Probiotics for use as anti-inflammatory agents in the oral cavity

Abstract

The present invention relates to certain microorganisms or mixtures thereof for use in the treatment and/or prevention of inflammation in the oral cavity, in particular for use in the treatment and/or prevention of dental caries and/or periodontal disease. In particular, the present invention relates to microorganisms or mixtures thereof for use as an anti-inflammatory agent in the oral cavity for reducing or inhibiting the release of certain inflammatory factors. Furthermore, the present invention provides oral pharmaceutical compositions, oral care products or products for nutrition or pleasure comprising one or more of the microorganisms as probiotic agents as well as a method of production thereof.

Claims

1. A method of treating gingivitis or periodontitis, comprising introducing a microorganism or mixture comprising two or more microorganisms into the oral cavity of a subject in need thereof, wherein the microorganism(s) is/are selected from the group consisting of Lactobacillus paracasei LPc-G110 (CCTCC M 2013691), Lactobacillus plantarum GOS 42 (DSM 32131), Lactobacillus delbrueckii subsp. lactis LL-G41 (CCTCC M 2016652), Lactobacillus plantarum Heal19 (DSM 15313), and Lactobacillus paracasei NS9 (NCIMB 8823).

2. The method of claim 1, wherein the microorganism(s) is/are (an) attenuated or (a) dead microorganism(s).

3. The method of claim 1, wherein the treating reduces or inhibits release of one or more inflammatory factors selected from the group consisting of interleukin 1 (IL-1), interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor (TNF), pros-taglandin E2 (PGE2), isoprostanes, matrix metallopeptidase 9 (MMP9), and NF-.sub.κB.

4. The method of claim 1, wherein from 1×10.sup.3 to 1×10.sup.11 colony forming units (CFU) of the microorganism(s) are introducted into the oral cavity.

5. The method of claim 4, wherein from 1×10.sup.5 to 1×10.sup.10 CFU of the microorganism(s) are introduced into the oral cavity.

6. A method of treating gingivitis or periodontitis, comprising introducing into the oral cavity of a person in need thereof, an effective amount of an oral pharmaceutical composition, oral care product, product for nutrition, chewing gum, or lozenge, comprising one or more microorganism(s) selected from the group consisting of Lactobacillus paracasei LPc-G110 (CCTCC M 2013691), Lactobacillus plantarum GOS 42 (DSM 32131), Lactobacillus delbrueckii subsp. lactis LL-G41 (CCTCC M 2016652), Lactobacillus plantarum Heal19 (DSM 15313), and Lactobacillus paracasei NS9 (NCIMB 8823).

Description

SHORT DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the anti-Inflammatory effects of Lactobacillus paracasei LPc-G110 (CCTCC M 2013691) in human primary monocytes on interleukin 1 beta (A), interleukin 6 (B), interleukin 8 (C), prostaglandin E2 (D), tumor necrosis factor alpha (E) and isoprostane (F). The left column refers to untreated cells, the right column to attenuated cells.

(2) FIG. 2 shows the anti-Inflammatory effects of Lactobacillus paracasei LPc-G110 (CCTCC M 2013691) in untreated (A) and attenuated (B) form on interleukin in human gingival fibroblasts. The left column refers to interleukin 6, the right column to interleukin 8.

(3) FIG. 3 shows the anti-Inflammatory effects of Lactobacillus plantarum GOS 42 (DSM 32131) in human primary monocytes on interleukin 1 beta (A), interleukin 6 (B), interleukin 8 (C), prostaglandin E2 (D), tumor necrosis factor alpha (E) and isoprostane (F). The left column refers to untreated cells, the right column to attenuated cells.

(4) FIG. 4 shows the anti-Inflammatory effects of Lactobacillus plantarum GOS 42 (DSM 32131) in attenuated form on interleukin in human gingival fibroblasts. The left column refers to interleukin 6, the right column to interleukin 8.

(5) FIG. 5 shows the anti-inflammatory effects of Lactobacillus paracasei (NCIMB 8823) in human primary monocytes on interleukin 1 beta (A), interleukin 6 (B), interleukin 8 (C), tumor necrosis factor alpha (D), prostaglandin E2 (E), isoprostane (F) and metallopeptidase 9 (MMP-9, G). The left column refers to untreated cells, the right column to attenuated cells.

(6) FIG. 6 shows the anti-inflammatory effects of Lactobacillus plantarum Heal19 (DSM 15313) in human primary monocytes on interleukin 1 beta (A), interleukin 6 (B), interleukin 8 (C), prostaglandin E2 (D), isoprostane (E) and matrix metallopeptidase 9 (MMP-9, F).

(7) FIG. 7 shows the anti-inflammatory effects of Lactobacillus delbrueckii LL-G41 (CCTCC M 2016652) in human primary monocytes on interleukin 1 beta (A), interleukin 6 (B), interleukin 8 (C), tumor necrosis factor alpha(D), prostaglandin E2 (E), isoprostane (F) and matrix metallopeptidase 9 (MMP-9, G). The left column refers to untreated cells, the right column to attenuated cells.

(8) FIG. 8 shows the anti-Inflammatory effects of attenuated Lactobacillus paracasei (NCIMB 8823) on interleukin 6 and interleukin 8 in human gingival fibroblasts. The left column refers to interleukin 6 , the right column to interleukin 8.

(9) The following examples are added to illustrate the present invention without being intended to limit the scope.

Example 1: Establishing the Cultivation and Handling of Probiotic Strains

(10) The strains of the invention were selected from amongst over 50 candidate probiotic strains tested, including strains of Bacillus subtilis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium lactis, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus LAFTI, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus bulgaricus, Lactobacillus gasseri, Lactobacillus fermentum, Lactobacillus brevis, Lactobacillus cellobiosus, Lactobacillus salivarius, Streptococcus thermophilus and Lactococcus lactis.

(11) In order to identify the optimal growth conditions and points of harvest and to determine the colony forming units (CFU) for the probiotic bacteria to be screened, first the log phase and the end of the growth phase were determined.

Bacterial Growth

(12) The frozen (−80° C.) pro-biotic stocks were thawed over-night at 4° C. and 6 ml of sterile 9% NaCl solution added to the 1.2 ml of bacteria at the next morning. The samples were centrifuged (5 min, 5000 rpm), the supernatant discarded, the pellet washed with 8 ml 9% NaCl and again centrifuged for 5 min at 5000 rpm. The pellet was then resuspended in 1.2 ml 9% NaCl and 1 ml of the sample added to 50 ml 37° C. warm media (MRS Bouillon, Carl Roth KG, Karlsruhe) and incubated at 37° C. The incubations were performed in a 50 ml sterile polypropylen tube (Greiner) and probes were harvested at different time points to evaluate the growth curve.

OD-Determination

(13) For determination of OD, 500 μl of the bacterial suspension were removed and diluted in 1 ml MRS Bouillon in a 1.5 ml-PS-cuvette (Brand). OD-determination was performed at 600 nm (ThermoScientific, Helios Epsilon) 1.5 ml MRS Bouillon were used as blanc.

Determination of CFU

(14) For determination of CFU, bacteria were diluted (1:10.000.000, 1:50.000.000 and 1:100.000.000), plated on MRS-agar-plates (MRS Agar, X924, Carl Roth) and incubated for 2 days at 37° C. The grown colonies were then counted and the CFU was calculated.

(15) The bacteria approached the log phase right from the beginning until 7 to 8 hours when they start to reach the plateau phase. The amount of bacteria to be seeded does not change the shape of the curve. 5 hours were chosen as the point at the steepest growing phase to harvest the bacteria in the log phase and 7 hours to harvest them at the end of the log phase.

Example 2: Establishing the Stimulation of Human Monocytes with Probiotics

(16) The stimulation of the monocyte cell cultures with the probiotics was established by using the strains obtained as powder. First, several application forms such as using the grown bacteria (picked from the log-phase), the supernatant of the grown bacterial cultures, direct application of the dissolved powder and powder supernatant were tested. According to the results, the bacteria at the end of the log phase were tested this with two batches of the frozen strain. Instead of using the supernatants, heat inactivation of these two strains was established and compared the inactivated bacteria with the activated bacteria.

Measurement of Cytokine; MMP-9 and PGE2 in Primary Human Monocytes

(17) Human primary monocytes were isolated from buffy coats of healthy human blood donors. Cells were seeded in 24-well-plates for ELISA experiments. Cells were incubated with LPS for 24 h. The probiotics (5 doses) were added 30 min before LPS treatment. After 24 h, supernatants were removed, centrifuged and investigated for IL-1beta, TNFalpha, IL-6, IL-8, MMP-9, isoprostane-8 and PGE.sub.2 concentrations in EIAs (PGE2, from AssayDesign, isoprostane, from Cayman) or ELISAs (all cytokines, Immunotools, MMP-9, GE Healthcare) using manufacturer's protocol. Each dose was investigated 2-3 times in two buffy coats from 2 different donors.

(18) First, different types of probiotic lyophilized powder preparations for the stimulation of human monocytes were tested.

(19) The probiotics were harvested and then centrifuged. The cells were dissolved in fresh media and applied to the human monocytes.

(20) The monocytes were then incubated with the probiotics for 30 minutes, then LPS was added and after 24 hours the supernatant removed and use for the determination of the inflammatory parameters.

Example 3: Testing of Heat Inactivated Strains

Establishment of heat inactivation

(21) Heat inactivation was established for two batches. At the end of the bacterial growth log phase, an aliquot of the bacterial suspension was removed, added to a fresh 50 ml tube and incubated for 5 min at 80° C. in a water bath. 5 minutes at 80° C. inactivated the bacteria and thus stopped their growth.

(22) Testing the heat inactivated batches of the strain, it was found that the enhancing effects on IL-1 and TNF were not affected by heat treatment Furthermore, heat activation did not or only slightly affect PGE2 inhibition by both strains.

Example 4: Screening of the Probiotics on Human Monocytes and Effects on NF-kappaB Activation

(23) Various probiotic strains were screened in their activated and attenuated (heat-inactivated form) on LPS-induced human primary monocytes (determining IL-1beta, IL-6, IL-8, TNFalpha, PGE2, 8-isoprostane, and MMP-9.

(24) A typical anti-inflammatory pattern of a strain according to Examples 1-4 is given in FIGS. 1 and 3.

(25) The experiments to test effects on NF-kappaB activation induced by TNFin NIH-3T3 fibroblasts were done in a fibroblast cell line that contained stably transfected the Luciferase gene drive by a NF-kappaB dependent promoter. The cells were stimulated with TNF in the presence or absence of the probiotics. After 6 h of stimulation the cells were lysed and the luciferase activity measured in a Luminometer. The most potent strains were selected.

Example 5: Screening of Selected Probiotics on Human Gingival Fibroblasts

(26) Selected strains were applied to human gingival fibroblasts. The fibroblast cultures were maintained as described in the manufacturer's protocol. Prior to stimulation, cells are seeded in 24-well plates for ELISA experiments. Cells were incubated without (unstimulated control) or with IL-1beta for 24 h. The probiotics (5 doses, depending on the outcome of screening assays) are added 30 min before IL-1 treatment. After 24 h, supernatants were removed, centrifuged and investigated for IL-6, IL-8, isoprostane, and PGE2 concentrations in EIA (PGE2, from AssayDesign, isoprostane form Cayman) or ELISA (IL-6, IL-8, Immunotools), using manufacturer's protocol. Each dose was investigated at least 2-3 times. The strains showed some IL-6 inhibiting effects.

(27) An anti-inflammatory pattern of a strain according to Example 5 is given in FIGS. 2 and 4.

Example 6: Probiotic Lozenge or Comprimate

(28) TABLE-US-00001 Isomalt Comprimates No Block Ingredients Placebo Probiotic only + Flavor 1 A Magnesium Stearate  1.800%  1.800%  1.800% 2 Acesulfam  0.050%  0.050%  0.050% 3 Sucralose  0.025%  0.025%  0.025% 4 Probiotic Material  1.000%  1.000% 5 Flavor (e.g. 134229  0.500% Optamint Peppermint s/d) 6 B Isomalt 98.125% 97.125% 96.625% Sum total 100.00% 100.00% 100.00%

Production Method

(29) Components 1 and 6 are dried in a vacuum compartment drier at 50° C. and a pressure of max. 10 mbar for 16 hours. All components are weight out exactly components 1, 2, 3, 4 and 5 combined and thoroughly mixed (block A). The probiotic material is applied in lyophilized form having an activity of about 10.sup.5 to 10.sup.12 colony forming units (CFU) per gram. Block A is subsequently added to component 6 and mixed thoroughly for 5 minutes. The powder mixture is pressed into tablets in a tablet press EK0 (Korsch AG, Berlin) at an adjusted pressure of 15-20 kN target parameters: tablet diameter: 20 mm tablet weight: 2.0 g. Storage at RT in sealed aluminum sachets. Per 5 lozenges 1 g of desiccant is used for dehumidification (activated by 3 h storage at 105° C. in a vacuum compartment drier).

Example 7: Powder Dentifrice

(30) TABLE-US-00002 Toothpowder No Block Ingredients Placebo Probiotic only + Flavor 1 A Magnesium  3.00%  3.00%  3.00% Carbonate 2 Sodium Bicarbonate  2.00%  2.00%  2.00% 3 Sodium Fluoride  0.25%  0.25%  0.25% 4 Sodium Saccharin  0.60%  0.60%  0.60% 5 B Probiotic Material  4.00%  4.00% 6 Flavor (e.g. 134229  2.00% Optamint Peppermint s/d) 7 C Calcium carbonate  94.15%  90.15%  88.15% Sum total 100.00% 100.00% 10000%

Production Method

(31) Component 7 is dried in a vacuum compartment drier at 50° C. and a pressure of max. 10 mbar for 16 hours. All components are weight out exactly. Components 1, 2, 3 and 4 are combined and thoroughly mixed together (block A). Components 5 and 6 are, if necessary, combined and thoroughly mixed (block B). The probiotic material is applied in lyophilized form having an activity of about 10.sup.5 to 10.sup.12 colony forming units (CFU) per gram. Blocks A and B are subsequently combined and thoroughly mixed together. The mixture is added to component 7 and mixed thoroughly for 5 minutes. The powder mixture is made up into portions of 0.5 g each storage at RT together with 1 g of desiccant per portion (activated by 3 h storage at 105° C. in a vacuum compartment drier) in sealed aluminum sachets.

Example 8: Powder Dentifrice

(32) TABLE-US-00003 No Block Ingredients Toothpaste tablets 1 A Magnesium Carbonate  3.00% 2 Sodium Bicarbonate  2.00% 3 Sodium Fluoride  0.25% 4 Sodium Saccharin  0.60% 5 Sodium Laurylsulphate  0.50% 6 Magnesium Stearate  1.00% 7 B Flavor (e.g. 134229  2.00% Optamint Peppermint s/d) 8 Probiotic Material  6.67% 9 C Calcium Carbonate  17.00% 10 Microcristalline Cellulose  66.98% Sum total 100.00%

Production Method

(33) Components 6, 9 and 10 are dried in a vacuum compartment drier at 50° C. and a pressure of max. 10 mbar for 16 hours. All components are weight out exactly. Components 1, 2, 3, 4, 5 and 6 are combined and thoroughly mixed together (block A). Components 7 and 8 are combined and thoroughly mixed together (block B). The probiotic material is applied in lyophilized form having an activity of about 10.sup.5 to 10.sup.12 colony forming units (CFU) per gram. Blocks A and B are subsequently combined and thoroughly mixed together. Components 9 and 10 are combined and thoroughly mixed together (block C). The two mixtures (Block A/B and Block C) are combined and mixed thoroughly for 5 minutes. The powder mixture is pressed into tablets in a tablet press EK0 (Korsch AG, Berlin) at an adjusted pressure of 15-20 kN target parameters tablet diameter: 9 mm tablet weight: 0.3 g. Storage at RT in sealed aluminum sachets. Per 3 tablets 1 g of desiccant is used for dehumidification (activated by 3 h storage at 105° C. in a vacuum compartment drier).

Example 9: Chewing Gum

(34) TABLE-US-00004 Chewing gum Chewing gum with Vegetable with Vegetable Oil, Probiotics Oil, Probiotics No Ingredients in Flavor in Oil 1 Gum Base 30.00% Block A 30.00% Block A (e.g. Geminis T) 2 Isomalt 65.00% Block B 65.00% Block B (here: Isomalt ST-PF) 3 Sucralose coated  1.00%  1.00% (10% in wax) 4 Deoiled Soy Lecithin  0.30%  0.30% (here: Emulpur IP) 5 Vegetable Oil -  1.60% Block C  1.60% Block C Triglyceride 6 Probiotic Material  0.80% Block D  0.80% 7 Flavor (e.g. 203191  1.30%  1.30% Block D Optamint Peppermint)

Production Method

(35) Component 2 is dried in a vacuum compartment drier at 50° C. and a pressure of max. 10 mbar for 16 hours. All components are weight out exactly. Component 1 is tempered to 45-59° C. in a chewing gum lab-kneader with the heating kneaded until a homogenous mass is obtained. The heating is on during the whole mixing process. Components 2,3 and 4 are added subsequently and kneaded until the mixture is homogenous and no powder is visible anymore. According to the formula component 6 is either worked into component 5 (block C) or component 7 (block D). The probiotic material is applied in lyophilized form having an activity of about 10.sup.5 to 10.sup.12 colony forming units (CFU) per gram. The components are mixed until an even suspension is obtained. First, block C is added to the chewing gum mass and kneaded again until a homogenous mass is obtained. Last, block D is processed accordingly. After addition the composition has to be kneaded until an even chewing gum mass is obtained. The mass is taken out of the mixer and is formed into mini-sticks by an embossing roller using the embossing set “slabs”. Storage at RT in sealed aluminum sachets. Per 7 chewing gums 1 g of desiccant is used for dehumidification (activated by 3 h storage at 105° C. in a vacuum compartment drier).

Example 10: Probiotic Beadlets

(36) TABLE-US-00005 probiotic probiotic beadlets beadlets with with probiotic probiotic high load, high load, beadlets beadlets without without with with aroma, aroma, low load, low load, without dye, without dye, without with without without aroma, aroma, gellan gellan with with gum, high gum, low dye, with dye, with water water gellan gum gellan gum content content components wt. % wt. % wt. % wt. % Alginate 1.75 1.65 1.44 1.57 Gum arabic 1.25 1.18 0.60 0.65 Wheat fiber 1.125 1.06 0.52 0.57 Dye 0.0125 0.018 — — Aroma — 1.41 — — Glycerol 0.1875 — — — probiotic 1.125 1.35 7.20 7.83 Gellan Gum 0.0625 0.059 — — Water Add to 100 Add to 100 Add to 100 Add to 100 load approx. 20% approx. 20% approx. 74% approx. 74%

Production Method

(37) Production of the calcium chloride bath for precipitation of the alginate beadlets: A 2% calcium chloride solution is produces from distilled water and calcium chloride. Care has to be taken that the CaCl.sub.2 is completely dissolved. Production of the alginate solution (instead of alginate also pectin or gellan gum may be used): In a reaction vessel with a stirrer and which is suitable to the batch size, water is provided. The stirrer is turned on and, while stirring at a high level, the respective amounts of alginate, gum arabic, wheat fiber and probiotic, as well as the optionally required gellan gum are added. The mixture is heated to 80° C. while stirring and kept at this temperature for 5 minutes—during this step the gel forming components are dissolved. Afterwards, the heating is turned off and the hot gel solution is further stirred for at least 30 minutes until it is free of lumps. Subsequently, the solution is cooled by refrigeration to 39-43° C. while stirring. In a further vessel, the aroma and the dye are provided if required and thoroughly mixed In case no aroma is used, the dye is mixed with glycerol. When the dye dispersion is mixed homogenously, it is added to the batch vessel with the alginate solution. The mixing vessel is washed several times with approx. 10% of the amount of alginate solution used of water and added to the dispersion. The alginate dispersion is stirred further for at least 5 minutes. Subsequently, the batch is stirred for further at least 15 minutes at a low speed to remove potentially present air.

Dripping of the Alginate Solution into the Calcium Chloride Solution for Precipitation of the Beadlets

(38) The alginate dispersion is moved to a tightly sealable pressure stable reaction vessel having two outlets. At one outlet pressurized air is applied. The second outlet leads to the nozzles of the dripping unit via a tube. The reaction vessel is tempered over a heating plate so that the alginate solution reaches a temperature of approx. 45° C. The solution is slightly stirred with a magnet stirrer. After application of pressure to the reaction vessel, alginate solution is pressed towards the nozzles, which are set to oscillation by an oscillator. By adaption of pressure and the frequency of the oscillator, the size of the resulting drops at the tips of nozzles may be adjusted. The drops of alginate solution forming at the tips of the nozzles fall into a collection vessel in the form of a funnel in which the calcium chloride solution prepared at the beginning circulates. The cured alginate beadlets pass with the calcium chloride solution through the funnel and are collected in a sieve, the collected calcium chloride solution is pumped back into the funnel below the dripping unit and thus recycled. The beadlets are dried in an Aeromatic flowbed-drier at an supply air temperature of 80° C. until an exhaust air temperature of 45° C. is reached.