CN102028224A - Antiallergic lactobacillus - Google Patents
Antiallergic lactobacillus Download PDFInfo
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Abstract
The invention relates to a novel lactobacillus strain and a composition containing one or more types of strains, wherein the strain(s) has/have the unknown capability of enhancing antianaphylaxis in the prior art, and the composition is in a form of composition of foods or medicines. The invention also discloses a method for screening and measuring the lactobacillus strain and a method for regulating and controlling hyperreactive Th2-type immunoreaction due to allergy by enhancing Th1-type immunoreaction.
Description
Technical field
The present invention relates to be applied in the antiallergy lactic acid bacteria strains composition in food or the medicine, and the purposes in improving the antiallergy ability.
Background technology
In recent years, anaphylactic disease had the trend that increases year by year, no matter be the situation that increase is all arranged in the incidence of Atopic dermatitis, allergic rhinitis and bronchial asthma.Think that at present the generation of anaphylactic disease may be relevant with civilization progress, comprise that the pollution of air and the minimizing of infection all are some possible causes that cause anaphylactic disease to increase.And the treatment of asthma mainly relies on drug therapy at present: as using steroids and suppressing medicine, trachea expanding agent and the immunotherapy etc. that mast cell discharges the inflammation material.But, comprise the change allergic immune response that the drug therapy of anti-inflammatory drug and trachea expanding agent still can't be real, so the method that can only say so and take stopgap measures.And unique can saying so changes the therapeutic modality of irritated immune physique, is so-called hyposensitization, but subtracting quick treatment needs long period of time usually, and occurs some side effects sometimes, therefore can not be used for all patients.
The increase of relevant anaphylactic disease is thought at present and is followed the variation of overall situation that substantial connection is arranged, and more obvious relation is particularly arranged aspect environment.Probio meeting stimulating immune system effect in the human body intestinal canal, if but the contained antibiotic of food or the content of steroids is too high in daily life, the capital causes the probio in the enteron aisle to reduce, thereby the generation of auxiliary type T cell 1 (Th1) in the immune stimulatory cell effectively, and the close ties that have of the generation of these auxiliary types T cell 1 and the relevant anaphylactic disease of auxiliary type T cell 2 (Th2).Therefore, if can utilize the health food stimulating immune system, the Th2 type immune response that excites the Th1 type immune response that can regulate in the allergic immune response to come balance allergy to be caused can reach the effect of improving allergic constitution.
On the history of human using microbe, the application of lactic acid bacteria origin quite early.Find that from the processing of early stage dairy products edible food with lactobacillus-fermented helps to improve human intestines and stomach disease incidence rate and prolongs average life span.Thus, cause many scholars and begin one's study the discussion lactic acid bacteria in the middle role that promotes health.Proposed in 1954 after the functional lactobacillus, constantly start the upsurge of research in the period of several, and formally propose the notion of Probiotic-probio by people such as Lilly DM, make it to become the general designation of functional lactobacillus in nineteen sixty-five.
The general edible product that contains lactic acid bacteria (LAB) only has the health effect of adjusting enteron aisle, though there is ten hundreds of lactic acid bacteria strains to be present in nature, only has the minority lactic bacteria strain to have antianaphylactic speciality.The ability of the acidproof and bile tolerance ability that these bacterial strains of minority are had, absorption mucodermis cell and the characteristics such as ability that still can survive after by intestines and stomach are the important evidence of screening when having the bacterial strain that promotes health effect.Even to this day, only have that several strains of lactic acid bacteria bacterial strains are verified to have an antianaphylactic health effect, and lactic acid bacteria is the specificity of bacterial strain (strain) but not bacterial classification (species) to healthy function, and this bacterial strain that human body health is had a special efficacy is called as functional probio (Guideline s for the evaluation of probiotics in food; Report of joint FAO/WHO working group on drafting guidelines for the evaluation of probiotics in food; " food probio assessment guide ", FAO/WHO joint working group is about drafting the report of food probio assessment guide, London Ontario, Canada, on April 30th, 2002 and May 1: 1-7).
Anaphylactic disease, for example the allergic rhinitis and the allergic asthma of allergic eczema, nettle rash, outbreak have repeatedly become serious social concern in Taiwan and other developed country.Cause the rising of anaphylactic disease popularity degree that reason is arranged, well known hygiene hypothesis is: the chance that the infant contacts the immunostimulation pathogen is fewer, causes irritated relevant disease (Strachan, 1989) more easily.When anaphylactic disease takes place, immune response meeting in the human body descends the Th1 cell quantity, produces the various kinds of cell hormone continuously and impels immune response towards the Th2 approach, forms humoral immune reaction, for example the generation of IgE and eosinophil are too much waited (Romagnani, 1994; Holt, 1995).
Find that in children's population of Estonia and these two countries of Sweden the Bacillus acidi lactici in children's enteron aisle of trouble anaphylactic disease lacks (Bjo ¨ rksten etc., 1999) than the child of no anaphylactic disease.Bacillus acidi lactici is considered to promote the immune response of Th1 and then improves allergic symptom (Cross etc., 2001).Moreover, in the animal experimental model that brings out mouse allergy with ovalbumin, find, cheese lactic acid bacteria Shitota strain (Lactobacillus casei strain Shitota) after allowing mouse with the edible heat treatment of oral way, the content (Matsuzaki etc., 1998) that can suppress IgE in the mice serum.In addition, prove in the animal experimental model that brings out mouse allergy with casein that the lactobacillus germ L-137 after the lumbar injection heat treatment (Lactobacillus plantarum L-137) also can suppress the generation (Murosaki etc., 1998) of IgE.In the allergic symptom that is caused by the hogweed pollen hypersensitivity, oral enterococcus faecalis (Enterococcus faecalis) FK-23 extract can make the eosinophil cell concentration of assembling in the peritonaeum reduce (Shimada etc., 2003).In the clinical testing of human body, treat to take between term sugar and Lactobacillus rhamnosus bacterial strain GG (Lactobacillus rhamnosus strain GG) in the pregnant woman, discovery is 2 years (Kallioma ¨ ki etc. behind baby due, 2001) (Kallioma ¨ ki etc. and after infancy, 2003), can reduce risk and the incidence that allergic eczema takes place the infant.Lactobacillus rhamnosus 19070-2 and lactobacillus reuteri (Lactobacillus reuteri) DSM122460 also can moderately improve the children's that suffer from Atopic dermatitis serious eczema allergic symptom (Rosenfeldt etc., 2003).
Summary of the invention
One aspect of the present invention broadly comprises the composition of being made up of following any biological pure culture and physiologically acceptable excipient or diluent: lactobacillus acidophilus (Lactobacillus acidophilus) PM-A0002 bacterial strain, be preserved in Chinese typical culture collection center, preserving number M 207,038 2007 on April 6; Jia Shi Bacillus acidi lactici (Lactobacillus gasseri) PM-A0005 bacterial strain is preserved in Chinese typical culture collection center, preserving number M 207,039 2007 on April 6; Saliva Bacillus acidi lactici (Lactobacillus salivarius) PM-A0006 bacterial strain is preserved in Chinese typical culture collection center, preserving number M 207,040 2007 on April 6; Yue Shi Bacillus acidi lactici (Lactobacillus johnsonii) PM-A0009 bacterial strain is preserved in Chinese typical culture collection center, preserving number M 207,041 2007 on April 6; Lactobacillus acidophilus (Lactobacillus acidophilus) PM-A0013 bacterial strain is preserved in Chinese typical culture collection center, preserving number M 207,042 2007 on April 6.
In a specific embodiment of the present invention, said composition contains one or more described bacterial strains, preferred physiologically acceptable excipient or diluent are a kind of food, and this food can be any in sour kefir milk, yogurt (yogurt), cheese, dairy drink, milk powder, tea beverage or the coffee.Perhaps, said composition is a pharmaceutical composition, and its excipient or diluent should be pharmaceutically acceptable excipient or diluent.
In another specific embodiment, adopted to have the biological pure culture that strengthens antiallergy of the same race or mutant variety ability, physiologically acceptable following any bacterial strain: lactobacillus acidophilus PM-A0002 bacterial strain, Jia Shi Bacillus acidi lactici PM-A0005 bacterial strain, saliva Bacillus acidi lactici PM-A0006 bacterial strain, Yue Shi Bacillus acidi lactici PM-A0009 bacterial strain or lactobacillus acidophilus PM-A0013 bacterial strain.
In addition, in another specific embodiment, but the present invention's broad sense comprises by promoting the immune response of il-1 2 (IL-12) or interferon gamma regulation and control Th1 type to suppress Immunoglobulin IgE, improve the method for the excessive allergic phenomena of Th2 type immune response, described method comprises and gives any aforementioned biological pure culture that mammal can reach stimulates Th1 type immune effect dosage.
Moreover, in another specific embodiment, aforementioned more than one the biological pure cultures in the bacterial strain that define have been adopted, preferably the form with composition gives this culture and physiologically acceptable excipient or diluent, wherein said physiologically acceptable excipient or diluent are preferably food, any in the preferably sour kefir milk of this food, yogurt, cheese, dairy drink, milk powder, tea beverage or the coffee.Perhaps, said composition is a pharmaceutical composition, and its excipient or diluent should be pharmaceutically acceptable excipient or diluent.
In another specific embodiment, provide above-mentioned arbitrary or multiple biological pure culture to be used for the purposes of the medicine of immune stimulatory emiocytosis antiallergy relevant cell hormone in preparation.
But the present invention is comprising in the present specification separately or part, its constitutive requirements and the feature of sum total explanation of broad sense also, reach any comprise any combination any or multiple in these parts, constitutive requirements and the feature or its whole combination, and when known coordinate having occurred in the correlation technique relevant with the present invention as if the clear and definite complete things that reaches described herein, these known coordinates will be considered as independent item and be incorporated herein.
Description of drawings
Fig. 1 shows: respectively with 10
6To 10
8Individual cfu lactobacillus acidophilus (Lactobacillus acidophilus) PM-A0002 bacterial strain, Jia Shi Bacillus acidi lactici (Lactobacillus gasseri) PM-A0005 bacterial strain, saliva Bacillus acidi lactici (Lactobacillus salivarius) PM-A0006 bacterial strain, Yue Shi Bacillus acidi lactici (Lactobacillus johnsonii) PM-A0009 bacterial strain or lactobacillus acidophilus (Lactobacillus acidophilus) PM-A0013 bacterial strain and 10
5To 10
7Individual human peripheral blood mononuclear cells (PBMC) co-incubation was collected cell conditioned medium liquid after 48 hours, detected the content of interferon gamma (IFN-gamma) in supernatant with enzyme-linked immunosorbent assay.Wherein, with the negative control group of lactic acid bacteria (cheese lactic acid bacteria BCRC12249, Lactobacillus casei BCRC12249-food Industry in Taiwan Institute of Development Studies) of no antiallergy function, the positive control group of PHA, the irriate concentration of detection interferon gamma.The result shows test group stimulating human PMBC (PBMC) secretion significantly interferon gamma, with negative control group significant difference is arranged.
Fig. 2 shows: respectively with heat-inactivated 10
6To 10
8Individual cfu lactobacillus acidophilus (Lactobacillus acidophilus) PM-A0002 bacterial strain, Jia Shi Bacillus acidi lactici (Lactobacillus gasseri) PM-A0005 bacterial strain, saliva Bacillus acidi lactici (Lactobacillus salivarius) PM-A0006 bacterial strain, Yue Shi Bacillus acidi lactici (Lactobacillus johnsonii) PM-A0009 bacterial strain or lactobacillus acidophilus (Lactobacillus acidophilus) PM-A0013 bacterial strain and 10
5To 10
7Individual human dcs (dendritic cell) co-incubation 48 hours is collected cell conditioned medium liquid, detects the content of interleukin I L-12 in supernatant with enzyme-linked immunosorbent assay.Wherein, with the negative control group of lactic acid bacteria (cheese lactic acid bacteria BCRC12249-food Industry in Taiwan Institute of Development Studies) of no antiallergy function, the positive control group of PHA, the irriate concentration of detection il-1 2 (IL-12).The result shows test group stimulating human BMDC secretion significantly il-1 2 (IL-12), with negative control group significant difference is arranged.
Fig. 3 shows: result of the test shows the irritated lactic acid bacteria of commercial anti after the activation of culture medium, and after handling via acidic buffer solution and cholate, the bacterium number obviously descends; And the present invention has the preservation lactic bacteria strain of the irritated effect of adjusting after the culture medium activation, handle with acidic buffer solution and cholate, PM-A0006, PM-A0009, PM-A0013 bacterium number are not changed by the hydrochloric acid in gastric juice choline to be influenced and sharply decline, PM-A0002 and PM-A0005 are not subjected to hydrochloric acid in gastric juice to influence (the bacterium number proves that it has tolerance to hydrochloric acid in gastric juice), and when bacterial strain handled via cholate, though the bacterium number has a little decline, but cholate is still had tolerance, prove that the antianaphylactic lactic bacteria strain of this five strain can be by the test of the strict environment of digestion.
Fig. 4 shows: according to the analytical method of scholars such as Jacobsen (1999), when the average bacterium number in each visual field is less than 40, be judged to be " non-cohesive (nonadhesive) "; When the average bacterium number in each visual field during, be judged to be " adhering to (adhesive) " between 41 to 100; When the average bacterium number in each visual field during, be judged to be " strong adhesion (strongly adhesive) " greater than 100.The result shows lactobacillus acidophilus (Lactobacillus acidophilus) PM-A0002 bacterial strain, Jia Shi Bacillus acidi lactici (Lactobacillus gasseri) PM-A0005 bacterial strain, saliva Bacillus acidi lactici (Lactobacillus salivarius)) four strain antiallergy bacterial strains such as PM-A0006 bacterial strain, Yue Shi Bacillus acidi lactici (Lactobacillus johnsonii) PM-A0009 bacterial strain all judge " strong adhesion ", lactobacillus acidophilus (Lactobacillus acidophilus) PM-A0013 bacterial strain then is judged to be " adhering to ".
Fig. 5 shows: the antiallergy lactic acid bacteria is improved the design cycle of allergic experiment, and stage of tube feed antiallergy lactic acid bacteria is divided into the fourth phase: the first phase is 0～22 day; The second phase is 23～36 days; The third phase is 37～50 days; The fourth phase is 51～53 days.With lumbar injection ovalbumin (OVA) sensitized mice, dividing four times sensitization therebetween, is the 2nd day of beginning tube feed for the first time; Be the 16th day of the beginning tube feed second time; Be beginning the 30th day of tube feed for the third time; The 4th time is the 44th day of beginning tube feed.To passing through lumbar injection ovalbumin (ovalbumin; OVA) mouse of sensitization is drawn blood every other week, checks the variation of IgE content in the mouse body.After continuous 2 days, detect the pulmonary respiration resistance response of mouse with schneiderian membrane suction sensitized mice in putting to death mouse preceding 4 days (the 54th day), put to death the detection that mouse carries out other biochemical values every other day.
Fig. 6 shows: when replenishing saliva Bacillus acidi lactici PM-A0006 lactic bacteria strain for small white mouse, with ovalbumin (OVA) specific antibody titres part in the mouse serum of egg albumen sensitization, along with feeding dosage increases, ovalbumin (OVA) specific IgE antibody in ovalbumin (OVA) the sensitized mice serum has the trend of minimizing.
Fig. 7 shows: when replenishing saliva Bacillus acidi lactici PM-A0006 lactic bacteria strain for small white mouse, small white mouse pulmonary respiration resistance part with egg albumen sensitization, with respect to control group, all can under stimulating, high concentration methacholine (methacholine) significantly lower mouse Penh value.
Fig. 8 shows: when replenishing saliva Bacillus acidi lactici PM-A0006 lactic bacteria strain for small white mouse, with cell part in the small white mouse lung flushing liquor of egg albumen sensitization, with respect to control group, significantly lower eosinophil (eosinophil) infiltration ratio is arranged.
Fig. 9 shows: with respect to control group, feeding PM-A0006 lactic acid bacteria can significantly reduce the secretory volume of chemotactic factor for eosinophils (eotaxin) in the lung flushing.
Figure 10 shows: with respect to control group, feeding PM-A0006 lactic acid bacteria can significantly reduce PGE in the lung flushing
2Secretory volume.
Figure 11 shows: spleen cell stimulates cultivation after 48 hours through ConA or ovalbumin (OVA), and under ConA stimulated, the interferon gamma of the spleen cell secretion of feeding PM-A0006 lactic acid bacteria group was significantly higher than control group.
The specific embodiment
But the present invention's broad sense comprise following any biological pure culture: lactobacillus acidophilus (Lactobacillus acidophilus) PM-A0002 bacterial strain is preserved in Chinese typical culture collection center, preserving number M 207,038 2007 on April 6; Jia Shi Bacillus acidi lactici (Lactobacillus gasseri) PM-A0005 bacterial strain is preserved in Chinese typical culture collection center, preserving number M 207,039 2007 on April 6; Saliva Bacillus acidi lactici (Lactobacillus salivarius) PM-A0006 bacterial strain is preserved in Chinese typical culture collection center, preserving number M 207,040 2007 on April 6; Yue Shi Bacillus acidi lactici (Lactobacillus johnsonii) PM-A0009 bacterial strain is preserved in Chinese typical culture collection center, preserving number M 207,041 2007 on April 6; Lactobacillus acidophilus (Lactobacillus acidophilus) PM-A0013 bacterial strain is preserved in Chinese typical culture collection center, preserving number M 207,042 2007 on April 6.
Many documents point out that the cytohormone that increases auxiliary type T cell 1 comprises that the secretion of il-1 2 (IL-12), interferon gamma (IFN-gamma) increases, and can effectively improve allergic symptom; Further Toll sample acceptor (Toll-like receptor) combination on specific lactic bacteria strain and the BMDC is also pointed out in research, the albumen of translating in the activating cell moves in the nuclear and discharges a large amount of cytohormones, a link that belongs to congenital immunity, therefore some specific lactic acid bacteria culturers is by its cell wall polysaccharides class material such as peptide glycan (peptidoglycan), lipopolysaccharides (lipopolysaccharide), polysaccharide (polysaccharide) etc., through innate immune system, growth that really can activating T cell.
The freeze drying culture of this five strains bacterial isolates has been deposited in Chinese typical culture collection center, and the address is: Chinese Wuhan City Wuhan University.The particulars of preservation are as shown in table 1:
Table 1
| The bacterial strain name | Numbering | Date |
| Lactobacillus acidophilus PM-A0002 | M?207038 | On 04 6th, 2007 |
| Jia Shi Bacillus acidi lactici PM-A0005 | M?207039 | On 04 6th, 2007 |
| Saliva Bacillus acidi lactici PM-A0006 | M?207040 | On 04 6th, 2007 |
| Yue Shi Bacillus acidi lactici PM-A0009 | M?207041 | On 04 6th, 2007 |
| Lactobacillus acidophilus PM-A0013 | M?207042 | On 04 6th, 2007 |
This five strains of lactic acid bacteria of having found preservation as listed above has antianaphylactic ability, comprises that the symptom for Atopic dermatitis, nettle rash, allergic rhinitis, food hypersenstivity and asthma has the function of slowing down and treating.
Embodiment
Morphology and the general aspects of 1: five strain antiallergy of embodiment lactic acid bacteria
Confirm the feature of bacterial strain on taxology according to 16S rDNA sequence analysis and API Bacteria Identification network analysis result.Find that eastern space strain number PM-A0002 is a lactobacillus acidophilus; East space strain number PM-A0005 is for adding the formula Bacillus acidi lactici; East space strain number PM-A0006 is the saliva Bacillus acidi lactici; East space strain number PM-A0009 is about formula Bacillus acidi lactici; And eastern space strain number PM-A0013 is a lactobacillus acidophilus.The feature of this five strains bacterial strain on morphology and general aspects listed in table 2 in detail:
Table 2
Embodiment 2: the antiallergy lactic acid bacteria is to regulating and control the effect (is external effect verification platform with PMBC) of Th1 type immunocompetence by the secretion that promotes interferon gamma
Detect five strain antiallergy lactic bacteria strains: lactobacillus acidophilus PM-A0002 bacterial strain, Jia Shi Bacillus acidi lactici PM-A0005 bacterial strain, saliva Bacillus acidi lactici PM-A0006 bacterial strain, Yue Shi Bacillus acidi lactici PM-A0009 bacterial strain or lactobacillus acidophilus PM-A0013 bacterial strain are to the enhancement effect of Th1 type immunocompetence, by measuring the secretory volume of interferon gamma after human peripheral blood mononuclear cell and the antiallergy lactic acid bacteria co-incubation, screen bacterial strain with antiallergy ability.Use following experimental procedure:
1. extract an amount of human blood, about at every turn 200ml.
2. the blood cell parting liquid (Ficoll-paque) of getting equal proportion and blood under 18-20 ℃ with the centrifugal 30-40 of 400g minute.
3. (Peripheral blood mononuclear cell, PBMC) layer is behind cushioning liquid cleaning cell 2-3 time, with suitable culture medium (for example RPMI-1640) suspension cell to get human peripheral blood mononuclear cells.
With cell with activated 3 days lactic bacteria strain with 1: 10 ratio co-incubation 48 hours.
5. collect clear liquid on the cell cultivation.Utilize enzyme-linked immunosorbent assay (ELISA) to detect the content of interferon gamma (IFN-gamma) in supernatant.
Data statistic analysis such as table 3 and shown in Figure 1 are represented with mean value ± SD.Figure 1 shows that: respectively with 10
6To 10
8Individual cfu lactobacillus acidophilus (Lactobacillus acidophilus) PM-A0002 bacterial strain, Jia Shi Bacillus acidi lactici (Lactobacillus gasseri) PM-A0005 bacterial strain, saliva Bacillus acidi lactici (Lactobacillus salivarius) PM-A0006 bacterial strain, Yue Shi Bacillus acidi lactici (Lactobacillus johnsonii) PM-A0009 bacterial strain or lactobacillus acidophilus (Lactobacillus acidophilus) PM-A0013 bacterial strain and 10
5To 10
7Individual human peripheral blood mononuclear cells (PBMC) co-incubation 48 hours is collected cell conditioned medium liquid, detects the content of interferon gamma (IFN-gamma) in supernatant with enzyme-linked immunosorbent assay.Wherein, with the negative control group of lactic acid bacteria (cheese lactic acid bacteria BCRC12249) of no antiallergy function, the positive control group of PHA (phyohenagglutimin) detects interferon gamma irriate concentration.The result shows test group stimulating human PMBC (PBMC) secretion significantly interferon gamma, with negative control group significant difference is arranged.Table 3 is for cultivating with human peripheral blood mononuclear cells with five strain antiallergy bacterial strains respectively, stimulate interferon gamma secretory volume (mean value ± SD):
Table 3
| Strain name | The secretory volume of interferon gamma (pg/ml) |
| Lactobacillus acidophilus PM-A0002 | 19833±2767 |
| Add formula Bacillus acidi lactici PM-A0005 | 46625±3624 |
| Saliva Bacillus acidi lactici PM-A0006 | 25850±2347 |
| Yue Shi Bacillus acidi lactici PM-A0009 | 25725±2008 |
| Lactobacillus acidophilus PM-A0013 | 17416±2803 |
| Negative control group (cheese lactic acid bacteria BCRC 12249) | 11±2.3 |
| Positive controls | 44666±2488 |
Embodiment 3: the antiallergy lactic acid bacteria is to the effect (is external effect verification platform with BMDC) of the Th1 type immunocompetence of the secretion regulation and control of interleukin (IL-12)
Detect five strain antiallergy lactic bacteria strains: lactobacillus acidophilus PM-A0002 bacterial strain, Jia Shi Bacillus acidi lactici PM-A0005 bacterial strain, saliva Bacillus acidi lactici PM-A0006 bacterial strain, Yue Shi Bacillus acidi lactici PM-A0009 bacterial strain or lactobacillus acidophilus PM-A0013 bacterial strain are to the enhancement effect of Th1 type immunocompetence, by the secretory volume of il-1 2 (IL-12) after mensuration human dcs and the antiallergy lactic acid bacteria co-incubation, screen bacterial strain with antiallergy ability.Use following experimental procedure:
1. extract an amount of human blood, about at every turn 200ml.
2. the blood cell parting liquid (Ficoll-paque) of getting equal proportion and blood under 18-20 ℃ with the centrifugal 30-40 of 400g minute.
3. get human peripheral blood mononuclear cells (PBMC) layer, behind cushioning liquid cleaning cell 2-3 time, with suitable culture medium (for example RPMI-1640) suspension cell.
4. with CD14
+CD14 in microballoon (MiniMACS system) the purifying human PMBC (PBMC)
+Monocyte.
5. be divided into BMDC with cytohormone (IL-4) and growth hormone GM-CSF irritation cell, behind 6-7 days the incubation time, collect the BMDC that has broken up.
6. with the activation in 3 days before co-incubation of antiallergy lactic bacteria strain, afterwards with 100 ℃ of hot deactivation lactic bacteria strains 30 minutes.
With BMDC and heat-inactivated lactic bacteria strain with 1: 10 ratio co-incubation 48 hours.
8. collecting cell culture supernatant.Utilize enzyme-linked immunosorbent assay (ELISA) to detect the content of interleukin (IL-12) in supernatant.
Data statistic analysis such as table 4 and shown in Figure 2 are represented with mean value ± SD.Figure 2 shows that: respectively with hot deactivation 10
6To 10
8Individual cfu lactobacillus acidophilus (Lactobacillus acidophilus) PM-A0002 bacterial strain, Jia Shi Bacillus acidi lactici (Lactobacillus gasseri) PM-A0005 bacterial strain, saliva Bacillus acidi lactici (Lactobacillus salivarius) PM-A0006 bacterial strain, Yue Shi Bacillus acidi lactici (Lactobacillus johnsonii) PM-A0009 bacterial strain or lactobacillus acidophilus (Lactobacillus acidophilus) PM-A0013 bacterial strain and 10
5To 10
7Individual human dcs (dentritic cell) co-incubation 48 hours is collected cell conditioned medium liquid, detects the content of interleukin (IL-12) in supernatant with enzyme-linked immunosorbent assay.Wherein, with the negative control group of no antiallergy function lactic acid bacteria (cheese lactic acid bacteria BCRC12249), the positive control group of PHA detects interleukin (IL-12) irriate concentration.The result shows test group stimulating human BMDC secretion significantly il-1 2 (IL-12), with negative control group significant difference is arranged.Table 4 is depicted as respectively and cultivates with BMDC with the hot deactivation antiallergy of five strains bacterial strain, the secretory volume of stimulation interleukin (IL-12) (mean value ± SD):
Table 4
| Strain name | The secretory volume (pg/ml) of interleukin (IL-12) |
| Lactobacillus acidophilus PM-A0002 | 15019±569 |
| Add formula Bacillus acidi lactici PM-A0005 | 19222±212 |
| Saliva Bacillus acidi lactici PM-A0006 | 18625±365 |
| Yue Shi Bacillus acidi lactici PM-A0009 | 18291±39 |
| Lactobacillus acidophilus PM-A0013 | 17836±168 |
| Negative control group (cheese lactic acid bacteria BCRC 12249) | 80±15 |
| Positive controls | 13786±341 |
Embodiment 4: the test of antiallergy lactic acid bacteria stomach juice-resistant cholate
Detect five strain antiallergy lactic bacteria strains: lactobacillus acidophilus PM-A0002 bacterial strain, Jia Shi Bacillus acidi lactici PM-A0005 bacterial strain, saliva Bacillus acidi lactici PM-A0006 bacterial strain, Yue Shi Bacillus acidi lactici PM-A0009 bacterial strain or lactobacillus acidophilus PM-A0013 bacterial strain, whether has ability by the test of hydrochloric acid in gastric juice cholate, thereby be able to successfully bring into play its antianaphylactic function at enteron aisle, experiment process is as follows:
1. with five strain antiallergy lactic acid bacterias activation 3 days.
2. get 1mL bacterium liquid and calculate original bacterium number, remaining lactic acid bacteria centrifugal 10 minutes with 500g adds the washed with de-ionized water lactic acid bacteria 2-3 time.
3. add and be deployed in the simulation hydrochloric acid in gastric juice solution of pH 2.5 with hydrochloric acid, lactic acid bacteria places 37 ℃ of incubators with after the culture medium of pH 2.5 fully mixes.
4. per hour take out 1mL bacterium liquid with washed with de-ionized water 2-3 time after, the calculating survivaling cell number is till 3 hours incubation times.
5. (ox gall, Hui Rong in simulation choline solution Sigma) is after fully mixing, in 37 ℃ of cultivations to contain 1.5% (w/V) fel bovis again for residue bacterium liquid centrifuged deposit thing.
6. the bacterium liquid that per hour takes out 1mL with washed with de-ionized water 2-3 time after, the calculating survivaling cell number is till 4 hours incubation times.
7. record lactobacter growth speed is calculated the tolerance of lactic acid bacteria to hydrochloric acid in gastric juice and cholate, and in the presence of hydrochloric acid in gastric juice and cholate, whether strain growth is suppressed with lactic acid bacteria in the comparative sample.
The capability result of stomach juice-resistant and analysis and arrangement Figure 3 shows that result of the test shows the irritated lactic acid bacteria of commercial anti after the activation of culture medium in table 5 and Fig. 3, via acidic buffer solution and cholate processing, the bacterium number obviously descends; Regulate irritated preservation lactic bacteria strain of the present invention after the culture medium activation, handle bacterial strain with acidic buffer solution and cholate, PM-A0006, PM-A0009, PM-A0013 bacterium number are not changed by the hydrochloric acid in gastric juice choline to be influenced and sharply decline, PM-A0002 and PM-A0005 are not subjected to hydrochloric acid in gastric juice to influence (the bacterium number proves that it has tolerance to hydrochloric acid in gastric juice), and when bacterial strain handled via cholate, though bacterial strain has a little decline, but cholate is still had tolerance, prove that antianaphylactic this five strains of lactic acid bacteria strain can be by the test of the strict environment of digestion.Table 5 is that the hydrochloric acid with pH value 2.5 mixes the result who tests its stomach juice-resistant ability with the antiallergy lactic acid bacteria:
Table 5:
| Strain name | Original bacterium number | pH2.5，1hr | pH2.5，2hr | pH2.5，3hr |
| Lactobacillus acidophilus PM-A0002 | 8.20×10 8 | 6.50×10 8 | 2.87×10 8 | 2.51×10 8 |
| Add formula Bacillus acidi lactici PM-A0005 | 2.65×10 9 | 2.06×10 9 | 1.19×10 9 | 7.10×10 8 |
| Saliva Bacillus acidi lactici PM-A0006 | 2.55×10 9 | 9.70×10 8 | 1.42×10 9 | 1.43×10 9 |
| Yue Shi Bacillus acidi lactici PM-A0009 | 6.87×10 8 | 4.05×10 8 | 4.15×10 8 | 4.35×10 8 |
| Lactobacillus acidophilus PM-A0013 | 1.78×10 9 | 1.83×10 9 | 1.86×10 9 | 1.73×10 9 |
The capability result of bile tolerance and analysis and arrangement are in table 6 and Fig. 3, and table 6 is to mix the result who tests its bile tolerance ability with the antiallergy lactic acid bacteria with 1.5% bile.
Table 6
Embodiment 5: the antiallergy lactic acid bacteria is to the adsorption capacity test of human intestine's epidermal cell (CaCo-2)
Adopt the cell line that has broken up to analyze the ability of five strain antiallergy lactic acid bacteria strains absorption human intestine's epidermal cells (CaCo-2) in vitro.Earlier human intestine's epidermal cell (CaCo-2) cell monolayer is inoculated on the cover glass, grow to cell monolayer after, insert in the porous cell culture plate.Then with cell: lactic acid bacteria is with 1: 200 ratio, co-incubation 1-3 hour, with 1 * PBS clean and with the methyl alcohol fixed cell and remaining adhere to the bacterium number after, carry out Gram dyeing and definite accompanying bacterium number.With reference to the analytical method of scholars such as Jacobsen (1999), 20 visuals field of counting when the average bacterium number in each visual field is less than 40, are judged to be " non-cohesive (nonadhesive) " under 1000 power microscopes; When the average bacterium number in each visual field during, be judged to be " adhering to (adhesive) " between 41 to 100; When the average bacterium number in each visual field during, be judged to be " strong adhesion (strongly adhesive) " greater than 100.
The statistical analysis of data is represented with mean value ± SD.On average, getting the calculated value in 45 visuals field. its result puts in order in table 7 and Fig. 4.Fig. 4 shows the analytical method according to scholars such as Jacobsen (1999), when the average bacterium number in each visual field is less than 40, is judged to be " non-cohesive (nonadhesive) "; When the average bacterium number in each visual field during, be judged to be " adhering to (adhesive) " between 41 to 100; When the average bacterium number in each visual field during, be judged to be " strong adhesion (strongly adhesive) " greater than 100.The result shows that four strain antiallergy bacterial strains such as lactobacillus acidophilus (Lactobacillus acidophilus) PM-A0002 bacterial strain, Jia Shi Bacillus acidi lactici (Lactobacillus gasseri) PM-A0005 bacterial strain, saliva Bacillus acidi lactici (Lactobacillus salivarius) PM-A0006 bacterial strain, Yue Shi Bacillus acidi lactici (Lactobacillus johnsonii) PM-A0009 bacterial strain all are judged as " strong adhesion ", and lactobacillus acidophilus (Lactobacillus acidophilus) PM-A0013 bacterial strain then is judged as " adhering to ".Table 7 is the result of the tests of five strain antiallergy lactic acid bacterias to the adsorption capacity of human intestine's epidermal cell (CaCo-2) cell line:
Table 7
| The bacterial strain name | Mean value ± SD |
| Lactobacillus acidophilus PM-A0002 | 115.4±4.4 |
| Add formula Bacillus acidi lactici PM-A0005 | 100.1±2.6 |
| Saliva Bacillus acidi lactici PM-A0006 | 108.3±1.4 |
| Yue Shi Bacillus acidi lactici PM-A0009 | 208.3±22.1 |
| Lactobacillus acidophilus PM-A0013 | 62.8±5.0 |
Embodiment 6: with the antiallergy lactic acid bacteria as a supplement feed produced the treatment or the effect of the allergic asthma of releiving
To give ovalbumin (ovalbumin; OVA) asthmogenic small white mouse feeding saliva Bacillus acidi lactici PM-A0006 is an example, carries out allergy by following experiment and improves assessment.
A. experimental design
1. animal used as test
Use BALB/c mouse, 6-8 big female six weeks of mouse feeding in week, 14 every group of control group and experimental group.Buy the SPF level from platform large animal center, six all big female mouse of BALB/c, every mouse is raised respectively in Rotating Stainless Steel Cage, room temperature is controlled in 22 ± 2 ℃, light respectively is 12 hours (light morning six, and at 6 in afternoon is dark) dark circulation timei, freely ingests, drinking-water and feed, with the body weight random packet, beginning gave tested lactic acid bacteria (2.6x10 in the tube feed mode when eight weeks of mouse were big
6To 2.6x10
7Cfu/ days) carry out sensitization simultaneously.
2. set up the respiratory inflammation zootype
The mouse sensitization step is summarized as follows: experiment beginning the 2nd day with 50 μ g ovalbumins (OVA) and adjuvant Alum 4mg lumbar injection to the mouse body, in experiment the 16th, 30,44 day again with 25 μ g ovalbumins (OVA) and 4mg Alum duplicate injection mouse.After the sensitization, experiment the 54th, 55 day, with mouse anesthesia, so that (intra-nasal, i.n.) mode that splashes into 100 μ g ovalbumins (OVA) is brought out the respiratory tract infection reaction in the nasal cavity.
3. experiment flow improves the design cycle of allergic experiment for antiallergy lactic acid bacteria as shown in Figure 5
The stage of tube feed antiallergy lactic acid bacteria is divided into the fourth phase: the first phase is 0～22 day; The second phase is 23～36 days; The third phase is 37～50 days; The fourth phase is 51～53 days.With the mode sensitized mice of lumbar injection ovalbumin (OVA), dividing four times sensitization therebetween, is the 2nd day of beginning tube feed for the first time; Be the 16th day of the beginning tube feed second time; Be beginning the 30th day of tube feed for the third time; The 4th time is the 44th day of beginning tube feed.Whole mouse to lumbar injection ovalbumin (OVA) sensitization are drawn blood every other week, check the content of IgE in the mouse body.In putting to death mode sensitized mice that mouse preceding 4 days (the 54th day) sucks with schneiderian membrane after continuous 2 days, detect the pulmonary respiration resistance response of mouse, put to death the detection that mouse carries out other biochemical values every other day.
B. experimental technique
1. the collection of execution of animal and experiment material and analysis
A. the collection of blood serum sample
When experiment was carried out the 0th, 23,37,51 day and put to death, mouse is carried out contrary eye socket blood sampling (retro-orbital), collect blood sample.Behind mouse anesthesia, rapidly with micro-capillary from eye socket venous sinus blood sampling, get 200-250 μ L blood approximately, 4 ℃ leave standstill 2-4 hour after, with the 12000rpm rotating speed centrifugal 20 minutes, collect serum, in-80 ℃ of preservations.
B. measure the ELISA (ELISA) of ovalbumin (OVA) specific antibody
0.5mg antigen is dissolved in 100 μ L carbonate bags is cushioned liquid (carbonate coating buffer, 0.1M NaHCO
3) add microtest plate, in 4 ℃ of standing over night.The 2nd day to contain the 1X PBS buffer solution for cleaning microtest plate of 0.05%Tween 20.Carry out the sealing more than two hours with lock solution (the 1X PBS buffer solution that contains 1%BSA) then, after cleaning 3 times, then add 100 μ L samples to be tested, react on 4 ℃ of standing over night, after cleaning 4 times, add the anti-mouse IgE of 100 μ L antibody, reaction is one to two hour under room temperature, cleans 5 times again.Add avidin-HRP (the avidin-horseradish peroxidase conjugated of 100 μ L through suitably diluting, the avidin of horseradish peroxidase combination) reaction is one hour, clean 6 times, add at last 100 μ L AB TS (2.2 '-Azino-bi s-3-Ethylbenzthiazoline-6-Sulfonic Acid, 2,2 '-azine-two-(3-ethyl benzo thiazole phenanthroline sulfonic acid) substrate solution, in color development at room temperature after about 30 minutes, with the 5%SDS cessation reaction of 100 μ L, and read light absorption value with microplate reader (microplate reader).Measurement result is expressed as follows in the mode of ELISA (ELISA) unit:
ELISA unit=(A
Sample-A
Blank)/(A
Positive control-A
Blank)
C. (airway hyperresponsiveness AHR) measures airway hyperreactivity
The allergic reaction that whether can slow down sensitized mice for research oral anti-allergy lactic acid bacteria after anaphylactogen stimulates in twice tracheae, is accepted the test of resistance of respiratory tract every other day.Test macro be Buxco system (Biosystem XA, Buxco Electronics Inc.Sharon, CT, USA).Utilize sensor in the system (differential pressure pickup, differential pressure transducer, Buxco) and preamplifier (preamplifier) (MAX II Buxco) collects the variable signal in mouse breathing road, thereby calculates the Penh value.The account form of Penh value is: intermittently * and PIF/PEF; Intermittently=(Te-Tr)/and Tr, (PIF: peak inspiratory flow (peak inspiratory flow); PEF: expiration amount peak value (peak expiratory flow); Te: expiratory duration (expiratory time); Tr: diastolic time (relaxation time)).Mouse is placed under the state of Consciousness in whole body volume scan instrument (the whole body plethysmograph) chamber, the physiological saline of ultrasonic concussion gasification was imported chamber (chamber) three minutes, write down the average pulmonary respiration Resistance Value of per minute Penh in the mouse three minutes then, again with the methacholine (Methacholine of cumulative concentration, 6.25,12.5,25,50mg/mL) atomization gives mouse stimulates, each concentration all after stimulating three minutes, writes down the physiological change in three minutes internal respiration roads.Write down the mean P enh value of each minute in three minutes.
D. lung flushing liquor and lung cells analysis
Test the pulmonary respiration resistance every other day, put to death mouse, cutting off neck skin and flesh exposes tracheae, put with vein and to stay pipe to insert tracheae, for the first time earlier with the aseptic HBSS buffer solution flushing of 1mL lung, second and third time be then with the HBSS that contains 2%BSA (Hanks ' balanced salt solution) buffer solution flushing lung, and the lung flushing liquor that obtains first is through 1500rpm after centrifugal 5 minutes, separation of supernatant is stored in-80 ℃, treats subsequent analysis.Cell is partly incorporated in the twice lung flushing liquor in back, with 1500rpm centrifugal 5 minutes, throw aside after the supernatant, contain the HBSS buffer solution suspension cell of 2%BSA with 1mL, and, get about 1 * 10 with trypan blue (trypan blue) dyeing counting
5Individual cell was made cell smear in centrifugal 3 minutes with cell centrifugation smearing machine (cytospin) 500rpm, after treating its natural air drying, successively with Liu A and Liu B dyeing, with a small amount of Arabic gum mounting, use oily mirror (1000x) to read cell or the cell of sum more than 300 at least five visuals field, calculate its medium size lymphocyte and coenocytic ratio.
E. the cultivation of spleen cell
Under aseptic condition, open mouse peritoneal and take out spleen, place in the 20mm culture dish (petri dish) that adds 3mL HBSS buffer solution, with aseptic syringe tail end spleen is ground, its medium size lymphocyte is disengaged, cell suspending liquid is sucked in the 15mL centrifuge tube, after leaving standstill 5 minutes, get supernatant centrifugal 5 minutes with 1500rpm, absorb supernatant after, add 1mL RBC lysis buffer (lysis buffer), leave standstill and made globulolysis in 1 minute, add 5mL HBSS buffer solution, with 1500rpm centrifugal 5 minutes, absorb supernatant; After repeating to wash twice with the HBSS buffer solution, add the RPMI-1640 complete medium that 5mL contains 5%FBS again cell is suspended.Calculate TCS with the trypan blue decoration method, adjust cell number to 1 * 10
7Individual cell/mL culture medium.Get cell liquid 0.5mL and add in 48 well culture plates (48-well plate), add the equal-volume nutrient solution again, contain the nutrient solution of fragmentation element or ovalbumin (OVA), making the cell ultimate density is 5 * 10
6Individual cell/mL culture medium.Place 37 ℃ of constant incubators, 5%CO
2Cultivated 48 hours, and collected supernatant and be stored in-80 ℃ and treat the wherein secretory volume of each cytohormone of later analysis.
F. the mensuration of cytohormone
Measure the content of the secreted cytohormone of spleen cell with sandwich ELISA method (ELISA).Be summarized as follows: earlier with the anti-cell hormone antibody with NaHCO
3Buffer solution (pH 9.6) dilution back adds 96 hole microtest plates, in 4 ℃ of standing over night.After culture plate being cleaned 3 times in second day, at room temperature react with the PBS that contains 1%BSA and to seal (blocking) at least in 1 hour.Can or place 4 ℃ of reactions to spend the night in 37 ℃ of reactions 2 hours after adding sample to be tested.Clean after 4 times anti-cell hormone antibody that the biotin that adds with the dilution of 1%BSA-PBS buffer solution engages (biotin-conjugated) in room temperature reaction 1 hour, clean 5 times, the peroxidase (streptavidin-conjugated peroxidase) that then adds the streptavidin joint reacted 1 hour again.At last after cleaning, add enzyme substrate solution tetramethyl benzidine (TMB) substrate (Tetramethylbenzidine substrate) of 100 μ L to every hole, reacted about 20 minutes in the room temperature lucifuge.Read the light absorption value of wavelength 450nm with microplate reader (microplate autoreader).
2. statistical method
Experimental result is represented (Means ± SEMs) with mean+/-standard error.Each group difference is analyzed with Student T check (t-test), p＜0.05 be considered as that there were significant differences (
*, p＜0.05;
*, p＜0.001).Be considered as p＜0.1 variant in addition.
Interpretation:
A. measure the result of the ELISA (ELISA) of ovalbumin (OVA) specific antibody
Ovalbumin in the serum (OVA) specific antibody titres part, the control group of feeding PM-A0006 lactic acid bacteria, ovalbumin (OVA) specific IgE antibody in ovalbumin (OVA) the sensitized mice serum has the trend of minimizing, and the result is illustrated in Fig. 6 in table 8.The measurement result of table 8 demonstration ovalbumin (OVA) specificity antibody IgE (mean value ± SEM, N=14, the P value=P)
Table 8
B. airway hyperreactivity (airway hyperresponsiveness, AHR) measurement result
Pulmonary respiration resistance part, with respect to control group, feeding PM-A0006 lactic acid bacteria is significantly lowered mouse Penh value under high concentration methacholine (Methacholine) stimulates.The results are shown in table 9, be illustrated among Fig. 7.Table 9 demonstration airway hyperreactivity measurement result (mean value ± SEM, N=14, the P value=P)
Table 9
C. lung flushing liquor and lung cells analysis result
Cell part in the lung flushing liquor, with respect to control group, the experimental group of feeding PM-A0006 lactic acid bacteria has significantly lower eosinophil (eosinophil) infiltration ratio.The results are summarized in table 10, be illustrated in Fig. 8.Table 10 demonstration lung flushing liquor analysis result (mean value ± SEM, N=14, the P value=P)
Table 10
D. lung flushing liquor cytohormone analysis result
With respect to control group, the experimental group of feeding PM-A0006 lactic acid bacteria can significantly reduce chemotactic factor for eosinophils's secretory volume and PGE in the lung flushing
2The trend that minimizing is also arranged.The results are summarized in table 11, be illustrated in Fig. 9 and Figure 10.Table 11 demonstration lung flushing cytohormone analysis result (mean value ± SEM, N=14, the P value=P)
Table 11
E. the supernatant cytohormone analysis result cultivated of spleen cell
Spleen cell stimulates cultivation after 48 hours through ConA or ovalbumin (OVA), and under ConA stimulated, the IFN-γ of the spleen cell secretion of the experimental group of feeding PM-A0006 lactic acid bacteria was significantly higher than control group.The results are summarized in table 12, be illustrated in Figure 11.Table 12 shows the supernatant that detects the spleen cell cultivation, and the influence that analysis different stimulated material is secreted the cytohormone of spleen cell (mean value ± SEM, N=14, the P value=P)
Table 12
Food compositions of the present invention, contain above-described any one or more than one various compositions that bacterial strain combined, for example acidified milk, yogurt, cheese, dairy drink milk powder, tea or coffee or the like, and bacterial strain can viable bacteria or the form of dead bacterium be present in the composition.
Because lactic acid bacteria will be brought into play antianaphylactic effect, except will finding out bacterial strain, to confirm that more can this bacterial strain also require this bacterial strain to have the good adsorption ability to the mucous membrane of small intestine epidermal cell by the environment of human body hydrochloric acid in gastric juice cholate with specific function.Exactly because also this specific character makes antiallergy lactic acid bacteria of the present invention that the medical application of the treatment or the allergic symptom of releiving just can be provided.The allergic reaction that antiallergy lactic acid bacteria described in the invention can strengthen the Th1 approach simultaneously and regulation and control Th2 crosses Sheng.A target of the present invention continues popular to provide except that steroids or antfhistamine new selection in the allergy treatment towards reaching these targets or being at least exactly, and the present invention finds out human body is had no side effect and wholesome antiallergy lactic acid bacteria is used as the new selection of irritated treatment.
The above is by embodiment characteristics of the present invention to be described, its purpose is to make those skilled in the art can understand content of the present invention and implements according to this, and non-limiting claim of the present invention.Therefore, all other do not break away from disclosed spirit and the equivalence finished is modified or revise, and must be included in the claim scope of the following stated.
Culture presevation
Five strains of lactic acid bacteria bacterial strains of the present invention are preserved in Chinese typical culture collection center (CCTCC, China, Wuhan City) on April 6th, 2007, and preserving number and information see Table 13.The viability of these five kinds of cultures is detected on April 27th, 2007 by the preservation center and finishes, and the result is survival.
In addition, the freeze drying culture of five strain bacterial isolateses of the present invention once was deposited in food Industry in Taiwan Institute of Development Studies, and the address is: No. 331, food road, Hsinchu City, Taiwan Province.The particulars of Taiwan preservation are as shown in table 13.
Claims (8)
1. food compositions, it comprises:
Jia Shi Bacillus acidi lactici (Lactobacillus gasseri) the PM-A0005:CCTCC NO:M 207039 of immune stimulatory emiocytosis antiallergy relevant cell hormone concentration, and any in physiologically acceptable excipient and the diluent.
2. food compositions as claimed in claim 1 is characterized in that, described bacterial strain is viable bacteria or dead bacterium.
3. food compositions as claimed in claim 1 is characterized in that, described physiologically acceptable excipient or diluent are a kind of food.
4. food compositions as claimed in claim 1 is characterized in that, described food is any in acidified milk, cheese, dairy drink, milk powder, the tea or coffee.
5. food compositions as claimed in claim 4 is characterized in that, described cheese is yogurt.
6. one kind is used for antianaphylactic pharmaceutical composition, it comprises Jia Shi Bacillus acidi lactici (Lactobacillus gasseri) the PM-A0005:CTCC NO:M 207039 of immune stimulatory emiocytosis antiallergy relevant cell hormone concentration, and pharmaceutically acceptable excipient or diluent.
7. pharmaceutical composition as claimed in claim 6 is characterized in that, described bacterial strain is viable bacteria or dead bacterium.
8. a biological pure culture is used for the purposes of the medicine of immune stimulatory emiocytosis antiallergy relevant cell hormone in preparation, it is characterized in that this biological pure culture is Jia Shi Bacillus acidi lactici (Lactobacillus gasseri) PM-A0005:CCTCC NO:M 207039.
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| CN104056259A (en) * | 2013-03-22 | 2014-09-24 | 东宇生物科技股份有限公司 | An effective component for treating or preventing allergic diseases |
| CN104862241A (en) * | 2014-02-21 | 2015-08-26 | 联亚益生生物科技股份有限公司 | Lactic acid bacterium having immunomodulatory and anti-allergic effects and pharmaceutical composition containing the same |
| CN108883141A (en) * | 2016-04-04 | 2018-11-23 | 龟甲万株式会社 | Composition and its manufacturing method for promoting interferon lambda to generate |
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