Method and Composition for Inhibiting Bacterial Adhesion of Oral Pathogens

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.

Claims

1. 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, wherein the Lactobacillus strain is selected from Lactobacillus paracasei GMNL-143, Lactobacillus helveticus GMNL-164 or Lactobacillus rhamnosus GMNL-464, wherein the Lactobacillus paracasei GMNL-143 is deposited at CGMCC-China General Microbiological Culture Collection with deposit number CCTCC No. M2014301, wherein the Lactobacillus helveticus GMNL-164 is deposited at CGMCC-China General Microbiological Culture Collection with deposit number CCTCC No. M2015609, wherein the Lactobacillus rhamnosus GMNL-464 is deposited at CGMCC-China General Microbiological Culture Collection with deposit number CCTCC No. M2015610, wherein the Lactobacillus strain is heat-inactivated at 90-121° C. for 15-30 minutes.

2. The method for inhibiting bacterial adhesion of oral pathogens according to claim 1, wherein the Lactobacillus strain is orally administered to the subject in need at dosage of 3×10.sup.8-1×10.sup.10 cells/time.

3. A composition for inhibiting bacterial adhesion of oral pathogens, comprising: a heat-inactivated Lactobacillus strain selected from a group consisting of Lactobacillus paracasei GMNL-143, Lactobacillus helveticus GMNL-164 and Lactobacillus rhamnosus GMNL-464, wherein the Lactobacillus paracasei GMNL-143 is deposited at CGMCC-China General Microbiological Culture Collection with deposit number CCTCC No. M2014301, wherein the Lactobacillus helveticus GMNL-164 is deposited at CGMCC-China General Microbiological Culture Collection with deposit number CCTCC No. M2015609, wherein the Lactobacillus rhamnosus GMNL-464 is deposited at CGMCC-China General Microbiological Culture Collection with deposit number CCTCC No. M2015610, wherein the Lactobacillus strain is heat-inactivated at 90-121° C. for 15-30 minutes; and a medical acceptable excipient.

4. The composition for inhibiting bacterial adhesion of oral pathogens according to claim 3, wherein the composition comprises the Lactobacillus paracasei GMNL-143, the Lactobacillus helveticus GMNL-164 and the Lactobacillus rhamnosus GMNL-464 in the ratio 1:1:1.

Description

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. 1a depicts a Giemsa stain showing the adhesion of S. mutans on the slide after treating by PBS in trial (A).

[0014] FIG. 1b 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. 1c 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. 2a depicts a Giemsa stain showing the adhesion of S. mutans on the slide after treating by PBS in trial (B).

[0017] FIG. 2b 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. 2c 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. 6a 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. 6b 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. 6c 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. 6d 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. 7a 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. 7b 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. 7c 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. 7d 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. 8a 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. 8b 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. 8c 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. 8d 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. 9a 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. 9b 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. 9c 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. 9d 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.

TABLE-US-00001 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.

TABLE-US-00002 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.

TABLE-US-00003 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.

TABLE-US-00004 TABLE 4 matrix dosage time mouthwash 1 × 10.sup.9 cells/time 30 seconds toothpaste 7 × 10.sup.8-1 × 10.sup.9 cells/time 1 minute troche 3 × 10.sup.8 cells/time 10-20 minutes chewing gum 2 × 10.sup.9 cells/time 10-30 minutes tooth powder 1 × 10.sup.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.

TABLE-US-00005 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. 1a, S. mutans (ATCC 25175) adheres on the slides in the presence of PBS (group A0). In contrast, as shown in FIGS. 1b and 1c, 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. 2a-2c, 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.

TABLE-US-00006 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.sup.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.sup.−ΔCT is calculated (as shown in FIG. 3).

TABLE-US-00007 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.sup.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.sup.−ΔCT is calculated (as shown in FIG. 4).

TABLE-US-00008 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.sup.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.sup.−ΔCT is calculated (as shown in FIG. 5).

TABLE-US-00009 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.sup.7 cells/mL, group F1), with a middle-dose Lactobacillus mixture (5×10.sup.8 cells/mL, group F2) and with a high-dose Lactobacillus mixture (5×10.sup.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. 6a-6c show relative amounts of S. mutans, P. gingivalis and F. nucleatum before and after gargling with the control mouthwash (group F0), respectively. FIG. 6d shows the total oral bacteria before and after gargling with the control mouthwash (group F0), respectively. Moreover, FIGS. 7a-7d, 8a-8d and 9a-9d 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.