CN103550258B - The purposes of lactobacillus strain immunity moderation reaction - Google Patents

The purposes of lactobacillus strain immunity moderation reaction Download PDF

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CN103550258B
CN103550258B CN201310481560.9A CN201310481560A CN103550258B CN 103550258 B CN103550258 B CN 103550258B CN 201310481560 A CN201310481560 A CN 201310481560A CN 103550258 B CN103550258 B CN 103550258B
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lactobacillus
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mouse
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陈庆源
邱雪惠
宋璧君
庄雅惠
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FOODSTUFF INDUSTRIAL DEVELOPMENT INST OF FINANCIAL GROUP LEGAL PERSONS
YI QING TECHNOLOGY Co Ltd
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Abstract

The invention relates to novel lactobacillus strain MP137, and the purposes of immunity moderation reaction.Particularly, lactobacillus strain of the present invention can promote Th1 to react and suppress Th2 reaction.

Description

The purposes of lactobacillus strain immunity moderation reaction
The purposes of the application's to be invention and created name be novel lactobacillus strain and immunity moderation reaction thereof, application number is 201110404611.9, and the applying date is the divisional application of the application for a patent for invention on December 7th, 2011.
Technical field
The invention relates to novel lactobacillus strain and the purposes for immunity moderation reaction thereof.
Background technology
Immunity system is the system of defense that health is used for resisting external microorganism or toxin.In immunity system, the 1st type helper cell (Th1), the 2nd type helper cell (Th2) and regulation type T cell (Treg) after T cell irriate, may be divided into.Wherein, Th1 cell dominates the immune response of Cell regulate, interferon-γ (IFN-γ) can be secreted, and promote that dendritic cell and macrophages secrete cytohormone-12 (IL-12) kill the lymphocytic hyperplasia of T with cell, contribute to antagonism virus infection and cancer cells.Th2 cell is leading humoral immune reaction then, can secretory cell hormone-4 (IL-4), cytohormone-5 (IL-5) and cytohormone-13 (IL-13), impel the generation of B cell hyperplasia and immune stimulatory sphaeroprotein E (IgE), free germ can be resisted; But excessive Th2 reaction may cause inflammation and anaphylaxis, such as, asthma, allergic rhinitis, eczema, urticaria and gastrointestinal illness etc.Separately, regulation type T cell is responsible for regulating the balance of Th1 and Th2 cell, can pass through secretion TGF-β or IL-10 to perform this regulating power.Regulation type T cell of pointing out existing report can suppress autoimmunity with irritated or pant and react, and thinks the prevention and treatment that can be used for anaphylactic disease at present.
Many research display milk-acid bacteria (Lactobacillus sp.) tool immunomodulatory effects, can suppress inflammation or relax the anaphylactic diseases such as atopic dermatitis, allergic rhinitis or asthma, its mechanism of action comprises the secretion of adjustment cytohormone, the balance of control Th1 and Th2 reaction and affects the generation etc. of antibody.But milk-acid bacteria effect has strain specificity (strain-specific effects), have differing appearance according to the difference of bacterial strain kind, and nature still there are many lactobacillus strains be not yet found or study fully.
Summary of the invention
The present invention isolates novel lactobacillus strain MP137 and MP108 from a Taiwan healthy infants ight soil corpse or other object for laboratory examination and chemical testing, and they are different from existing known bacterial strain.Novel lactobacillus strain of the present invention has immunomodulatory effect, particularly makes individual immune response trend towards Th1 immune response, suppresses Th2 immune response, contributes to antagonism courses of infection and reduces anaphylaxis.
Therefore, on the one hand, the invention provides a kind of separated lactobacillus strain, wherein this lactobacillus strain has the characteristic of the bacterial strain being selected from following formed group: MP137 bacterial strain, be deposited at TaiWan, China Foodstuff Industrial Development Inst. of Financial Group Legal Persons, deposit and be numbered BCRC910484; And MP108 bacterial strain, be deposited at TaiWan, China Foodstuff Industrial Development Inst. of Financial Group Legal Persons, deposit and be numbered BCRC910483.Particularly, the invention provides novel lactobacillus strain MP137 and MP108.
On the other hand, the invention provides a kind of composition comprising aforementioned lactobacillus strain.Said composition can be medicine or food, can be used for regulating individual immune response, particularly impels individual immune response to trend towards Th1 immune response, avoids Th2 immune response, more especially reduce individual anaphylaxis.Composition tool of the present invention treatment asthma or allergic rhinitis or effect that anti-enterovirus is infected.
In another, the present invention also provides a kind of purposes for the preparation of the medicine for regulating individual immune response, treatment asthma or allergic rhinitis or infect anti-enterovirus or food of lactobacillus strain described herein.
The present invention also comprises a kind of in required individual immunity moderation reaction, the method that promotes intestinal immunity, Immunosuppression reaction, treatment asthma or allergic rhinitis or infect anti-enterovirus, and it comprises the described lactobacillus strain administration of significant quantity in this individuality.
The specification specified of each specific embodiment of the present invention is as rear.Other features of the present invention will be known via the detailed description in each specific embodiment following and claim and present.
Further need not set forth, all believe that the technical field of the invention technician can utilize the present invention to the widest degree based on aforementioned explanation.Therefore, be appreciated that the following description is only the use illustratively illustrated, but not limit remaining disclosure by any way.
Accompanying drawing explanation
For object of the present invention is described, the specific embodiment of graphic middle display is best at present.It is to be understood, however, that the invention is not restricted to shown specific embodiment.
The kenel that Fig. 1 system strain isolated MP137 of the present invention examines under a microscope.
The kenel that Fig. 2 system strain isolated MP108 of the present invention examines under a microscope.
Fig. 3 shows the mouse test flow process of embodiment 3.2.
Fig. 4 A and Fig. 4 B show the result of the Respiratory Tract of Mice change in resistance mensuration through MP108 bacterial strain of the present invention and MP137 process.
Fig. 5 shows inflammatory cells floristic analysing result in the lung lavage of mouse, and Mon represents monocyte, Eos represents it is acid white cell, Neu represents it is that neutral leukocyte and Lym represent lymphocytes.
Fig. 6 A and Fig. 6 B shows the eliminating experimental result of embodiment 4, and wherein Fig. 4 A is for the experimental result of Salmonellas, and Fig. 4 B is for colibacillary experimental result.
Fig. 7 A and Fig. 7 B shows the displacement experimental result of embodiment 4, and wherein Fig. 7 A is for the experimental result of Salmonellas, and Fig. 7 B is for colibacillary experimental result.
Embodiment
Unless otherwise stated, whole technology used herein and scientific term and usual the understood same meaning of the technical field of the invention technician.
Article as used herein " one " refers to that the syntax of more than one or one (that is, at least one) of this article are by word.
The invention provides novel lactobacillus strain MP137 and MP108, its tool immunomodulatory effect.MP137 bacterial strain is deposited at German Organism Depositary (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH on November 19th, 2010, DSMZ, Inhoffenstra β e 7B, 38124 Brunswick cities, Germany), deposit and be numbered DSM24230.MP108 bacterial strain is deposited at German Organism Depositary (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH on November 19th, 2010, DSMZ, Inhoffenstra β e 7B, 38124 Brunswick cities, Germany), deposit and be numbered DSM24229.
Lactobacillus strain MP137 of the present invention and MP108 system are originated by Taiwan healthy infants ight soil and obtain through separation.Through tentative experiment, these bacterial strains are Gram-positive bacillus (Fig. 1 and 2), and do not have catalase, oxidase activity and a mobility, do not produce statospore, all can grow under aerobic and anaerobic environment, carry out 16S rDNA sequential analysis and API 50CHL system identification more further, result is as shown in SEQ ID NO:3 and SEQ ID NO:4 and table 1 and table 2 (embodiment 2).Comprehensive identification result, MP137 confirms as a kind of secondary cheese subspecies (Lactobacillus paracasei subsp.paracasei) bacterial strain of Lactobacillus paracasei of novelty, and MP108 confirms as a kind of rhamnose lactic acid bacteria (Lactobacillus rhamnosus) bacterial strain of novelty.
MP137 and MP108 bacterial strain of the present invention can exist by any way, comprises viable bacteria or dead bacterium form, also comprises the equivalent bacterial strain of tool same characteristic features and derived thalline or the product of gained by these bacterial strains.
" immunomodulatory " used herein word, when for describing a material, refer to that this material has the ability changing or adjust the immune function of at least one, include but not limited to, change or adjust quantity (or content) and/or the activity of immune member cells or effect molecule (such as, cytohormone and antibody).Association area has developed various testing method, assesses a kind of immunoloregulation function of material.
" treatment " used herein one word comprise object in order to cure, healing, alleviate, releive, change, correct, improve, improve or affect this disease, the symptom of this disease, deformity that this disease causes or the tendency of suffering from this disease, and the composition comprising one or more promoting agent is used or administration to suffer from this disease, this disease symptom or have the individuality of the tendency suffering from this disease, or other process are carried out to these individualities.Such as, according to the present invention, treatment asthma or allergic rhinitis comprise individuality one activeconstituents bestowing needs, to reach the symptom reducing or slow down asthma or allergic rhinitis, such as, reduce respiratory tract shrinkage phenomenon, reduce lung inflammation and improve sniffle (as runny nose, nasal obstruction, nose are itched, sneezed) or non-sniffle (as eyes or ear itch, throat is itched, eyes are red, drop tears).
Lactobacillus strain tool immunomodulatory effect of the present invention.In one embodiment, the generation of the adjustable cytohormone of lactobacillus strain of the present invention, such as, promotes the generation of individual IL-12, IL-10 or IFN-γ, and the generation of IL-4, IL-5 or IL-13 of suppression individuality.In another specific embodiment, the generation of the immunoglobulin (Ig) of the adjustable individuality of lactobacillus strain of the present invention, such as, promotes the generation of individual IgG2a and suppression IgE.The generation that those skilled in the art can understand cytohormone IL-12 and IFN-γ and Immunoglobulin IgG2 a represents Th1 immune response; The generation of cytohormone IL-4, IL-5 and IL-13 and Immunoglobulin IgE represents Th2 immune response; And the generation of cytohormone IL-10 represents the activation of adjustment type T cell, it is responsible for the balance regulating Th1 and Th2 reaction, can be used for prevention and the treatment of anaphylactic disease.
Therefore, lactobacillus strain of the present invention can impel individual immune response to trend towards Th1 immune response, suppresses Th2 immune response, contributes to antagonism courses of infection and reduces anaphylaxis.
Again in certain embodiments of the invention, lactobacillus strain of the present invention can promote the generation of individual IgA.The known IgA of those skilled in the art plays an important role at intestinal tract immune system, and it can be secreted into outside intestinal mucosa, forms immunocomplex (immune complex), prevent bacterium intrusion in this way with antigen.Therefore, lactobacillus strain of the present invention can promote individual intestinal immunity.
In another specific embodiment, lactobacillus strain of the present invention to the infection of anti-enterovirus, can include but not limited to Salmonellas and intestinal bacteria.In a particular embodiment, lactobacillus strain of the present invention can suppress or replace pathogen enterobacteria and be adsorbed on enteron aisle, therefore, has effect of prevention or the infection for the treatment of pathogen enterobacteria.
In addition, in another specific embodiment, lactobacillus strain of the present invention can reduce the generation of resistance of respiratory tract and/or reduce lung inflammation.Therefore, lactobacillus strain of the present invention still can be used for treatment asthma.Asthma is a kind of inflammatory airway disease, and segmental bronchus increases because of inflammatory cells and mucus, and tube wall oedema, therefore, causes resistance of respiratory tract to increase, sufferer is had difficulty in breathing, uncomfortable in chest, causes sudden death time serious.This area has developed standard animal pattern test resistance of respiratory tract, can see following examples.
Moreover, it be known to those skilled in the art that allergic rhinitis presents the trend of Th2 cytosis, and Th1/Th2 immunomodulator is for preventing and one of primary study for the treatment of of allergic rhinitis in recent years.According to the present invention, lactobacillus strain described herein still can be used for resisting allergic rhinitis.
In one embodiment, lactobacillus strain of the present invention can be used with other Claritins.Claritin described herein can field known can be used for, treats irritated medicine for this reason, particularly treatment of allergic rhinitis medicine, include but not limited to antihistaminic, go congested agent and anti-inflammatory drug (e.g., leukotriene antagonist, steroid, mast cell stabilizers, Sodium Cromoglicate, ketotifen etc.).In a specific embodiment, lactobacillus strain of the present invention and antihistaminic use.Lactobacillus strain of the present invention optionally can use with Claritin simultaneously or sequentially successively use.
On the other hand, the present invention also provides a kind of composition comprising aforementioned lactobacillus strain.In one embodiment, composition of the present invention comprises MP137 bacterial strain.In another embodiment, composition of the present invention comprises MP108.
Particularly, composition of the present invention can be used for regulating individual immune response.In one embodiment, the generation of the adjustable cytohormone of composition of the present invention, such as, promotes the generation of individual IL-12, IL-10 or IFN-γ, and the generation of IL-4, IL-5 or IL-13 of suppression individuality.In another specific embodiment, the generation of the immunoglobulin (Ig) of the adjustable individuality of composition of the present invention, such as, promotes the generation of individual IgG2a and suppression IgE.Therefore, composition of the present invention can impel individual immune response to trend towards Th1 immune response, suppresses Th2 immune response, contributes to antagonism courses of infection and reduces anaphylaxis.
Separately, composition of the present invention still can suppress the generation of individual resistance of respiratory tract and/or reduce lung inflammation, can in order to treatment asthma.Composition of the present invention also can in order to treatment of allergic rhinitis.
In another specific embodiment, composition of the present invention can promote the generation of individual IgA, and tool promotes effect of individual intestinal immunity.
Composition of the present invention also can be used for the infection to anti-enterovirus, includes but not limited to Salmonellas and intestinal bacteria.
Typically, composition of the present invention includes the lactobacillus strain of effective amount, to reach effect described herein.The visual various factors of this significant quantity and changing, such as, the approach that comes into operation, individual body weight and kind, and cast object.The method that technician can set up according to announcement herein and this prior art empirically determines the dosage of case.Preferably, composition of the present invention contains 10 8the lactobacillus strain of more than cfu.In one embodiment, composition of the present invention contains 10 9-10 12the lactobacillus strain of cfu; Particularly, composition of the present invention contains 2.5x10 9-5x10 11the lactobacillus strain of cfu; More particularly, composition of the present invention contains 5x10 9the lactobacillus strain (cfu represents colony-forming unit (colony-forming unit)) of cfu.
For being conducive to reaching described effect and/or transmission object, composition of the present invention can be deployed into desired form with physiologically acceptable supporting agent further." physiologically acceptable supporting agent " means that in this supporting agent and the present composition, contained effective constituent is compatible herein, and it is preferably can stablize this activeconstituents and to the individuality for casting or environmentally friendly for what use.Composition of the present invention can utilize various known ordinary method and suitable supporting agent to be deployed into desired form.
The embodiment of physiologically acceptable supporting agent includes but not limited to vehicle or thinner, such as, and Xylo-Mucine, Sorbitol Powder, talcum, dextran, lactose, sucrose, N.F,USP MANNITOL and analogue thereof; Cakingagent, such as, gum arabic, sodium alginate, ethyl cellulose, agar, gelatin, starch, hydroxylated cellulose, hydroxypropylcellulose and analogue thereof; Lubricant, comprises stearic acid, calcium stearate, Magnesium Stearate, talcum, winterized stearin, wax and analogue thereof; Wetting agent; Emulsification and suspension agent etc.
The form of the present composition can be lozenge, pill, powder, lozenge, tablet, suspension, emulsion, solvent, syrup and soft hard gelatin capsule.In a specific embodiment, combination system powder type of the present invention.In another specific embodiment, combination system capsule form of the present invention.In addition, the present composition is better casts with oral way.
Composition of the present invention can be made into medicine or food, such as, and yogurt, cheese and lactobacillus powder etc.Composition of the present invention also can comprise other additives, includes but not limited to, antioxidant, such as, and fertility alcohol, butylated hydroxytoluene, butylhydroxy methoxy benzene, xitix; Sweeting agent, such as, stevioside, generation sugar, asccharin; Tinting material, such as, beet red, gardenia blue, curcumine; And sanitas, such as, sodium benzoic acid salt, sulphite, phenylformic acid, oneself two diluted acids etc.
In one embodiment, composition of the present invention comprises one or more other Claritins further, include but not limited to antihistaminic, go congested agent and anti-inflammatory drug (e.g., leukotriene antagonist, steroid, mast cell stabilizers, Sodium Cromoglicate, ketotifen etc.).
The present invention also provides a kind of cover group, and it comprises one or more the first dose unit, and it includes the lactobacillus strain of the present invention of effective amount, and one or more second dose unit, and it includes other Claritins of effective amount.Particularly, lactobacillus strain wherein can use with Claritin simultaneously or sequentially successively use.
The present invention also comprises a kind of in required individual immunity moderation reaction, the method that promotes intestinal immunity, Immunosuppression reaction, treatment asthma or allergic rhinitis or infect anti-enterovirus, and it comprises the described lactobacillus strain administration of significant quantity in this individuality.In one embodiment, described lactobacillus strain is MP137 bacterial strain.In another embodiment, described lactobacillus strain is MP108.Preferably, described milk-acid bacteria strain is with in oral administration to required individuality.In the method for the invention, taking dose can optionally be adjusted.Preferably, taking dose is every day 10 8the lactobacillus strain of more than cfu.In one embodiment, taking dose is every day 10 9-10 12the lactobacillus strain of cfu; In another specific embodiment, taking dose is 2.5x10 every day 9-5x10 11the lactobacillus strain of cfu; In another specific embodiment, taking dose is 5x10 every day 9the lactobacillus strain of cfu.
Those skilled in the art can, based on the content disclosed, use any known method and technology to allocate composition of the present invention according to need herein.
The present invention will be described further by the following example, but actual invention is not limited thereto the embodiment that specification sheets is stated.
More clearly the present invention is described referring now to the specific embodiment of the following unrestricted object as explanation.
Embodiment 1: the separation of bacterial strain and cultivation
Gather a Taiwan healthy infants ight soil corpse or other object for laboratory examination and chemical testing, at 37 DEG C, cultivate 48 to 72 hours with the Rogosa plate culture medium of lactobacillus (Lactobacillus) selective medium, cultivate to obtain each bacterium colony of the doubtful bacterial strain of lactobacillus.Getting this culture coats on MRS flat board, Anaerobic culturel 48 to 72 hours at 37 DEG C, selects the single bacterium colony be grown on flat board and is further purified, and carries out identification of strains according to describing of embodiment 2, obtains isolated strains MP137 and MP108.
Isolated strains is inoculated on MRS flat board, Anaerobic culturel is after 48 to 72 hours, in the single colony inoculation of picking to fresh MRS nutrient solution, treat strain growth (the interpretable turbidity of naked eyes) in order, getting 1 ﹪ bacterium liquid is again transferred in another fresh MRS nutrient solution, cultivate 18 to 24 hours in applicable temperature, repeating this step activation thalline secondary, cultivating gained bacterium liquid for carrying out follow-up test.Embodiment 2: identification of strains
2.1 initial analysis
Initial analysis is carried out according to standard manner, it is Gram-positive bacillus (Fig. 1 and 2) that result shows isolated strains MP137 and MP108 of the present invention, and do not have catalase, oxydase and a mobility, do not produce statospore, all can grow under aerobic and anaerobic environment.
2.2 16S rDNA pcr analysis
16S rDNA pcr analysis is carried out for isolated strains MP137 and MP108 of the present invention.Use business cover group (AxyPrep Bacterial Genomic DNA Miniprep Kit, Anxygen Bioscience) extract the genomic dna of bacterial strain as masterplate, in PCR centrifuge tube, add positive and negative introduction (16S-F:GGAGTTTGATCCTGGCTCAG (SEQ ID NO:1); And 16S-R2:AAGGAGGTGAT CCAGCCGCA (SEQ ID NO:2)), the reagent such as archaeal dna polymerase, damping fluid, dNTPs, content is as follows:
16S rDNA PCR reaction conditions comprises step 1:95 DEG C, 3 minutes; Step 2:95 DEG C, 30 seconds; 60 DEG C, 30 seconds, 72 DEG C, 45 seconds, carry out 30 circulations; And step 3:72 DEG C, 10 minutes, be finally placed in 4 DEG C of termination reactions.After PCR reaction terminates, with agar gel electrophoresis PCR primer, again the colloid of the PCR primer fragment containing prediction size is cut, with business cover group Gel/PCR DNA Fragments Extraction Kit (Geneaid Co.) purifying, then carry out sequence analysis.
Will by sequencing gained DNA sequence dna, via commercially available computer program (the config program of Vector NTI, Invitrogen Co.) arrange combination, obtain correct DNA sequence dna, be sent on NCBI (http://www.ncbi.nim.nih.gov/) website again, compare with Nucleotide blast program, choose the preliminary evaluation result of the highest strain name of 16S rDNA sequence alignment similarity as bacterial strain.
SEQ ID NO:3 and SEQ ID NO:4 is the 16S rDNA partial sequence of isolated strains MP137 and MP108.Comparison result shows, strain isolated MP108 is closest to rhamnose lactic acid bacteria (Lactobacillus rhamnosus), corn milk bacillus (Lactobacillus zeae), kelvin Bacterium lacticum (Lactobacillus casei), secondary cheese subspecies (Lactobacillus paracasei subsp.paracasei) of Lactobacillus paracasei and the tough and tensile subspecies of lactobacillus paracasei (Lactobacillus paracasei subsp.tolerans), and similarity reaches more than 98%; And strain isolated MP137 is closest to the secondary cheese subspecies of Lactobacillus paracasei, the tough and tensile subspecies of lactobacillus paracasei, corn milk bacillus, kelvin Bacterium lacticum and rhamnose lactic acid bacteria, similarity reaches more than 98%.
2.3API 50CHL system identification
In addition, also API 50 CHL system identification is carried out for isolated strains MP137 and MP108 of the present invention, the Physiology and biochemistry test result of table 1 and 2 difference display separation bacterial strain MP137 and MP108.
According to API identification systems analytical results, in 49 test events, isolated strains MP137 and secondary cheese subspecies (the Lactobacillus paracasei subsp.paracasei) BCRC 12248 of reference culture Lactobacillus paracasei tclose, the project met has 43 (table 1).
The API identification systems analytical results of table 1 isolated strains MP137
In addition, according to API identification systems analytical results, in 49 test events, strain isolated MP108 and reference culture rhamnose lactic acid bacteria (Lactobacillus rhamnosus) BCRC10940 tclose, the project met has 44 (table 2).
The API identification systems analytical results of table 2 strain isolated MP108
Comprehensive the above results display, strain isolated MP108 of the present invention belongs to rhamnose lactic acid bacteria, and strain isolated MP137 is then the secondary cheese subspecies of Lactobacillus paracasei.
Embodiment 3: immunomodulatory analysis
3.1 cell experiment
3.1.1 the preparation of the dead thalline liquid of heat kill
Get the cultivation bacterium liquid of aforementioned strain isolated MP108 and MP137, load in centrifuge tube, be placed in water-bath and boil heating 30 minutes, be prepared into the dead thalline liquid of heat kill.Bacterium body-fluid concentration is 1x10 10cfu/ml, is placed in 4 DEG C for subsequent experimental.
3.1.2CD3+T the purifying of cell
The venous blood collecting healthy human body is about 100ml, after getting 25ml dilution, blood slowly adds the centrifuge tube that 20ml Ficoll-Hypaque (Ficoll-Hypaque) is housed, centrifugal 400xg, 40 minutes, utilize density variation, peripheral blood mononuclear glomus cell is separated, again with phosphoric acid buffer cleaning, and calculate cell quantity.The peripheral blood mononuclear white cell (PBMCs) human peripheral's blood separation gone out adds MACS damping fluid and CD3+ microballon according to suitable proportion, 4 DEG C left standstill after 15 minutes, add 10-20ml MACS buffer by centrifugation 300xg, 10 minutes, wash away unnecessary microballon, finally, with a small amount of MACS damping fluid back dissolving cell, prepare to start purifying.First soak MACS tubing string with MACS damping fluid 3ml, after drop to be buffered is dry, add the cell needing purifying, 3ml MACS wash buffer tubing string is used again after drip-dry, the cell of CD3-and CD3+ can be obtained respectively, wherein the cell of CD3+ is with containing the RPMI-1640 nutrient solution back dissolving of 2% serum, add 10%DMSO cell is frozen preserve in liquid nitrogen, the cell of CD3-then can continue to be used in the cultivation of dendritic cell.
3.1.3 the cultivation of human dendritic cell
The CD3-cell that is left by purified T cell uses the RPMI-1640 nutrient solution containing 10% serum to cultivate, and adds 800U/ml rhGM-CSF (rhGM-CSF) in addition and 400U/ml recombinant human IL-4 is divided into prematurity dendritic cell to impel monocyte.By cell cultures 5%CO2,37 DEG C of incubators 6 to 7 days, then these suspension cells are swept away from culture plate, the dendritic cell of purity about 95% can be obtained.
3.1.4 thalline sample is for the adjustment of human dendritic cell's immunologic function
Collect aforementioned dendritic cell, calculate cell count.The cell count ratio of adjustment thalline sample and dendritic cell is 1:1,10:1 or 100:1, and be used as positive controls with lipopolysaccharides (LPS) 100ng/ml, cultivate and accept nutrient solution after 48 hours, IL-10 and the IL-12 content secreted by dendritic cell is measured with enzyme immunoassay (ELISA, eBiosience commercial reagents).Use other milk-acid bacterias in contrast, comprise Jia Shi lactobacillus (L.gasseri), Yue Shi lactobacillus-1 (L.johnsonii-1) and Yue Shi lactobacillus-2 (L.johnsonii-2).
Result represents with the standard deviation of mean value sample distribution (Mean ± SEM), and * p<0.05 indicates and shows difference, and * * p<0.01 indicates the difference shown very much.Statistical is non-paired t calibrating.
Table 3: thalline sample stimulates the IL-12 amount of dendritic cell secretion
NC represents nutrient solution control group, compares with NC, if * is p<0.05, * * p<0.01 representative has statistically meaning.
Table 4: thalline sample stimulates the IL-10 amount of dendritic cell secretion
NC represents nutrient solution control group, compares with NC, if * is p<0.05, * * p<0.01 representative has statistically meaning.
Table 5: thalline sample stimulates dendritic cell to produce the ratio of IL-12 and IL-10 secretory volume
NC represents nutrient solution control group, compares with NC, if * is p<0.05, * * p<0.01 representative has statistically meaning.
If table 3 is to 5 displays, strain isolated MP137 and MP108 of the present invention can stimulate dendritic cell to produce more IL-12 and IL-10, display can be facilitated Th1 to react and bring out the generation of regulatory T cells (Treg), and the ratio of IL-12/IL-10 is higher, represent that Th1 reaction is better than Treg reaction.
3.1.5 bacterial strain affects the immunologic function of T cell by dendritic cell
Then, confirm that bacterial strain affects the immunologic function of T cell by dendritic cell, analyze the secretory volume of IFN-γ, IL-10 and IL-4 further.
These dendritic cell are utilized gamma-radiation process to stop its Reproductive activity, cell count are adjusted to 1x10 by dendritic cell and each bacterial strain Co stituation 48 hours 4cell/groove, simultaneously by previous ready CD3 +t cell number is adjusted to 1x10 5cell/groove, by two kinds of cell co-cultures 48 hours, the supernatant liquor of collecting cell, utilize ferment immunoassay cover group (ELISA, eBiosience commercial reagents) measure the secretory volume of cytohormone IFN-γ, IL-10 and IL-4, wherein with the stimulation of mitogen (phytoh(a)emagglutinin, PHA10g/ml) as a control group, also use other milk-acid bacterias as a control group, comprise Jia Shi lactobacillus, Yue Shi lactobacillus-1 and Yue Shi lactobacillus-2.Statistical as hereinbefore.
Table 6: the dendritic cell of bacterial strain impact stimulate T cell to produce the situation of IFN-γ
NC represents nutrient solution control group, and T represents to only have T cell, and DC represents dendritic cell, and T+PHA represents that T cell adds phytoh(a)emagglutinin.Compare with NC, if * is p<0.05, * * p<0.01 representative has statistically meaning.
Table 7: the dendritic cell of bacterial strain impact stimulate T cell to produce the situation of IL-10
NC represents nutrient solution control group, and T represents to only have T cell, and DC represents dendritic cell, and T+PHA represents that T cell adds phytoh(a)emagglutinin.Compare with NC, if * is p<0.05, * * p<0.01 representative has statistically meaning.
Table 8: the dendritic cell of bacterial strain impact stimulate T cell to produce the situation of IL-4
NC represents nutrient solution control group, and T represents to only have T cell, and DC represents dendritic cell, and T+PHA represents that T cell adds phytoh(a)emagglutinin.Compare with NC, if * is p<0.05, * * p<0.01 representative has statistically meaning.
Table 9: bacterial strain affects dendritic cell stimulates T cell to produce the ratio of IFN-γ and L-10 secretory volume
NC represents nutrient solution control group, and T represents to only have T cell, and DC represents dendritic cell, and T+PHA represents that T cell adds phytoh(a)emagglutinin.Compare with NC, if * is p<0.05, * * p<0.01 representative has statistically meaning.
If table 6 is to 9 displays, strain isolated MP137 and MP108 of the present invention can affect dendritic cell stimulates T cell secretion of gamma-IFN and IL-10, but IL-4 performance amount is all very low, and expression can facilitate T cell move towards Th1 reaction and stimulate IL-10 secretion, suppresses Th2 reaction.
3.2 experimentation on animals
3.2.1 the preparation of bacterium powder
The cultivation bacterium liquid of test bacterium is given centrifugal after, remove supernatant liquor, leave pellet fraction, add suitable protective material, it is overnight to be placed in-80 DEG C of pre-freezes.Again sample is inserted Freeze Drying Equipment and carry out lyophilize, obtain bacterium powder, comprise MP137 bacterium powder (3.2x10 of the present invention 11cfu/g) with MP108 bacterium powder (1.8x10 11cfu/g).
3.2.2 process fed by animal-origin and pipe
Select the female mouse of Balb/c, support in Taipei Medical University's Animal House generation.Be divided into 10 groups of mouse, often organize mouse and separately raise in cage, freely ingest drinking-water and feed.Bacterium powder sample is additionally given, sky on every Fridays in pipe mode of feeding; Pipe is fed and is then sacrificed after six weeks, to carry out the irritated immunoreactive both effectiveness evaluation test of every adjustment.
To converse than dose,equivalent according to laboratory animal and body surface area that manage hello dosage needed for mouse be 2.6x10 8cfu/ time/mouse, once, this dosage is the dosage of 1 times to one day feeding, is called 1 multiple dose group, and also carry out 0.5 multiple dose group, 5 multiple dose group and control groups, each dosage group profile is as follows:
0.5 multiple dose group: every day, pipe fed 0.2ml containing 1.3x10 8the bacterium powder of the bacterium amount of cfu (is equivalent to adult human dose 5x10 10);
1 multiple dose group: every day, pipe fed 0.2ml containing 2.6x10 8the bacterium powder of the probiotic bacterium amount of cfu (is equivalent to adult human dose 1x10 11);
5 multiple dose groups: every day, pipe fed 0.2ml containing 1.3x10 9the bacterium powder of the probiotic bacterium amount of cfu (is equivalent to adult human dose 5x10 11); And
Control group: every day feeds operation with pipe equally, the sterile distilled water of same volume 0.2ml fed by pipe.
* the ratio of mouse and body surface area is that the adult of 0.0026,70 kg body weight ingests 1 every day
G substances, be equivalent to the dosage of 20 g of mouse feedings every day 0.0026 g.3.2.3 the asthma zootype irritated to Protalbinic acid (OVA) is set up
Every two weeks, abdominal injection is carried out to mouse, give the solution mixed by OVA antigen and adjuvant.Period can carry out canthus blood sampling, and centrifugal blood was collected serum after 12,000rpm, 5 minutes, is kept at-20 DEG C to carry out the enzyme immunoassay analysis of follow-up antibody.Pipe fed bacterium powder sample after six weeks, gave mouse imbedibility OVA antigenic stimulation.Sacrifice mouse afterwards and gather lung lavage and spleen cell, and carry out the detections such as the secretory volume of the cytohormone of cytohormone and spleen in lung lavage.Fig. 3 shows mouse test flow process.
3.2.4 body weight detects
During animal experiment, every two weeks record Mouse Weight increase and decrease situations, to assess the impact of feeding probiotic bacterium on mouse growth.Table 10 is record result.
Table 10: Mouse Weight measuring result
Result shows, and compare with control group, the bacterium powder of feeding strain isolated of the present invention can not affect Mouse Weight, without affecting mouse appetite or causing the anxiety that loses weight.
3.2.5 specific immunoglobulin concentration determination in blood
In the process setting up mouse species pattern, carry out canthus blood sampling respectively at different time points, measure the antibody titer of IgA, IgE, IgG2a of anti ova antigen in serum.The mensuration of antibody will detect with ELISA reagent, survey light absorption value calculate each antibody concentration with ELISA plate reading.Table 11 is to 13 display measurement results.
Table 11: the IgA assay result of anti ova antigen in mice serum
A, b and c represent have statistical meaning (a, p<0.05 compared with control group; B, p<0.01; C, p<0.001).
As shown in Table 11, mouse is after the bacterium powder of feeding strain isolated of the present invention, and having to show for the output of the Specific IgA antibody of OVA antigen in body increases, and indicates the effect of lifting intestinal immunity.
Table 12: the IgE assay result of anti ova antigen in mice serum
A, b and c represent have statistical meaning (a, p<0.05 compared with control group; B, p<0.01; C, p<0.001).
As shown in Table 12, mouse is after the bacterium powder of feeding strain isolated of the present invention, and the generation for the specific IgE antibody of OVA antigen in body is suppressed, and indicates and suppresses anaphylactoid effect.
Table 13: the IgG2a assay result of anti ova antigen in mice serum
A, b and c represent have statistical meaning (a, p<0.05 compared with control group; B, p<0.01; C, p<0.001).
As shown in Table 13, mouse is after the bacterium powder of feeding strain isolated of the present invention, and having to show for the output of the specific IgG 2a antibody of OVA antigen in body increases, and indicates the effect of Th1 cell immune response in promotion Mice Body.
3.2.6 IL-13 assay in mouse lung washing fluid
Collect lung lavage with the IL-13 content in ELISA detecting lung lavage.The antibody getting anti-cell hormone is respectively dissolved in damping fluid, be placed in room temperature overnight, rinse with lavation buffer solution every other day, add and fill damping fluid at room temperature standing 2 hours, then rinse with lavation buffer solution, add lung lavage to be measured at room temperature to act on, then rinse with lavation buffer solution, add the antibody of the anti-cell hormone of vitamin H connection, left at room temperature is after 2 hours, rinse with lavation buffer solution, add ferment again to act under room temperature, rinse with lavation buffer solution afterwards, finally add photoghraphic coupler colour generation, the concentration contained by liquid to be measured is conversed with ELISA plate reading survey specific wavelength light absorption value.Table 14 shows measurement result.
Table 14: IL-13 assay result in mouse lung washing fluid
A, b and c represent have statistical meaning (a, p<0.05 compared with control group; B, p<0.01; C, p<0.001).
As shown in Table 14, mouse, after the bacterium powder of feeding strain isolated of the present invention, can dramatically the secretory volume reducing IL-13 in lung lavage, represents that Th2 reaction is suppressed.
3.2.7 the cytohormone secretion of Mouse spleen cells measures
Sacrifice and to be taken out by spleen after mouse and to be prepared into single cell suspension, further utilize damping fluid to be removed by red blood corpuscle, white cell carries out next step experiment again after then utilizing the cleaning of HBSS solution.After isolated cell is mixed up suitable concentration, be placed in culture plate, utilize the allergy of quantitative mistake originally to stimulate these lymph corpuscles.By centrifugal for the upper liquid amount manufactured to measure its cytohormone IL-5 of getting off after the cultivation of 96 hours.
Table 15: the cytohormone secretion assay result of Mouse spleen cells (PHA stimulation)
A, b and c represent have statistical meaning (a, p<0.05 compared with control group; B, p<0.01; C, p<0.001).
As shown in Table 15, mouse is after the bacterium powder of feeding strain isolated of the present invention, and the IL-5 secretory volume of spleen cell shows and reduces, and represents that Th2 reaction is suppressed.
3.2.8 Respiratory Tract of Mice change in resistance measures
Mouse feeding MP108 bacterium powder, feeding dosage is as shown in 3.2.2 paragraph.Mouse is anesthesia after six weeks, with autogenous cutting mode intubate, gives the atomizing gas that mouse sucks containing different concns mecholyl (methacholine) cause asthma via pipeline, and recording respiration change in resistance situation simultaneously.Result is expressed as follows with comparative resistance:
Comparative resistance=(resistance-tube wall resistance of mecholyl)/(resistance-tube wall resistance of salt solution)
Result as shown in Figure 4 A, the resistance of respiratory tract of feeding normal saline solution sensitized mice increases along with suction mecholyl dosage and rises, the resistance of respiratory tract of the mouse of the MP108 bacterium powder of feeding 1 multiple dose to be compared with feeding normal saline solution group and is shown lower under 100mg/ml mecholyl stimulates, its resistance of respiratory tract presented of mouse of feeding 5 multiple dose MP108 bacterium powder be then show very much land lower, even lower than not by sensitization ( ) resistance of respiratory tract (*, the p<0.05 of mouse; *, p<0.01; * *, p<0.001).
Separately carry out mouse with MP137 bacterium powder and inhale road resistance measurement, feeding dosage is as follows:
0.5 multiple dose group: every day, pipe fed 0.2ml containing 6.5x10 6the bacterium powder of the bacterium amount of cfu (is equivalent to adult human dose 2.5x10 9);
1 multiple dose group: every day, pipe fed 0.2ml containing 1.3x10 7the bacterium powder of the probiotic bacterium amount of cfu (is equivalent to adult human dose 5x10 9);
5 multiple dose groups: every day, pipe fed 0.2ml containing 6.5x10 7the bacterium powder of the probiotic bacterium amount of cfu (is equivalent to adult human dose 2.5x10 10); And
Control group: every day feeds operation with pipe equally, the sterile distilled water of same volume 0.2ml fed by pipe.
* the ratio of mouse and body surface area is that the adult of 0.0026,70 kg body weight ingests the substances of 1 g every day, is equivalent to the dosage of 20 g of mouse feedings every day 0.0026 g.
This experiment utilizes Buxco system with under the operation of Noninvasive, measures the change in resistance of Respiratory Tract of Mice.Change in resistance is by Computer Analysis and do mathematical operation according to the take off data of collecting Respiratory Tract of Mice change and obtain, and result represents with index (Penh value): Penh value=interval (pause) × PIF/PEF; Interval=(Te-Tr)/Tr (PIF: the highest inspiratory flow rate (peak inspiratory flow); PEF: maximum expiratory flow rate (peak expiratory flow rate)).The mode stimulating Respiratory Tract of Mice is the stimulation first making mouse accept normal saline solution, again sequentially acceptor concentration from low toward high mecholyl (6.25,12.5,25,50 and 100mg/mL), each concentration records respiratory tract physiology change after stimulating 3 minutes, and mean P enh value.
Result as shown in Figure 4 B, increases along with suction mecholyl dosage at the respiratory tract function pointer value of water control group mice and rises.The mouse finding feeding 0.5 times, 1 times and 5 multiple doses is compared with the irritated mouse of water control group, its Penh value is lower, represent that the respiratory tract situation of being obstructed is improved, especially can dramatically reduce to be subject to 50 and 100mg/mL stimulate under respiratory tract that mouse is caused to be obstructed situation.
3.2.9 inflammatory cells floristic analysing in lung lavage
This experimental analysis feeding MP137 bacterium powder is on the impact of inflammation Species distributing in mouse washing fluid, and feeding dosage is as shown in 3.2.8 paragraph.After accepting respiratory tract anaphylaxis primary stimuli, sacrifice the collection that mouse carries out lung lavage.Lung lavage is centrifugal, and supernatant liquor takes out and is placed in-20 DEG C of preservations.Cell is then beaten after on wave plate air-dry in order to cytospine instrument by the cell developed, and then carries out Liu's Albert'stain Albert, and air-dry slide after being rinsed by stain, afterwards by microscopic counting cell.Judge according to coloration result and cell kenel, cell is divided into four large classes: monocyte (monocyte), lymphocytes (lymphocyte), eosinocyte (eosinophil) and neutrophilia Archon (neutrophil).Fig. 5 display analysis result.
Result show, unsensitized mouse ( group), because of without inflammation phenomenon in its lung, so there is monocyte cell, almost without the gathering of eosinocyte and neutrophilia Archon more.And the mouse (water control group) of sensitization, the inflammation phenomenon caused because there being Th2 cell to dominate in its lung, so the gathering having obvious acid white cell and neutrophilia Archon.Experimental group and water control group are made comparisons, observe the mouse having feeding bacterium powder of the present invention, eosinocyte and the neutrophilia Archon situation infiltrated in lung have the trend of minimizing, especially have in 1 multiple dose group the difference shown, and represent that lung inflammation phenomenon is improved.
3.3 clinical experiments (MP108 merges antihistaminic Zyrtec therapy)
3.3.1 group is observed
Observe group to be 100 and to seek medical advice to primary care clinic, suffer from above allergic rhinitis one year and nasal obstruction, runny nose, cough or the throat severity of itching more than moderate 6-13 year children, but get rid of following illness:
(1) serious asthma is suffered from;
(2) acute asthma is had to show effect in past three months;
(3) acute or chronic sinusitis is suffered from;
(4) long-acting type antihistaminic was once used in past 10 days;
(5) past 3 days was used in nose or the short-acting type antihistaminic of systemic effect;
(6) leukotriene antagonist was used in past 7 days;
(7) used in nose in past 1 month, the steroid medicine of imbedibility or systemic effect;
(8) nasal congestion agent is in the past used in past 3 days; And
(9) anti-quick probiotic bacterium is used in past 3 months.
3.3.2MP108 anti-quick probiotics capsule
MP108 bacterium powder produces according to front aforesaid way and makes capsule, and dosage is 5 × 10 9cfu/cap.3.3.2 test design
The first month course for the treatment of (0-30 days, treatment phase): every day, an antihistaminic 10mg Zyrtec added that a MP108 resists quick probiotics capsule; And
The 2 to 3 month course for the treatment of (30 to 90 days, maintenance phase): antihistaminic of stopping using, take anti-quick probiotics capsule every day one.
3.3.3 evaluation item
The assessment of sufferer comprises:
(1) full symptom score, is divided into sniffle: runny nose, nasal obstruction, nose are itched, sneezed; And non-sniffle: eyes or ear are itched, throat is itched, eyes are red, drop tears.Be divided into 4 grades as follows:
The completely dissolve of 0=symptom.
1=slight (have symptom to occur, but do not affect individual).
2=moderate (symptom is obvious, impact individual, but does not affect sleep or daily life).
3=serious (interference daily life or sleep, even cannot work and rest).
(2) nasal meatus resistance measurement: left side and right side.
(3) consciously scale evaluation is improved.Be divided into 5 grades as follows:
0=is improved greatly.
1=improves some.
2=does not improve.
3=worsens some.
4=obviously worsens.
Sufferer carried out above-mentioned (1) full symptom score and (2) nasal meatus resistance measurement in the 30th, 60 and 90 day, and wherein the 60th and within 90 days, additionally carry out that (3) are conscious improves scale evaluation.
3.3.4 test-results
This test has 100 children and participates in, and finally has 59 and completes and within 3 months, test and include assessment in.
Table 16:
Table 17: full symptom score
Table 17 shows, and includes in the children of analysis at 59, and participate in this test sniffle mark minimizing 39% after three months, non-sniffle mark reduces 48%, and total symptom score (nose+non-nose) reduces 43%.
Table 18: nasal meatus resistance mark
Table 18 shows, and includes in the children of analysis at 59, participates in this test average nasal meatus resistance improvement nearly 40% after three months.
Embodiment 4: the determination and analysis that anti-enterovirus is infected
Effect that the present embodiment infects anti-enterovirus to get rid of (exclusion) and displacement (displacement) measuring isolated strains MP108 and MP137 of the present invention.The pathogen enterobacteria of the present embodiment test comprises Salmonellas (Salmonella enterica subsp.Enterica, BCRC10744), and intestinal bacteria (Escherichia coli, BCRC15372), and with commercially available lactobacillus strain (Lactobacillus casei variety rhamnosus, Lcr35 and Lactobacillus acidophilus, L.a) as a control group.
In eliminating experiment, by intestinal cell strain Caco-2 cell (BCRC 60182) in substratum (Dulbecco ' s modified Eagle ' s Medium, DMEM) cultivate after stablizing in, add milk-acid bacteria to be measured, wherein the ratio of milk-acid bacteria and Caco-2 cell is 10:1 (10MOI, multiplicity of infection).At 37 DEG C, co-cultivation is after 1.5 hours, washes away with phosphoric acid buffer (PBS) milk-acid bacteria do not adhered to.Then, add pathogenic infection Caco-2 cell, both ratios are pathogenic bacterium: Caco-2 cell is 10:1.Co-cultivation 1.5 hours at continuing at 37 DEG C, after washing away the bacterial strain do not adhered to, carries out gramstaining with PBS, counts the pathogenic bacterium bacterium number be attached on Caco-2 cell.Fig. 6 A and Fig. 6 B shows the result getting rid of experiment, and wherein Fig. 6 A is for the experimental result of Salmonellas, and Fig. 6 B is for colibacillary experimental result.
Separately, in displacement experiment, culture condition is tested identical with aforementioned the eliminating, but first adds pathogenic infection intestinal cell strain Caco-2 cell (ratio is pathogenic bacterium: Caco-2 cell=10:1), at 37 DEG C, co-cultivation is after 1.5 hours, washes away the pathogenic bacterium do not adhered to PBS; And then add milk-acid bacteria (ratio is for entering milk-acid bacteria: Caco-2 cell=10:1), co-cultivation 1.5 hours at continuing at 37 DEG C, after washing away with PBS the bacterial strain do not adhered to, carry out gramstaining, count the pathogenic bacterium bacterium number be attached on Caco-2 cell.Fig. 7 A and Fig. 7 B shows the result of displacement experiment, and wherein Fig. 7 A is for the experimental result of Salmonellas, and Fig. 7 B is for colibacillary experimental result.Symbol " # " represents to have compared to control group and shows difference P<0.05, and symbol " * " represents that two groups are compared to have and show difference P<0.05.
As Fig. 6 A-6B and Fig. 7 A-7B shows, isolated strains MP108 and MP137 of the present invention can successfully suppress or replace pathogen enterobacteria and be adsorbed on enteron aisle, therefore has the effect infected anti-enterovirus.
Confirmed by above result, strain isolated MP108 and MP137 of the present invention has immunity moderation function, can promote that Th1 reacts, suppress Th2 reaction, attenuating is irritated, bring out regulatory T cells reaction, promote intestinal immunity and reduce the effects such as individual resistance of respiratory tract of panting, also there is the effect infected anti-enterovirus.
Those skilled in the art can not deviate under spirit of the present invention, carry out changing and revising according to embodiment.It should be noted that the present invention is not limited to the scope that embodiment in specification sheets discloses, and be covered by the form of all changes that other disclose according to claim.

Claims (5)

1. a separated lactobacillus strain is for the preparation of the purposes reducing irritated medicine, wherein this lactobacillus strain has the characteristic of following bacterial strain: MP137 bacterial strain, be deposited at German Organism Depositary (Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH, DSMZ), deposit and be numbered DSM24230.
2. a separated lactobacillus strain is for the preparation of the purposes of the medicine infected anti-enterovirus, wherein this lactobacillus strain has the characteristic of following bacterial strain: MP137 bacterial strain, be deposited at German Organism Depositary (Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH, DSMZ), deposit and be numbered DSM24230.
3. purposes as claimed in claim 2, wherein this pathogen enterobacteria is Salmonellas or intestinal bacteria.
4. a separated lactobacillus strain is for the preparation of the purposes of antasthmatic medicine, wherein this lactobacillus strain has the characteristic of following bacterial strain: MP137 bacterial strain, be deposited at German Organism Depositary (Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH, DSMZ), deposit and be numbered DSM24230.
5. purposes as claimed in claim 4, wherein this medicine can reduce the individual resistance of respiratory tract of asthma.
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CN114149937A (en) * 2021-07-27 2022-03-08 中国海洋大学 Strain with function of regulating allergic constitution and application thereof
TWI801057B (en) * 2021-12-23 2023-05-01 豐華生物科技股份有限公司 Enhancement of gastrointestinal barrier function with a combination of lactic acid bacteria strains
JP7503857B2 (en) * 2022-04-11 2024-06-21 緑茵生技股▲ふん▼有限公司 Antiallergic peptides and their uses in immunomodulation and antiallergy

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101328468A (en) * 2007-06-21 2008-12-24 东宇生物科技股份有限公司 Antiallergic lactic acid bacteria
CN101541194A (en) * 2006-09-27 2009-09-23 利特尔卡鲁梅控股有限公司 Probiotic oral dosage forms
CN102028224A (en) * 2007-06-21 2011-04-27 东宇生物科技股份有限公司 Antiallergic lactobacillus
CN102046777A (en) * 2008-04-25 2011-05-04 龟田制菓株式会社 Novel lactic acid bacterium having anti-allergic activity, anti-allergic agent, food and pharmaceutical composition each comprising the lactic acid bacterium, and process for production of the anti-allergic agent

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100479719B1 (en) * 2005-01-29 2005-03-31 주식회사 프로바이오닉 -65Novel acid tolerant Lactobacillus sakei Probio-65 with the ability of growth suppression of pathogenic microorganisms and the anti-allergic effect
ZA200802934B (en) * 2005-10-06 2009-09-30 Probi Ab Use of lactobacillus for treatment of virus infections
FR2928935B1 (en) * 2008-03-19 2011-05-20 Gervais Danone Sa STRAIN OF LACTOBACILLUS RHAMNOSUS.
FR2930264B1 (en) * 2008-04-18 2013-02-22 Gervais Danone Sa NEW STRAIN OF LACTOBACILLUS PARACASEI SUBSP. PARACASEI WITH ANTIMICROBIAL AND IMMUNOMODULATORY PROPERTIES

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101541194A (en) * 2006-09-27 2009-09-23 利特尔卡鲁梅控股有限公司 Probiotic oral dosage forms
CN101328468A (en) * 2007-06-21 2008-12-24 东宇生物科技股份有限公司 Antiallergic lactic acid bacteria
CN102028224A (en) * 2007-06-21 2011-04-27 东宇生物科技股份有限公司 Antiallergic lactobacillus
CN102046777A (en) * 2008-04-25 2011-05-04 龟田制菓株式会社 Novel lactic acid bacterium having anti-allergic activity, anti-allergic agent, food and pharmaceutical composition each comprising the lactic acid bacterium, and process for production of the anti-allergic agent

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
乳酸菌对肠道病原菌的抑菌试验;李彦等;《哈尔滨商业大学学报(自然科学版)》;19891130(第02期);66-69 *
抗过敏乳酸菌的临床实证研究;苏伟志等;《中国乳业》;20080525(第05期);34-37 *

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