Abstract:
A method for inhibiting bacterial adhesion of oral pathogens, comprising administering a heat-inactivated  Lactobacillus  strain to a subject in need to inhibit bacterial adhesion of oral pathogens is disclosed. The  Lactobacillus  strain is selected from  Lactobacillus paracasei  GMNL-143,  Lactobacillus helveticus  GMNL-164 or  Lactobacillus rhamnosus  GMNL-464. A composition for inhibiting bacterial adhesion of oral pathogens is also disclosed.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims the benefit of Taiwan application serial No. 105110614, filed on Apr. 1, 2016, and the subject matter of which is incorporated herein by reference. 
       BACKGROUND OF THE INVENTION 
     Field of the Invention 
       [0002]    The present invention generally relates to a method for inhibiting bacterial adhesion of oral pathogens and, more particularly, to a method for inhibiting bacterial adhesion of oral pathogens by administering a heat-inactivated  Lactobacillus  strain. The present invention further relates to a composition for inhibiting bacterial adhesion of oral pathogens. 
       Description of the Related Art 
       [0003]    The mouth harbors a diverse, abundant and complex microbiota, which probably accumulates on both the hard and soft oral tissues in biofilms. The microbiota may not only vary according to change in oral environment such as the appearance of the teeth, but also be different because of the dietary and living habits. 
         [0004]    Oral microbiota plays an important role in the dental diseases. For example,  Streptococcus mutans  produces acidic substances following fermentation of dietary sugars, destroying the tooth surface (enamel) and resulting in dental caries. Biofilms surrounding the gingival crevice area formed by anaerobic bacterium such as  Porphyromonas gingivalis  or  Prevotella intermedia  may result in inflammation and periodontal disease. Moreover, the volatile sulfur compounds (VSCs) produced by breakdown of sulfur containing amino acids by  P. gingivalis, P. intermedia  or  Fusobacterium nucleatum  are the major reason of halitosis. Therefore, regulating the distribution of oral microbiota by probiotics can prevent from dental diseases, such as dental caries, periodontal disease and halitosis. 
         [0005]    In light of this, it is necessary to provide a method and a composition for inhibiting bacterial adhesion of oral pathogens. 
       SUMMARY OF THE INVENTION 
       [0006]    It is therefore the objective of this invention to provide a method with a heat-inactivated  Lactobacillus  strain to inhibit bacterial adhesion of oral pathogens. 
         [0007]    It is another objective of this invention to provide a composition including a heat-inactivated  Lactobacillus  strain to inhibit bacterial adhesion of oral pathogens. 
         [0008]    A first embodiment of the invention discloses a method for inhibiting bacterial adhesion of oral pathogens. The method comprises administering a heat-inactivated  Lactobacillus  strain to a subject in need to inhibit bacterial adhesion of oral pathogens. The  Lactobacillus  strain is selected from  Lactobacillus paracasei  GMNL-143,  Lactobacillus helveticus  GMNL-164 or  Lactobacillus rhamnosus  GMNL-464. The  L. paracasei  GMNL-143 is deposited at CGMCC-China General Microbiological Culture Collection with deposit number CCTCC No. M2014301. The  L. helveticus  GMNL-164 is deposited at CGMCC-China General Microbiological Culture Collection with deposit number CCTCC No. M2015609. The  L. rhamnosus  GMNL-464 is deposited at CGMCC-China General Microbiological Culture Collection with deposit number CCTCC No. M2015610. The  Lactobacillus  strain is heat-inactivated at 90-121° C. for 15-30 minutes. 
         [0009]    The  Lactobacillus  strain is orally administered to the subject in need thereof at dosage of 3×10 8 -1×10 10  cells/time. 
         [0010]    A second embodiment of the invention discloses a composition for inhibiting bacterial adhesion of oral pathogens. The composition includes a heat-inactivated  Lactobacillus  strain and a medical acceptable excipient. The heat-inactivated  Lactobacillus  strain is selected from a group consisting of  Lactobacillus paracasei  GMNL-143,  Lactobacillus helveticus  GMNL-164 and  Lactobacillus rhamnosus  GMNL-464. The  L. paracasei  GMNL-143 is deposited at CGMCC-China General Microbiological Culture Collection with deposit number CCTCC No. M2014301. The  L. helveticus  GMNL-164 is deposited at CGMCC-China General Microbiological Culture Collection with deposit number CCTCC No. M2015609, wherein the  L. rhamnosus  GMNL-464 is deposited at CGMCC-China General Microbiological Culture Collection with deposit number CCTCC No. M2015610. The  Lactobacillus  strain is heat-inactivated at 90-121° C. for 15-30 minutes. 
         [0011]    The composition includes the  L. paracasei  GMNL-143, the  L. helveticus  GMNL-164 and the  L. rhamnosus  GMNL-464 in the ratio 1:1:1. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0012]    The present invention will become more fully understood from the detailed description given hereinafter and the accompanying drawings which are given by way of illustration only, and thus are not limitative of the present invention, and wherein: 
           [0013]      FIG. 1 a    depicts a Giemsa stain showing the adhesion of  S. mutans  on the slide after treating by PBS in trial (A). 
           [0014]      FIG. 1 b    depicts a Giemsa stain showing the adhesion of  S. mutans  on the slide after treating by  L. paracasei  GMNL-33 in trial (A). 
           [0015]      FIG. 1 c    depicts a Giemsa stain showing the adhesion of  S. mutans  on the slide after treating by  L. paracasei  GMNL-143 in trial (A). 
           [0016]      FIG. 2 a    depicts a Giemsa stain showing the adhesion of  S. mutans  on the slide after treating by PBS in trial (B). 
           [0017]      FIG. 2 b    depicts a Giemsa stain showing the adhesion of  S. mutans  on the slide after treating by  L. paracasei  GMNL-33 in trial (B). 
           [0018]      FIG. 2 c    depicts a Giemsa stain showing the adhesion of  S. mutans  on the slide after treating by  L. paracasei  GMNL-143 in trial (B). 
           [0019]      FIG. 3  depicts a bar chart showing the adhesion of  P. gingivalis  on SG cells after treating by PBS (group C0),  L. paracasei  GMNL-33 (group C1) and  L. helveticus  GMNL-164 (group C2) in trial (C). 
           [0020]      FIG. 4  depicts a bar chart showing the adhesion of  P. intermedia  on SG cells after treating by PBS (group D0),  L. paracasei  GMNL-33 (group D1) and  L. helveticus  GMNL-164 (group D2) in trial (D). 
           [0021]      FIG. 5  depicts a bar chart showing the adhesion of  F. nucleatum  on SG cells after treating by PBS (group E0),  L. paracasei  GMNL-33 (group E1) and  L. rhamnosus  GMNL-464 (group E2) in trial (E). 
           [0022]      FIG. 6 a    depicts a point chart showing the relative level of oral  S. mutans  before and after gargling with control mouthwash (group F0) in trial (F). 
           [0023]      FIG. 6 b    depicts a point chart showing the relative level of oral  P. gingivalis  before and after gargling with control mouthwash (group F0) in trial (F). 
           [0024]      FIG. 6 c    depicts a point chart showing the relative level of oral  F. nucleatum  before and after gargling with control mouthwash (group F0) in trial (F). 
           [0025]      FIG. 6 d    depicts a point chart showing the relative level of total oral bacteria before and after gargling with control mouthwash (group F0) in trial (F). 
           [0026]      FIG. 7 a    depicts a point chart showing the relative level of oral  S. mutans  before and after gargling with the low-dose mouthwash (group F1) in trial (F). 
           [0027]      FIG. 7 b    depicts a point chart showing the relative level of oral  P. gingivalis  before and after gargling with the low-dose mouthwash (group F1) in trial (F). 
           [0028]      FIG. 7 c    depicts a point chart showing the relative level of oral  F. nucleatum  before and after gargling with the low-dose mouthwash (group F1) in trial (F). 
           [0029]      FIG. 7 d    depicts a point chart showing the relative level of total oral bacteria before and after gargling with the low-dose mouthwash (group F1) in trial (F). 
           [0030]      FIG. 8 a    depicts a point chart showing the relative level of oral  S. mutans  before and after gargling with the middle-dose mouthwash (group F2) in trial (F). 
           [0031]      FIG. 8 b    depicts a point chart showing the relative level of oral  P. gingivalis  before and after gargling with the middle-dose mouthwash (group F2) in trial (F). 
           [0032]      FIG. 8 c    depicts a point chart showing the relative level of oral  F. nucleatum  before and after gargling with the middle-dose mouthwash (group F2) in trial (F). 
           [0033]      FIG. 8 d    depicts a point chart showing the relative level of total oral bacteria before and after gargling with the middle-dose mouthwash (group F2) in trial (F). 
           [0034]      FIG. 9 a    depicts a point chart showing the relative level of oral  S. mutans  before and after gargling with the high-dose mouthwash (group F3) in trial (F). 
           [0035]      FIG. 9 b    depicts a point chart showing the relative level of oral  P. gingivalis  before and after gargling with the high-dose mouthwash (group F3) in trial (F). 
           [0036]      FIG. 9 c    depicts a point chart showing the relative level of oral  F. nucleatum  before and after gargling with the high-dose mouthwash (group F3) in trial (F). 
           [0037]      FIG. 9 d    depicts a point chart showing the relative level of total oral bacteria before and after gargling with the high-dose mouthwash (group F3) in trial (F). 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0038]    A  Lactobacillus  strain according to the present invention is selected from a group consisting of a  Lactobacillus paracasei  GMNL-143, a  Lactobacillus helveticus  GMNL-164 and a  Lactobacillus rhamnosus  GMNL-464. 
         [0039]    In detail, the  L. paracasei  GMNL-143 is isolated from human intestine with morphology after culture at 37° C. for 48 hours includes: circular, retractile, entire edge, 1.0 mm×1.0 mm, milky-white color, smooth surface and convex elevation. Gram-staining shows the  L. paracasei  GMNL-143 is a gram-positive strain. The  L. paracasei  GMNL-143 is a rod type bacilli, which cannot form spore and has no motility. The physiological properties of the  L. paracasei  GMNL-143 includes: growth temperature is 25-45° C., growth pH is pH 4.0-10.0 and belongs to facultative anaerobic bacteria. 
         [0040]    Moreover, the  L. paracasei  GMNL-143 has a partial 16S rDNA sequence set forth as SEQ ID NO: 1 and is identified to  L. paracasei  compared to NCBI database. The sugar availability of the  L. paracasei  GMNL-143 is shown in TABLE 1. 
         [0000]    
       
         
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 carbohydrates substrate 
                 result 
                 carbohydrates substrate 
                 result 
               
               
                   
               
             
             
               
                 control 
                 − 
                 esculin ferric citrate 
                 + 
               
               
                 glycerol 
                 − 
                 salicin 
                 + 
               
               
                 erythritol 
                 − 
                 D-cellobiose 
                 + 
               
               
                 D-arabinose 
                 − 
                 D-maltose 
                 + 
               
               
                 L-arabinose 
                 − 
                 D-lactose 
                 + 
               
               
                 D-ribose 
                 + 
                 D-melibiose 
                 − 
               
               
                 D-xylose 
                 − 
                 D-sucrose 
                 + 
               
               
                 L-xylose 
                 − 
                 D-trehalose 
                 + 
               
               
                 D-adonitol 
                 + 
                 inulin 
                 − 
               
               
                 methyl-β-D-xylopyranoside 
                 − 
                 D-melezitose 
                 + 
               
               
                 D-lactosegalactose 
                 + 
                 D-raffinose 
                 − 
               
               
                 D-glucose 
                 + 
                 starch 
                 − 
               
               
                 D-fructose 
                 + 
                 glycogen 
                 − 
               
               
                 D-mannose 
                 + 
                 xylitol 
                 − 
               
               
                 L-sorbose 
                 + 
                 gentiobiose 
                 + 
               
               
                 L-rhamnose 
                 − 
                 D-turanose 
                 + 
               
               
                 lactosedulcitol 
                 − 
                 D-lyxose 
                 + 
               
               
                 inositol 
                 + 
                 D-tagatose 
                 + 
               
               
                 D-mannitol 
                 + 
                 D-fucose 
                 − 
               
               
                 D-sorbitol 
                 + 
                 L-fucose 
                 − 
               
               
                 methyl-α-D-mannopyranoside 
                 − 
                 D-arabitol 
                 − 
               
               
                 methyl-α-D-glucopyranoside 
                 − 
                 L-arabitol 
                 − 
               
               
                 N-acetyl glucosamine 
                 + 
                 potassium gluconate 
                 + 
               
               
                 amygdalin 
                 + 
                 potassium 
                 − 
               
               
                   
                   
                 2-ketogluconate 
               
               
                 arbutin 
                 + 
                 potassium 
                 − 
               
               
                   
                   
                 5-ketogluconate 
               
               
                   
               
             
          
         
       
     
         [0041]    The  L. helveticus  GMNL-164 is isolated from human intestine with morphology after culture at 37° C. for 48 hours includes: circular, undulated edge, 1.2 mm×1.2 mm, gray-white color, dull surface and flat elevation. Gram-staining shows the  L. helveticus  GMNL-164 is a gram-positive strain. The  L. helveticus  GMNL-164 is a rod type bacilli, which cannot form spore and has nomotility. The physiological properties of the  L. helveticus  GMNL-164 includes: growth temperature is 25-45° C., growth pH is pH 4.0-10.0 and belongs to facultative anaerobic bacteria. 
         [0042]    Moreover, the  L. helveticus  GMNL-164 has a partial 16S rDNA sequence set forth as SEQ ID NO: 2 and is identified to  L. helveticus  compared to NCBI database. The sugar availability of the  L. helveticus  GMNL-164 is shown in TABLE 2. 
         [0000]    
       
         
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 carbohydrates substrate 
                 result 
                 carbohydrates substrate 
                 result 
               
               
                   
               
             
             
               
                 control 
                 − 
                 esculin ferric citrate 
                 − 
               
               
                 glycerol 
                 − 
                 salicin 
                 − 
               
               
                 erythritol 
                 − 
                 D-cellobiose 
                 − 
               
               
                 D-arabinose 
                 − 
                 D-maltose 
                 − 
               
               
                 L-arabinose 
                 − 
                 D-lactose 
                 + 
               
               
                 D-ribose 
                 − 
                 D-melibiose 
                 − 
               
               
                 D-xylose 
                 − 
                 D-sucrose 
                 − 
               
               
                 L-xylose 
                 − 
                 D-trehalose 
                 − 
               
               
                 D-adonitol 
                 − 
                 inulin 
                 − 
               
               
                 methyl-β-D-xylopyranoside 
                 − 
                 D-melezitose 
                 − 
               
               
                 D-lactosegalactose 
                 − 
                 D-raffinose 
                 − 
               
               
                 D-glucose 
                 + 
                 starch 
                 − 
               
               
                 D-fructose 
                 + 
                 glycogen 
                 − 
               
               
                 D-mannose 
                 + 
                 xylitol 
                 − 
               
               
                 L-sorbose 
                 − 
                 gentiobiose 
                 − 
               
               
                 L-rhamnose 
                 − 
                 D-turanose 
                 − 
               
               
                 lactosedulcitol 
                 − 
                 D-lyxose 
                 − 
               
               
                 inositol 
                 − 
                 D-tagatose 
                 − 
               
               
                 D-mannitol 
                 − 
                 D-fucose 
                 − 
               
               
                 D-sorbitol 
                 − 
                 L-fucose 
                 − 
               
               
                 methyl-α-D-mannopyranoside 
                 − 
                 D-arabitol 
                 − 
               
               
                 methyl-α-D-glucopyranoside 
                 − 
                 L-arabitol 
                 − 
               
               
                 N-acetyl glucosamine 
                 + 
                 potassium gluconate 
                 − 
               
               
                 amygdalin 
                 − 
                 potassium 
                 − 
               
               
                   
                   
                 2-ketogluconate 
               
               
                 arbutin 
                 − 
                 potassium 
                 − 
               
               
                   
                   
                 5-ketogluconate 
               
               
                   
               
             
          
         
       
     
         [0043]    The  L. rhamnosus  GMNL-464 is isolated from human intestine with morphology after culture at 37° C. for 48 hours includes: circular, glistening, entire edge. 1.5 mm×1.5 mm, milky-white color, smooth surface and convex elevation. Gram-staining shows the  L. rhamnosus  GMNL-464 is a gram-positive strain. The  L. rhamnosus  GMNL-464 is a rod type bacilli, which cannot form spore and has no motility. The physiological properties of the  L. rhamnosus  GMNL-464 includes: growth temperature is 25-45° C., growth pH is pH 4.0-10.0 and belongs to facultative anaerobic bacteria. 
         [0044]    Moreover, the  L. rhamnosus  GMNL-464 has a partial 16S rDNA sequence set forth as SEQ ID NO: 3 and is identified to  L. rhamnosus  compared to NCBI database. The sugar availability of the  L. rhamnosus  GMNL-464 is shown in TABLE 3. 
         [0000]    
       
         
               
               
               
               
             
           
               
                 TABLE 3 
               
               
                   
               
               
                 carbohydrates substrate 
                 result 
                 carbohydrates substrate 
                 result 
               
               
                   
               
             
             
               
                 control 
                 − 
                 esculin ferric citrate 
                 + 
               
               
                 glycerol 
                 + 
                 salicin 
                 + 
               
               
                 erythritol 
                 − 
                 D-cellobiose 
                 + 
               
               
                 D-arabinose 
                 − 
                 D-maltose 
                 + 
               
               
                 L-arabinose 
                 − 
                 D-lactose 
                 + 
               
               
                 D-ribose 
                 − 
                 D-melibiose 
                 − 
               
               
                 D-xylose 
                 − 
                 D-sucrose 
                 + 
               
               
                 L-xylose 
                 − 
                 D-trehalose 
                 + 
               
               
                 D-adonitol 
                 − 
                 inulin 
                 − 
               
               
                 methyl-β-D-xylopyranoside 
                 − 
                 D-melezitose 
                 + 
               
               
                 D-lactosegalactose 
                 + 
                 D-raffinose 
                 − 
               
               
                 D-glucose 
                 + 
                 starch 
                 + 
               
               
                 D-fructose 
                 + 
                 glycogen 
                 − 
               
               
                 D-mannose 
                 + 
                 xylitol 
                 − 
               
               
                 L-sorbose 
                 − 
                 gentiobiose 
                 + 
               
               
                 L-rhamnose 
                 + 
                 D-turanose 
                 + 
               
               
                 lactosedulcitol 
                 + 
                 D-lyxose 
                 − 
               
               
                 inositol 
                 + 
                 D-tagatose 
                 − 
               
               
                 D-mannitol 
                 + 
                 D-fucose 
                 − 
               
               
                 D-sorbitol 
                 + 
                 L-fucose 
                 − 
               
               
                 methyl-α-D-mannopyranoside 
                 − 
                 D-arabitol 
                 − 
               
               
                 methyl-α-D-glucopyranoside 
                 + 
                 L-arabitol 
                 − 
               
               
                 N-acetyl glucosamine 
                 + 
                 potassium gluconate 
                 + 
               
               
                 amygdalin 
                 + 
                 potassium 
                 − 
               
               
                   
                   
                 2-ketogluconate 
               
               
                 arbutin 
                 + 
                 potassium 
                 − 
               
               
                   
                   
                 5-ketogluconate 
               
               
                   
               
             
          
         
       
     
         [0045]    The  L. paracasei  GMNL-143 is deposited at CGMCC-China General Microbiological Culture Collection with deposit number CCTCC No. M2014301. The  L. helveticus  GMNL-164 is deposited at CGMCC-China General Microbiological Culture Collection with deposit number CCTCC No. M2015609. The  L. rhamnosus  GMNL-464 is deposited at CGMCC-China General Microbiological Culture Collection with deposit number CCTCC No. M2015610. 
         [0046]    Moreover, the  Lactobacillus  strain can be heat-inactivated, followed by applying to inhibit adhesion of oral pathogens. In this embodiment, for example, the  Lactobacillus  strain is heat-inactivated at 90-121° C. for 15-30 minutes. 
         [0047]    Moreover, the  Lactobacillus  strain can be mixed with a medical acceptable excipient to form a composition of inhibiting adhesion of oral pathogens. Alternatively, the  L. paracasei  GMNL-143, the  L. helveticus  GMNL-164 and the  L. rhamnosus  GMNL-464 can be first mixed to form  Lactobacillus  mixture having  L. paracasei  GMNL-143, the  L. helveticus  GMNL-164 and the  L. rhamnosus  GMNL-464 in the ratio 1:1:1, followed by forming the composition of inhibiting adhesion of oral pathogens. It is worthy to note that the  Lactobacillus  strain can also be added into a matrix such as mouthwash, toothpaste, troche, chewing gum and tooth powder to increase the ability of inhibiting adhesion of oral pathogens of the matrix thereof. Specifically, the matrix including the  Lactobacillus  strain can be applied in a dosage and time listed in TABLE 4, which is appreciated by a person having ordinary skill in the art. 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 4 
               
               
                   
               
               
                 matrix 
                   
                 dosage 
                 time 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 mouthwash 
                 1 × 10 9   
                 cells/time 
                 30 
                 seconds 
               
               
                 toothpaste 
                 7 × 10 8 -1 × 10 9   
                 cells/time 
                 1 
                 minute 
               
               
                 troche 
                 3 × 10 8   
                 cells/time 
                 10-20 
                 minutes 
               
               
                 chewing gum 
                 2 × 10 9   
                 cells/time 
                 10-30 
                 minutes 
               
               
                 tooth powder 
                 1 × 10 10   
                 cells/time 
                 1 
                 minute 
               
               
                   
               
             
          
         
       
     
         [0048]    To evaluate the  Lactobacillus  strain can inhibit the adhesion of oral pathogens, the  Lactobacillus  strain is heat-inactivated at 90-121° C. for 15-30 minutes, followed by cooling to room temperature to carry out the following trials. 
         [0049]    Trial (A):  L. paracasei  GMNL-143 inhibits adhesion of  S. mutans  (ATCC 25175) 
         [0050]    Referring to TABLE 5,  S. mutans  (ATCC 25175) is used in trail (A).  L. paracasei  GMNL-143 (0.5 mL) and  S. mutans  (ATCC 25175, 0.5 mL) is mixed and stood for 5 minutes. The upper solution (500 μL) is added into a 24-well plate with sterile slides and incubated at 37° C. for 2 hours. The upper solution is then removed and methanol is added after the slide is washed by phosphate buffer saline (PBS). Finally, the slide is stained by Giemsa stain to evaluate the number of  S. mutans  adhering on the slide. 
         [0000]    
       
         
               
               
               
             
           
               
                   
                 TABLE 5 
               
               
                   
                   
               
               
                   
                 groups 
                 condition 
               
               
                   
                   
               
             
             
               
                   
                 A0 
                 PBS +  S. mutans  (ATCC 25175) 
               
               
                   
                 A1 
                   L. paracasei  GMNL-33 +  S. mutans  (ATCC 25175) 
               
               
                   
                 A2 
                   L. paracasei  GMNL-143 +  S. mutans  (ATCC 25175) 
               
               
                   
                   
               
             
          
         
       
     
         [0051]    With reference to  FIG. 1   a, S. mutans  (ATCC 25175) adheres on the slides in the presence of PBS (group A0). In contrast, as shown in  FIGS. 1 b  and 1 c   , treatment of  L. paracasei  GMNL-33 and GMNL-143 can reduce the adhesion of  S. mutans  (ATCC 25175) on the slides with the  L. paracasei  GMNL-143 according to the present invention showing a better effect. 
         [0052]    Trial (B):  L. paracasei  GMNL-143 inhibits adhesion of  S. mutans  (BCRC 16002) 
         [0053]    Referring to TABLE 6,  S. mutans  (BCRC 16002) is used in trial (B). With respect to  FIGS. 2 a -2 c   , similar to the results mentioned above, treatment of  L. paracasei  GMNL-33 and GMNL-143 can reduce the adhesion of  S. mutans  (BCRC 16002) on the slides with the  L. paracasei  GMNL-143 according to the present invention showing a better effect. 
         [0000]    
       
         
               
               
               
             
           
               
                   
                 TABLE 6 
               
               
                   
                   
               
               
                   
                 groups 
                 condition 
               
               
                   
                   
               
             
             
               
                   
                 B0 
                 PBS +  S. mutans  (BCRC 16002) 
               
               
                   
                 B1 
                   L. paracasei  GMNL-33 +  S. mutans  (BCRC 16002) 
               
               
                   
                 B2 
                   L. paracasei  GMNL-143 +  S. mutans  (BCRC 16002) 
               
               
                   
                   
               
             
          
         
       
     
         [0054]    Trial (C):  L. helveticus  GMNL-164 inhibits adhesion of  P. gingivalis  (ATCC 33277) 
         [0055]    Smulow-Clickman gingival cells (SG cells) are used in trial (C). Referring to TABLE 7, same amount of  L. helveticus  GMNL-164 and  P. gingivalis  (ATCC 33277) are mixed. The upper solution (100 μL) is added into a 24-well plate coated SG cells (3×10 5  cells/well) and incubated at 37° C. for 2 hours. The upper solution is then removed. The SG cells in each well are collected and bacterial DNA adhering on the SG cells is extracted. Real-time PCR using primer pairs set forth as SEQ ID NOS: 4 and 5 is finally carried out and 2 −ΔCT  is calculated (as shown in  FIG. 3 ). 
         [0000]    
       
         
               
               
             
           
               
                 TABLE 7 
               
               
                   
               
               
                 groups 
                 condition 
               
               
                   
               
             
             
               
                 C0 
                 PBS +  P. gingivalis  (ATCC 33277) 
               
               
                 C1 
                   L. paracasei  GMNL-33 +  P. gingivalis  (ATCC 33277) 
               
               
                 C2 
                   L. helveticus  GMNL-164 +  P. gingivalis  (ATCC 33277) 
               
               
                   
               
             
          
         
       
     
         [0056]    Referring to  FIG. 3 , both  L. paracasei  GMNL-33 and  L. helveticus  GMNL-164 inhibit  P. gingivalis  adhering on SG cells, with  L. helveticus  GMNL-164 according to the invention shows a better effect. 
         [0057]    Trial (D):  L. helveticus  GMNL-164 inhibits adhesion of  P. intermedia  (ATCC 25611) 
         [0058]    The SG cells are also used in trial (D). Referring to TABLE 8, same amount of  L. helveticus  GMNL-164 and  P. intermedia  (ATCC 25611) are mixed. The upper solution (100 μL) is added into a 24-well plate coated SG cells (3×10 5  cells/well) and incubated at 37° C. for 2 hours. The upper solution is then removed. The SG cells in each well are collected and bacterial DNA adhering on the SG cells is extracted. Real-time PCR using primer pairs set forth as SEQ ID NOS: 6 and 7 is finally carried out and 2 −ΔCT  is calculated (as shown in  FIG. 4 ). 
         [0000]    
       
         
               
               
             
           
               
                 TABLE 8 
               
               
                   
               
               
                 groups 
                 condition 
               
               
                   
               
             
             
               
                 D0 
                 PBS +  P. intermedia  (ATCC 25611) 
               
               
                 D1 
                   L. paracasei  GMNL-33 +  P. intermedia  (ATCC 25611) 
               
               
                 D2 
                   L. helveticus  GMNL-164 +  P. intermedia  (ATCC 25611) 
               
               
                   
               
             
          
         
       
     
         [0059]    Referring to  FIG. 4 , both  L. paracasei  GMNL-33 and  L. helveticus  GMNL-164 inhibit  P. intermedia  adhering on SG cells, with  L. helveticus  GMNL-164 according to the invention shows a better effect. 
         [0060]    Trial (E):  L. rhamnosus  GMNL-464 inhibits adhesion of  Fusobacterium nucleatum  (ATCC 25586) 
         [0061]    The SG cells are also used in trial (E). Referring to TABLE 9, same amount of  L. rhamnosus  GMNL-464 and  F. nucleatum  (ATCC 25586) are mixed. The upper solution (100 μL) is added into a 24-well plate coated SG cells (3×10 5  cells/well) and incubated at 37° C. for 2 hours. The upper solution is then removed. The SG cells in each well are collected and bacterial DNA adhering on the SG cells is extracted. Real-time PCR using primer pairs set forth as SEQ ID NOS: 8 and 9 is finally carried out and 2 −ΔCT  is calculated (as shown in  FIG. 5 ). 
         [0000]    
       
         
               
               
             
           
               
                 TABLE 9 
               
               
                   
               
               
                 groups 
                 condition 
               
               
                   
               
             
             
               
                 E0 
                 PBS +  F. nucleatum  (ATCC 25586) 
               
               
                 E1 
                   L. paracasei  GMNL-33 +  F. nucleatum  (ATCC 25586) 
               
               
                 E2 
                   L. rhamnosus  GMNL-464 +  F. nucleatum  (ATCC 25586) 
               
               
                   
               
             
          
         
       
     
         [0062]    Referring to  FIG. 5 , both  L. paracasei  GMNL-33 and  L. rhamnosus  GMNL-464 inhibit  F. nucleatum  adhering on SG cells, with  L. rhamnosus  GMNL-464 according to the invention shows a better effect. 
         [0063]    Trial (F): Synergetic effects of  L. paracasei  GMNL-143,  L. helveticus  GMNL-164 and  L. rhamnosus  GMNL-464 
         [0064]    In trial (F), same amount of  L. paracasei  GMNL-143,  L. helveticus  GMNL-164 and  L. rhamnosus  GMNL-464 is mixed to form the  Lactobacillus  mixture. Sterile water is used to further form a mouthwash with a low-dose  Lactobacillus  mixture (5×10 7  cells/mL, group F1), with a middle-dose  Lactobacillus  mixture (5×10 8  cells/mL, group F2) and with a high-dose  Lactobacillus  mixture (5×10 9  cells/mL, group F3). The sterile water without the  Lactobacillus  mixture is used as the control (group F0). 
         [0065]    In detail, sterile cotton swabs are used to collect oral bacteria before gargling with the mouthwash (T0), 1 hour after gargling with the mouthwash (T1) and 2.5 hours gargling with the mouthwash (T2), respectively. The cotton swab with oral bacteria is put into sterile water (0.5 mL), vortexed for 3-5 seconds, stood for 1 hour at room temperature, and centrifugated at 13,000 rpm for 10 minutes. The cotton swab is then removed and the sterile water with oral bacteria is then centrifugated at 13,000 rpm for 10 minutes. After discarding the supernatant, DNA of the pellet is extracted. Finally, real-time PCR is carried out using the primers with DNA sequence set forth as SEQ ID NOS: 10 and 11, the primers with DNA sequence set forth as SEQ ID NOS: 3 and 4, or the primers with DNA sequence set forth as SEQ ID NOS: 8 and 9 to quantify  S. mutans, P. gingivalis  or  F. nucleatum  in oral microbiota, respectively. Moreover, the primers with DNA sequence set forth as SEQ ID NOS: 12 and 13 are used to quantify the total oral bacteria. 
         [0066]      FIGS. 6 a -6 c    show relative amounts of  S. mutans, P. gingivalis  and  F. nucleatum  before and after gargling with the control mouthwash (group F0), respectively.  FIG. 6 d    shows the total oral bacteria before and after gargling with the control mouthwash (group F0), respectively. Moreover,  FIGS. 7 a -7 d , 8 a -8 d  and 9 a -9 d    show the results before and after gargling with the low-dose, middle-dose and high-dose mouthwash (groups F1, F2 and F3), respectively. That is, the dose of the  Lactobacillus  mixture is positive correlated to the decrease of  S. mutans, P. gingivalis  and  F. nucleatum . Furthermore, the mouthwash of group F3 shows a significant bacteriostatic effect. 
         [0067]    In view of the foregoing, the heat-inactivated  Lactobacillus  strain according to the present invention can prevent oral pathogens from adhesion on oral cavity, adjusting distribution of the oral microbiota. Therefore, the  Lactobacillus  strain can be used to improve oral health. 
         [0068]    Moreover, the composition for inhibiting bacterial adhesion of oral pathogens according to the present invention includes the  L. paracasei  GMNL-143, the  L. helveticus  GMNL-164 and the  L. rhamnosus  GMNL-464. The  L. paracasei  GMNL-143, the  L. helveticus  GMNL-164 and the  L. rhamnosus  GMNL-464 show synergetic effect on preventing oral pathogens from adhering on oral cavity. Therefore, the composition for inhibiting bacterial adhesion of oral pathogens according to the present invention shows a better effect on adjusting the distribution of oral microbiota, improving oral health. 
         [0069]    Although the invention has been described in detail with reference to its presently preferable embodiment, it will be understood by one of ordinary skill in the art that various modifications can be made without departing from the spirit and the scope of the invention, as set forth in the appended claims.