Patent Publication Number: US-2004047849-A1

Title: Use of some lactobacillus strains in treating allergy

Description:
BACKGROUND OF THE INVENTION  
       [0001] 1. Field of the Invention  
       [0002] The invention mainly relates to a new use of some Lactobacillus strains in treating allergy.  
       [0003] 2. Description of the Related Art  
       [0004] Allergy refers to an acquired potential to develop immunologically mediated adverse reaction to normally innocuous substances. Allergic reaction provokes symptoms such as itching, coughing, wheezing, sneezing, watery eyes, inflammation and fatigue. It is normally believed that allergic reaction includes an early specific immune response and a late inflammatory reaction. It is reported that allergens (e.g. pollens and mite dust) mediate the early phase of allergy by stimulating high affinity immunoglobulin (IgE) receptors. For instance, mast cells and basophils, when stimulated by allergens, will release histamine and cytokines. The cytokines released from mast cells and basophils then mediate the late phase of allergy by recruiting inflammatory cells. It is also reported that the influx of eosinophils, macrophages, lymphocytes, neutrophils and platelets starts the vicious inflammatory cycle. This late phase of allergy amplifies 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. Respir Res 2000;1:54-61).  
       [0005] Various therapies have been pursued in order to treat the symptoms of allergies. Among them, anti-allergics and histamine H-receptor antagonists (anti-histamines) have been used. Histamine antagonists are administered to antagonize the action of histamine released from mast cells in response to the presence of allergens. They reduce the redness, itching and swelling caused by the action of histamine on the target tissues, and serve to prevent or alleviate many of the symptoms resulting from degranulation of mast cells. However, anti-histamines have also been associated with adverse reactions such as diminished alertness, slowed reaction times and somnolence (U.S. Pat. No. 6,225,332).  
       [0006] There are also some reports on the treatment of allergies by regulating cytokines. Among them, interferon-γ (INF-γ) was found to inhibit the over-expression of cytokines in Th2 lymphocytes, especially the secretion of IL-4 to lower the proliferation of B cells. Besides, INF-γ could stimulate the immune response of Th1 and repress the synthesis of IgE (Sareneva T et al. Influenza A virus-induced INF-α/β 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 culture.  Int Arch Allergy Immunol  1998;115:278-287). Since INF-γ can repress B cell proliferation and IgE secretion, it is believed that INF-γ is effective in treating allergy.  
       [0007] Lactic acid bacteria, which are gram-positive bacteria, are commonly used in industrial food fermentations. In recent studies, lactic acid bacteria were shown to stimulate INF-γ secretion of cells (Contractor NV et al. Lymphoid hyperplasia, autoimmunity and compromised intestinal intraepithelial lymphocyte development in colits-free gnotobiotic IL-2-deficient mice.  J Immunol  1998; 160:385-394). Some specific lactic acid bacteria, such as  Bifidobacterium lactis  and  Lactobacillus brevis  subsp., were found to stimulate INF-γ secretion of lymphocytes in blood derived from mice and humans (U.S. patent Publication Ser. No. 2002/0,031,503 A1; U.S. Pat. No. 5,556,785). It was also reported that lactic acid bacteria could stimulate lymphocytes derived from humans or mice to secret Interleukin-12 (IL-12), which was a T cell stimulatory cytokine activating T cells and NK cells to secrete INF-γ (Hessle et al. Lactobacilli from human gastrointestinal mucosa are strong stimulators of IL-12 production.  Clin Exp Immunol  1999; 116:276-282).  
       SUMMARY OF THE INVENTION  
       [0008] The invention provides a new use of some Lactobacillus strains in treating allergy.  
       [0009] One subject of the invention is to provide a method for treating allergy in a subject comprising administrating said subject with a medicament comprising a lactic acid bacterial strain stimulating INF-γ secretion, which is selected from the group consisting of  Lactobacillus plantarum  CCRC 12944,  Lactobacillus acidophilus  CCRC 14079,  Lactobacillus rhamnosus  CCRC 10940,  Lactobacillus paracasei  subsp.  paracasei  CCRC 14023,  Lactobacillus delbrueckii  subsp.  bulgaricus  CCRC 12297,  Lactobacillus delbrueckii  subsp.  bulgaricus  CCRC 14007, and  Lactobacillus delbrueckii  subsp.  bulgaricus  CCRC 14069.  
       [0010] In another aspect, the invention provides a composition for treating allergy comprising a lactic acid bacterial strain stimulating INF-γ secretion in a therapeutically effective amount to treat allergy, which strain is selected from the group consisting of  Lactobacillus plantarum  CCRC 12944,  Lactobacillus acidophilus  CCRC 14079,  Lactobacillus rhamnosus  CCRC 10940,  Lactobacillus paracasei  subsp.  paracasei  CCRC 14023,  Lactobacillus delbrueckii  subsp.  bulgaricus  CCRC 12297,  Lactobacillus delbrueckii  subsp.  bulgaricus  CCRC 14007, and  Lactobacillus delbrueckii  subsp.  bulgaricus  CCRC 14069. 
     
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
     [0011]FIG. 1 illustrates the secretion of INF-γ in the co-culture of a Lactobacillus strain and lymphocytes. The secretions of INF-γ were detected with ELISA after the 12 and 36-hour co-culture of the lactic acid bacterium and lymphocytes, respectively. The amounts of INF-γ were expressed by the absorbance values (O.D. values). In the figure, “PC” represents  Lactobacillus casei  CCRC 10697 as positive control; “NC” represents  Lactobacillus delbrueckii  subsp.  bulgaricus  CCRC 14071 as negative control; 1 represents  Lactobacillus plantarum  CCRC 12944; 2 represents  Lactobacillus acidophilus  CCRC 14079; 3 represents  Lactobacillus rhamnosus  CCRC 10940; 4 represents  Lactobacillus paracasei  subsp.  paracasei  CCRC 14023; 5 represents  Lactobacillus delbrueckii  subsp.  bulgaricus  CCRC 12297; 6 represents  Lactobacillus delbrueckii  subsp.  bulgaricus  CCRC 14007; and 7 represents  Lactobacillus delbrueckii  subsp.  bulgaricus  CCRC 14069.  
     [0012]FIG. 2 illustrates the secretion of INF-γ in the co-culture of a Lactobacillus strain and peripheral blood mononuclear cells (PBMC). The secretions of INF-γ were detected with ELISA after the 12, 48, and 72-hour co-culture of the lactic acid bacterium and PBMCs, respectively. The amounts of INF-γ were expressed by the absorbance values (O.D. values). In the test,  Lactobacillus casei  CCRC 10697 was used as positive control;  Lactobacillus delbrueckii  subsp.  bulgaricus  CCRC 14071 was used as negative control;  Lactobacillus paracasei  subsp.  paracasei  CCRC 14023 was tested. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
     [0013] According to the invention, some Lactobacillus strains stimulating INF-γ secretion are unexpectedly found, and can be used for treating allergy.  
     [0014] In one aspect, the invention provides a method for treating allergy in a subject comprising administrating said subject with a medicament comprising a lactic acid bacterial strain stimulating INF-γ secretion, which is selected from the group consisting of  Lactobacillus plantarum  CCRC 12944,  Lactobacillus acidophilus  CCRC 14079,  Lactobacillus rhamnosus  CCRC 10940,  Lactobacillus paracasei  subsp.  paracasei  CCRC 14023,  Lactobacillus delbrueckii  subsp.  bulgaricus  CCRC 12297,  Lactobacillus delbrueckii  subsp.  bulgaricus  CCRC 14007, and  Lactobacillus delbrueckii  subsp.  bulgaricus  CCRC 14069, which are all deposited at the Food Industry Research and Development Institute (FIRDI), Hsinchu, Taiwan. The above-mentioned strains became available to the public from the FIRDI. They are safe, natural, nontoxic, and meet the G.R.A.S. (General Regarded as Safe) standard. The strains are commonly used in food and are not harmful to humans.  
     [0015] According to the invention, the strains were proved to have the ability to stimulate INF-γ secretion when co-incubated with lymphocytes. One of the strains was proved to stimulate INF-γ secretion of the peripheral blood mononuclear cells (PBMCs). In the most preferred embodiment of the invention,  Lactobacillus paracasei  subsp.  paracasei  CCRC 14023 was found to have a better (four-fold) ability to stimulate INF-γ secretion than  Lactobacillus casei  CCRC 10697 as positive control.  
     [0016] According to the invention, the lactic acid bacterial strain used in the treatment of allergy can be live or inactive. For instance, the live bacterial strains can be treated with a heating step or other treatments commonly used in the art for killing the lactic acid bacterial strains to obtain inactive strains.  
     [0017] The term “allergy” used herein refers to INF-γ mediated allergy. The allergic disorders include rhinitis, sinusitis, asthma, hypersensitive pneumonia, extrinsic allergic alveolitis, conjunctivitis, urticaria, eczema, dermatitis, anaphylaxis, angioedema, allergic and migraine headache, and certain gastrointestinal disorders. It has been proven that atopic eczema is treatable with the probiotics stimulating INF-γ secretion (Isolauri E et al. Probiotics in the management of atopic eczema.  Clinical and experimental Allergy  2000; 30:1604-1610; Sutas Y et al. Suppression of lymphocyte proliferation in vitro by bovine caseins hydrolyzed with  Lactobacillus casei  GG-derived enzyme.  J Allergy Clin Immunol  1996; 98:216-224; Kalliomaki M et al. Probiotics in primary prevention of atopic disease: a randomized placebo-controlled trial.  Lancet  2001; 357:1076-79).  
     [0018] In another aspect, the invention provides a composition for treating allergy comprising a lactic acid bacterial strain stimulating INF-γ secretion in a therapeutically effective amount to treat allergy, which strain is selected from the group consisting of  Lactobacillus plantarum  CCRC 12944,  Lactobacillus acidophilus  CCRC 14079,  Lactobacillus rhamnosus  CCRC 10940,  Lactobacillus paracasei  subsp.  paracasei  CCRC 14023,  Lactobacillus delbrueckii  subsp.  bulgaricus  CCRC 12297,  Lactobacillus delbrueckii  subsp.  bulgaricus  CCRC 14007, and  Lactobacillus delbrueckii  subsp.  bulgaricus  CCRC 14069.  
     [0019] According to the invention, the lactic acid bacterial strain can be included in a pharmaceutical composition, dietary supplement, food or the components thereof, which are normally administrated by people. In a preferred embodiment of the invention, the lactic acid bacterial strain can be delivered in food form, such as in a coagulated milk product that prepared through the fermentation of lactic acid in milk. The food products prepared according to the invention can be conveniently administrated to infants or children.  
     [0020] The following Examples are given for the purpose of illustration only and are not intended to limit the scope of the present invention.  
     EXAMPLE 1  
     [0021] Screening of Lactic Acid Bacterial Strains Stimulating INF-γ Secretion in Lymphocytes  
     [0022] Bacterial Culture: Sixty-seven lactic acid bacterial strains listed in Table 1 were pre-selected. The strains as positive control (PC) and negative control (NC) were also illustrated. All strains were purchased from the FIRDI.  
                       TABLE 1                       No.   Lactic acid bacterial strain   CCRC No.                  PC     Lactobacillus casei     10697       NC     Lactobacillus delbrueckii  subsp.  bulgaricus     14071        1     Lactobacillus plantarum     10069        2     Lactobacillus plantarum     10357        3     Lactobacillus plantarum     11697        4     Lactobacillus plantarum     12250        5     Lactobacillus plantarum     12251        6     Lactobacillus plantarum     12327        7     Lactobacillus plantarum     12944        8     Lactobacillus plantarum     14059        9     Lactobacillus plantarum     15478       10     Lactobacillus johnsonii     14004       11     Lactobacillus acidophilus     14026       12     Lactobacillus rhamnosus     14029       13     Lactobacillus acidophilus     14064       14     Lactobacillus acidophilus     14065       15     Lactobacillus acidophilus     14079       16     Lactobacillus  sp.   16000       17     Lactobacillus acidophilus     16092       18     Lactobacillus acidophilus     16099       19     Lactobacillus acidophilus     17009       20     Lactobacillus acidophilus     17064       21     Lactobacillus acidophilus     10695       22     Lactobacillus casei  subsp.  casei     10358       23     Lactobacillus rhamnosus     10940       24     Lactobacillus casei  subsp.  casei     11197       25     Lactobacillus rhamnosus     11673       26     Lactobacillus paracasei  subsp.  paracasei     12193       27     Lactobacillus paracasei  subsp.  paracasei     12248       28     Lactobacillus casei  subsp.  casei     12249       29     Lactobacillus casei  subsp.  casei     12272       30     Lactobacillus paracasei  subsp.  paracasei     14001       31     Lactobacillus paracasei  subsp.  paracasei     14023       32     Lactobacillus casei  subsp.  casei     14025       33     Lactobacillus casei  subsp.  casei     14073       34     Lactobacillus casei  subsp.  casei     14074       35     Lactobacillus casei  subsp.  casei     14080       36     Lactobacillus casei  subsp.  casei     14082       37     Lactobacillus casei  subsp.  casei     14083       38     Lactobacillus casei  subsp.  casei     14084       39     Lactobacillus casei  subsp.  casei     14705       40     Lactobacillus casei  subsp.  casei     16093       41     Lactobacillus casei  subsp.  casei     16094       42     Lactobacillus paracasei  subsp.  paracasei     16100       43     Lactobacillus casei  subsp.  casei     17001       44     Lactobacillus casei  subsp.  casei     17002       45     Lactobacillus casei  subsp.  casei     17004       46     Lactobacillus casei  subsp.  casei     17005       47     Lactobacillus delbrueckii  subsp.  bulgaricus     10696       48     Lactobacillus helveticus     11052       49     Lactobacillus delbrueckii  subsp.  bulgaricus     12255       50     Lactobacillus delbrueckii  subsp.  bulgaricus     12297       51     Lactobacillus delbrueckii  subsp.  bulgaricus     14007       52     Lactobacillus delbrueckii  subsp.  bulgaricus     14008       53     Lactobacillus delbrueckii  subsp.  bulgaricus     14009       54     Lactobacillus delbrueckii  subsp.  bulgaricus     14010       55     Lactobacillus delbrueckii  subsp.  bulgaricus     14069       56     Lactobacillus delbrueckii  subsp.  bulgaricus     14075       57     Lactobacillus delbrueckii  subsp.  bulgaricus     14077       58     Lactobacillus delbrueckii  subsp.  bulgaricus     14090       59     Lactobacillus delbrueckii  subsp.  bulgaricus     14091       60     Lactobacillus delbrueckii  subsp.  bulgaricus     14098       61     Lactobacillus deibrueckii  subsp.  bulgaricus     16050       62     Lactobacillus delbrueckii  subsp.  bulgaricus     16051       63     Lactobacillus delbrueckii  subsp.  bulgaricus     16052       64     Lactobacillus delbrueckii  subsp.  bulgaricus     16053       65     Lactobacillus paracasei  subsp.  paracasei     12188       66     Lactobacillus brevis     12247       67     Lactobacillus brevis     14060                  
 
     [0023] Among them, thirty-eight strains were safe, natural, nontoxic, and met the G.R.A.S. (General Regarded as Safe) standard. All of the strains were cultured in Lactobacillus MRS broth (DIFCO 0881) at 37° C. to the stationary phase, and collected by centrifuging at 3000 g for 15 minutes and washed with 2 mL and 1 mL PBS (phosphate buffered saline, pH 7.2). The cultures of the strains were re-suspended in 1 mL PBS and then heated at 95° C. for 30 minutes, and then were autoclaved and stored in PBS at −20° C.  
     [0024] Lymphocyte Culture: HL-60 CCRC 60273 (Clone 15 HL-60) cells (purchased from the FIRDI) were treated according to the method described by Fischkoff (Fischkoff S. A. Graded increase in probability of eosinophilic differentiation of HL-60 promyelocytic leukemia cells induced by culture under alkaline condition.  Leukemia Research  1988; 12(8): 679-686). The HL-60 cells were subcultured in RPMI 1640 (pH 7.2) and induced to differentiate to eosinophils and then subcultured with RPMI 1640 (pH 7.7) for several generations to obtain lymphocyte samples. In each lymphocyte sample, the cell density was adjusted to 5×10 6  cells per sample. The lymphocyte samples were incubated in 2 mL RPMI 1640 (pH 7.7) for 6 hours.  
     [0025] Stimulating INF-γ Secretion: The lymphocyte samples were co-cultured with a given amount of the above-mentioned bacterial strains.  Lactobacillus casei  CCRC 10697 was taken as a positive control and  Lactobacillus delbrueckii  subsp.  bulgaricus  CCRC 14071 as a negative control. After the 12, 36 and 60-hour co-culture, the cells in each sample were collected, respectively. The collected cells were re-suspended and centrifuged at 2000 rpm for 5 minutes. The supernatant were taken for the determination of the INF-γ level in each sample.  
     [0026] Determination of INF-γ Level: The method for determining INF-γ level by ELISA was described by Shida et al. (Shida K., Makino K., Morishita A., Takamizawa K., Hachimura S., Ametani A., Takehito S., Kumagai Y., Habu S., Kaminogawa S.  Lactobacillus casei  inhibits antigen induced IgE secretion through regulation of cytokine production in murine splenocyte cultures.  Int Arch Allergy Immunol  1998; 115:278-287) comprising the steps of:  
     [0027] adding 150 μL of 2.5 μg/mL purified mouse anti-human INF-γ antibodies in coating buffer (8.00 g NaCl, 0.20 g KCl, 1.44 g Na 2 HPO 4 , 0.24 g KH 2 PO 4 , 30.0 g bovine serum albumin, and 0.50 g NaN 3  per liter, pH 7.4) into each well of an ELISA plate;  
     [0028] shaking the plate at 40 rpm at room temperature;  
     [0029] incubating the plate at 4° C. overnight;  
     [0030] discarding the coating buffer in the wells;  
     [0031] washing each well of the plate with wash buffer (8.00 g NaCl, 0.20 g KCl, 1.44 g Na 2 HPO 4 , 0.24 g KH 2 PO 4 , 0.5 mL Tween 20, and 0.50g NaN 3  per liter, pH 7.4) for 3 minutes twice;  
     [0032] washing the wells with distilled water;  
     [0033] adding 200 μL block buffer into each well of the plate;  
     [0034] incubating the plate at room temperature for at least 2 hours;  
     [0035] discarding the block buffer in the wells;  
     [0036] washing each well of the plate with wash buffer for 3 minutes three times;  
     [0037] washing each well of the plate with distilled water;  
     [0038] taking the supernatant of the lymphocyte sample and adding it to each well of the plate;  
     [0039] shaking the plate at 40 rpm at 4° C. overnight;  
     [0040] discarding the samples in the wells;  
     [0041] washing each well of the plate with wash buffer for 3 minutes three times and then with distilled water;  
     [0042] adding 150 μL biotin mouse anti-human INF-γ antibodies diluted with dilute buffer into each well of the plate;  
     [0043] incubating the plate for 2 hours at room temperature;  
     [0044] washing each well of the plate with wash buffer for 3 minutes three times and then with distilled water;  
     [0045] adding 150 μL Streptavidin-Alkaline phosphatase (Streptavidin-AKP) diluted with dilute buffer into each well of the plate;  
     [0046] incubating the plate for 1 hour at room temperature;  
     [0047] washing each well of the plate with wash buffer for 3 minutes four times and then with distilled water;  
     [0048] adding 150 μL of substrate p-Nitrophenyl phosphate (pNpp) into each well of the plate;  
     [0049] incubating the plates at room temperature until the substrate reaction is completed;  
     [0050] measuring the absorbance of each well of the plate at 405 nm (i.e. OD405).  
     [0051] Result: The results of INF-γ level stimulated by the 68 lactic acid bacterial strains are listed in Table 2.  
                           TABLE 2                       CCRC No.   12 hours (OD)   36 hours (OD)   60 hours (OD)                  Positive Control   0.156   0.295   0.106       Negative Control   0.117   0.241   0.103       10069   0.117   0.304   0.107       10357   0.129   0.267   0.104       11697   0.112   0.397   0.104       12250   0.122   0.335   0.156       12251   0.177   0.293   0.110       12327   0.131   0.289   0.111       12944   0.152   0.427   0.092       14059   0.111   0.363   0.102       15478   0.157   0.385   0.109       14004   0.162   0.399   0.106       14026   0.115   0.405   0.103       14029   0.131   0.272   0.110       14064   0.114   0.337   0.164       14065   0.159   0.244   0.110       14079   0.142   0.342   0.099       16000   0.123   0.255   0.105       16092   0.127   0.254   0.114       16099   0.114   0.262   0.114       17009   0.111   0.276   0.117       17064   0.147   0.272   0.114       10695   0.131   0.274   0.118       10358   0.148   0.271   0.119       10697   0.160   0.340   0.098       10940   0.336   0.335   0.109       11197   0.150   0.293   0.104       11673   0.109   0.298   0.106       12193   0.116   0.305   0.111       12248   0.160   0.284   0.112       12249   0.142   0.267   0.112       12272   0.120   0.276   0.112       14001   0.173   0.410   0.108       14023   0.120   0.538   0.125       14025   0.142   0.339   0.110       14073   0.157   0.398   0.104       14074   0.125   0.455   0.117       14080   0.124   0.308   0.116       14082   0.148   0.248   0.113       14083   0.129   0.203   0.116       14084   0.153   0.335   0.121       14705   0.159   0.277   0.122       16093   0.131   0.328   0.127       16094   0.160   0.309   0.114       16100   0.158   0.316   0.121       17001   0.219   0.252   0.123       17002   0.155   0.207   0.120       17004   0.236   0.112   0.119       17005   0.125   0.320   0.104       10696   0.122   0.373   0.122       11052   0.142   0.316   0.107       12255   0.118   0.325   0.116       12297   0.121   0.418   0.105       14007   0.122   0.502   0.110       14008   —   0.359   0.100       14009   0.224   0.293   0.103       14010   0.150   0.312   0.100       14069   0.146   0.440   0.161       14071   0.144   0.270   0.099       14075   0.152   0.319   0.100       14077   0.163   0.342   0.102       14090   0.203   0.302   0.106       14091   0.184   0.288   0.097       14098   0.147   0.242   0.101       16050   0.136   0.264   0.098       16051   0.135   0.250   0.103       16052   0.132   0.386   0.104       16053   0.132   0.314   0.113       12188   0.150   0.263   0.101       12247   0.137   0.246   0.103       14060   0.167   0.328   0.103                  
 
     [0052] Among the 67 strains, the following seven strains were found to be capable of stimulating INF-γ secretion in lymphocyte cells:  Lactobacillus plantarum  CCRC 12944,  Lactobacillus acidophilus  CCRC 14079,  Lactobacillus rhamnosus  CCRC 10940,  Lactobacillus paracasei  subsp.  paracasei  CCRC 14023, and  Lactobacillus delbrueckii  subsp.  bulgaricus  CCRC 12297, 14007 and 14069. The results are shown in FIG. 1. The OD405 values of  Lactobacillus paracasei  subsp.  paracasei  CCRC 14023, and  Lactobacillus delbrueckii  subsp.  bulgaricus  CCRC 12297, 14007 and 14069 are higher than the positive control, and even higher than the negative control by four folds. Besides, the OD405 values of the strains except  Lactobacillus rhamnosus  (CCRC 10940) collected after the 36-hour co-culture are 3-fold higher than those after 12-hour co-culture.  
     EXAMPLE 2  
     [0053] INF-γ Secretions in Peripheral Blood Mononuclear Cells by Stimulation of Lactic Acid Bacteria  
     [0054] Isolation of Peripheral Blood Mononuclear Cells: Five mL blood samples derived from healthy volunteers were added with 5 mL Ficoll-Hypaque (17-1400-02, Pharmacia) and then centrifuged at 500 g for 30 minutes. The peripheral blood mononuclear cells (PBMCs) were taken from the interface of the samples, and washed twice with PBS. The PBMCs (10 5  cells/mL) were transferred to the wells of a six-well plate wherein each well contained 2 mL RPMI 1640 medium of pH 7.7.  
     [0055] Stimulating INF-γ Secretion: Using the analogous method described in Example 1, the PBMCs were co-cultured with  Lactobacillus plantarum  CCRC 12944,  Lactobacillus acidophilus  CCRC 14079,  Lactobacillus rhamnosus  CCRC 10940,  Lactobacillus paracasei  subsp.  paracasei  CCRC 14023, and  Lactobacillus delbrueckii  subsp.  bulgaricus  CCRC 12297, 14007 and 14069 (10 7  cells/mL).  Lactobacillus casei  CCRC 10697 was taken as a positive control and  Lactobacillus delbrueckii  subsp.  bulgaricus  CCRC 14071 was taken as a negative control. The cells were collected after the 24, 48 and 72-hour co-culture, and re-suspended and centrifuged at 2000 rpm for 5 minutes. The supernatant were taken for determining the INF-γ levels by the same method described in Example 1.  
     [0056] Results: The Results of the amount of INF-γ of PBMCs stimulated by the seven strains are listed in Table 3, and especially, the result by  Lactobacillus paracasei  subsp.  paracasei  CCRC 14023 is shown in FIG. 2.  
                                   TABLE 3                                               INF-γ conc.           Time   CCRC Nos.   OD   (ng/ml)                                                            24 hours   Positive control   0.1945   861.5               12944   0.1685   731.5               14079   0.1895   836.5               10940   0.223   1004               14023   0.23   1039               12297   0.195   864               14007   0.165   714               14069   0.2015   896.5           48 hours   Positive control   0.2095   936.5               12944   0.1605   691.5               14079   0.244   1109               10940   0.305   1414               14023   0.267   1224               12297   0.1555   666.5               14007   0.141   594               14069   0.165   714           72 hours   Positive control   0.2575   1176.5               12944   0.159   684               14079   0.17   739               10940   0.193   854               14023   0.1895   836.5               12297   0.147   624               14007   0.133   554               14069   0.17   739                      
 
     [0057] The OD405 value of the sample collected after the 24-hour co-culture is 1.2-fold higher than the negative control; that collected after the 48-hour co-culture is 1.8-fold higher than the negative control and 1.3-fold higher than the positive control; and that collected after the 72-hour co-culture is 1.3-fold higher than the negative control.  
     [0058] While embodiments of the present invention have been illustrated and described, various modifications and improvements can be made by persons skilled in the art. It is intended that the present invention is not limited to the particular forms as illustrated, and that all the modifications not departing from the spirit and scope of the present invention are within the scope as defined in the appended claims.