1段 * |
| 鼠李糖乳杆菌功能特性的研究进展;贺璟等;《农产品加工·学刊》;20120331(第3期);第117-120页 * |
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