TW200532018A - Novel microorganism strain gmnl-32 of lactobacillus paracasei and its use for treating allergy related diseases - Google Patents

Abstract

The present invention provides an isolated microorganism strain, Lactobacillus paracasei GMNL-32, which is found to be effective in treating allergy. The use of the Lactobacillus paracasei GMNL-32 in treating allergy related disease is also provided.

Description

200532018 发明. Description of the invention: The technical field to which Liaoming belongs] The present invention relates to a novel microorganism strain Lactobacillus paracasei GMNL-32 and its use to stimulate the secretion of IPN_γ and treat allergy-related diseases :: Uses. [Previous technology] Allergy refers to the acquired potential for adverse reactions to immune regulation of normal harmless substances. Allergic reactions cause symptoms such as itching, coughing, asthma, sneezing, tearing, inflammation and fatigue. Allergic reactions are generally considered to include early specific immune responses and late inflammatory reactions. Allergens (such as pollen and dust mites) have been reported to modulate early allergies by stimulating high-affinity immunoglobulin (JgE) receptors. For example, when mast cells and basophils are stimulated by allergens, they will release histamine and cytokine. Then, the cytokines released by mast cells and Han test cells regulate later stage allergies by restoring inflammatory cells. It has also been reported that the influx of f-erythrocytes, giant pheasant cells, lymphocytes, peripheral neutrophils, and platelets has begun a malignant inflammatory cycle. Allergies in this later period expand the initial immune response, which in turn triggers the release of more inflammatory cells (Blease et al., Chemokines and their role in airway hyper-reactivity o Respir Res 2000; 1 54-61). In order to treat the allergic symptoms, various treatments have been sought. Among them, antiallergic agents and histamine receptor antagonists (antihistamines) have been used. Histamine antagonists are administered to counteract the effects of histamine released by mast cells in response to the presence of allergens. They reduce redness, pain and swelling caused by histamine action on target tissues and are used to prevent or alleviate O: \ 90 \ 908ll.DOC -6- 200532018 many caused by mast cell degranulation Symptoms. However, antihistamines are also associated with adverse reactions such as reduced agility, slowed response times, and lethargy (US Patent No. 6,225,332). There are also reports of cytokines (Cytokines) to treat allergies. Among them, interferon-γ (IFN-γ) has been found to inhibit the over-expression of cytokines in Th2 lymphocytes, especially to suppress the secretion of IL-4 to reduce the proliferation of B cells. IFN-γ can also stimulate the immune response of Thl and inhibit the synthesis of IgE (Sareneva T et al., Influenza A virus-induced IFN-a; // 5 and IL-18 synergistically enhance IFN-γ gene expression in human T cells, J Immunol 1998; 160: 6032-6038; Shida K et al., Lacobacillus casei inhibits antigen-induced IgE secretion through regulation of cytokine production in murine splenocyte cultures 5 Int Arch Allergy Immunol 1998; 115: 278-287). Because IFN-γ can inhibit B cell proliferation and IgE secretion, it is believed that IFN-γ is effective in the treatment of allergies. Lactic acid bacteria (which are Gram-positive bacteria) are commonly used in industrial food fermentation. Recent studies have shown that lactic acid bacteria can stimulate IFN-γ secretion of cells (Contractor NV et al., Lymphoid hyperplasia, autoimmunity and compromised intestinal intraepithelial lymphocyte development in colitis-free gnotobiotic IL-2-deficient mice. J Immunol 1 998; 1 60: 3 85 -3 94). Certain special lactic acid bacteria (such as Bifidobacterium lactis and Lactobacillus brevis subsp.) Have been found to stimulate IFN-γ secretion from lymphocytes in blood from mice and humans ( U.S. Patent Publication No .: 200532018 US 2002/003 1 503A1; U.S. Patent No. 5,556,785). It has also been reported that lactic acid bacteria can stimulate the secretion of interleukin-12 (IL-12) from lymphocytes from humans or mice, which is a T-cell stimulating cytokine (Hessle) that can activate T cells and NK cells to secrete IFN-γ. Et al., Lactobacilli from human gastrointestinal mucosa are strong stimulators of IL-12 production, Clin Exp Immunol 1 999; 1 16: 276-282). Lactobacillus paracasei has been used for a long time in the manufacture of Cheddar and Italian mother cheese. Feta cheese. It was found that it can grow and maintain high vigor in cheese during maturity (Gardiner, G., Ross, R.P., Collins, JK, Fitzgerald, G., Stanton, C. Development of a probiotic cheddar cheese containing human-derived Lactobacillus paracasei strains.Appl Environ Microbiol. 1998; 64: 2192-2199; Angelis, M, Corsetti, A., Tosti, N., Rossi, J., Corbo, MR, Gobbetti, M. Characterization of non-starter lactic acid bacteria

from Italian ewe cheeses based on phenotypic, genotypic, and cell wall protein analyses. Appl Environ Microbiol. 2001; 67: 201 1-2020). Lactobacillus paracasei has been noted to produce antibacterial and anti-yeast compounds, such as H202 and protein actives in the human vagina and oral cavity (Atanassova, M ·, Choiset, Y ·, Dalgalarrondo, M., Chobert, J_-M. , Dousset, X ·, Ivanova, I., HaertkS, T · Isolation and partial biochemical characterization of a proteinaceous anti-bacteria and anti-yeast compond produced by Lactobacillus paracasei subsp. Paracasei strain M3. Int. J. Food Microibiol. 2003 87: 63-73; Ocafia, VS,

O: \ 90 \ 908ll.DOC 200532018

Holgado, AA P. de R, Nader-Macias, M E. Growth inhibition of Staphylococcus aureus by H2〇2_producing Lactobacillus paracasei subsp.paracasei isolated from the human vagina. FEMS Immunol. Med. Microbol. 1999; 23: 87-92 Sookkhee S., Chulasiri, M, Prachyabrued. W., Lactic acid bacterial form healthy oral cavity of Thai volunteers: inhibition of oral pathogens. Journal of Applied Microbiology 2001; 90: 1.72-179). [Summary of the Invention] The present invention provides a novel microorganism strain Lactobacillus paracasei GMNL-32. In another aspect, the present invention provides a composition comprising the microorganism strain Lactobacillus paracasei GMNL-32. In another aspect, the present invention provides a method for treating an allergy-related disease in a patient, comprising administering to the patient a composition comprising a microbial strain of Lactobacillus paracasei GMNL-32; wherein the rickets are preferably selected from Groups: overtracheal reaction and inflammation, atopic dermatitis, allergic conjunctivitis, rhinitis, sinusitis, hypersensitive pneumonia, external allergic alveolitis ), Ramie therapy, hygrotherapy, anaphylaxis, angioedema, allergic migraine, specific gastrointestinal disorders and asthma. In yet another aspect, the present invention provides a method for stimulating IFN-γ secretion in a patient, comprising administering to the patient a combination comprising a microorganism strain Lactobacillus paracasei O: \ 90 \ 90811.DOC -9- 200532018 GMNL-32 Thing. [Embodiment] The present invention provides a novel microorganism strain Lactobacillus paracasei GMNL-32, which can be used to treat allergies. This strain of GMNL-32 was deposited with the Food Industry Research and Development Institute (FIRDI) of Hsinchu, Taiwan on March 19, 2003, and the accession number was BCRC 9 10220. Lactobacillus paracasei GMNL-32 is isolated from the healthy human gastrointestinal tract (GI tract). Tissue samples taken from the stomach, intestine or duodenum were suspended in MRS broth medium containing 100 gg / mL ampicillin cultured at _ 37 ° C for 2 days, and then drawn on an agar plate Streak plating. The lactic acid bacteria colonies grown on the plate can be pre-screened under a microscope. The candidate strain is then cultured with spleen cells. The amount of IFN-? Thus produced by splenocytes in the broth was measured. Next, GMNL-32 was selected for its high IFN-γ productivity. The mycological characteristics of GMNL-32 are as follows: _ (a) Morphological characteristics: (1) Cell shape and size: After the cells were cultured overnight in MRS broth at 37 ° C, the bacilli were observed through a microscope. It has a rod-like shape with rounded edges. (2) Motility: Active (3) Flagella: None (4) Spore formation: No spore formation (5) Gram stain: positive O: \ 90 \ 90811.DOC -10- 200532018 (b) Culture characteristics: ( 1) Medium: MRS broth (DIFCO®0881), final ρΗ6 · 5 soil 0 · 2 (2) Culture conditions: 37 ° C anaerobic or aerobic culture

(3) Antibiotic resistance · Ampicillin 100 / ig / mL (c) Physiological characteristics: (1) Catalase · Positive (2) Oxidase: negative (3) API 50 CHL test · API The 50 CHL system is used for identification of lactic acid bacteria. By analyzing the reaction of a series of enzymes, lactic acid properties are established. GMNL-32's API 50 CHL test results are listed in Table 1: Table 1: Reference: GMNL-32 excellent identification of this genus_

Strip · API 50CHL * ^ 7 ^ Profile, ----- + +++++ — ++ — +-++++++-++ ------ + +- + + — 0-GLY-ERY-DARA-LARA-RIB + DXYL-LXYL-ADO-MDX-GAL + GLU + FRU + MNE + SBE + RHA-DUL-INO -MAN + SOR + MDM-MDG- NAG + AMY -ARB + ESC + SAL + CEL + MAL + LAC + MEL-SAC + TRE + INU-MLZ-RAF-AMD-GLYG-XLT-GEN + TUR + LYX-TAG + DFUC- LFUC-DARL-LARL-GNT + 2KG -5KG---- Major categories-% Id ·-__τ ___-Comparative test-Lacto.para.paracasei 1 94.9 0.74 2 Lacto.para.paracasei 3 5.0 0.59 5 Next select O: \ 90 \ 90811.DOC -11 -200532018 Lactobacillus rhamnosus_0J_0.39 4

Lac to .para.paracasei 1: 2 Contrast J try Apricotin (AMY) 98% melezitose (MLZ) 93%

Lac to. Para.paracasei 3: 5 pairs of daggers try L-sorbose (SBE) 20% D-sorbitol (S0R) 20% almond (AMYGDALINE) (AMY) 99% D-melosyl (TUR) 20% Gluconate (GNT) 20% Secondly choose Lactobacillus rhamnosus: 4 Comparative test L-rhamnose (RHA) 100% Amino-D-CLUCOSIDE (MDG) 85% Almond (AMY) 99% pine Trisaccharide (MLZ) 99% (d) Genetic characteristics: Determine the 16S rDNA sequence analysis of GMNL-32. The results showed that the GMNL-32 line was highly homologous to other Lactobacillus paracasei strains (as shown in Figure 2). In addition, the germline spacing tree is shown in FIG. 3. In addition, randomly amplified polymorphic DNA was shown (RAPD analysis). It indicates that GMNL_32 belongs to Lactobacillus paracasei, but has a specific 16S rDNA sequence. In summary, GMNL-32 is a novel Lactobacillus paracasei strain. (e) Cell wall protein of GMNL-32: When compared with other known strains of Lactobacillus paracasei, cell wall protein of GMNL-32 showed a similar pattern. The SDS-PAGE pattern of the cell wall protein of GMNL-32 is shown in FIG. 4. O: \ 90 \ 90811.DOC -12- 200532018 (f) Standardized detection system for identifying GMNL-32: The standardized detection system for identifying microorganisms was filed in US Patent No. 10 / 446,781 on May 29, 2003 It is disclosed in the number that it utilizes the difference in gene expression between a specific microorganism culture and a test cell line not cultured with a specific microorganism as a marker for identification. The genes tested are listed in Table 2. Table 2

Gene Gene Gene Gene FHR-4 FGF19 FKBPIB-a FGF20 FGF13-C FGF10 FGF14 FGF11 FGF5-b FGF1--FGF6 FGFl-b FCGBP FCAR-f FCGR1A FCAR-g FADD ELK3 FCAR-a ENG ELA2 CXCR4 EGR1 CXCL16 CX3R1SF1SF -a CRL3 COL3A1 CRTAM CR1 CMRF-35H CHUK CNRl-a CKTSF1B1 CDC25A CD 163 CDH3 CD 164 CD97-b CD81 CD109 CD83 CD79A-a CD58 CD79A-b CD59 CD37 CD22 CD38 CD24 CD7 CD3G CD8A CD3Z CD2-a CC1 CCR4 CCL25 CCR5 CCL26 CCL19 CCL8 CCL20 CCL11 CAMK4 C9 CCBP2 CABIN1 C5 CIS C6 C2 C1 QTNF2 BTNL2 C1QTNF3 BY55 BLRl-b BCL2-a BLRl-c BCL2-b APISl-b ALDH1A1 APlSl-cd-cd-AOA FKBPIB-b FGF21 FLJ14639 FGF22 FGF16 FGF12-a FGF17 FGF12-b FGF7 FGF2 FGF8-a FGF3 FCGR2A FCAR-h FCGR2B FCER1A FCAR-b EP300 FCAR-c EPO O: \ 90 \ 90811.DOC -13-200532018 EGR2 CCYCR3 CXCL10 CSF3R-b CXCL13 CTLA1 CSNK2A1 CR2 CSNK2B CREBl-a CNRl-b CIAS1 CPA3 CIS4 CDKN1A CD200R CDKN2B-a CD209 CD151-a CD84 CD151-b CD84-H1 CD79B-a CD63 CD79B-b CD68 CD44 CD33 CD44 CD33 CD44 CD33 a CD8B1 CD4 CD9 CD5 CD2AP CD1B CD2BP2 CD1C CCR6 CCL27 CCR8 CCL28 CCL21 CCL13 CCL23-a CCL16 CCL1 CALM1 CCL2 CALM2 C7 C3 C8A C3AR1 C1QTNF2 AC1 ACRB1 ACRB1 ACRB1 ACRB1 -b ACE-a ACVR1 ACE-b F0G2 FGF23 FOS FHOD2 FGF18-a FGF13-a FGF18-b FGF13-b FGF8-b FGF4 FGF9 FGF5-a FCGR3A FCER1G FCGRT FCER2 FCAR-d ETEA FCAR-e EPX EGR4 DAF ELK1 EK1 CTLA4 CXCL6 CTRP5 CSF1R CREBl-b CSF2RA CREBBP C0L1A1 CMA1 COL1A2 CMRF35 CDKN2B-b CD209L CER1 CD244 CD151-C CD86-a CD151-d CD97-a CD79B-C CD72 CD80-a CD74 CD48 CD19 CD36 CD3 CD5 CD3 CD3 CD5 CD3 CD1D CD3E CD1E CCR9-a CCR1 CCR9_b CCR3 CCL23-b CCL17 CCL24 CCL18 CCL5 CALM3 CCL7 CAMK2B C8B C4BPA C8G C4BPB C1QTNF7 C1QBP C1R C1QR1 BMPR2-a BF BMPRA-BAD-BLR-BLR ACVRL1 ALDH1A2 ADRB1 O: \ 90 \ 90811.DOC -14- 200532018

ACVRIB-a ACE2 ACVRIB-b ACHE-a NCAM2 MUC4-c NCF2 MYC MORF MIF MUC1 MMD MEF2B MAPK14-a MEF2D MAPK14-b MAPK8 MAP3K14 MAPK9 MAP3K7-a MAF MADH3 MAP2K7-a MADH4 LY L6B1 LT6 LBR 6B -a LOCI 63702 LOCI 39429 LOC201595 LOC145314 LILRB5 LILRA2 LOCI 22687 LILRA3 KPNA5 JAK3 KPNB3 JUN ITGBl-a ITGA10 ITGBl-b ITGA11 ITGA3-b IRF6 ITGA4 IRF7 IRAK3 ILF2 IRAK4 ILF3-a IL19 IL-20 IL-17RE-b -17RC-b IL16 IL-17RC-C IL17 IL11 IL3RA ILllRA-a IL4I1 IRAK2-a IGSF6 IL1F8 IGSF8 IGFBP3 IFNW1 IGLL1 IFRD1 IFNA4 IFIT2 IFNA8 IFIT4 IFI16 ICOS IFI27 ICAM3 HCGIX GPR84 NFAT1 FO2 GF1 5-GB1 ITGB3BP NCF4-a MYD88 NCF4-b MYF5 MUC2 MME-a MUC3B MME-b MHCBFB MCP-a MHC2TA MCP-b MAPKIO-a MAP3K7-b MAPKIO-b MAP3K7-C MAP2K7-b MADH5 MAP3K1 MADH6 LY6 LY9 LYB 6C LTB4R2-a LTB4R2-b LAG3-b LOC205360 LOC145355 LOC221937 LOC145497 LOCI 28342 LILRB1 LOCI 36520 LILRB2 LAG3-a JUNB LAT JUND ITGBL1 ITGB4 ITK ITGB4BP ITGBl-c ITGAE ITG Bl-d ITGAL ITGA5 IRTA1 ITGA6 IRTA2 IRF2 ILF3-b IRF3 ILF3-C IL21 IL-17RE-d IL21R IL-17RE-e IL-17RC-d IL17C IL-17RC-e IL17F ILllRA-b IL7 ILllRA-c IL8 IL1F7 IGSF9 IL2RA IKBKB O: \ 90 \ 90811.DOC -15-200532018 IGSF1 IFRD2 IGSF2 IGBP1 IFNAR1 IFITM1 IFNAR2 IFNA14 IFI30 ICAM4-a IFI35 ICAM4-b HM74 GSCL HOXAl-a GSK3A GFI1 FST GPR2 FY NGB5-FAT NGB5-FAT c NBL1 NFAT5-a NCAM1 MUC4-a MMEL2 MUC4-b MMP9 MICA MCP-c MICB MEF2A MAPKIO-c MAP3K7-d MAPKIO-d MAPK3 MAP3K2 MADH7 MAP3K7IP1 MADH9 MADH1 LY6G6D MADH2 LY6 LOB-C6C1137C LOC-G6E6C LOC136531 LILRB3 LOCI 36535 LILRB4 LEP-a KITLG-a LILRA1 KITLG-b IVL ITGB5 JAK2 ITGB6 ITGB2 ITGAM ITGB1BP2 ITGAV ITGA7 ITGA2 ITGA8 ITGA3-a IRF5-a IRAKI IRF5-b IRAK2-b IL22R IL18BP IL-23R IL-23R IL17R IL-17RE-a IL-17RC-a IL14 IL8RA IL15RA IL8RB IL2RB IKBKG IL2RG IKKE IGSF3 IGHMBP2 IGSF4 IGF1 IFNGR1 IFNA2 IFNGR2 IFNA21 IFI44 ICAM5 IFIT1 IF HOXAl-b GSK3B HRAS HCC-1 GPR31 GPR31 GATA6 IL5 IL1R1 IFNA1 IL6ST IL10RB IL10RA ICAM1 IL13RA2 GATA3 IL1B IL10 IL2 MIP-A LOC126133 Unease HNF4A LOCI 61823 PGK1 G6PT1 NT5C1A DHFR PPARG-b PGK2 LOC200895 LOC132RP2 SCF PDH-PA P2-GPA2- MTHFR VLDLR LOC205855 YY1 NP PPAT VAV3 TRPV6-C VEGF TSA1902 TRAF4-a TRAF1 TRAF4-b TRAF2-a TNFRSF7 TNFSF5 TNFRSF8-a TNFSF6 O: \ 90 \ 9081l.DOC -16- 200532018

TLR10 TLR6 TNFAIP3 TLR7 TLR3 TGIF-b TLR4-a TGIF-c TGFB2 TBX21 TGFB3 TCF8 STAT2 SOCS5-a STAT3 SOCS5-b SERPING1 SEMA4B SFN SEMA4C SE20-4 RPL13A SEMA3A RUNXl REL PRA PLA2 PRLC2 POLA REUPL2 PLA NFKBIB NFATC2 NFKBIE NFATC3 IL5RA IL1R2 None IL9-a STATl-c STATl-b ITGB7-b CCR2-c IL13RA1 CCR2-a IL18 CD69 TGFB1 IL27 CD28 ILIA VCAMl-b JAK1 TNF-b CSF3 IL6R STATl-a IL12RB2 IL12RB2 GBP1 15MD-1 LOCI 69330 S100A8 IMPDH1 S100A9 MTHFD2 HDLBP G6PC LRP8 PRPS2 HPRT1 PRPSAP1 APRT XCL1 TSC22 XCR1 TYK2 TRAF5 TRAF2-b TRAF6 TRAF2-C TNFRSF8-b TNFRSF11A TNFRSF9 TNFRSb4 TLR1 TRF1 TR1 -C TIMP1 TGFBR1 TCP10 TGFBR2 TDGF1 STAT4 SOCS4 STATI2 SSI-1 SIVA-a SEMA4D SIVA-b SEMA4F SEMA3B RUNX2 SEMA3C SCYA3 RELB PTPRC-a RIPK1 PTPRC-b PPP3CC PIGR PPP3R1214 PILR-ALPHBUP PALDE-PDB NFKBIL1 NFATC4 NFKBIL2 NFKB1 CSF1 IL9-b CD80-b IL13 CCR2-b CD86 IL4 IFNB1 CEBPB TIM3 IRF1 IL4R TP53 IL12B TNF-a S ERPINA3 VCAMl-a SCYA4 CCR7 IL12A IL12RB1 CSF2 ADSS STAT6 IMPDH2 IL6 LGALS9 IFNG UMOD PTGS2 LOC223071 TCF2-c PRPS1 APOE ZNF144 APOB O: \ 90 \ 9081I.DOC -17- 200532018

XP05 ADA TRPV6-b DPP4 TRPV6-a VAV1 TPSD1 VAV2 RSF21 TRAF3-a TNFSF4 TRAF3-b TNFSFll-c .TNFRSF1B TLR5 TNFRSF21 TLR4-d TLR9-a TGIF-a TLR9-b TGFBR3 TLR1 TBXA2 RTFACTA TLR2 TLR2 3 SEMA3F SUDD SEMA3E SEMA4G RNASE3 SEMA7A RNASE2 SCYE1 PRG2 SDF2 PRKG1 PTPRC-c PDPK1 RDC1 PDGFB-b PILR (BETA) NOS2A-a PINNI NMA OPRD1 negative ORM1 ACTB NFKB2 G6PD NFKBIA standard cell detection GMNL Line as test cell line. When comparing the expression patterns of Jurkat cell lines cultured with GMNL-32 and without GMNL-32, the genes listed in Table 3 were significantly different. In addition, the detection results of other strains of Lactobacillus paracasei, CCRC 12193 & CCRC 12 188 are also shown in Table 3. It states that these strains are all L. paracasei, but belong to different strains. Table 3 Gene name Para cheese CCRC12193 GMNL-32 Para cheese CCRC12188 ADA ++++++ ++++ ++ BAD-3. ++ +++ + BCL3 + +-BLRl-c — + — BMPR2-a + + ++ + CCL2-+-CD2AP ++ ++ + CD2-b ++ ++ + CD38 ++ ++-CD3G ++++ ++++++ ++++++ CD48 ++ ++ + COL1A2---O: \ 90 \ 90811.DOC -18- 200532018 CR2 CREBl-a CREBl-b CX3CR1 ~~ DAF ETEA FCAR-h ~~ FGF23 FHOD2 HOXAl-a IFNAR1 IFNGR1 ~~ IKKE IL14 IL17R IL4R IL7 JAK1 LEP-a LOC200895 LY117 MADH4 ~~ MADH5 " " " MAP3K14 ++ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ++ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + MAPK14-a MAPK3 ~~ MCP-a MCP-c PDPK1 REL RIPK1 SEMA3C TGFBR2 TLR3 TNFSF4 ~ TRAF3_a TRAF6 TSC22 ++ ++ ++ ++ ++ ++ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

O: \ 90 \ 90811.DOC -19- 200532018 +: 2-fold increase in gene expression 1: 2-fold decrease in gene expression GMNL-32 was active after treatment with HC1 solution_2.0) for 3 hours and then with bile treatment for 4 hours. Therefore GMNL-32 is considered to remain active during digestion. GMNL-32 is isolated from a healthy subject, and is safe, natural, non-toxic, and meets aR.A.s (GeneraUyμ Safe, recognized safety level) standards. In addition, GMNL-32 strongly adheres to intestinal epithelial cells. In summary, GMNL-32 can stay in the intestine for a longer period of time to adjust physiological functions. By occupying the adhesion site of intestinal epithelial cells, GMNL_32 will also prevent other pathogenic bacteria from adhering to the intestine. GMNL-32 is considered a good probiotic. According to the present invention, GMNL-32 was found to stimulate IFN-γ secretion and can be used to treat allergy-related diseases. One aspect of the present invention provides a composition comprising GMNL-32. Preferably, the composition containing GMNL_32 is used to stimulate IFN 彳 secretion, which is helpful for the treatment of allergy-related diseases. As used herein, the term π allergy-related disease "refers to a disease in which a systemic response to a generally environmentally friendly antigen is caused by the interaction of the antigen with antibodies or T cells produced by earlier exposure to the same antigen The term "allergic reaction" as used herein refers to a response to environmentally friendly antigens or allergens due to pre-existing antibodies or T cells. There are various immune mechanisms for allergic reactions, but the most common type is allergens to hypertrophy The binding of IgE antibodies on cells' causes asthma, hay fever, and other common allergic reactions. Allergy-related diseases include excessive airway reactions and O: \ 90 \ 9081I.DOC -20- 200532018 inflammation, atopic Dermatitis, allergic conjunctivitis, rhinitis, treasure inflammation, allergic pneumonia, exogenous allergic alveolitis, ramie, _, allergic reactions, hemorrhagic edema, allergic migraine, specific gastrointestinal disorders and asthma. According to this In a preferred embodiment of the invention, the allergy-related diseases are overreaction and inflammation of the trachea. In another aspect, the allergy-related diseases are various Such as pollen, emblem bacteria, animal scales and insects are related to airborne allergens (airborne allergens). The term "airborne allergens" as used herein is defined as having at least the following characteristics: can trigger an active reactin response Specific antigen grouping, and environmental exposure levels that can induce significant tissue changes in sensitive bodies. Airborne allergens are airborne particles that can cause respiratory, skin or conjunctival allergies. Water solubility of ragweed pQllen Some (for example) films absorb the conjunctival mucosa, and the fat-soluble allergens of ragweed pollen can cause typical contact dermatitis on exposed skin. GMNL_32 was selected when it interacted with spleen cells and peripheral blood mononuclear cells (PBMC). The ability to stimulate IFN ^ secretion when co-cultivated in vitro. In addition, in the model according to the present invention, it was observed that animals activated by airborne allergens and subsequently treated with GMNL-32 increased IFN々 secretion. In addition, treatment The amount of airborne allergen-specific IgE was significantly reduced after treatment. On the other hand, the amount of allergen-specific IgG did not show significant before and after treatment. In addition, the number of eosinophils in bronchoalveolar lavage fluid (BALF) has decreased sharply; however, the number of macrophages and lymphocytes in BALF has increased sharply. It has been proven to reduce inflammation. According to the present invention, it is used for allergy treatment The GMNL-32 can be live or energy-reduced. The energy-reduced GMNL-32 is preferred. For example, live bacterial strains can be heated O: \ 90 \ 90811.DOC -21-200532018 steps or in this technology It is usually treated by other treatment methods used to kill lactic acid bacteria into energy-reducing strains. According to the present invention, the lactic acid strains can be included in medical compositions, dietary supplements, foods, health foods, medical foods or components thereof, They are generally taken by humans. In a preferred embodiment of the present invention, the lactic acid strain can be delivered in the form of food, for example, in the form of a coagulated milk product made by fermentation of lactic acid in milk. The food products prepared according to the present invention can be conveniently taken by infants or children. Yet another aspect of the present invention provides a method for stimulating IFN-secretion in a patient, the method comprising administering to the patient a composition comprising an isolated microorganism, GMNL-32. The scope of the present invention is not intended to be limited for the purpose of illustration only, and the following examples are given. Example 1: Isolated sample of Lactobacillus paracoccus GMNL-32 ·· A piece of human stomach, intestine or duodenum tissue obtained by endoscope at 37 ° C, 2mL of Lactobacillus containing 100Mg / mL ampicillin MRS broth (DIFCO⑧0881) was cultured for about 2 days. The broth was placed on agar containing CaC ... on agar and incubated for two days at 37 ° C. Individual colonies growing on the plate were selected and subjected to Gram staining. Then select Gram-positive fine g. All strains were cultured to a stationary phase in Lactobacillus MRS broth at 37 t, and collected by centrifugation at 3000 g for 15 minutes, and 2 mL and 1 mL of PBS (phosphate buffered saline, ρΗ7.2) Come and rinse. A culture of these strains was resuspended in 1 mL of pbs, and then at 95 ° C. Heating for 30 minutes, then receiving autoclave effect and storing in _2 (rcpBs. O: \ 90 \ 90811.DOC -22- 200532018 splenocyte isolation: adding 5 mL Ficoll to 5 mL blood samples from healthy volunteers -Hypaque (17-1400-02, Pharmacia) and then centrifuged at 500 g for 30 minutes. Spleen cells were obtained. In each spleen cell sample, the cell density was adjusted to 5x10 cells per sample. These spleen cell samples Incubate in 2 mL of RPMI1640 (pH 7.7) for 6 hours. Isolation of peripheral blood mononuclear cells: 5 mL of Ficoll-Hypaque (17-1400-02, Pharmacia) was added to 5 mL of blood samples from healthy volunteers and then at 500 Centrifuge for 30 minutes at g. The peripheral blood mononuclear cells (PBMC) were taken from the interface of the sample and washed twice with PBS. The PBMCs (105 cells / mL) were transferred to the wells of a six-well plate, where each well Contains 2 mL of RPMI 1640 medium containing ρΗ7 · 7. Stimulates IFN-γ secretion: Splenocytes or PBMC samples are co-cultured with a specific amount of Gram-positive bacteria. After 36 hours of co-cultivation, cells in each sample are collected separately Collected cells were resuspended and incubated at 2000 r Centrifuge at pm for 5 minutes. Take the supernatant to determine the IFN-γ content in each sample. Determination of IFN-γ content: Determine the IFN-γ content by ELISA, which includes the following steps:-Add 30pL 2.5gg / mL Purified mouse anti-human IFN-γ antibody (Cat. Nol8181D, PharMingen®, USA) into 10 mL of coating buffer (0.1MNa2HP04, pH9 · 0), and 100 μL of antibody solution was added to each well of the ELISA plate -Shake the plate at 4 ° C-Wash each well of the plate with washing buffer (0.05% Tween20 in PBS); O: \ 90 \ 90811.DOC -23-200532018-Add 300 / xL blocking buffer Agent (1% BSA in PBS) into each well of the plate;-Shake the plate for at least 2 hours at room temperature;-Add 100 gL of the supernatant of the spleen cell sample to each well of the plate -Shake the plate overnight at 4 ° C;-Rinse each well of the plate with washing buffer;-Add 150 ^ L biotin mouse anti-human IFN-γ antibody (Cat. Nol8112D, PharMingen®, USA ) Into each well of the plate;-incubate the plate for 1 hour at room temperature;-rinse each well of the plate with a washing buffer;

-Add 150 / xL diluted with dilution buffer (1: 1000)

Streptavidin-AKP into each well of the plate;-Shake the plate for 1 hour at room temperature;-Rinse each well of the plate eight times with a washing buffer;-Add 200 gL of substrate pNpp to the plate In each well;-Incubate the plate at room temperature until completion of the mass reaction;-Measure the absorbance of each well of the plate at 405 nm (meaning OD4G5). Results: Among Gram-positive bacteria, GMNL-32 was selected to have the strongest ability to stimulate IFN-γ secretion in splenocytes and PBMCs. Example 2: 16s rDNA sequence determination DNA extraction: The genomic DNA of GMNL-32 and other bacteria CCRC12913, CCRC14001, and CCRC16100 were extracted by using QIAamp® DNA Stool Mini Kit (Qiagen®, cat No. 5 1504). Purification was performed according to the steps listed below: O: \ 90 \ 90811.DOC -24- 200532018-Add 1.4 mL of ABS buffer to the culture and vortex for 1 minute;-Heat at 70 ° C from the aforementioned The solution obtained in the step is 5 minutes;-the solution is vortexed for about 15 seconds and then centrifuged at about 13,000 rpm for 1 minute;-the supernatant is transferred to a new centrifuge tube;-the InhibitEx vortex is added to the supernatant And shake it to dissolve the lozenge, and then incubate at room temperature for 1 minute;-centrifuge the solution at about 13,000 rpm for 3 minutes to adhere the bacteria to InhibitEx;-transfer the supernatant to a new centrifuge tube and Then centrifuge at about 13,000 rpm for 3 minutes;-take 200 / xL supernatant into a new centrifuge tube and add proteinase K;-add 200 / xL buffer AL and vortex for 15 minutes to obtain a homogeneous solution;-add 15 mL of proteinase K into the homogeneous solution and vortex for 15 seconds;-incubate the solution at 70 ° C for 10 minutes;-add 200 # L96-100% ethanol and vortex;-move the solution to a QIAamp spin tube Spin column and centrifuge at about 13,000 rpm for 1 minute;-spin the QIAamp The column was moved to a new centrifuge tube and 500 / xL buffer AW1 was added, and then centrifuged at about 13,000 rpm for 1 minute;-the QIAamp spin column was moved to a new centrifuge tube and 500 gL buffer was added AW2 and then centrifuge it at about 13,000 rpm for 1 minute; O: \ 90 \ 90811.DOC -25- 200532018-Move the QIAamp spin column into a new centrifuge tube and add 200 / xL buffer AE, and then It was allowed to incubate at room temperature for 1 minute; and-it was centrifuged at about 13,000 rpm for 1 minute to dissociate the DNA. 16s rDNA fragment amplification: The primers used to amplify the L region are designed according to the following: Lactobacillus paracasei 16S rRNA VI region, 5. CAC CGA GAT TCA ACA TGG-3 '(SEQ ID NO. · 1) and Lactobacillus conserved ( conserved) 16S rRNA, 5'-CCC ACT GCT GCC TCC CGT AGG AGT-3, (SEQ ID NO 2) (Ward, LJH, and Timmins, MJ (1999) Differentiation of Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus by polymerase Chain reaction. Lett. Appl. Microbiol. 29: 90-92). The genomic DNA of GMNL-32, CCRC12913, CCRC14001, and CCRC16100 are used as templates for performing PCR reactions. The 16s rDNA PCR amplification program is as follows: (1) 10 minutes at 95 ° C; (2) 45 seconds at 95 ° C; (3) 45 seconds at 46 ° C; (4) 72 minutes at 1 minute; (5) 7 minutes at 72 ° C; steps 2 to 5 are repeated 30 cycles. 16s rDNA sequence determination: PCR products of GMNL-32, CCRC12913, CCRC14001 and CCRC16100 were subjected to agarose gel electrophoresis (Figure 2) and sorted. Sequence alignment by ARB sequence editor (release 8.1) against multiple sequence alignment dataset (NCBI blastn, http://www.ncbi.nlm.nih.gov/BLAST) . It also shows that the 16s rDNA sequence of Lactobacillus paracasei strains PB4, AY 186046; F31, AF243147; KLB5 8, AF243 168 is similar to the 16s rDNA sequence of GMNL-32 as shown in Figure 3 (produced with VectorNTITM, InforMax® Inc. ). In addition, as shown in Figure 4, the 16s rDNA germline occurred O: \ 90 \ 90811.DOC -26- 200532018 spacing tree was generated with EMBL-EBI ClustalW (http://www.ebi.ac.uk/clustalw) . According to the 16s rDNA analysis, GMNL-32 is highly related to KLB58, but it is completely different from KLB58. In summary, GMNL-32 belongs to Lactobacillus paracasei. Example 3: Randomly amplified polymorphic DNA (RAPD analysis) GMNL-3 2. 畐] Lactobacillus aeruginosa ATCC25 5 98, 25302, 335, 11582 and 27216 DNA extraction was performed as described in Example 2. Development of a probiotic cheddar cheese containing human-derived Lactobacillus paracasei strains for random amplification of y-ATGTAACGCC-3 乂 Gardiner, G., Ross, R_P., Collins, JK, Fitzgerald, G ·, Stanton, C. (Appl Environ Microbiol. 1998; 64: 2192-2199). RAPD results are shown in Figure 5. According to this RAPD analysis, GMNL-32 is very different from the conventional strain of Lactobacillus paracasei. In summary, GMNL-32 is a novel strain of Lactobacillus paracasei. Example 4: GMNL-32 cell wall protein extraction and analysis according to the method described by Angelis (Angelis, MD, Corsetti, A, Tosti, N., Rossi, J., Corbo, MR., And Gobbetti, M. (2001) Characterization of Non-Starter Lactic Acid Bacteria from Italian Ewe Cheeses Based on Phenotypic, Genotypic, and Cell Wall Protein Analyses. Appl. Environ. Microbiol. 67: 201 1-2020) to purify cell wall proteins. The cells were harvested overnight in MRS broth (Difco®), and then rinsed twice with 0.05M Tris-HCl (pH 7.5) containing 0.1M CaCl2 and resuspended in OD600 O at 10.0: 90 \ 90811.DOC -27- 200532018 1! ^ In the same buffer. After centrifugation at 8000 \ 8 for 5 minutes, the cell wall proteins were extracted from the centrifuge block with 1.0 ml extraction buffer (pH 8.0) containing 0.01 ^ 1 EDTA, 0.01M NaCn, and 2% (wt / vol) SDS. . The suspension was stored at room temperature for 60 minutes, heated at 100 ° C for 5 minutes', and centrifuged at 11600 xg for 10 minutes at 4C. The supernatant was analyzed by 12% SDS-PAGE and stained with Comassie Blue. The results are shown in Figure 6. The pattern of GMNL-32 has three specific bands, P1, P2, and P3, which are similar to the pattern of Lactobacillus paracasei reported in a previous study (Angelis et al., 2001). Therefore, GMNL-32 was confirmed to belong to L. paracasei. Example 5: Standard detection system to identify GMNL-32 Stimulation: Jurkat cells were renewed by adding fresh medium and incubating for 16 hours. Subsequently, the cells were divided into two groups, one for culture using lactic acid bacteria and the other for culture without lactic acid bacteria. When the cell concentration reached 1 × 107/10 mL, the cells were stimulated for 24 hours with or without lx10 7 different lactic acid bacteria (CCRC12193, GMNL-3 2 or CCRC12188). After stimulation, cells were collected and washed twice with PBS and used for RNA isolation. RNA isolation and labeling: RNA was extracted from cells using Trizol Reagent (Life Technologies®, Gaithersburg, Md.) According to the manufacturer's instructions. 8L of RNA (10 / xg) and 2L of agglomerated-dT (12-18mer, lg / L) were thoroughly mixed and kept at 70 ° C for 10 minutes and then cooled with ice for 2 minutes. In the dark, mix RNA with a reverse transcription labeling mixture and 3L Cy3-dUTP (lmM), 2L Superscript III (200U / L), and RNasin (lL) O: \ 90 \ 908ll.DOC -28- 200532018. The mixture was incubated at 50 ° C for 2 hours for reverse transcription, and the reaction was stopped by adding 1.5 L of 20 mM EDTA. After labeling, RNA was removed by NaOH treatment and neutralized with HC1. Immediately purify cDNA with the YM30 purification kit. Microarray manufacturing: Many selected genes are amplified by polymerase chain reaction and quantified by 260 nm spectrophotometry. All purified PCR products were adjusted to a concentration of 0.1 Mg / μΙ in 50% dimethylsulfoxide and spotted on UltraGAPSTM coated glass slides (Corning®, Inc., Corning) in duplicate. , NY ·). After printing, the microarrays were UV cross-linked at 700 mJoulesand and stored in glass slide containers in a desiccator at room temperature. The genes are listed in Table 2 as described above. Microarray hybridization: Denaturing the fluorescently labeled cDNA in a hybridization solution (5x SSC, 0.1% SDS, and 25% amidine) at 100 ° C for 5 minutes, cooling to ambient temperature, and depositing on a glass slide. Hybridization was performed at 55 ° C for 18 hours. After hybridization, successively rinse with low stringency (lx SSC and 0.1% SDS), medium stringency (0.1x SSC and 0.1% SDS), and high stringency (0.1x SSC) buffers, and finally by compression N2 was dried. Signal detection and data analysis · N2 dried slides were immediately scanned at the GenePix 4000B scanner (Axon Instruments®) with the same photomultiplier laser intensity and sensitivity level for each slide. , Inc.). Obtain raw fluorescence data (10nm resolution) and perform subsequent processing and data visualization in Microsoft Excel ™. To compare the results of independent hybridization experiments, the local background signal is subtracted from the hybridization signal at each independent point, and then divided by the custody gene gene actin. Every O: \ 90 \ 90811.DOC -29- 200532018 the final expression of a gene is expressed as the average of duplicates. Gene expression maps of Jurkat cells cultured with lactic acid bacteria and without lactic acid bacteria were obtained. Jurkat cells cultured with lactic acid bacteria (CCRC12193, GMNL-32, or CCRC 12188) were selected to regulate up to one-fold more genes than Jurkat cells cultured without such bacteria. The results are shown in Table 3. This difference means that different species or strains can turn on or turn off different genes in the cell. Therefore, from the gene expression profile, the CCRC12193, GMNL-32 and CCRC 12 188 lines are Lactobacillus paracasei, but belong to different strains. Example 6: Adhesion of GMNL-32 to intestinal epithelial cells In this example, Caco-2 cells were treated as epithelial cells. Caco-2 cells have functional microvilli and hydrolases attached to them, which demonstrate the distinct morphology and function of mature epithelial cells in the living intestine. Cells · At 37 ° C, Caco-2 was cultured in a Minimum Essential Medium (MEM, GIBCO®) supplemented with 5% FBS with 5% CO 2/95% air. For adhesion analysis, 2 ml monolayer Caco-2 cells (3 x 105 cells / ml) were prepared on glass coverslips placed in 6 孑 L plates. The culture medium was replaced every other day, and the monolayers were used for adhesion analysis after 2 weeks of incubation. Immediately before use, these monolayers were washed twice with PBS and 1.5 ml of MEM was added to each well and incubated at 37 ° C for 1 hour before bacterial inoculation. Adhesion: 1.5 ml (4 x 108 CFU / ml) GMNL-32, which was rinsed once with PBS and resuspended in 1.5 ml MEM medium, was added to Caco-2 cells. After 1 hour incubation at 37 ° C, the monolayer cells were washed with PBS buffer 4 O: \ 90 \ 90811.DOC -30- 200532018 times, mixed with 3 ml of methanol and incubated at room temperature for 5 to 10 minutes. Rinse 3 times with PBS, dry in air and Gram stain. Detect (X100) adherent bacteria under oil immersion with a microscope, count 15 random areas on each cover glass, and determine the mean ± SD of adherent bacteria in each area. Results: After counting, 102 ± 23 · 6 GMNL-32 bacteria adhered to Caco-2 cells. Therefore, according to the standards established by Jacobsen et al. (Jacobsen, CN, Nielsen, RV, Hayford, AE, Moller, PL, Michaelsen, K, F., Paerregarrd, A., Sandstrom, B., Tvede, M. A Jakobsen , M.Screeing of probiotic activities of forty-seven strains of Lactobacillus spp.by in vitro techniques an evaluation of the colonization ability of five selected strains in human. Appl.Environ. Microbiol. 1 999; 65: 4949-4956) J GMNL -32 is thought to have strong adhesion to Caco-2 cells. Example 7: Live acid of GMNL_32 and other lactic acid bacteria in a simulated gastrointestinal tract in an environment: 9 ml of PBS with different pH values (2.0, 2.5 and 3.2) were added to GMNL-32, L. plantarum cultured overnight ), L. acidophilus, L. casei, and L. bulgaricus, and then further cultured at 37 ° C. for 3 hours. After incubation, 1 mL of cells was serially diluted with 9 mL of PBS, pH 7.4. Cell counts were estimated before and after acid treatment, and are shown in Table 4 below. Bile: 9 ml PBS with a pH value (2.0) was added to GMNL-32, Lactobacillus embryonicus, Lactobacillus acidophilus, Lactobacillus flavus, and Perga O: \ 90 \ 9081l.DOC -31- 200532018 Lactobacillus liabilis, and then further cultured at 37 ° C for 3 hours. After the culture, 1 mL of cells was centrifuged at 6,000 rpm for 10 minutes. The pellet was resuspended in 100 ML PBS (pH 7.2). To this solution was further added 10 mL of MRS broth containing 0.3% (w / v) beef bile. Culture the cells and take 1 mL samples at 3, 12 and 24 hours. The samples were serially diluted with 9 mL of PBS, pH 7.4. Cell counts were estimated before and after bile treatment, and are also shown in Table 4. It shows that these lactic acid bacteria maintain their activity in an environment that mimics the gastrointestinal tract. Table 4 Cell count (Log CFU / mL) Strain treated with HC1 for 3 hours before treatment with bile and 4 hours after treatment with bile for Lactobacillus 9.003 8.114 7.097 Lactobacillus acidophilus 9.114 8.097 8.176 Lactobacillus casei 8.889 8.653 5.658 GMNL-32 9.029 7.699 6.602 Bacillus 9.230 9.076 7.447 Example 8: Animal Model Animals · Female BALB / c mice were obtained from National Laboratory Animal Center of Taiwan ff (National Laboratory Animal Center) and kept for 2 weeks in a room with controlled light and temperature . Allergen purification: Dust mite allergen Der p 5 is expressed in E. coli containing a PGEX-2T expression vector as a recombinant Der p 5-glutathione S-transferase fusion protein, which can be obtained via glutathione agar Pure O: \ 90 \ 9O811.DOC -32- 200532018 by sugar binding chromatography. Culture and introduction of specific E. coli strains that express the desired allergen Bacteria were collected and rinsed with TBS (pH 7.5), and 0.1 μm of methyl methylfluoride was added. The cells were disrupted by adding Dnase I, Tween 20 and lysing enzymes, and by the freeze-thaw method. EDTA was added to the mixed solution, and the residue was removed by centrifugation to obtain a supernatant containing recombinant Der p% glutathione S-transferase fusion protein. The supernatant was subjected to a glutathione lipoprotein affinity column to absorb the fusion protein. The column was then rinsed with TBS buffer at 4 ° C and then with reduced glutathione in Tris base (pH 8.0) to separate the protein from the column. The molecular weight of the protein was estimated by SDS-PAGE, and the concentration was analyzed. Sensitization: Mice are automatically sensitized by intraperitoneal injection of 10 VgiDa p 5 and 4111§ aluminum hydroxide. 14 and 21 days after initial sensitization, mice were exposed to 0.1% purified Der p 5 aerosol (aerosoli) for 30 minutes to perform the inhalation challenge. ⑺ Treatment · The sensitized mice were divided into three groups for experiments. MRS mice were fed with MRS broth 10 times during two weeks as a control group. Mice in Group B were given Lactobacillus casei for 10 times over a two-week period, with 10 CFU bacteria each time. Since Lactobacillus casei has been shown to be effective in suppressing IgE secretion, six groups were used as positive controls. The mice in group C were given GMNL-3210 times within two weeks, and each time they were given 109 CFU bacteria. Example 9: IgG and igE secretion

Der p5 specific igG and IgE measurements: 18 hours after the last inhalation challenge '500 pL blood samples were taken from the tail. Keep the blood sample in the chamber

O: \ 90 \ 90811.DOC • 33- 200532018 at 1 hour and then subjected to centrifugation. Store the serum at _80 ° C. The amounts of Derp5-specific IgG2a and IgE were determined by ELISA. A 96-well protein high binding plate (Protein high) was coated with 200 pL purified Der p5 diluted to a concentration of 10 pg / mL in a coating buffer (0.1 M NaHC〇3, pH 9.6). -binding plate). In; 4. (: After the next overnight incubation, rinse the plates with PBS-Tween 20 and then add 300 pL blocking buffer (3% BSA). After shaking at room temperature for 2 hours, rinse the plates again with PBS-Tween 20 The serum was diluted 1:10 for IgG measurement and 1: 4 for IgE measurement. The sample was shaken at room temperature for 2 hours. After overnight incubation at 4 ° C, PBS-Tween was used. Rinse the plates and add 200 pL of biotinylated rat anti-mouse IgE monoclonal antibody, or rat anti-mouse IgG mAb. The samples are shaken at room temperature for 2 hours, and then rinsed with PBS-Tween 20 Then 200 pL of streptavidin-alkaline phosphatase (1: 1000) was added and the sample was shaken at room temperature for 1 hour. After 6 washes, 200 pL was added Phosphatase began to react with p-nitrophenyl phosphate and disodium salt (pNPP) (Sigma® N-2770, USA). The plate was read using a microplate reader (Metertech®, Taiwan) at 405 nm. Take the absorbance value of the plate. Data analysis: In order to analyze the changes in IgE and IgG content, perform a one-way ANOVA analysis to repeat the measurement to Compare the differences between the groups. After the change analysis, Duncan's multi-range test was used to distinguish the difference between the experimental group and the control group. The value of ρ < 0.05 was used to indicate a statistically significant difference. Results: The results are as follows Figure 7 shows that IgE secretion in sera of animals treated with GMNL-32 is drastically reduced, and only O: \ 90 \ 9081l.DOC -34- 200532018 in sera of untreated animals

25% of IgE secretion. On the other hand, the IgG secretion in the serum of animals treated with GMNL-32 increased by a factor of two. Because IgG secretion represents a Thl T cell response, GMNL-32 is designed to eliminate allergies associated with allergic diseases! gE secretion. Example 10: Cell counting sample preparation of bronchoalveolar lavage fluid: After 18 hours of sensitization, through a polyethylene tube introduced by tracheostomy, 0.9% of a 5x0.5 ml aliquot Rinse mice with saline. Centrifuge (500 g, 4 minutes at 4 ° C) lavage solution, and resuspend the cell pellet in 1 ml PBS solution. Fractionated cell counts were prepared from cell centrifuge preparations stained with Leu's stain. Negative analysis • In order to analyze changes in cell counts, perform a one-way anova analysis repeat measurement to compare differences between groups. After the change analysis, Duncan's multi-range test was used to distinguish the difference between the experimental group and the control group. The value of ρ < 0.05 was used to represent a statistically significant difference. Results: The results are shown in Figure 8. The blood cell type contribution in BALF represents the degree of inflammation. In addition, the main symptoms of allergic asthma are chronic inflammation of the trachea and eosinophil infiltration. It proved that the eosinophils in BALF of animals treated with GMNL-32 drastically decreased from 5% to 1%. On the other hand, macrophages and lymphocytes in BALF of animals treated with GMNL-32 increased significantly. Example 11: Preparation of IFN-γ secretion sample in bronchoalveolar lavage fluid: After 24 hours of sensitization, a polyethylene tube introduced by tracheotomy was used, and 0.9% of a 5 x 0.5 ml aliquot was sterile. Mice were rinsed with saline. Centrifuge (500 g, 10 minutes at 4 ° C) lavage solution and let the upper layer

O: \ 90 \ 90811.DOC -35-200532018 The serum was subjected to quantitative analysis of IFN-γ, as described in Example 1. Results · The results are shown in Fig. 9. It was shown that animals fed GMNL-32 produced about 00 pg / mL of IFN-γ in BALF. On the other hand, the control group produced only 20 to 40 pg / mL of IFN-γ in BALF. GMNL-32 is effective in suppressing allergic inflammation. Example 12 · Preparation of energy-reducing GMNL-32 for the treatment of allergic energy-reduction GMNL-32 · Before feeding the mice, the GMNL-32 powder of Donggan was suspended in distilled water and subjected to the action of an autoclave: 2pc: ,15 minutes). Mice and sensitization · Female BALB / c mice (6-8 weeks old) were purchased from National Laboratory Animal Breeding and Research Center (Taipei, Taiwan). All animals were individually kept in cages with controlled temperature (24 ± 2 ° C) and humidity (60 ± 5%), and maintained for 12 hours day / night cycle under specific-pathogen-free conditions . BALB / c mice were injected intraperitoneally (i.p.) with 10 g of reconstituted Dermatophagoides pieronyssinus adsorbed to 4 mg of aluminum hydroxide. Oei: chlorophyllin. The mice were recruited at a daily rate of 107, 109, and 101 iFU GMNL-32 per mouse for three weeks. The mice were stimulated on the 14th day with the same dose of allergen as the sensitization effect, and after 21 days of sensitization, they were challenged with 0.1% Der p5-6xHis diluted in PBS. . The inhalation challenge was performed in an 11 ^ chamber connected to a DeVilbissTM pulmosonic nebulizer (Model 2512; 0. \ ^ 11 ^ 35 @ (1: 01 ^., 3〇11 ^ 1 ^ € 18). The nebulizer generates an aerosol mist. After 18 hours, O: \ 90 \ 90811.DOC -36- 200532018 serum was collected by tail vein bleeding, and IgE was measured by ELIS A, as described in Example 9. Results : The results are shown in Table 10. It shows that BALB / c mice challenged with dust allergy Der p-5 have significantly higher serum IgE content than the naive group (ρ < 0.05). This suggestion may be A mouse model of allergic sensitization was successfully established. After feeding GMNL-32 with different doses daily for 21 days, the serum IgE in the GMNL-32 group was significantly lower than that in the control group (ρ < 0.05). GMNL-32 can reduce allergic reactions by reducing allergen-specific IgE. Although embodiments of the present invention have been illustrated and described, those skilled in the art can make various modifications and improvements. The present invention is not intended to be limited to The specific form described, and all modifications that do not depart from the spirit and scope of the present invention are attached Please refer to the scope defined in the patent scope. [Schematic description] Figure 1 illustrates a 1000 X micrograph of GMNL-32 subjected to Gram staining. Figure 2 illustrates agarose gel of a 16s rDNA fragment ) As a result of analysis, the fragment was amplified by PCR of GMNL_32 and lactic acid strains CCRC12913, CCRC14001, and CCRC16100; M represents a molecular marker; 1 represents GMNL-32; 2 represents CCRC12913; 3 represents CCRC14001; and 4 represents CCRC16001.

Figure 3 illustrates GMNL-32 and lactic acid strains CCRC12913 and CCRC14001 'CCRC16100 λ KLB58' PB4> 5.F31 ^ 16s rDNA sequence alignment. Figure 4 illustrates a 16s rDNA phylogenetic distance tree comparing GMNL-32 of the present invention with related lactic acid bacteria. Figure 5 illustrates RAPD analysis of GMNL-32 and conventional lactic acid strains; -

O: \ 90 \ 90811.DOC 200532018 100-bp ladder, Lane 1: GMNL-32; Lane 2: Lactobacillus paracasei ATCC 25598; Lane 3: Lactobacillus paracasei ATCC 25302; Lane 4: Para cheese Lactobacillus ATCC 335; Lane 5: Lactobacillus paracasei ATCC 1 1 582; Lane 6: 畐 J Lactobacillus pluvialis ATCC 272 16. Figure 6 illustrates SDS-PAGE patterns of cell wall proteins of GMNL-32, conventional Lactobacillus paracasei, and Lactobacillus yeast strains; where M represents the molecular weight of the protein; Lane 1 represents Lactobacillus paracasei; Lane 2 represents Lactobacillus paracasei GMNL-32; Lane 3 represents Lactobacillus yeast / ermeniwm); F1 represents the specific band of Lactobacillus yeast; and PI, P2 and P3 represent the specific band of Lactobacillus paracasei. Figure 7 illustrates the content of Der p 5 specific IgG (white bars) and IgE (black bars) in the serum of Der p 5 sensitized BALB / c mice receiving the challenge of Der p 5 challenge; A represents the group treated with MRS broth; B represents the group treated with Z. ewe /; and C represents the group treated with GMNL-32. Figure 8 illustrates the cell counts of macrophages, lymphocytes, and erythrocytes in brochoalveolar lavage of Der p5 sensitized mice; A represents the group treated with MRS broth; B represents Lactobacillus paracasei Treatment group; and C represents the treatment group with GMNL-32. Figure 9 illustrates IFN-γ secretion in bronchoalveolar lavage of Der p5 sensitized mice; A represents the group treated with MRS broth; B represents the group treated with Lactobacillus paracasei; and C represents the treatment with GMNL-32 Group. Figure 10 illustrates the effect of inactive GMNL-3 2 on IgE production in Der p5 sensitized BALB / c mice. Der p5 sensitized mice were orally administered different doses of GMNL-32 or distilled water (control group) daily for three weeks. By O: \ 90 \ 90811.DOC -38- 200532018 ELISA was used to determine the specific content of IgE in serum Der p5. When compared with the control group, the Kruskal-WallisH test * (p < 0.1) and ** (ρ < 0.05) were significantly different, and the Dunnettt test used a posteri comparison. O: \ 90 \ 90811.DOC -39- 200532018 Sequence Listing < 110 > GenMont Biotech Inc. < 120 > Novel microorganism strain GMNL-32 Lactobacillus casei and its use for treating allergy-related diseases < 130> None <; 160 > 2 < 170 > Patentlnversion 3.2 < 210〉 1 < 211 > 18 < 212〉 DN A < 213 > Artificial < 220 > < 223 > Lactobacillus paracasei specific 16S rRNA < 400〉 1 caccgagatt caacatgg 18 < 210 > 2 < 211 > 24 < 212 > DNA < 213 > artificial < 220〉 < 223 > 16S rRNA stored < 400〉 2 cccactgctg cctcccgtag gagt 24 O : \ 90 \ 90811.DOC -40-

Claims (1)

200532018 The scope of patent application: 1. An isolated microbial strain Lactobacillus paracasei gmnl_32, which is deposited at the F_induStry Research and Development Institute (FIRm) in Hsinchu, Yiwan, and the deposit number is BCRC910020. 2. A composition comprising a microbial strain as described in item 1 of the patent application. 3. The composition of claim 2 as stated in the patent scope, which is used to stimulate ifn_y secretion. 4. The composition according to item 2 of the patent application, which is used for treating allergy-related diseases. 5. The composition according to item 4 of the scope of patent application, wherein the allergy-related disease is selected from the group consisting of: overtracheal reaction and inflammation, atopic dermatitis, allergic conjunctivitis, rhinitis, sinusitis, allergic pneumonia , Exogenous allergic alveolitis, measles, eczema, allergic reactions, angioedema, allergic migraine, specific gastrointestinal disorders and asthma. 6_ The composition according to item 5 of the scope of patent application, wherein the allergy-related disease is an overreaction of the trachea and inflammation. 7. The composition of claim 4 in which the allergy-related disease is related to exposure to an airborne allergen. 8. The composition according to item 2 of the patent application range, wherein the microorganism strain is alive or energy-reduced. 9. The composition of claim 8 in the scope of patent application, wherein the microorganism strain is reduced in energy. 10. The composition of item 2 in the scope of patent application is in the form of a pharmaceutical composition, a dietary supplement, a food, or a component thereof. OA90 \ 90811.DOC