TWI284149B - 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

1284149 发明Invention Description: TECHNICAL FIELD The present invention relates generally to a novel microorganism strain, Lactobacillus paracasei GMNL-32, and its use for stimulating ifn_t secretion and treating allergy-related diseases. [Prior Art] Allergies refer to the acquired/reactive effects of adverse reactions to immune regulation of normal and harmless substances. Allergic reactions cause symptoms such as itching, coughing, wheezing, sneezing, tearing, inflammation and fatigue. Allergic reactions are generally considered to include early specific immune responses and late inflammatory responses. As directed, allergens (such as pollen and dust mites) regulate early allergies by stimulating high-affinity immunoglobulin (IgE) receptors. For example, when mast cells and basophils are stimulated by allergens, histamine and cytokines (Cyt〇kine) are released. Next, the cytokines released by mast cells and basophils regulate late allergies by restoring inflammatory cells. It has also been reported that the influx of eosinophils, macrophages, lymphocytes, neutrophil A and platelets begins a cycle of malignant inflammation. This late allergy augments the initial immune response, which in turn triggers the release of more inflammatory cells (Blease et al, Chemokines and theirrole in airway hyper-reactivity. Respir Res 2000; 1 : 54-61). Seek a variety of treatments. Among them, anti-allergic agents and histamine quinone receptor antagonists (antihistamines) have been used. The amine antagonist is administered to counteract the action of histamine released by mast cells due to the presence of a reactive allergen. They reduce redness, itching and swelling caused by histamine on the target tissue and are used to prevent or alleviate O:\90\90811.DOC -6- 1284149 caused by degranulation of mast cells Symptoms. However, antihistamines are also associated with adverse reactions such as reduced agility, slowed response times, and narcolepsy (US Patent No. 6,225,332). There are also reports of treating allergies by adjusting cytokines (Cytokines). Among them, interferon-γ (IFN-γ) was found to inhibit Th2 lymphocytes

The over-expression of cytokines, especially the secretion of IL-4, reduces the proliferation of B cells. IFN-γ can also stimulate the immune response of Th 1 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, Lactobacillus casei inhibits antigen-induced IgE secretion through regulation of cytokine production in murine splenocyte cultures ? Int Arch Allergy Immunol 1998; 1 15:278-287). Since IFN-γ can inhibit B cell proliferation and IgE secretion, sensitization of IFN-γ is effective for treating allergies. Lactic acid bacteria (which are Gram-positive bacteria) are commonly used in industrial foods. Recent studies have shown that lactic acid bacteria can stimulate IFN-γ secretion in cells (Contractor NV et al, Lymphoid hyperplasia, autoimmunity and compromised intestinal intraepithelial lymphocyte development in colitis-free gnotobiotic IL-2-deficient mice. J Immunol 1998 ; 160 : 3 85-394 ° Some special lactic acid bacteria (such as Bifidobacterium lactis and Lactobacillus brevis subsp) have been found to stimulate IFN-γ from lymphocytes derived from blood of mice and humans. Secretion (US Patent Bulletin:

O:\90\90811.DOC 1284149 US 2002/0031503A1; US Patent No. 5,556,785). It has also been reported that lactic acid bacteria can stimulate the secretion of interleukin 12 (IL-12) from human or mouse lymphocytes, which is a T cell stimulating cytokine that activates T cells and NK cells to secrete IFN_7 (Hessle et al. Lactobacilli from human gastrointestinal mucosa are strong stimulators of IL-12 production, Clin Exp Immunol 1999; 116 · 276-282) Lactobacillus paracasei has long been used in the manufacture of Cheddar and Italian ewe cheese. It was found to grow and maintain high viability in cheese during ripening (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;67:2011-2020). Lactobacillus paracasei has been noted to produce antibacterial and anti-yeast compounds such as H202 and protein active substances in the human vagina and mouth (Atanassova, M_, Choiset, Y., Dalgalarrondo, Μ. ^ Chobert, J.-M. 'Dousset, X. ^ Ivanova, I. ^ Hasolke, 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\90811.DOC 1284149

Holgado, AA P.de R, Nader-Macias, M Ε·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: 172-179) 〇 [Summary] The invention provides a novel microorganism strain, Lactobacillus paracasei GMNL-32. In another aspect, the invention provides a composition comprising the microorganism strain Lactobacillus paracasei GMNL-32. In another aspect, the present invention provides a method of treating an allergy-related disease in a patient, comprising causing the patient to take a composition comprising the microorganism strain Lactobacillus paracasei GMNL-32; wherein the sputum is preferably selected from the following conditions Groups of: tracheal hyperreactivity and inflammation, atopic dermatitis, allergic conjunctivitis, rhinitis, sinusitis, hypersensitive pneumonia, exogenous allergic alveolitis (extrinsic allergic alveolitis) ), urticaria, moist therapy, allergic reactions (anaphylaxis), angioedema, allergic migraine, specific gastrointestinal disorders and asthma. In still another aspect, the present invention provides a method of stimulating secretion of IFN-γ in a patient comprising administering to the patient a combination comprising the microorganism strain Lactobacillus paracasei O:\90\90811.DOC -9- 1284149 GMNL-32 Things. [Embodiment] The present invention provides a novel microorganism strain, Lactobacillus paracasei GMNL-32, which can be used for the treatment of allergy. The GMNL-32 strain was deposited on March 19, 2003 by the BCRC at the Food Industry Research and Development Institute (FDI) in Hsinchu, Taiwan, under the accession number BCRC 910220. 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/xg/mL ampicillin at 37 ° C for 2 days, followed by agar plates Streak plating. Lactic acid bacteria colonies grown on the plate can be pre-screened under microscopy. The candidate strain is then cultured with spleen cells. The amount of IFN-? thus produced by the spleen cells in the broth was measured. Subsequently, GMNL-32 was selected for its high IFN-γ productivity. The mycological features of GMNL-32 are as follows: (a) Morphological characteristics: (1) Cell shape and size: After cells were cultured overnight at 37 ° C in MRS broth, the bacilli were observed by microscopy. It has a rod shape with a rounded edge. (2) Activity: active (3) Flagella: none (4) sporulation: no sporulation (5) Gram stain: positive O:\90\90811.DOC -10- 1284149 (b) Culture characteristics: ( 1) Medium: MRS broth (DIFCO® 0881), final ρΗ6·5 ± 0.2 (2) Culture conditions: anaerobic or aerobic culture at 37 °C

(3) Antibiotic resistance · Ampicillin 100pg/mL (c) Physiological characteristics: (1) Catalase: positive (2) Oxidase: negative (3) API 50 CHL test: API 50 CHL system Identification of lactic acid bacteria. The lactic acid properties are established by analyzing a series of enzyme reactions. The API 50 CHL test results for GMNL-32 are listed in Table 1: Table 1: Reference: GMNL-32 Excellent determination for this genie_

Strip: API 50CHL ( ) · ------1-----+ + + + H-----l· + 1 h + + + + + —H----- ---l· H 1------1 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 - ----1 λ FI class negative - % Id ---T---tb ----

Lacto.para.paracasei 1 94.9 0.74 2 Lacto.para.paracasei 3 5.0 0.59 5 Second choice O:\90\90811.DOC -11 - 1284149 Lactobacillus rhamnosus _ 〇·1_0-39 4

Lacto.para.paracasei 1:2 Comparative test Apricotin (AMY) 98% Pine sucrose (MLZ) 93%

Lacto.para.paracasei 3 : 5 Contrast test L-sorbose (SBE) 20% D-sorbitol (SOR) 20% Almond (AMYGDALINE) (AMY) 99% D-pinediose (TUR) 20% gluconic acid Salt (GNT) 20% Secondly, select Lactobacillus rhamnosus···4 comparison test L-rhamnose (RHA) 100% methyl-D-CLUCOSIDE (MDG) 85% almond (AMY) 99% pine triose (MLZ) 99% (d) Genetic characteristics: 16S rDNA sequence analysis of GMNL-32 was determined. The results indicate that the GMNL-32 line is highly homologous to other Lactobacillus paracasei strains (as shown in Figure 2). In addition, the germline spacing tree is shown in Figure 3. In addition, polymorphic DNA (RAPD analysis) was randomly amplified. 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 proteins of GMNL-32: Cell wall proteins of GMNL-32 showed similar patterns when compared to other conventional Lactobacillus paracasei strains. The SDS-PAGE pattern of the cell wall protein of GMNL-32 is shown in Figure 4. O:\90\90811.DOC -12- 1284149 (f) Standardized Detection System for Screening GMNL-32: US Patent No. 1 to the Standardized Detection System for Identifying Microorganisms, May 29, 2003 It is disclosed in /446,781 that it utilizes a difference in gene expression between a specific microbial culture and a test cell line cultured without a specific microorganism as a marker for identification. The genes tested are listed in Table 2. Table 2

Gene gene gene FHR-4 FGF19 FKBPIB-a FGF20 FGF13-C FGF10 FGF14 FGF11 FGF5-b FGFl-a FGF6 FGFl-b FCGBP FCAR-f FCGR1A FCAR-g FADD ELK3 FCAR_a ENG ELA2 CXCR4 EGR1 CXCL16 CX3CR1 CSF2RB CXCL1 CSF3R-a CRL3 COL3A1 CRTAM CR1 CMRF-35H CHUK CNRl-a CKTSF1B1 CDC25A CD 163 CDH3 CD164 CD97-b CD81 CD109 CD83 CD79A-a CD58 CD79A-b CD59 CD37 CD22 CD38 CD24 CD7 CD3G CD8A CD3Z CD2 - a CCRL2 CD2-b CD1A CCR4 CCL25 CCR5 CCL26 CCL19 CCL8 CCL20 CCL11 CAMK4 C9 CCBP2 CABIN1 C5 CIS C6 C2 C1QTNF2 BTNL2 C1QTNF3 BY55 BLRl-b BCL2 - a BLRl-c BCL2-b APISl-b ALDH1A1 APISl-c AOAH ADRB2 ACVRIB-c ATF2-a ACVRIB-d 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- 1284149 EGR2 CXCR3 EGR3 CYSLTR1 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 CD47 CD34-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 C1QTNF4 C1QA C1QTNF6 C1QB BMPR1A BCL2-c BMPR1B BCL3 AP1S2 AMH ATF2-b AMHR2 AGT ACVR2 AIFl-a ACVR2B ACHE- b ACE-a ACVR1 ACE-b FOG2 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 E48 CXCL5 CTLA4 CXCL6 CTRP5 CSF1R CREBl-b CSF2RA CREBBP COL1A1 CMA1 COL1A2 CMRF35 CDKN2B-b CD209L CER1 CD244 CD151-C CD86-a CD151-d CD97-a CD79B-C CD72 CD80-a CD74 CD48 CD34-b CD53 CD36 CD14 CD5L CD19 CD6 CD3D 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 BMPR2-b BLRl-a BAD-a ANXA3 BAD-b APISl-a AIFl- b ACVRL1 ALDH1A2 ADRB1 O:\90\90811.DOC -14- 1284149

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 LY6H LY6E LY75 LY6G5B LTB-b LLT1 LTBR LTB4R-a LOC163702 LOC139429 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-17RE-b IL20 IL-17RE-C IL-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 HF1 GRLF1 GDF10 FOSL1 GBP2 FOSL2 NFAT5-b ITGB3 NFAT5-C ITGB3BP NCF4-a MYD88 NCF4 -b MYF5 MUC2 MME-a MUC3B MME-b MHCBFB MCP-a MHC2TA MCP-b MAPKIO-a MAP3K7-b MAPK10 Seven MAP3K7-C MAP2K7-b MADH5 MAP3K1 MADH6 LY9 LY6G5C LYL1 LY6G6C LTB4R-b 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-HRC-d IL17C IL_17RC_e IL17F ILllRA-b IL7 ILllRA-c IL8 IL1F7 IGSF9 IL2RA IKBKB O: \90\90811.DOC -15- 1284149 IGSF1 IFRD2 IGSF2 IGBP1 IFNAR1 IFITM1 IFNAR2 IFNA14 IFI30 ICAM4-a IFI35 ICAM4_b HM74 GSCL HOXAl-a GSK3A GFI1 FST GPR2 FY NFAT5-d ITGB7-a NFATC1 ITGB8 NCF4-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 LY6G6E LY117 LTA LY64 LTB-a LOC221938 LOC147137 LEP-b LOC149620 LOC136531 LILRB3 LOC136535 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 IL18R1 IL-17RC-f 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-4 GPR31 GATA1 GPR44 GATA6 IL5 IL1R1 IFNA1 IL6ST IL10RB IL10RA ICAM1 IL13RA2 GATA3 IL1B IL10 IL2 MIP-A LOC126133 Uricase HNF4A LOCI 61823 PGK1 G6PT1 NT5C1A DHFR PPARG-b PGK2 LOC200895 LOCI 32198 XDH PPARG-a GDA TCF2-a SLC22A12-a TCF2-b SLC22A12-b ALDH2 PRPSAP2 MTHFR VLDLR LOC205855 YY1 NP PPAT VAV3 TRPV6-C VEGF TSAI 902 TRAF4-a TRAF1 TRAF4-b TRAF2-a TNFRSF7 TNFSF5 TNFRSF8-a TNFSF6 O:\90\90811.DOC -16- 1284149

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 RUNX1 REL PRL RELA PTGER2 PLAU PECAM1 PPP3CB PFC P2RX7 NOS2A-b ΡΑΚΙ NPPB 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 STAT1 - a IL12RB2 IL15 15MD2 HNF-1B GBP1 15MD-1 LOC169330 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 TNFRSF1A TNFSFll-a TLR8-a TNFSFll-b TLR8-b TLR4-b TH1L TLR4-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 PPP3R1 PILR(ALPHA) PDE4B NUP214-a PDGFB-a NUP214- b NFKBIL1 NFATC4 NFKBIL2 NFKB1 CSF1 IL9-b CD80-b IL13 CCR2-b CD86 IL4 IFNB1 CEBPB TIM3 IRF1 IL4R TP53 IL12B TNF-a SE RPINA3 VCAMl-a SCYA4 CCR7 IL12A IL12RB1 CSF2 ADSS STAT6 IMPDH2 IL6 LGALS9 IFNG UMOD PTGS2 LOC223071 TCF2-c PRPS1 APOE ZNF144 APOB

O:\90\90811.DOC -17- 1284149

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 TBXA2R TLR2 TACTILE TFCP2 SLAM TGFA SLA SSI- 3 SEMA3F SUDD SEMA3E SEMA4G RNASE3 SEMA7A RNASE2 SCYE1 PRG2 SDF2 PRKG1 PTPRC-c PDPK1 RDC1 PDGFB-b PILR(BETA) NOS2A-a ΡΙΝΙ NMA 0PRD1 Negative ORM1 ACTB NFKB2 G6PD NFKBIA Standard Detection System for Identifying GMNL-32 Jurkat Cells As a test cell line. The genes listed in Table 3 were significantly different when comparing the expression patterns of Jurkat cell lines cultured with GMNL-32 and those not cultured with GMNL-32. In addition, the detection results of other Lactobacillus paracasei, CCRC 12193 & CCRC 121 88 are also shown in Table 3. It is pointed out that these strains are both Lactobacillus paracasei but are subordinate to different strains. Table 3 Gene name cheese CCRC12193 GMNL-32 cheese CCRC12188 ADA ++++++ ++++ ++ BAD_a ++ +++ + BCL3 + + BLRl-c — + — BMPR2-a ++ ++ + CCL2 + - CD2AP ++ ++ + CD2-b ++ ++ + CD38 ++ ++ - CD3G ++++++ ++++++ ++++++ CD48 ++ ++ + COL1A2 — - — O:\90\90811.DOC -18- 1284149 CR2 ++ ++ + CREBl-a ++ ++ + CREBl-b +++ +++ + CX3CR1 ++ +++ ++ DAF ++ + ++ + ETEA ++ ++ + FCAR-h +++ +++++ ++ FGF23 + ++ + FH0D2 ++ ++ + 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 - 1284149 + : Gene expression increased by a factor of 2: Gene expression decreased by 2 times GMNL-32 in H The C1 solution (ρΗ2·0) was treated for 3 hours and then treated with bile for 4 hours. Therefore, GMNL-32 is considered to remain active in digestion. GMNL-32 is isolated from a healthy subject and is safe, natural, non-toxic and meets the G.R.A.S. (Generally Regarded as Safe) standard. 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 sites of intestinal epithelial cells, GMNL-32 also prevents other pathogenic bacteria from sticking to the intestines. GMNL-32 is considered a good probiotic. According to the present invention, GMNL-32 was found to stimulate IFN-γ secretion and was useful for treating allergy-related diseases. One aspect of the present invention provides a composition comprising GMNL-32. Preferably, the composition comprising GMNL-32 is used to stimulate IFN-γ secretion, which is useful for treating allergy-related diseases. The term π allergy-related disease as used herein refers to a disease in which a systemic response to a general environmentally sound antigen is caused by an interaction between an antigen and an antibody or a sputum cell produced by an earlier exposure to the same antigen. The term "allergic reaction" as used herein refers to a reaction to an environmentally harmful antigen or allergen due to pre-existing antibodies or sputum cells. There are various immune mechanisms for allergic reactions, but the most common type is allergen to hypertrophy. The binding of IgE antibodies on cells, which trigger asthma, hay fever, and other common allergic reactions. Allergic-related diseases include hyperventilation and O:\90\90811.DOC -20- 1284149, allergic conjunctivitis, rhinitis Sinusitis, allergic

inflammation. In another aspect, the allergy-related disease is associated with airborne allergens (airborne allergens) such as pollen, mold, animal scales, and insects. Inflammation, atopic dermatitis, pneumonia, vascularity The term "airborne allergen" as used herein is defined as a group of specific antigens having at least the following characteristics 叮 narration/lingual responsiveness, and the conductance The level of environmental exposure that is sensitive to significant tissue changes in the body. Airborne allergens are airborne particles that can cause beer, skin or conjunctival allergy. The water-soluble portion of ragweed pollen (for example) affects the respiratory and conjunctival mucosa, while the fat-soluble allergen of ragweed pollen causes typical contact dermatitis on the exposed skin. GMNL-32 was selected to have the ability to stimulate secretion of ΙΙ Ν γ when it was co-cultured with spleen cells and peripheral blood mononuclear cells (PBMC) in vitro. Furthermore, in the model according to the present invention, it was observed that animals activated by airborne allergens and then treated with GMNL-32 increased IFN_T secretion. In addition, 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 a significant difference before and after treatment. In addition, the eosinophil count in bronchoalveolar lavage fluid (BALF) decreased dramatically; however, macrophage and lymphocyte counts in BALF increased dramatically. It has been shown to reduce inflammation. According to the present invention, GMNL-32 for allergy treatment can be live or depleted. Preferably, the GMNL-32 is energy-reduced. For example, live bacterial strains can be treated by heating the O:\90\90811.DOC-21 to 1284149 step or other treatments commonly used in the art to kill lactic acid bacteria as reduced strains. According to the present invention, the lactic acid strain can be included in a medical composition, a dietary supplement, a food, a health food, a medical food or a component thereof, which are generally administered by a human. In a preferred embodiment of the invention the lactic acid strain can be delivered in the form of a food product, for example in the form of a condensed milk product prepared by lactic acid fermentation in milk. Food products made in accordance with the present invention are conveniently administered to 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 the isolated microorganism (?)^^^32. For the purpose of illustration only Without intending to limit the scope of the invention, the following examples are given. Example 1 · Isolated sample of Lactobacillus paracasei GMNL-32: A human stomach, intestine or duodenal tissue obtained by endoscopy at 37 C, 2 mL containing 1 〇〇/ig/niL of Lactobacillus ampicillin MRS broth (DIFCO® 0881) was cultured for about 2 days. The broth was covered on CaC〇3iMRS qiongyue and incubated at 37 C for two days. A single colony on the plate was subjected to Gram staining. Gram-positive bacteria were then selected. All strains were grown to a stationary phase at 37 ° C in Lactobacillus MRS broth, and by Collect by centrifugation at 3000 g for 15 minutes, and rinse with 2 mL and 1 mL of PBS (phosphate buffered saline, ρΗ7·2). Resuspend the culture of this special strain in pmL of ! mL, then at 95. Heated for 30 minutes and then subjected to autoclave and stored in _2 〇 it pBs .

O:\90\90811.DOC -22- 1284149 Splenocyte isolation: 5 mL of Ficoll-Hypaque (17-1400-02, Pharmacia) was added to a 5 mL blood sample from healthy volunteers and then centrifuged at 500 g for 30 minutes. . Obtain spleen cells. In each spleen cell sample, the cell density was adjusted to 5 x 106 cells per sample. The spleen cell samples were incubated for 6 hours in 2 mL RPMI 1640 (pH 7.7). 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 centrifuged at 500 g for 30 minutes. These 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 wells of a six-well plate containing 2 mL of RPMI 1640 medium in ΡΗ7·7. Stimulation of IFN-γ secretion: Spleen cells or PBMC samples are co-cultured with specific amounts of Gram-positive bacteria. After co-cultivation for 36 hours, the cells in each sample were collected separately. The collected cells were resuspended and centrifuged at 2000 rpm for 5 minutes. The supernatant was taken to determine the IFN-γ content in each sample. Determination of IFN-γ content: The IFN-γ content was determined by ELISA, which included the following steps: - Add 30 μί 2.5 ptg/mL of purified mouse anti-human IFN-γ antibody (Cat. Nol 8181D, PharMingen®, USA) to 10 The mL was coated with a gentle inscription (0·1Μ Na2HP04, ρΗ9·0), and the lOOpL antibody solution was added to each well of the ELISA plate; - the plate was shaken at 4 ° C - rinse buffer (0.05 in PBS) %Tween20) Rinse each of the plates; O:\90\90811.DOC >23- 1284149 - Add 300 μί blocking buffer (1% BSA in PBS) to each well of the plate; Shake the plate gently for at least 2 hours; - Add 10 0 /iL spleen cell sample supernatant to each well of the plate; - Shake the plate overnight at 4 ° C; - Rinse with rinse buffer Each well of the plate; - 150/xL biotin mouse anti-human IFN-γ antibody (Cat. Nol8112D, PharMingen®, USA) was added to each well of the plate; - the plate was incubated for 1 hour at room temperature - flushing each well of the plate with a rinse buffer;

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

Streptavidin-AKP into each well of the plate; - shaking the plate for 1 hour at room temperature; - flushing each well of the plate eight times with a rinse buffer; - adding 200 gL of the substrate pNpp to the plate In each well; - incubate the plate at room temperature until completion of the substrate reaction; - measure the absorbance of each well of the plate at 405 nm (ie OD4G5). RESULTS: Among Gram-positive bacteria, GMNL_32 was selected to have the strongest ability to stimulate IFN-γ secretion in splenocytes and PBMC. Example 2: 16s rDNA sequencing DNA extraction: The genomic DNA of GMNL-32 and other bacteria CCRC12913, CCRC14001 and CCRC16100 was extracted by using QIAamp® DNA Stool Mini Kit (Qiagen®, cat 51·51504). Purification was carried out according to the procedure listed below: O:\90\90811.DOC -24- 1284149 - Add 1.4 mL of ABS buffer to the culture and vortex for 1 minute; - Heat at 70 ° C by the aforementioned The solution obtained in the step is for 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; - InhibitEx is added to the supernatant: And shaking it to dissolve the rotatory agent, followed by incubation at room temperature for 1 minute; - allowing the solution to be centrifuged at about 13,000 rpm for 3 minutes to adhere the bacteria to InhibitEx; - moving the supernatant to a new centrifuge tube and then Centrifuge at about 13,000 rpm for 3 minutes; - Take 200 / xL of supernatant to a new centrifuge tube and add proteinase K; - Add 200 μl of buffer AL and vortex for 15 minutes to obtain a homogeneous solution; - Add 15 g of protease Immersed into the homogeneous solution and vortexed it for 15 seconds; - incubated the solution at 70 ° C for 10 minutes; - added 200 / XL 96 · 100% ethanol and vortex; - moved the solution to the QIAamp rotating column ( Spin column) and centrifuge it at about 13,000 叩 111 for 1 minute; - spin the QIAamp Transfer the turret to a new centrifuge tube and add 500 /xL buffer AW1 and then centrifuge at 13,000 rpm for 1 minute; - Move the QIAamp spin column to a new centrifuge tube and add 500 pL of buffer Agent AW2, and then centrifuge it at about 13,000 rpm for 1 minute; O:\90\90811.DOC -25- 1284149 - Move the QIAamp spin column into a new centrifuge tube and add 200 μί buffer buffer, and then It was incubated at room temperature for 1 minute; and - it was centrifuged at about 13,000 rpm for 1 minute to dissolve the DNA. 16s rDNA fragment amplification: The primer used to amplify the L region was designed according to the following: Lactobacillus paracasei 16S rRNA VI region, 5'-CAC CGA GAT TCA ACA TGG-3f (SEQ ID Νο·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). Genomic DNA of GMNL-32-CCRC12913, CCRC14001 and CCRC16100 was used as a template for performing PCR reactions. The 16s rDNA PCR amplification procedure is as follows: (1) 10 minutes at 95 ° C; (2) 45 seconds at 95 ° C; (3) 45 seconds at 46 ° C; (4) 72 ° (: 1 minute; 5) 7 minutes at 72 ° C; steps 2 to 5 were repeated for 30 cycles. 16s rDNA sequence determination: PCR products of GMNL-32, CCRC12913, CCRC14001 _ and CCRC16100 were subjected to agarose gel electrophoresis (Fig. 2) and sorted Sequence alignments were performed by the ARB sequence editor (release 8.1) against the multiple sequence alignment dataset (NCBI blastn, http://www.ncbi.nlm.nih.gov/BLAST). It also indicates that the 16s rDNA sequence of Lactobacillus paracasei strains PB4, AY186046; F31, AF243147; KLB5 8 and AF243168 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 O:\90\90811.DOC -26- 1284149 spacing tree is generated with EMBL-EBI ClustalW (http://www.ebi.ac.uk/clustalw) According to the 16s rDNA analysis, GMNL-32 is highly correlated with KLB58, but it is completely different from KLB58. In summary, GMNL-32 belongs to the deputy. Lactobacillus casei. Example 3: Random amplified polymorphic DNA (RAPD analysis) DNA extraction of GMNL-32, Chang J. lactis ATCC 25598, 25302, 335, 11582 and 27216 was performed as described in Example 2. The primers for amplification are S'-ATGTAACGCdCGardinei^G·, Ross, RP·, 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). The RAPD results are shown in Figure 5. According to the RAPD analysis, GMNL_32 is distinct from the conventional Lactobacillus strain. 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, Ν·, Rossi, J., Corbo, MR·, and Gobbetti, Μ. (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: 2011-2020) to purify cell wall proteins. The cells cultured overnight in MRS broth (Difco®) were harvested and then washed twice with 0.05 M Tris-HCl (pH 7.5) containing 0.1 M CaCl 2 and resuspended in OD600 O of 10.0: 90\90811.DOC -27- 1284149 in 1 mL of the same buffer. After centrifugation at 8000 xg for 5 minutes, the cell wall proteins were extracted from the centrifugation block with l_0 ml extraction buffer (ρΗ8·0) containing o.oiM EDTA, 0_01 M 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 x g for 10 minutes at 4 °C. The supernatant was analyzed by 12% SDS-PAGE and stained with Comassie Blue. The result is 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 previous studies (Angelis et al., 2001). Therefore, GMNL-32 was confirmed to belong to Lactobacillus paracasei. Example 5: Standard Detection System for Identification of 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 using lactic acid bacteria. When the cell concentration reached lxl07/10 mL, the cells were stimulated with or without lxlO7 different lactic acid bacteria (CCRC12193, GMNL-32 or CCRC12188) for 24 hours. After stimulation, cells were harvested 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. 8 L of RNA (10 jLtg) and 2 L of oligo-dT (12_18mer, lg/L) were thoroughly mixed and kept at 70 ° C for 10 minutes and then cooled by ice for 2 minutes. The RNA was mixed with the reverse transcription marker mixture and 3L Cy3-dUTP (lmM), 2L Superscript III (200 U/L) and RNasin (lL) O:\90\90811.DOC -28-1284149 in the dark. The mixture was incubated at 50 °C for 2 hours for reverse transcription, and the reaction was terminated by the addition of 1.5 L of 20 mM EDTA. After labeling, RNA was removed by NaOH treatment and neutralized with HC1. The cDNA was purified immediately with the YM30 purification kit. 〇 Microarray fabrication: Many selected genes were amplified by multi-polymerase chain reaction and quantified by 260 nm spectrophotometry. All purified PCR products were adjusted to a concentration of 0.1 Zxg/μΐ in 50% dimethyl sulfoxide and spotted on UltraGAPSTM coated slides (Corning®, Inc., Corning) , NY). After printing, the microarrays were UV crosslinked under 700 m Joulesand and stored in a glass slide container in a desiccator at room temperature. This gene is listed in Table 2 as described above. Microarray hybridization: Fluorescently labeled cDNA was denatured in a hybridization solution (5x SSC, 0.1% SDS and 25% proguanamine) for 5 minutes at 100 ° C, cooled to ambient temperature, and deposited on glass slides. Hybridization was performed at 55 ° C for 18 hours. After hybridization, continuous rinsing with low stringency (lx SSC and 0.1% SDS), medium stringency (0_lx SSC and 0.1% SDS), high stringency (O.lx SSC) buffer, and finally by compression N2 Dry. Signal Detection and Data Analysis: N2 dried slides were immediately scanned on the GenePix 4000B scanner (Axon Instruments®, with the same photomultiplier laser intensity and sensitivity level for each slide). Inc.). Obtain original landing data (1 Onm resolution), and perform subsequent processing and data visualization in Microsoft ExcelTM. To compare the results of independent hybridization experiments, the local background signal was subtracted from the hybridization signals at each individual point and then divided by the gene silencing actin. The final expression of a gene per O:\90\90811.DOC -29- 1284149 is expressed as the average of the duplicates. A gene expression map of Jurkat cells cultured with lactic acid bacteria and cultured without lactic acid bacteria was obtained. Jurkat cells cultured using lactic acid bacteria (CCRC12193, GMNL-32 or CCRC12188) were selected to up- or down-regulated more than 2 times the group of genes in Jurkat cells cultured without using these bacteria. The results are shown in Table 3. This distinction indicates that different species or strains can turn on or turn off different genes of the cell. Therefore, from the gene expression profile, it is pointed out that CCRC12193, GMNL-32 and CCRC 12188 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 that exhibit distinct morphological and functional properties of mature epithelial cells in the living colon. Cells: Caco_2 was cultured in a minimal essential medium (MEM, GIBCO®) supplemented with 5% FBS at 5% CO 2 /95% air at 37 °C. For adhesion analysis, 2 ml of monolayer Caco-2 cells (3 X 105 cells/ml) were prepared on glass coverslips placed in 6-well 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, the 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 10 CFU/ml) GMNL-32, which was washed once with PBS and resuspended in 1.5 ml of MEM medium, was added to Caco-2 cells. After incubating for 1 hour at 37 ° C, the monolayer cells were washed with PBS buffer 4 O: \90 \90811.DOC -30 - 1284149 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 stained with Gram. The bacteria were adhered by microscopy (xl 00) under oil immersion, 15 random areas on each coverslip were counted, and the mean ± SD of adherent bacteria in each area was determined. RESULTS: After counting, 1〇2±23.6 GnNL-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, KF, 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· 1999;65:4949-4956), GMNL-32 It is believed to have strong adhesion to Caco-2 cells. Example 7: Active acid of GMNL-32 and other lactic acid bacteria in an environmental simulated gastrointestinal tract: 9 ml PBS with different pH values (2.0, 2.5, and 3.2) was added to GMNL-32, Lactobacillus plantarum incubated overnight. L. plantarum), L. acidophilus, L. casei, and L. bulgaricus, and then further cultured at 37 ° C for 3 hours. After the incubation, 1 mL of cells were serially diluted with 9 mL of PBS pH 7.4. Cell counts before and after acid treatment were estimated and are listed in Table 4 as listed below. Bile: 9 ml PBS with pH (2.0) was added to overnight cultured GMNL-32, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus licheniformis and Paula O:\90\90811.DOC -31 - 1284149 Lactobacillus licheniformis, and then further incubated at 37 ° C for 3 hours. After the incubation, 1 mL of the cells was centrifuged at 6,000 rpm for 10 minutes. The pellet was resuspended in 100 μL of PBS (pH 7.2). To this solution was further added 10 mL of MRS broth containing 0.3% (w/v) bovine bile. The cells were cultured and 1 mL samples were taken at 3, 12 and 24 hours. The samples were serially diluted with 9 mL of PBS pH -7. Cell counts before and after bile treatment were estimated and are also shown in Table 4. It shows that these lactic acid bacteria retain activity in the environment simulating the gastrointestinal tract. Table 4 Cell Count (Log CFU/mL)

The strain was treated with HC1 for 3 h and treated with bile for 4 h and then treated with B. lactis 9.03 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 Lactobacillus bulgaricus 9.230 9.076 7.447 Example 8: Animal model animals Female BALB/c mice were obtained from the National Laboratory Animal Center in Taiwan and kept in a room with controlled light and temperature for 2 weeks. Allergen Purification · Dust worm allergen Der p 5 is expressed in Escherichia coli containing PGEX-2T expression vector as recombinant Der p 5-glutathione S-transferase fusion protein, which can be expressed by glutathione Pure O:\90\90811.DOC -32-1284149 was obtained by agarose-binding chromatography. A specific E. coli strain capable of expressing the desired allergen is cultured and introduced. The bacteria were collected and washed with TBS (pH 7.5), and 〇·ιμ benzyl myrazine fluorene was added. The cells were disrupted by the addition of Dnase I, Tween 20 and lysozyme, and by the ice thawing method. EDTA was added to the mixed solution, and the residue was removed by centrifugation to obtain a supernatant containing the recombinant Der p 5-glutathione S-transferase fusion protein. The supernatant was subjected to an affinity column of glutathione nopalyx to absorb the fusion protein. The column was then rinsed with TBS buffer at 4 °C and then rinsed 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: Automated sensitization by intraperitoneal injection of 1 〇 of Der p 5 and 4 mg of hydrazine. At 14 and 21 days after the initial sensitization, the mice were exposed to 0.1% purified Der p 5 aerosol for 30 minutes to perform an inhalation challenge. Treatment: The sensitized mice were divided into three groups for experiments. Group A mice were fed MRS broth 10 times during the two weeks as a control group. Group B mice were administered Lactobacillus casei 1 week twice a week and 1 〇 9 CFU of bacteria per administration. Since Lactobacillus casei has been shown to be effective in inhibiting IgE secretion, group B was used as a 11⁄4 axillary control. Group C mice were administered GMNL-3210 times in two weeks, and 10 〇 9 CFU of bacteria per sputum. Example 9: lgG and IgE secretion er P5 specific IgG and IgE assay: After 1 hour of the last inhalation challenge, 50 sputum blood samples were taken from the tail. Keep the blood sample in the room

O:\90\90811.DOC -33- 1284149 Warm for 1 hour and then subject it to centrifugation. Serum was stored at _80 °c. The amount of 〇61?5 specific 1§(^ and 1§£) was determined by £1^ by the coating buffer (0.1 MNaHC03, ρη9·6) diluted to a concentration of 10 pg/mL of 200 μM The purified Der p5 was coated with a 96-well protein high-binding plate. After 4 overnight incubations, the plates were rinsed with PBS-Tween 20, followed by 300 #blocking buffer. (3% BSA). After shaking for 2 hours at room temperature, the plates were washed again with PBS-Tween 20. Serum was diluted 1:10 for IgG measurement and diluted 1:4 for IgE measurements Shake the sample for 2 hours at room temperature. After 4: overnight incubation, rinse the plates with PBS-Tween 20 and add 200 μί biotinylated rat anti-mouse IgE monoclonal antibody, or large Mouse anti-mouse IgG mAb. The sample was shaken at room temperature for 2 hours and then rinsed with PBS-Tween 20. Next, 200 pL of Streptavidin-alkaline phosphatase (l: 1000) and shaking the sample for 1 hour at room temperature. After 6 rinses, the nitrophenyl phosphate and disodium phosphate were supported by the addition of 200 μί phosphatase. The color reaction was started with pNPP) (Sigma® N-2770, USA). The plate was read at 405 nm using a microplate reader (Metertech®, Taiwan). Data analysis: For analysis of IgE and IgG The change in content was performed by performing a one-way ANOVA analysis to measure the difference between groups. After the change analysis, the Duncan multi-range test was used to distinguish the difference between the experimental group and the control group. The value of ρ<〇·〇5 It was used to indicate a statistically significant difference. Results·· The results are shown in Figure 7. It demonstrates that the secretion of IgE in the serum of GMNL-32-treated animals is drastically reduced and is only in the serum of untreated animals: \90\90811.DOC -34- 1284149

25% of IgE secretion 〇 On the other hand, IgG secretion in the serum of GMNL-32-treated animals was increased by a factor of two. Since IgG secretion represents a Th1 T cell response, GMNL-32 aims to eliminate IgE secretion associated with allergy-related diseases. Example 10: Bronchoalveolar lavage fluid cell count Sample preparation: After 18 hours of sensitization, a polyethylene tube introduced by tracheostomy, 0.9% sterility in a 5x0.5 ml aliquot Brine was used to irrigate the mice. Centrifuge (500 g, 10 min at 4 °C) the lavage solution and resuspend the cell pellet in 1 ml PBS solution. Self-bundled cell counts were prepared from cell centrifuge preparations stained with Leu's stain. Data analysis: To analyze changes in cell counts, one-way ANOVA analysis was performed to measure repeated measures to compare differences between groups. After the change analysis, the Duncan multi-range test was used to distinguish the difference between the experimental group and the control group. The value of Ρ<〇·〇5 is used to indicate a statistically significant difference. Results: The results are shown in Figure 8. The blood cell type composition in BALF represents the degree of inflammation. In addition, the main symptoms of allergic asthma are chronic tracheal inflammation and eosinophil infiltration (infiitrati〇n). It was confirmed that eosinophils in BALF of animals treated with GMNL-32 were drastically reduced from 5% to 1%. On the other hand, macrophages and lymphocytes in BALF of GMNL-32-treated animals increased significantly. Example 11: IFN-γ secretion sample preparation in bronchoalveolar lavage fluid: After sensitization for 24 hours, the polyethylene tube introduced by tracheotomy was filled with 0.9% sterile saline in 5x0_5 ml aliquots. Wash the mice. Centrifuge (500 g, 10 min at 4 ° C) lavage and let the upper layer

O:\90\90811.DOC -35- 1284149 The serum was subjected to quantitative analysis of IFN-γ as described in Example 1. Results: The results are shown in Figure 9. It was shown that animals fed GMNL-32 produced approximately 1 〇〇 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 inhibiting allergic inflammation. Example I2: Inactivation of GMNL-32 for the treatment of allergy-reduction GMNL-32: The lyophilized GMNL-32 powder was suspended in distilled water and subjected to autoclave before feeding the mice (at 121 ° C, 15 minute). • Mice and sensitization: Female BALB/c mice (6-8 weeks old) were purchased from the National Laboratory Animal Breeding and Research Center (Taipei, Taiwan). All animals were individually maintained in cages controlled at temperature (24 ± 2 °C) and humidity (60 soil 5%) and maintained for 12 hours day/night cycle under specific-pathogen-free conditions. . BALB/c mice were intraperitoneally injected (i.p.) to 10 g of chlorinated 10 g of recombinant house dust worm φ (Dermatophagoides pteronyssinus) to defeat the Der: p5-6xHis hostile protein. The mice were fed an amount of 107, 109 and 1011 CFU GMNL-32 per mouse per day for three weeks. The mice were boosted on the 14th day with the same dose of allergen as sensitization, and after 21 days of sensitization, the mice were challenged with 0.1% Der p5-6xHis diluted in PBS. . The inhalation challenge is performed in a 11^ chamber connected to a DeVilbissTM pulmosonic nebulizer (Model 2512; 〇. \^11^88@(1:0., 8011861861:, 八), which produces a float Mist. After 18 hours, O:\90\90811.DOC-36-1284149 serum was collected by tail vein bleeding, and IgE was determined by ELISA as described in Example 9. Results: The results are shown in Table 10. It shows that the BALB/C mice challenged by the dust allergic Derp-5 have a significantly higher serum IgE content than the untested (naive) group (ρ<〇·〇5). This suggests that allergic sensitization can be successfully established. Mouse model. Serum IgE was significantly lower in the GMNL-32 group than in the control group after 21 days of daily feeding with different doses of GMNL-32 (ρ < 0.05). The results showed that the depletion of GMNL-32 was achieved by Allergen-specific IgE is reduced to reduce allergic reactions.Although embodiments of the invention have been illustrated and described, various modifications and improvements can be made by those skilled in the art, and the invention is not intended to be limited to the particular form illustrated. And all amendments that do not depart from the spirit and scope of the present invention belong to Within the scope defined in the scope of the patent application. [Simplified Schematic] Figure 1 illustrates a 1000 X micrograph of GMNL_32 subjected to Gram staining. Figure 2 illustrates a 16s rDNA fragment agarose gel analysis. As a result, the fragment was amplified by PCR of GMNL-32 and lactic acid strains CCRC12913, CCRC14001 and CCRC16100; Μ represents molecular marker; 1 represents GMNL-32; 2 represents CCRC12913; 3 represents CCRC14001; and 4 represents CCRC16001. 16s rDNA sequence alignment of GMNL-32 and lactic acid strains CCRC12913, CCRC14001, CCRC16100, KLB5 8, ΡΒ4 and F31 s Figure 4 illustrates the comparison of GMNL-32 of the present invention with related lactic acid bacteria for 16s The phylogenetic distance tree of the rDNA germ line. Figure 5 illustrates the RAPD analysis of GMNL-32 and the conventional lactic acid strain; Μ: O:\90\90811.DOC -37- 1284149 100_bp ladder, Lane 1 : GMNL-32 ; Lane 2 : Lactobacillus paracasei ATCC 25598 ; Lane 3 : Lactobacillus paracasei ATCC 25302 ; Lane 4 : Lactobacillus paracasei ATCC 335 ; Lane 5 : Lactobacillus paracasei ATCC 1 1582 ; Lane 6 : vice Buttermilk Lactobacillus ATCC 27216. Figure 6 illustrates the SDS-PAGE pattern of cell wall proteins of GMNL-32, a conventional Lactobacillus paracasei and Lactobacillus yeast strain; wherein Μ represents protein molecular weight; Lane 1 represents Lactobacillus paracasei; Lane 2 represents Lactobacillus paracasei GMNL-32; Lane 3 represents Lactobacillus yeast; F1 represents a specific band of Lactobacillus yeast; and P1, P2 and P3 represent a specific band of Paracasei lactic acid bacteria. Figure 7 illustrates Der p 5 specific IgG (white bars) and IgE (black bars) levels in serum of Der ρ 5 sensitized BALB/c mice receiving the inhalation of Der p 5 challenge; A represents the MRS broth treatment group; B represents the group treated with Lactobacillus paracasei (Z. cwd); and C represents the group treated with GMNL-32. Figure 8 illustrates the cell counts of giant sputum cells, lymphocytes and eosinophils in brochoalveolar lavage of Der p5 sensitized mice; A represents MRS broth treatment group; B represents Lactobacillus paracasei The treatment group; and C represents the treatment group with GMNL-32. Figure 9 illustrates IFN-γ secretion in bronchoalveolar lavage in Der p5 sensitized mice; A represents MRS broth treatment; B represents Lactobacillus paracasei treatment group; and C represents GMNL_32 treated group . Figure 10 illustrates the effect of inactive GMNL_32 on IgE production in Der p5 sensitized BALB/c mice. Derp5 sensitized mice were orally administered different doses of GMNL-32 or distilled water (control) daily for three weeks. By using O:\90\90811.DOC -38- 1284149 serum Der p5 specific IgE content was determined by ELISA. When compared with the control group, the Kruskal-WallisH test *(ρ<0·1) was significantly different from **(ρ<0·05), and the Dunnettt test used posteriori comparison. O:\90\90811.DOC -39- 1284149 Sequence Listing <110> GenMont Biotech Inc. <120> Novel Microbial Plant GMNL-32 Lactobacillus paracasei and its use for treating allergy-related diseases <130>;160〉 2 <170〉Effective version (Patentlnversion) 3.2 <210> 1 <211> 18 <212>DNA <213> Artificial <220><223> Lactobacillus paracasei specific 16S rRNA < 400> 1 caccgagatt caacatgg 18 <210〉 2 <211> 24 <212>DNA <213> Man Made <220><223> Saved 16S rRNA <400> 2 cccactgctg cctcccgtag gagt 24 O: \90\90811.DOC -40-

Claims (1)

1284149 Picking up and applying for patent Fangu: K kinds of isolated microbial strains of Lactobacillus paracasei GMNL-32, which are deposited in the F〇〇d Industry Research and Development Institute (FIRDI), Taiwan. The number is BCRC910020. 2. A composition comprising a microorganism strain as in claim 1 of the patent application. 3. A composition as claimed in claim 2, which is for use in stimulating IFN_? secretion. The invention claims a composition of the second aspect of the patent for the treatment of allergy-related diseases. 5. The composition of claim 4, wherein the allergy-related disease is selected from the group consisting of: tracheal hyperreactivity and inflammation, atopic dermatitis, allergic conjunctivitis, rhinitis, sinusitis, allergic pneumonia, Exogenous allergic alveolitis, urticaria, eczema, allergic reactions, angioedema, allergic migraine, specific gastrointestinal disorders and asthma. 6. The composition of claim 5, wherein the allergy-related disease is caused by excessive tracheal reaction and inflammation. 7. The composition of claim 4, wherein the allergy-related disease is associated with exposure to a gas-borne allergen. 8. The composition of claim 2, wherein the microbial strain is viable or depleted. 9. The composition of claim 8 wherein the microbial strain is depleted. 10. The composition of claim 2, in the form of a pharmaceutical composition, dietary supplement, food or a component thereof. O:\90\90811.DOC