Use of heat-treated lactobacillus paracasei gmnl-143

Abstract

The use of a heat-treated lactobacillus, which is Lactobacillus casei GMNL-143, Lactobacillus helveticus GMNL-164 or Lactobacillus rhamnosus GMNL-464, for the preparation of a medicament for inhibiting the adhesion of oral pathogenic bacteria, and a composition for inhibiting the adhesion of oral pathogenic bacteria.

Description

Technical Field

[0001] The present invention relates to a use of a lactobacillus strain, in particular to a use of a lactobacillus strain for preparing a medicine for inhibiting the adhesion of oral pathogens. The present invention also relates to a composition for inhibiting the adhesion of oral pathogens. Background Art

[0002] The oral cavity contains a large and complex microbial flora, which often adheres to the hard and soft tissues of the oral cavity in the form of biofilms. This microbial flora may change with changes in the oral environment, such as individual growth and tooth development, and may also vary depending on the dietary and lifestyle habits of individuals or families.

[0003] Oral flora plays a crucial role in oral diseases. For example, Streptococcus mutans metabolizes various sugars to produce acids, which erode tooth enamel and cause dental caries. Anaerobic bacteria such as Porphyromonas gingivalis and Prevotella intermedia form biofilms around the gums, potentially leading to gingival inflammation and periodontal disease. Halitosis is caused by the foul-smelling volatile sulfur compounds produced during the metabolism of bacteria such as Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum. Therefore, regulating the oral flora through the use of probiotics can effectively reduce the likelihood of oral diseases. Summary of the Invention

[0004] The invention provides a use of heat-treated Lactobacillus paracasei GMNL-143 for preparing a medicine for inhibiting the adhesion of oral pathogens.

[0005] The present invention further provides a composition for inhibiting the adhesion of oral pathogens, which inhibits the adhesion of oral pathogens by using heat-treated lactobacilli.

[0006] The use of the heat-treated Lactobacillus paracasei GMNL-143 of the present application is to prepare a medicine for inhibiting the adhesion of Streptococcus mutans, the Lactobacillus paracasei GMNL-143 is deposited in the Food Industry Development Institute of Taiwan, China, with the deposit number BCRC 910626, and deposited in the China Center of Type Culture Collection, with the address of the deposit unit being Wuhan University, Er Yi Road, Wuchang District, Wuhan City, Hubei Province, China, the deposit date being June 29, 2014, the classification name being Lactobacillus paracasei GMNL-143, the deposit number being CCTCC No.: M2014301, the Lactobacillus helveticus GMNL-164 is deposited in the Food Industry Development Institute of Taiwan, China, with the deposit number BCRC 910695, and deposited in the China Center of Type Culture Collection, with the address of the deposit unit being Wuhan University, Er Yi Road, Wuchang District, Wuhan City, Hubei Province, China, the deposit date being March 6, 2016, the classification name being Lactobacillus helveticus GMNL-164, the deposit number being CCTCC No.: M2015609, the Lactobacillus rhamnosus GMNL-464 is deposited in the Food Industry Development Institute of Taiwan, China, with the deposit number BCRC 910696, and deposited in the China Center of Type Culture Collection, with the address of the deposit unit being Wuhan University, Er Yi Road, Wuchang District, Wuhan City, Hubei Province, China, the deposit date being March 6, 2016, the classification name being Lactobacillus rhamnosus GMNL-464, the deposit number being CCTCC No.: M2015610, and the Lactobacillus is heat-treated at 90-121 DEG C for 15-30 minutes, the heat-treated Lactobacillus can prevent oral pathogenic bacteria from adhering to the oral cavity, adjust the distribution of oral flora, and achieve the purpose of maintaining oral health.

[0007] The use of the heat-treated Lactobacillus paracasei GMNL-143 of the present application, the Lactobacillus is orally administered to a desired individual at a dose of 3x10 8 ~1x10 10 cells per time, the dose is effective in preventing oral pathogenic bacteria from adhering to the oral cavity, adjusting the distribution of oral flora, and achieving the purpose of maintaining oral health.

[0008] The composition for inhibiting oral pathogenic bacteria adhesion of the present application comprises: a heat-treated Lactobacillus selected from the group consisting of Lactobacillus paracasei GMNL-143 deposited in the Food Industry Development Institute, Taiwan, ROC, with the accession number BCRC 910626, and deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, Hubei, China, on June 29, 2014, with the accession number CCTCC No.: M2014301, and named Lactobacillus paracasei GMNL-143, Lactobacillus helveticus GMNL-164 deposited in the Food Industry Development Institute, Taiwan, ROC, with the accession number BCRC 910695, and deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, Hubei, China, on March 6, 2016, with the accession number CCTCC No.: M2015609, and named Lactobacillus helveticus GMNL-164, and Lactobacillus rhamnosus GMNL-464 deposited in the Food Industry Development Institute, Taiwan, ROC, with the accession number BCRC 910696, and deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, Hubei, China, on March 6, 2016, with the accession number CCTCC No.: M2015610, and named Lactobacillus rhamnosus GMNL-464, which is heat-treated at 90-121°C for 15-30 minutes, and a pharmaceutically acceptable excipient, so as to effectively prevent oral pathogenic bacteria from adhering to the oral cavity by using Lactobacillus paracasei GMNL-143, Lactobacillus helveticus GMNL-164, and Lactobacillus rhamnosus GMNL-464, to adjust the distribution of oral bacteria, and to achieve the purpose of maintaining oral health.

[0009] The composition for inhibiting adhesion of oral pathogenic bacteria of the present application, wherein the composition comprises: the Lactobacillus paracasei GMNL-143, the Lactobacillus helveticus GMNL-164 and the Lactobacillus rhamnosus GMNL-464, and the ratio of the amount of the Lactobacillus paracasei GMNL-143, the Lactobacillus helveticus GMNL-164 and the Lactobacillus rhamnosus GMNL-464 is 1:1:1, by the synergistic effect of the Lactobacillus paracasei GMNL-143, the Lactobacillus helveticus GMNL-164 and the Lactobacillus rhamnosus GMNL-464, the adhesion of oral pathogenic bacteria in the oral cavity is effectively prevented, the distribution of oral bacteria phase is adjusted, and the purpose of maintaining oral health is achieved.

[0010] The use of the heat-treated Lactobacillus paracasei GMNL-143 of the present application, the heat-treated Lactobacillus can prevent the adhesion of oral pathogenic bacteria in the oral cavity, adjust the distribution of oral bacteria phase, and achieve the purpose of maintaining oral health.

[0011] The composition for inhibiting adhesion of oral pathogenic bacteria of the present application, by the Lactobacillus paracasei GMNL-143, the Lactobacillus helveticus GMNL-164 and the Lactobacillus rhamnosus GMNL-464, the adhesion of oral pathogenic bacteria in the oral cavity is effectively prevented, the distribution of oral bacteria phase is adjusted, and the purpose of maintaining oral health is achieved. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1a : Test (A) results of the inhibitory ability of Streptococcus mutans adhesion of the A0 group.

[0013] Figure 1b : Test (A) results of the inhibitory ability of Streptococcus mutans adhesion of the A1 group.

[0014] Figure 1c : Test (A) results of the inhibitory ability of Streptococcus mutans adhesion of the A2 group.

[0015] Figure 2a : Test (B) results of the inhibitory ability of Streptococcus mutans adhesion of the B0 group.

[0016] Figure 2b : Test (B) results of the inhibitory ability of Streptococcus mutans adhesion of the B1 group.

[0017] Figure 2c : Test (B) results of the inhibitory ability of Streptococcus mutans adhesion of the B2 group.

[0018] Figure 3 : Test (C) results of the inhibitory ability of Porphyromonas gingivalis adhesion of each group.

[0019] Figure 4Results of the ability of each group in test (D) to inhibit the adhesion of Prevotella intermedia.

[0020] Figure 5 Results of the ability of each group in test (E) to inhibit the adhesion of Fusobacterium nucleatum.

[0021] Figure 6a Results of the change in the amount of Streptococcus mutans in the oral cavity before and after gargling with the mouthwash of group F0 in test (F).

[0022] Figure 6b Results of the change in the amount of Porphyromonas gingivalis in the oral cavity before and after gargling with the mouthwash of group F0 in test (F).

[0023] Figure 6c Results of the change in the amount of Fusobacterium nucleatum in the oral cavity before and after gargling with the mouthwash of group F0 in test (F).

[0024] Figure 6d Results of the change in the total amount of bacteria in the oral cavity before and after gargling with the mouthwash of group F0 in test (F).

[0025] Figure 7a Results of the change in the amount of Streptococcus mutans in the oral cavity before and after gargling with the mouthwash of group F1 in test (F).

[0026] Figure 7b Results of the change in the amount of Porphyromonas gingivalis in the oral cavity before and after gargling with the mouthwash of group F1 in test (F).

[0027] Figure 7c Results of the change in the amount of Fusobacterium nucleatum in the oral cavity before and after gargling with the mouthwash of group F1 in test (F).

[0028] Figure 7d Results of the change in the total amount of bacteria in the oral cavity before and after gargling with the mouthwash of group F1 in test (F).

[0029] Figure 8a Results of the change in the amount of Streptococcus mutans in the oral cavity before and after gargling with the mouthwash of group F2 in test (F).

[0030] Figure 8b Results of the change in the amount of Porphyromonas gingivalis in the oral cavity before and after gargling with the mouthwash of group F2 in test (F).

[0031] Figure 8c Results of the change in the amount of Fusobacterium nucleatum in the oral cavity before and after gargling with the mouthwash of group F2 in test (F).

[0032] Figure 8d Results of the change in the total amount of bacteria in the oral cavity before and after gargling with the mouthwash of group F2 in test (F).

[0033] Figure 9a Results of the change in the amount of Streptococcus mutans in the oral cavity before and after gargling with the mouthwash of group F3 in test (F).

[0034] Figure 9b : Test (F) for the change in the content of Porphyromonas gingivalis in the oral cavity before and after gargling with the F3 group mouthwash.

[0035] Figure 9c : Test (F) for the change in the content of Fusobacterium nucleatum in the oral cavity before and after gargling with the F3 group mouthwash.

[0036] Figure 9d : Test (F) for the change in the total bacterial content in the oral cavity before and after gargling with the F3 group mouthwash. DETAILED DESCRIPTION

[0037] In order to make the above and other objects, features and advantages of the present application more comprehensible, preferred embodiments will be described below with reference to the accompanying drawings, in which:

[0038] The Lactobacillus described in the present application is selected from the group consisting of Lactobacillus paracasei GMNL-143, Lactobacillus helveticus GMNL-164 and Lactobacillus rhamnosus GMNL-464.

[0039] In detail, the Lactobacillus paracasei GMNL-143 is isolated from human intestinal tract, and the colony morphology after incubation at 37°C for 48 hours is round, compact, complete edge, 1.0 mm x 1.0 mm, milky white, smooth surface, intermediate protrusion, Gram-positive bacteria by Gram staining, bacillus in shape, non-spore forming, non-motile, and the physiological characteristics are as follows: survival temperature is 25-45°C, survival pH is pH 4.0-10.0, and it is a facultative anaerobic Lactobacillus.

[0040] In addition, 16S rDNA molecular identification is performed, and the 16S rDNA partial sequence is shown as SEQ ID NO: 1, which is confirmed as Lactobacillus paracasei by comparison with the NCBI database, and the sugar utilization result is shown in Table 1.

[0041] Table 1: Sugar utilization test results of Lactobacillus paracasei GMNL-143

[0042]

[0043]

[0044]

[0045] Moreover, the Lactobacillus helveticus GMNL-164 is isolated from human intestine, and the colony morphology after incubation at 37°C for 48 hours is round, wavy edge, 1.2 mm x 1.2 mm, off-white, slow surface, flat projection, gram-positive bacteria in gram staining, bacillus in morphology, non-spore formation, non-mobility, and the physiological characteristics are as follows: survival temperature is 25-45°C, survival pH is pH 4.0-10.0, and it belongs to facultative anaerobic lactobacillus.

[0046] In addition, the 16S rDNA molecular identification is carried out, the 16S rDNA partial sequence is shown as SEQ ID NO: 2, and it is confirmed to be Lactobacillus helveticus by comparison with the NCBI database; and the sugar utilization result is shown in Table 2.

[0047] Table 2 Sugar utilization test result of Lactobacillus helveticus GMNL-164

[0048]

[0049]

[0050] The Lactobacillus rhamnosus GMNL-464 is isolated from human intestine, and the colony morphology after incubation at 37°C for 48 hours is round, reflective, complete edge, 1.5 mm x 1.5 mm, milky white, smooth surface, intermediate projection, gram-positive bacteria in gram staining, bacillus in morphology, non-spore formation, non-mobility, and the physiological characteristics are as follows: survival temperature is 25-45°C, survival pH is pH 4.0-10.0, and it belongs to facultative anaerobic lactobacillus.

[0051] In addition, the 16S rDNA molecular identification is carried out, the 16S rDNA partial sequence is shown as SEQ ID NO: 3, and it is confirmed to be Lactobacillus rhamnosus by comparison with the NCBI database; and the sugar utilization result is shown in Table 3.

[0052] Table 3 Sugar utilization test result of Lactobacillus rhamnosus GMNL-464

[0053]

[0054]

[0055] The Lactobacillus paracasei GMNL-143 is deposited with the Taiwan Food Industry Research and Development Institute under accession number BCRC 910626 and with the China Center of Type Culture Collection under accession number CCTCC No.: M2014301, the Lactobacillus helveticus GMNL-164 is deposited with the Taiwan Food Industry Research and Development Institute under accession number BCRC 910695 and with the China Center of Type Culture Collection under accession number CCTCC No.: M2015609, and the Lactobacillus rhamnosus GMNL-464 is deposited with the Taiwan Food Industry Research and Development Institute under accession number BCRC 910696 and with the China Center of Type Culture Collection under accession number CCTCC No.: M2015610.

[0056] In addition, the Lactobacillus can be subjected to a heat treatment procedure to lose physiological activity and then applied to inhibit the adhesion of oral pathogenic bacteria; for example, in the present embodiment, the heat treatment procedure is performed at a temperature of 90-121°C for 15-30 minutes.

[0057] In addition, the Lactobacillus can be mixed with a pharmaceutically acceptable excipient to form a composition for inhibiting the adhesion of oral pathogenic bacteria, or the Lactobacillus paracasei GMNL-143, the Lactobacillus helveticus GMNL-164, and the Lactobacillus rhamnosus GMNL-464 are preferentially mixed in a ratio of 1:1:1 (bacterial amount ratio) to form a complex Lactobacillus, and then the complex Lactobacillus is mixed with the pharmaceutically acceptable excipient to form the composition for inhibiting the adhesion of oral pathogenic bacteria. It is worth noting that the Lactobacillus can also be added to a mouthwash, toothpaste, lozenge, chewing gum, tooth powder, or the like to improve the ability of the mouthwash, toothpaste, lozenge, chewing gum, tooth powder, or the like to inhibit the adhesion of oral pathogenic bacteria; in detail, when added to the mouthwash, the Lactobacillus is used in an amount of 1 x 10 9 cells per time, and the use time is 30 seconds; when added to the toothpaste, the Lactobacillus is used in an amount of 7 x 10 8 ~1 x 10 9 cells per time, and the use time is 1 minute; when added to the lozenge, the Lactobacillus is used in an amount of 3 x 10 8 cells per time, and the use time is 10-20 minutes; when added to the chewing gum, the Lactobacillus is used in an amount of 2 x 10 9 cells per time, and the use time is 10-30 minutes; when added to the tooth powder, the Lactobacillus is used in an amount of 1 x 10 10 cells per time, and the use time is 1 minute; however, the above-mentioned amount and use time can be adjusted by those skilled in the art according to needs, which are not limited herein.

[0058] To confirm that the lactobacillus of the present application can indeed inhibit the adhesion of oral pathogenic bacteria, the lactobacillus was heat-treated at 90-121°C for 15-30 minutes, and then cooled to room temperature, and the following test was performed:

[0059] (A) Inhibition of adhesion of Streptococcus mutans (ATCC 25175) by Lactobacillus paracasei GMNL-143

[0060] As shown in Table 4, Streptococcus mutans (ATCC 25175) was tested, 0.5 mL of Lactobacillus paracasei GMNL-143 and 0.5 mL of Streptococcus mutans (ATCC 25175) were mixed, and after standing for 5 minutes, 500 μL of the upper solution was taken and added to a 24-well plate in which a sterile glass slide had been placed, and incubated at 37°C for 2 hours. After removing the upper solution, the glass slide was washed with phosphate buffered saline (PBS), 0.5 mL of methanol was added, and finally Giemsa stain was added for staining, and the number of Streptococcus mutans adhering to the glass slide was observed.

[0061] Table 4 Test conditions for each group in this experiment

[0062] Group Test condition Group A0 PBS + Streptococcus mutans (ATCC 25175) Group A1 Lactobacillus paracasei GMNL-33 + Streptococcus mutans (ATCC 25175) Group A2 Lactobacillus paracasei GMNL-143 + Streptococcus mutans (ATCC 25175)

[0063] As shown in Table 4, Streptococcus mutans (ATCC 25175) was tested, 0.5 mL of Lactobacillus paracasei GMNL-143 and 0.5 mL of Streptococcus mutans (ATCC 25175) were mixed, and after standing for 5 minutes, 500 μL of the upper solution was taken and added to a 24-well plate in which a sterile glass slide had been placed, and incubated at 37°C for 2 hours. After removing the upper solution, the glass slide was washed with phosphate buffered saline (PBS), 0.5 mL of methanol was added, and finally Giemsa stain was added for staining, and the number of Streptococcus mutans adhering to the glass slide was observed. Figure 1a Figure 1b 1c As shown in Table 4, Streptococcus mutans (ATCC 25175) was tested, 0.5 mL of Lactobacillus paracasei GMNL-143 and 0.5 mL of Streptococcus mutans (ATCC 25175) were mixed, and after standing for 5 minutes, 500 μL of the upper solution was taken and added to a 24-well plate in which a sterile glass slide had been placed, and incubated at 37°C for 2 hours. After removing the upper solution, the glass slide was washed with phosphate buffered saline (PBS), 0.5 mL of methanol was added, and finally Giemsa stain was added for staining, and the number of Streptococcus mutans adhering to the glass slide was observed.

[0064] (B) Inhibition of adhesion of Streptococcus mutans (BCRC 16002) by Lactobacillus paracasei GMNL-143

[0065] As shown in Table 5, the same test was performed using Streptococcus mutans (BCRC 16002), and the results were substantially the same. Treatment with Lactobacillus paracasei GMNL-33 and GMNL-143 both reduced the number of Streptococcus mutans (BCRC 16002) adhering to the glass slide, and Lactobacillus paracasei GMNL-143 of the present application was the most effective. Figures 2a-2c

[0066] Table 5 Test conditions for each group in this experiment

[0067] Group Test condition Group B0 PBS + Streptococcus mutans (BCRC 16002) Group B1 Lactobacillus paracasei GMNL-33 + Streptococcus mutans (BCRC 16002) Group B2 Lactobacillus paracasei GMNL-143 + Streptococcus mutans (BCRC 16002) ​​​

[0068] (C) Lactobacillus helveticus GMNL-164 inhibits the adhesion of Porphyromonas gingivalis (ATCC 33277)

[0069] This experiment was conducted using human normal oral gingival cell lines (Smulow-Clickman gingival cells, referred to as SG cells). Please refer to Table 6. Equal amounts of Lactobacillus helveticus GMNL-164 and Porphyromonas gingivalis (ATCC33277) were mixed. 100 μL of the upper layer solution was taken and added to the 3×10 SG cells attached. 5 The cells were cultured in a 24-well plate at 37°C for 2 hours. The supernatant was removed and the SG cells in each well were collected. The bacterial DNA attached to the SG cells was extracted and real-time polymerase chain reaction was performed using a primer pair having SEQ ID NOS: 4 and 5. The 2 -ΔCT , the results are as follows Figure 3 shown.

[0070] Table 6 Test conditions for each group in this experiment

[0071] Group Test condition Group C0 PBS + Porphyromonas gingivalis (ATCC 33277) Group C1 Lactobacillus paracasei GMNL-33 + Porphyromonas gingivalis (ATCC 33277) Group C2 Lactobacillus helveticus GMNL-164 + Porphyromonas gingivalis (ATCC 33277)

[0072] Please refer to Figure 3 As shown, both Lactobacillus paracasei GMNL-33 and Lactobacillus helveticus GMNL-164 have the ability to inhibit the adhesion of Porphyromonas gingivalis to SG cells, among which Lactobacillus helveticus GMNL-164 of the present invention is particularly preferred.

[0073] (D) Lactobacillus helveticus GMNL-164 inhibits the adhesion of Prevotella intermedia (ATCC 25611)

[0074] This experiment was conducted with the SG cells described above. Please refer to Table 7. Equal amounts of Lactobacillus helveticus GMNL-164 and Prevotella intermedia (ATCC 25611) were mixed. 100 μL of the upper layer solution was added to the SG cells (3×10 5 The cells were cultured in a 24-well plate at 37°C for 2 hours. The supernatant was removed and the SG cells in each well were collected. The bacterial DNA attached to the SG cells was extracted and real-time polymerase chain reaction was performed using a primer pair having SEQ ID NOS: 6 and 7. The 2 -ΔCT , the results are as follows Figure 4 shown.

[0075] Table 7 Test conditions for each group in this experiment

[0076] Group Test condition Group D0 PBS + Prevotella intermedia (ATCC 25611) Group D1 Lactobacillus paracasei GMNL-33 + Prevotella intermedia (ATCC 25611) Group D2 Lactobacillus helveticus GMNL-164 + Prevotella intermedia (ATCC 25611)

[0077] Please refer toFigure 4 As shown, both Lactobacillus paracasei GMNL-33 and Lactobacillus helveticus GMNL-164 have the ability to inhibit the adhesion of Prevotella intermedia to SG cells, among which Lactobacillus helveticus GMNL-164 of the present invention is particularly preferred.

[0078] (E) Lactobacillus rhamnosus GMNL-464 inhibits the adhesion of Fusobacterium nucleatum (ATCC 25586)

[0079] This experiment was still conducted using the aforementioned SG cells. Please refer to Table 8. Mix equal amounts of Lactobacillus rhamnosus GMNL-464 and Fusobacterium nucleatum (ATCC 25586). Take 100 μL of the upper layer solution and add it to the SG cells (3×10 5 The cells were cultured in a 24-well plate at 37°C for 2 hours. The supernatant was removed and the SG cells in each well were collected. The bacterial DNA attached to the SG cells was extracted and real-time polymerase chain reaction was performed using a primer pair having SEQ ID NOS: 8 and 9. The 2 -ΔCT , the results are as follows Figure 5 shown.

[0080] Table 8 Test conditions for each group in this experiment

[0081] Group Test condition Group E0 PBS + Fusobacterium nucleatum (ATCC 25586) Group E1 Lactobacillus paracasei GMNL-33 + Fusobacterium nucleatum (ATCC 25586) Group E2 Lactobacillus rhamnosus GMNL-464 + Fusobacterium nucleatum (ATCC 25586)

[0082] Please refer to Figure 5 As shown, both Lactobacillus paracasei GMNL-33 and Lactobacillus rhamnosus GMNL-464 have the ability to inhibit the adhesion of Fusobacterium nucleatum to SG cells, among which Lactobacillus rhamnosus GMNL-464 of the present invention is particularly preferred.

[0083] (F) Synergistic effect of Lactobacillus paracasei GMNL-143, Lactobacillus helveticus GMNL-164, and Lactobacillus rhamnosus GMNL-464

[0084] In this experiment, equal amounts of Lactobacillus paracasei GMNL-143, Lactobacillus helveticus GMNL-164, and Lactobacillus rhamnosus GMNL-464 were mixed to form the composite lactobacillus, and a low-dose mouthwash containing the composite lactobacillus was prepared with sterile water (containing a concentration of 5×10 7 cells / mL of complex lactobacillus, group F1), a medium-dose complex lactobacillus mouthwash (containing a concentration of 5×10 8 cells / mL of complex lactobacillus, group F2), high-dose complex lactobacillus mouthwash (containing a concentration of 5×10 9 cells / mL of the composite lactobacillus, the F3 group) was used as a standby, and sterile water without the addition of the composite lactobacillus was used as a control group of mouthwash (the F0 group).

[0085] In detail, the present experiment takes the aforementioned F0-F3 group of mouthwash before (T0) and 1 hour after (T1) and 2.5 hours after (T2) mouthwash, respectively, with sterile cotton swab to take the oral flora, the cotton swab is placed in 0.5 mL of sterile water and shaken for 3-5 seconds, and then left at room temperature for 1 hour. Then, it is centrifuged at 13,000 rpm for 10 minutes. After the cotton swab is removed and centrifuged at 13,000 rpm for 10 minutes, the supernatant is removed, the DNA of the cell pellet is extracted, and finally, real-time polymerase chain reaction is performed to quantify the content of Streptococcus mutans with a primer pair having the sequence shown in SEQ ID NOS: 10 and 11, to quantify the content of Porphyromonas gingivalis with a primer pair having the sequence shown in SEQ ID NOS: 4 and 5, and to quantify the content of Fusobacterium nucleatum with a primer pair having the sequence shown in SEQ ID NOS: 8 and 9. At the same time, the total amount of bacteria in the oral cavity is quantified by real-time polymerase chain reaction as an indicator of the growth and decline of the oral flora

[0086] Figures 6a-6c The relative amount of Streptococcus mutans, Porphyromonas gingivalis, and Fusobacterium nucleatum before and after mouthwash of the F0 group is shown in the sixth graph, and the total amount of bacteria in the oral cavity before and after mouthwash of the F0 group is shown in the seventh graph. The seventh to ninth graphs show the test results before and after mouthwash of the F1, F2, and F3 groups, respectively. The results show that the additive amount of the complex Lactobacillus is positively correlated with the ability to inhibit Streptococcus mutans, Porphyromonas gingivalis, and Fusobacterium nucleatum, and the mouthwash of the F3 group has a significant bacteriostatic effect.

[0087] In summary, the present application uses heat-treated Lactobacillus to prevent oral pathogenic bacteria from adhering to the oral cavity, adjust the distribution of oral flora, and achieve the purpose of maintaining oral health.

[0088] Furthermore, the composition of the present application for inhibiting the adhesion of oral pathogenic bacteria effectively prevents oral pathogenic bacteria from adhering to the oral cavity by using the Lactobacillus paracasei GMNL-143, the Lactobacillus helveticus GMNL-164, and the Lactobacillus rhamnosus GMNL-464, adjusts the distribution of oral flora, and achieves the purpose of maintaining oral health.

[0089] Although the present application has been described with reference to the preferred embodiments, the application is not intended to be limited to the details described in the specification. Without departing from the spirit and scope of the application, various modifications and changes can be made by those skilled in the art. The scope of the application is defined by the appended claims.

Claims

1. A heat-treated Lactobacillus paracasei ( Lactobacillus paracasei GMNL-143 is used to prepare a medicament for preventing and treating dental caries caused by Streptococcus mutans. The Lactobacillus paracasei GMNL-143 is deposited with the Taiwan Food Industry Development Research Institute under the registration number BCRC 910626 and the China Center for Type Culture Collection under the registration number CCTCC No.: M2014301. The Lactobacillus paracasei GMNL-143 is heat-treated at 90-121°C for 15-30 minutes.

2. the purposes of heat-treated Lactobacillus paracasei GMNL-143 as claimed in claim 1, is characterized in that, The Lactobacillus paracasei GMNL-143 was used at 3×10 8 ~1×10 10 A dose of granulocytes is orally administered to a subject in need thereof.

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