TW201121553A - Lactobacillus paracasei strain LT12 as immunity regulatory agent - Google Patents

Abstract

An isolated Lactobacillus paracasei strain LT12 that possesses immune regulating activity and uses thereof for regulating immune responses and treating allergy.

Description

201121553 VI. Description of the Invention: [Technical Field of the Invention] The present invention relates to a novel vice cheese lactic acid rod [Prior Art] Base, 'Daily immune system foreign matter' such as bacteria, pollen or dust, etc., ίίί Excessive immune response, the foreign substance Wei is called "allergic n ΐΐ ΐΐ 、 有时 有时 有时 有时 有时 有时 有时 有时 有时 有时 有时 有时 有时 有时 有时 有时 有时 有时 有时 有时 有时 有时 有时 有时 有时 有时 有时 有时 有时 有时 有时 有时 有时 有时 有时 有时 有时 有时 有时When the human body causes free scales, histamine and adenosine may be followed by ^, which causes allergic symptoms, including inflammatory reactions, ectopic skin fire, allergic conjunctivitis, #麻疗, 归, specific gastrointestinal Disease or asthma, reports indicate that children in developed countries are more likely to suffer from allergies, because this country is too focused on public health, and often vaccinated or antibiotics, so reduce the feeling (four) the opportunity to 'cry _ often against allergies. In the case of medical death, this phenomenon is called the "hygiene hypothesis". It mainly discusses the lack of exposure to the original infection in childhood and will lead to an immune response to Th_2, such as: Overproduction of IL-4 (mterleukin-4), IL-5 and IL-13. These Th-2 cytokines attract e嗜sin〇phils, basophils, and mastceu to the site of inflammation, either alone or with immunoglobulin E ( IgE) works together to produce an allergic reaction. In addition, IL 4 and IL-13 can promote b cell cell type switching (is〇 type switch) to increase the amount of _ in the blood. Inducing immune responses in Th-1 (T helper 1) cells according to the "hygiene hyp〇thesis", for example, promoting the expression of IFN-γ may effectively alleviate allergic reactions. Many studies have pointed out that lactic acid bacteria (sinking plus sp.) have the ability to regulate immune response in 201121553, including promoting the secretion of cytokines, activating macrophages and natural killer cells and antibody production (Madsen is β/. , 2 〇 () 1, 仏 price sink her ro / illusion; 121: 580-591). Collins et al. (uS Pat. No: 7,390,519) have shown that strains of Lactobacillus saliva (Zac secret ac///wji stomach (4) ΑΗ102, ΑΗ103, ΑΗ105, ΑΗ109 or AH110 can be effective as an anti-inflammatory biotherapeutic agent. Prevention and/or treatment of inflammatory reactions. Hsu et al. (US Pat. No. 6,994,848) also discloses that Lactobacillus paracasei GM_080 promotes the secretion of IFN_y and can be used to treat allergy-related diseases. However, various lactic acid bacteria present in nature It has not been fully discovered and fully studied to date. The present invention provides a composition comprising a variety of Lactobacillus paracasei and its application to immunomodulation, and its associated prior The literature has revealed different strains. [Inventive content] ^ The present invention - the aspect of providing - the novel immunophilic function of the new Lactobacillus paracasei to the LT12, or its genetic engineering 'cross-strain' In the case of exogenous gene (ex〇gen〇us gene), the gene is engineered to be used in the case of 'the mutant strain can be obtained by the following steps - LT12 (9) to exogenous gene and promoter (3) 〇 (4) After the connection, the expression ex (expressi〇n casstte) is transferred to the LT12 strain. The surface composition is a composition comprising the above-mentioned sub-dry lactic acid 3 变异 mutant and a carrier. The composition may be a food (for example) : yoghurt, wheat bran, chocolate and thin sticks), lean (eg tea, non-alcoholic drinks, milk and preserved fruit), or contain finely acceptable drugs. - Aspects provide - _ section of the immune system (such as · congenital dismissal ty) or adaptive immunity (10) 叩 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ · Human patients) may be patients with IgE-mediated dysbiosis (10) mediate hypersensitivity or tau cell-mediated delayed onset 4 201121553 delayed hypersensitivity. Still another aspect of the present invention also encompasses the use of the aforementioned by-product Lactobacillus LT12 to prepare a medicament for modulating an immune system or treating allergy. One or more specific embodiments of the invention are described below. Other features and advantages of the present invention will be apparent from the following drawings, embodiments and claims. [Embodiment] The present invention provides a novel Lactobacillus paracasei strain, designated LT12, which can be used to modulate an immune response and treat allergy. The strain was deposited at the American Agricultural Stem Breeding Center (1815N.) on September 21, 2009.

University Street, Peoria, imnois, 61601, U.S.A.), registration number NRRL-B50327. The LT12 strain of Lactobacillus strain 刎12 is isolated from human feces and must be separated within 12 hours after sampling. Weighing (about one gram) of the specimens were placed in a mixture of WStomacher 4, Seward, UK for 3 sec to homogenize, and then pre-reduced brain heart extract culture (brain) Heart infusion broth) is diluted and then cultured in a suitable selective medium. Appropriate dilutions are applied to the lactic acid bacteria acetate medium (R〇g〇sa Acetaie)

Difco USA) ' or lactic acid bacteria acetate medium containing vancomycin (vancomyCin, sigma, USA) (R〇g〇sa eight (2), so ton batch, so as not to. 'US A). ' _ to cultivate all the reduction Genus and lactic acid bacteria that can resist the eternal fresh, such as μ Tian j dry breast milk g; f dry bacteria group (£ 尸 沉 四 . . 〇叩 〇叩 , , , , , , , , , , , , , , , , , , , , , , , , , , , Bacillus (3) such as > and Lactobacillus brevis A (four), etc. API50 CHL system (four) tender _, heart usa) is used to identify strains resistant to vancomycin (four) dry Weier (4). The identification results are shown in Table 1. The microbiological characteristics of LT12 are as follows: (1) Morphological characteristics (a) Cell shape and size: The cells are at 37. (:下下]^11^ culture medium 201121553, after night culture, observed under a microscope _ shape scaly, the edge is round. (b) Activity: there are (4) flagella: no (d) sporulation: no (8) Gram stain: positive (2) Culture characteristics: (4) Medium: MRS medium (Difco, USA), final pH 6 2~6 5. (9) Culture conditions: 3Tt facultative anaerobic (3) Physiological characteristics: (8) Catalase: positive (9) oxidase: negative (c) API 50 CHL test: test results are shown in Table 1. (4) Genetic characteristics: As shown in Example 2, random amplified polymorphic DNA identification (Rand〇m

Amplification P〇lymorphic DNA, RAPD) method for LT12,

(6) (Cell biotech Co., Ltd., Korea) (hereinafter referred to as LP-CBT), Lfl gamma 0flcz7 / Nateng / GMNL32 (hereinafter referred to as: GMNL32) 'Lactobacillus paracasei subsp. paracasei BCRC 14023 (below For short, BCRC 14023) and B3, subsp BCRC 12188 (hereinafter referred to as BCRC 12188) perform mpd analysis. The 12 random nucleic acid primers are listed in Table 2. Electropherogram results show that LT12 has different strip modes (as shown in Table 3). Based on the above identification results, LT12 is a novel strain of Lactobacillus. The present invention also discloses an LT12 genetically engineered strain in which one or more foreign genes are transferred to LT12 by genetic engineering recombination. The variant strain can be prepared by a general molecular biology technique. For example, the LT 〖2 strain can be cultured under appropriate conditions'. The foreign gene of interest is then ligated to a suitable promoter and recombined by repeated methods, for example electroporation can be used. The transgenic method (e]ectrotransfomiation, as described by openwetware.org/ 6 201121553 wiki/Lactobacillus transformation), who transferred to τ τι ο 兹 her. After transfection, the protein of the foreign gene can be measured for successful transfection, for example, using an enzyme immunoassay (ELISA), or Western blotting or other analysis of the activity of the gene product. A promoter is a nucleotide sequence that contains a sequence necessary for initiation of transcription. The promoter must contain at least one binding site capable of binding to RNA polymerase, and may further comprise one or more enhancers that promote transcription 'or one or more regulatory dements that control the promoter switch. . "Exogenous genes" as used herein include, but are not limited to, 'immunoregulatory factors (eg, cytokines or superantigens), a gene encoding a therapeutic protein, or a gene encoding a secondary cheese. A protein that grows in lactic acid. According to a particular embodiment of the invention, LT12 or a functional variant thereof in the form of a living cell can be used. According to another embodiment of the invention, a form of non-viable cells, such as a heat lethal cell, comprising a beneficial factor produced by the strain 12 can be used. The term "r functional variant" as used herein refers to a strain having the same characteristics as LT12, which achieves the same efficacy. The present invention provides an LT12 strain and a variant thereof which have an activity of modulating an immunological activity and/or an antiallergic reaction. As used herein, the term "modulating immune activity" refers to the immune system that enhances immune activity (immunostimulatory activity) from its stable state or suppresses excess or excessive immune responses to return to an appropriate level ( Inhibition of immunological activity) In terms of H, this is used to regulate the balance of immune cells (eg, T cells, B cell gamma cells) to stabilize the overall immune response. For example, but not limited to, stimulating or inhibiting the production of cytokines, living = lymphocytes, balancing Th1/Th2 activity, and inhibiting allergic reactions. The present invention also provides a LT12 strain or a functional variant thereof which can be used for the treatment of allergies. The term "allergy" as used herein refers to an immune response caused by a physiological protective plague that harms the host. The two most common allergies are immediate allergic reactions mediated by 1gE_ and T-cell-mediated delayed allergic reactions. Allergic bed symptoms include occupational rhinitis, occupational asthma, food allergies, 201121553, allergic dermatitis, eye allergies, and systemic allergic reactions (anaphylaxis). The term "treatment of allergy" as used herein refers to a series of immune responses that reverse the aforementioned immune symptoms, such as, but not limited to, blocking Th2-mediated immune responses and reducing Th-2 cells. Hormones (eg, IL-4, IL-5, IL-13) increase Th-1 type cytokines (such as ΙΝΐ·_γ) and inhibit the activation or differentiation of IgE+ memory b cells and eosinophils. According to a specific embodiment of the present invention, it was confirmed by ELISA analysis that LT12 can stimulate peripheral blood mononuclear cells (PBMCs) secreted by donor blood to secrete INF(R) (see Table 4). INF-γ is a cytokine that is important for innate immunity and adaptive immunity against viral and bacterial infections. It is a representative of Th-Ι cytokines, which can inhibit the differentiation of Th-2 cells, thus reducing the production of L_4, IL_5, IL_13, which stimulates the conversion of immunoglobulins into IgE, as well as amplification of the original and The number of IgE+ memory B cells. Therefore, the promoted performance may be effective in treating allergies. According to another embodiment of the present invention, LT12 can also stimulate peripheral blood cells to secrete other cytokines, including jL_ia, IL_6, IL-, which are analyzed by human cytokine antibody array. 1〇, IFN-y, MCP_2, and D-NF-a (see Figure 1A_1E and Table 6). Accordingly, the present invention provides a method of modulating immunity comprising administering to a subject an effective amount of a side effect, a red T12, and a medically acceptable carrier. The present invention also provides a method of treating allergies, comprising administering an "individual", a sour red bar, and a medically acceptable carrier. The "supply acceptable carrier" herein includes, but is not limited to, physiological chyme 1 glucosinolate, water, glycerin, acetylene-^t, external 疋 'present invention--composition, including TM2 and II also have immunomodulatory and/or antiallergic activities. The second sub-dry sputum Lactobacillus strain is LP-CBT, and the ratio of LH2 to its halogen number is 1〇〇:1. The preferred ratio of bacteria is 2:1. As shown in Example 4, the solid surface of the 4th solid nodules, the composition can also stimulate peripheral 8 201121553 blood mononuclear cells to secrete other cytokines, including IL analyzed by the human cytokine antibody array. -1 a, IL-6, IL-10, IFN-γ, MCP-2 and TNF-α. In addition, MCP-3 is produced only by peripheral blood mononuclear cells stimulated by the LT12 composition. According to the present invention, 'LT12 provides excellent immunomodulation and anti-allergy activity. According to one embodiment of the invention, LT 12 or a combination thereof can be used as an active ingredient of a composition. The composition can be made into a food, drink or a drug. In addition, various additives may be included, and the types of the additives are exemplified by, but not limited to, artificial colors (for example, carotene, annatto red, turmeric, paprika, pharmaceuticals, and cosmetic dyes). , seasonings, spices, sweeteners, emulsifiers and/or thickeners, preservatives, vitamins and antioxidants (eg vitamins A, C, D, Ε, Β-1, Β-5, Β-6, Zinc, selenium, about, alpha-vitamin, giutathione, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and cysteine (cysteine) ). The invention will be further illustrated by the following examples, but the actual invention is not limited to the examples set forth in the specification. Example 1: Separation of Lactobacillus paracasei LT12 a Lactobacillus paracasei LT12 was isolated from human feces and subjected to separation within 12 hours of sample sampling. The sample weighed (about one gram) was placed in a homogenizer (Stomacher 400, Seward, UK) and homogenized for 30 seconds, and then pre-reduced brain heart extract culture (brain, time) ^fusion broth) Dilution' followed by incubation in a suitable selective medium. Appropriate dilutions are applied to lactic acid bacteria acetate medium (Rogosa Acetal agar, Difco USA)' or lactic acid bacteria acetate medium containing vancomycin (vang〇mycin, sigma, USA) (R〇g〇sa into (3) shirt & Cards, for example, f culture all lactic acid bacteria, and vancomycin-resistant lactic acid bacteria, for example: go to buttermilk to g group (X Qing (four) (10) gIOup), & with Conghe acid rod to do fine, casein lactic acid Bacillus (Z_Hidden zM Lactobacillus brevis. 201121553 followed). The API 50 CHL system (bioM0rieux, Inc. USA) was used to identify vancomycin-resistant strains of Lactobacillus paracasei. Bayesian Example 2. Identification of the lactic acid bacteria in the API 50 CHL system. The characteristics of the lactic acid bacteria strain are established by a series of enzyme reactions. The API 50 CHL system identification results for LT12 are listed in Table 1. Table 1 LT12 API 50 CHL system identification results reference: LT12 good identification test strip: API50CHLV5.1 - raw 4 匕 6 pu + + ++ +++-++ + +-+++++++++ -++++----++++----_) CIKL - GLY - ERY GAL + GLU + FRU MDM - MDG + NAG MEL - SAC -l· ΊΚΕ DARA - LARA MNE • Bu SBE AMY + ARB 1NU + ML2

RB + DXYL Κϊ-ΙΛ A DUL ESC + SAL RAF AMD LXYL - ADO - MDX - INO + MAN + SOR + CEL + MAL + LAC + OLYG - XLJ - GEN +

TUR 4- LYX + TAG + DRJC

LFUC - DARL

LARL - GNT -h 2KG - 5KG Meaningful classification unit identification % Inconsistent Ding test Lactobacillus paracasei ssp paracasei 1 95.2 0.69 DUL 13% INO 6% LYX 20% Next classification unit identification % Inconsistent test Lactobacillus paracasei ssp paracasei 2 4.6 0.6 LAC 0% LYX 16% Random amplified polymorphic DNA (RAPD analysis) identification:

The following strains were cultured for 40 hours and then subjected to DNA extraction: LT12, aracosez. (Cell biotech Co" Ltd., Korea) (LP-CBT for short), /w like wez· GMNL32 (abbreviation: GMNL32) ' Lactobacillus paracasei Subsp. paracasei BCRC 14023 (abbreviation .BCRC 14023) paracosez·subsp. BCRC 12188 (abbreviation: BCRC 12188), RAPD analysis was performed with 12 random polymorphic nucleic acid primers (Table 2) after chromosome extraction. The results of RAPD analysis are shown in the table. 3. It can be observed by electrophoresis that LT12 has different banding patterns from the other four strains 10 201121553. One-to-one API 50 CHL identification and RAPD analysis, LT12 is different from the typical Lactobacillus casei strain. Therefore, LT12 is A novel paracasei lactic acid strain. Table 2

.^5 DNA sequence 歹iJ(5'-3') SEQ ID NO: ΟΡΟοΓ^ ACCGCGAAGG 1 ΙΑ OPD-03 GTCGCCGTCA 2 IB 〇PD^〇7 TTGGCACGGG 3 1C OPD-20 ACCCGGTCAC 4 ID 〇PS^〇3 ^1 CAGAGGTCCC 5 IE 〇PS^〇7~~ TCCGATGCTG 6 IF 〇PSq〇~~~~ OPS^n ACCGTTCCAG 7 1G AGTCGGGTGG 8 1H 〇^Λ3 〇PS^7~~ CTGGGTGAGT 9 11 TGGGGACCAC 10 1J TGGGGACCAC 11 IK OPS-19 GAGTCAGCAG 12 1L Table 3 -------- LP-CBT GMNL-32 BCRC 14023 BCRC 12188 uljD^0l ————_ 〇———〇〇OPD-07, s /ΛΤ)Τν "XT--- 〇〇 〇 〇 UPD-2D OP OP OPS^^— 0^^07-~~~ 〇〇 — 〇 — 〇 — — ---- 〇〇〇〇 UPS-i 1 — 〇 — — —— 〇pgrr^-H r\T\f> :·~*————————— 〇〇 — 〇〇〇〇 (JPS-17 — 〇〇〇〇 OPS-iq — distinguishable 〇〇 — 〇 9 10 5 7 11 201121553 Example 3: Preparation of allergic regulation function of LT12 and other lactic acid bacteria Experimental preparation of LB12, LP-CBT and Lac'o0acz//(10) Coffee (hereinafter referred to as LGG) cultured in lactic acid bacteria MRS medium at 37 ° C 24 hours, pick up The bacterial solution was centrifuged at 3000 rpm for 15 minutes. After the chamber was washed twice with 1 ml of sterile pBS (ph〇sphate buffered saline), it was then heated to 95. The mixture was heated for 3 minutes to prepare a heat-killing liquid. Preparation of nuclear cells (PBMC) Fresh blood comes from healthy donors (two men and three women, ages 24 to 40 years old). Centrifuge at 2000 rpm for 1 minute at room temperature, then collect the skin color layer (buffy coat丨ayer). A sample of the collected sample was added to an equal amount of liquid (Dilute medium containing RPMI-1640, 2% fetal bovine serum) and slowly transferred to a 4 ml solution of Histopaque-1077 ficoll plus. After centrifugation at 12 〇〇φιη for 30 minutes at room temperature, '8 ml of Wash medium (containing RPMI-1640, 2% fetal bovine serum, 1% PBS) was mixed and thoroughly washed twice and collected from the interface of the sample. PMBC. The collected PMBC was resuspended in complete broth (containing RPMI-1640, 10% fetal bovine serum, 1% PBS) and cultured in a 24-well plate at a concentration of 106 cells/ml. Stimulation of IFN-τ secretion and cytokine IFN-γ quantification of PBMCs with three strains (LT12, LP-CBT and LGG) at a given concentration (5 X 106, 5 X 1〇7 or 5 X 108 CFU/ml) Co-cultivation. After 48 hours of co-cultivation, the cell supernatants of each well were collected, and the cytokine lFN-γ was quantitatively analyzed by an enzyme immunoassay kit (ELISA kit) as follows. Among them, 5 pg/ml phytohemagglutinin PHA (phytohemagglutinir〇 and 10 pg/ml lipopolysaccharides (LPS) were used as positive control group. Add 1 μμ] 7 well 4pg/ml anti-drug in 96-well plate. IFN-γ antibody

(RayBio® Human IFN-γ ELISA, RayBiotech hc., USA), allowed to stand overnight at 4 °C. The liquid in the well was removed, washed with 250 pL/well of washing buffer (wash 201121553 buffer), and the liquid in the well was removed, and the above washing steps were repeated 5 times. Add 200 μ! 7 well assay diluent and let stand for 1 hour at room temperature. Add 250 μΙ7 well to the washing buffer and repeat the above washing steps for 5 times. The PMBCs sample from 100 μM well was then added to react at room temperature for two hours. After washing five times with 250 μ! 7 well wash buffer, the winter well was added to the secondary antibody of 1 μ μμ well for one hour. After washing five times with 250 μΙ 7 well wash buffer, then add 100 μΙ 7 well of horseradish peroxidase (Avidin-HRP) (1:250) for 30 minutes. Add 7 times of washing buffer with 250 μM/well. Add the substrate solution of 1 〇〇 μίν well. After standing at room temperature for 3 to 15 minutes, the reaction was stopped by adding 2NH2S〇4 of 50 μ!7 well. The wells of each well were measured for absorbance at a wavelength of 450 nm, and the concentration of cytokine (IFN-γ) in the sample was converted according to a standard curve. The test results are shown in Table 4. Among the strains, LT12 has the strongest ability to stimulate IFN-γ production at different cell concentrations. Table 4 Cell concentration (CFU/mL) Source of PBMC

A B C D E test sample

Mean soil SD IFN-γ (pg/mL) LT12 5*106 5*107 5*108 7822 11058 4507 756 9981 8397 4717 32180 14169 3 236 4109 1991 11439 15079 3058 Soil 3210 12979 ±11685 9252 ±5192 lgg 5*106 2197 91 1036 15 293 726 ±916 5*107 5884 2562 14595 96 3399 5307 ±5589 5*108 1643 1326 2313 1399 1424 1621 ±404 lp-cbt 5*106 2273 81 456 .2 74 577 ±965 5*107 6916 1623 3904 31 2010 2897 ±2636 5*108 11261 7188 8956 221 3181 6162+4445 Positive Control PHA 5 pg/mL 15533 733 361 289 170 3417+ 6776 LPS 10 pg/mL 119 30 8 87 44 57 ±45 Negative Control PBS *' 0 0 7 4 3+3 Culture Medium 0 0 5 ' 5 2 3 ±3 13 201121553 Cell-producing cells Example 4: LT12 and its age for peripheral blood mononuclear hormones The functional evaluation of the heat-killing LT12, and its composition, including the g-number ratio of 2^ and LP-CBT, were prepared in the manner described above. pBMCs (source '(5 X(10)ml) and (10) composition (5 χ 1〇7/ml) co-cultured 48):;, after 'human cytokine antibody microarray (RayBi〇®Hu_ antibody array, Cat. AAH- CTT-1, RayBiotech Inc., USA) evaluated the induced cytokine secretion profile. The analysis steps are as shown in the description of the analysis kit (http://www.raY.biotech.com/cytokine antibody. She p 〇 simply say 'the packaging is the case, when the head is careful, the 所附 set is attached to the eight In the well plate, inject 2 mL/well of blocking buffer along the wall (w〇cking buffej^ on a shaker, shake at room temperature for 3 minutes. Remove the blocking buffer and inject 1 mL/well of the sample. After gently shaking for 2 hours on a shaker, wash twice with 2 claws/well of Wash Buffer I, and then 3 times with 2 mL/well of Wash Buffer π. Force σ into 1 mL/well The biotin-antibody solution was shaken on a shaker for 2 hours. After removing the antibody solution, it was washed twice with 2 mL/well of Wash Buffer I and then with 2 mL/well of Wash Buffer π. 3 times. Inject 2 mL/well of horseradish peroxidase avidin (HRp_Streptavidin), shake it for 2 hours on a shaker, carefully pry the membrane with tweezers, drip dry residual liquid, and then remove the removed liquid. The orifice plate was injected into a detection buffer of mL·5 mL/well, and the reaction was protected from light for 2 minutes. The membrane was clamped to the sleeve with tweezers. Attach the two pieces of plastic film or plastic wrap, and place the film together with the plastic film in a dark box for 40 seconds in the darkroom and develop it. The cytokine antibody array is on the film. The location map is shown in Table 5. ^ Table 5 cytokine antibody microarray location map GRO-a GRO-a IL-10 IL-10 cytokine cytokine positive control positive control (RANTES) (RANTES) group 14 201121553

The results of the analysis are shown in Table 6 and a group of 'BBMC, respectively, / _E. The experimental analysis group consisted of 5 blank groups co-cultured with LT12 (Fig. 1 A), PBMC source C with the culture group (Fig. ic), PBH), PBMC source C and LT12 compositions co-sourced with LT12 Shame ^ source E blank group (Figure 1D) and PBMC to cytokine secretion 1 Ε). In each co-culture test group, it was observed that tnfm ', where the two cells _,, 'I hormone IL-1 α, INF-γ, MCP-2 and TNF-a were only stimulated by the LT12 and LT12 compositions, were induced. produce. Comparing the groups of the same PBMC source C, it was found that the ability of the LT12 composition to induce GRO-α, IL-10 and MCP-2 production was higher than that of LT12. In addition, only LT12 compositions can induce PMBCs to exhibit MCP-3. Table 6 Cytokine PBMC Source C PBMCs Source E (5*107)-LT12 (5*107)-LT12 Composition (5*107)-LT12 Composition GCSF 0 0 0 GM-CSF 0 0 0 — + GRO JU— - GRO-α A— + + IL-l〇t 〇—► + 0-> + 0-> + IL-2 0 0 0 IL-3 0 0 0 15 201121553

And: no difference before and after the test +: increase ++: a large increase 〇-+: only appear in the test group. Those who are well-known in the art will be able to change and modify the case. It should be noted that the present invention is not limited to the scope disclosed in the embodiment, but is also encompassed by other applications according to the application. The place where the road is revealed in the month of the month [Simplified description of the drawings] The foregoing description and the implementation mode can achieve better results by adding additional drawings. To enhance the description of the invention, the drawings of the appropriate embodiments are set forth herein. It is to be noted that the present invention is not limited to the descriptions set forth herein. Figure 1 is a photograph showing the analysis of the breakdown of each group using the human cytokine antibody Array (w〇t): peripheral blood

Liquid monocyte-derived C blank group (Fig. 1A), peripheral blood mononuclear cell source C (Fig. 1B) was stimulated with LT12 alone, peripheral blood mononuclear cell source C was stimulated with LT12 and the second lactobacillus. Figure 1C), peripheral blood mononuclear cell source E 16 201121553 blank group (Figure ID) and peripheral blood mononuclear cell source E (Figure 1E) stimulated with LTl2 and the second Lactobacillus paracasei. The positional map of the cytokine antibody array on the membrane is shown in Table 5. [Explanation of main component symbols] No 201121553 Sequence Listing <110> Lite Co., Ltd. <120> Can be used as an immunomodulator for Lactobacillus paracasei LT12 <140> 098144461 <141> 2009-12-23 <160>12<170> Patentln version 3.3 <210> 1 <211> 10 <212> DNA <213> Synthetic <220><223> Introduction <400> 1 accgcgaagg 10 <210&gt 2 <211> 10 <212> DNA <213> Synthetic <220><223> Introduction <400> 2 gtcgccgtca 10 <210> 3 <211> 10 <212> DNA <;213> Synthetic <220> 201121553 <223> Introduction <400> 3 ttggcacggg 10 <210> 4 <211> 10 <212> DNA <213> Synthetic <220><223> introduction <400> 4 acccggtcac 10 <210> 5 <211> 10 <212> DNA <213> Synthetic <220><223> Introduction <400> 5 cagaggtccc 10 <210> 6 <211> 10 <212> DNA <213> Synthetic <220><223> Introduction <400> 6 tccgatgctg 10 <210> 7 <211> 10 201121553 <212> DNA <213> Synthetic <220><223> Introduction <400> 7 accgttccag 10 <210> 8 <211&gt 10 <212> DNA <213> Synthetic <220><223> Initiative <400> 8 agtcgggtgg 10 <210> 9 <211> 10 <212> DNA <213> Synthetic <220><223> Introduction <400> 9 ctgggtgagt 10 <210> 10 <211> 10 <212> DNA <213> Synthetic <220><223> Introduction <400> 10 201121553 tggggaccac 10 <210> 11 <211> 10 <212> DNA <213>synthetic <220><223> Introduction <400> 11 tggggaccac 10 <210> 12 <211> 10 <212> DNA <213> Synthetic <220><223> Introduction <400> 12 gagtcagcag 10

Claims (1)

201121553 VII. Scope of application for patents: 1 The vice-drug lactic acid rod g of the $1 has been deposited in the relevant basic engineering strain. The storage is corrected to be dirty ' or the bacterium as described in 2', wherein the ancestor-B is simple. . The sub-Yugus agricultural species preservation center, the registration number is 3·acid compound, and the composition of the sub-dry milk 4 ΊΐίΓΐτ1 described in item 1 is the result of the towel. Lactic acid mash; bacterium 2: deposited in the US Agricultural Cultures Preservation Center, registered in the composition described in Item 3, which has anti-allergic activity. _ Sex ° and ^ job = the 31st rider composition, its financial immune regulation live 7 · fine 3rd composition of the composition, its towel off the dry lactic acid _ is a living body. The composition of claim 3, wherein the sub-dry lactic acid® is non-activated. 9. The composition of claim 3, which may be a food, a beverage, or a musical. 10. The composition of claim 3, which may further comprise a second Lactobacillus paracasei strain. The composition of claim 10, wherein the ratio of the number of bacteria of the lactic acid to囷1 to the second strain of Lactobacillus paracasei may be between 2:1 and 12, and the patent is patented. The composition of claim 5, which has immunomodulatory activity and anti-allergic activity. 201121553 13· Ι ί ί 1 1 1 1 1 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 2011 Requires a receptor for a combination of a strain of Bacillus and a drug in the medicine = the range of the first line of the lactic acid as described in item 1. The scope of the patent is in the 14th item. The strain is m2, the deposit is two dry; The method described in Item No. 14, wherein the composition is capable of regulating (1 called immUnity) or adaptive immunity, and the method described in item 14, wherein the combination is The substance can promote the production of cytokines. 疋18· The method described in item 17, wherein the cytokine is selected; -α, IL_6, IL-10, ΙΝρ-γ, MCP-2 and TNF-α成之群. " 19_ A method for treating allergies, which comprises administering a dry-dried strain A medically acceptable carrier. 20. The method of claim 19, wherein the sub-dried lactobacillus strain is LT12, deposited in the US Agricultural Stem Conservation Center, NRRL-B50327. The method of claim 19, wherein the receptor is an immunoglobulin E (IgE)-mediated immediate allergy (imediate h-ity) or a tau cell-mediated delayed allergic reaction ( Patient of paper _ hypersensitivity. 22. A genetically engineered strain of Lactobacillus paracasei LT12 comprising: providing a strain of LT12 deposited in the American Agricultural Stem Breeding Center with the accession number NRRL-B50327; And 201121553, an expression cassette comprising an exogenous gene linked to a promoter is introduced into the strain, thereby producing a genetically engineered strain.