Self-film-forming composition for oral care

Abstract

A self-film-forming composition in powder form, a reconstituted formula and a kit for oral use are provided, which allow an adequate colonization of the probiotic in the oral cavities together with a sufficient residence time to allow beneficial effects against the pathogens related with oral conditions. Thus, the items provided are useful for the prevention and/or treatment of a condition related to alterations of the oral microbiota, and specifically for peri-implantitis. The self-film-forming composition in powder form comprises at least a gelifier agent and/or at least a bioadhesive agent, and at least one lactic acid bacteria strain.

Claims

1. An oral composition comprising: (i) at least one gelifier agent in powder form, (ii) at least one bioadhesive agent in powder form, and (iii) at least one lactic acid bacteria strain belonging to genus Pediococcus in powder form, wherein the (i) at least one gelifier agent is in an amount of 0.05-90% w/w of the composition in powder form to provide viscosity to the composition, and is selected from the group consisting of: (a) a starch, (b) a gum, (c) an algal polysaccharide, (d) a polysaccharide selected from the group consisting of pectin and maltodextrin, (e) a cellulose derivative, (f) a polypeptide selected from the group consisting of gelatin, collagen, and casein; wherein the (ii) at least one bioadhesive agent is in an amount of 0.05%-90% w/w of the composition in powder form to provide adhesiveness to the composition, and is selected from the group consisting of: (a) a gum, (b) an algal polysaccharide, (c) a cellulose derivative, (d) a polysaccharide selected from the group consisting of pectin and maltodextrin, and (e) a polymer selected from the group consisting of an acrylate-based polymer, a vinyl-based polymer and a cationic polysaccharide; and wherein the composition is a powder and subsequently forms a film under agitation in the presence of a liquid medium upon topical administration to an oral cavity.

2. The oral composition of claim 1, wherein the gelifier agent and the bioadhesive agent have no bactericidal effect against the at least one lactic acid bacteria.

3. The oral composition of claim 2, wherein the gelifier agent and the bioadhesive agent have no bacteriostatic effect against the at least one lactic acid bacteria.

4. The oral composition of claim 3, wherein the gelifier agent or the bioadhesive agent have prebiotic effect on the at least one lactic acid bacteria.

5. The oral composition of claim 1, wherein the gelifier agent is selected from the group consisting of a gum and an algal polysaccharide and the bioadhesive agent is selected from the group consisting of a cellulose derivative and a vinyl-based polymer.

6. The oral composition of claim 1, wherein the Pediococcus strain is selected from the group consisting of: strain deposited under accession number CECT 8903, strain CECT 8904, strain CECT 8905, and strain CECT 8906.

7. The oral composition of claim 1, wherein the oral composition and the liquid medium are in a single or in separate containers.

8. The oral composition according to claim 7, wherein the bacteria strain is selected from the group consisting of: strain deposited under accession number CECT 8903, strain CECT 8904, strain CECT 8905, strain CECT 8906.

9. The oral composition of claim 1, wherein the at least one gelifier agent is not a cellulose derivative and the at least one bioadhesive agent is selected from a cellulose derivative and a vinyl-based polymer.

10. The oral composition of claim 1, wherein the amount of gelifier agent and bioadhesive agent in the composition is from 0.05 to 20% (w/v) for each agent.

11. A process for preparing a reconstituted formula comprising mixing under agitation the oral composition of claim 1 with a liquid medium.

12. A reconstituted formula obtained by the process of claim 11.

13. The reconstituted formula according to claim 12, wherein the amount of gelifier agent and bioadhesive agent in the reconstituted formula is from 0.05 to 20% (w/v) for each agent.

14. The reconstituted formula according to claim 13, wherein the amount of gelifier agent is from 1 to 5% (w/v) and the amount of bioadhesive agent is from 4 to 10% (w/v).

15. A method of using the oral composition of in claim 1, comprising a step of administering the oral composition to a subject.

16. A method of treating a subject having a condition selected from the group consisting of: peri-implantitis, mucositis, periodontitis, gum disease, caries, oral candidiasis, cold sores and blisters, said method comprises administering the oral composition of claim 1 to the subject.

17. The method of claim 16 wherein the bacteria strain is selected from the group consisting of: strain deposited under accession number CECT 8903, strain CECT 8904, strain CECT 8905, and strain CECT 8906.

18. A kit for oral use, comprising: 1) the oral composition of claim 1; and 2) means to apply to the buccal cavity the oral composition.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: Growth (G) of probiotic strains in artificial saliva supplemented with guar gum (GG) compared to non-supplemented artificial saliva.

(2) FIG. 2: Growth (G) of probiotic strains in artificial saliva supplemented with hydroxyethylcellulose (HEC) compared to non-supplemented artificial saliva.

(3) FIG. 3: Growth (G) of probiotic strains in artificial saliva supplemented with sodium alginate (SA) compared to non-supplemented artificial saliva.

(4) FIG. 4: Growth (G) of probiotic strains in artificial saliva supplemented with methylcellulose (MC) compared to non-supplemented artificial saliva.

(5) FIG. 5: Inhibitory activity (In) of probiotic candidates against Aggregatibacter actinomycetemcomitans.

(6) FIG. 6: Strain genotyping by random amplified polymorphic DNA (RAPD). Patterns obtained after random amplification for 1, PERI1; 2, PERI2; 3, PERI3; 4, PERI4.

EXAMPLES

Example 1. Isolation of the Microorganisms

(7) Lactic acid bacteria candidates were isolated from fresh stools and oral hyssops from 0-9 year-old children. Samples were dissolved in PBS buffer (pH 7.4), aliquoted and plated on MRS supplemented with various antibiotic combinations. Strains were cultured under microaerophilic conditions (5% CO.sub.2) at 37 or 30° C. Incubation time depended on the growth rate, but ran normally from 24 hours to 3 days. Isolation of individual strains proceeded with the same selection media, and then Gram staining was carried out in order to get a first identification. Once grown, isolated strains were stored by freeze-drying in PBS 0.1× with 15% skim milk powder.

Example 2. Genus and Species Identification

(8) Identification to species level was performed by sequencing 16S rRNA gene. Briefly, the DNA of the strains was extracted with Chelex® 100 resin from Bio-Rad Laboratories (Barcelona, Spain). Complete sequence of 16S rRNA gene was amplified by polymerase chain reaction (PCR) using the universal primers for eubacteria 27F and 1492R as previously described [Weisburg, W. G. et al. 1991; Muyzer, G. et al. 1998]. The integrity of PCR products was checked in an agarose gel using SYBR green dye (Invitrogen, Life Technologies, Madrid, Spain). PCR products were sequenced using 27F, 357F, 907R and 1492 primers [Weisburg, W. G. et al. supra; Muyzer, G. et al. supra], a v3.1 Cycle Sequencing kit and an 3130 XL Genetic Analyzer (from Applied Biosystems, Life Technologies, Madrid, Spain). The resulting sequences were aligned and compared with those presents in the National Center for Biotechnology Information (NCBI) and RDP (Ribosomal Database Project). The strains were identified based on the highest hit scores.

(9) The 16S rRNA sequences corresponded to the Pediococcus genus. When compared with NCBI and RDP databases, the sequence of PERI1 corresponded to P. pentosaceus (100% identity); the sequence of PERI2 corresponded to P. acidilactici (100%); the sequence of PERI3 corresponded with a 100% of identity to either P. pentosaceus or P. acidilactici; and the sequence of PERI4 corresponded with a 99% of identity to either P. pentosaceus or P. acidilactici. They were deposited in the Spanish Type Culture Collection (CECT) under the accession numbers CECT 8903, CECT 8904, CECT 8905 and CECT 8906, respectively.

Example 3. Strain Genotyping

(10) Strain genotyping was performed by random amplified polymorphic DNA (RAPD) for confirming that the four strains of Pediococcus sp. deposited were different between them. RAPD was performed as described by Nigatu et al. 1998. RAPD patterns of the strains are depicted in FIG. 6, demonstrating that the four strains were different.

Example 4: Survival to Oral Conditions

(11) Survival of the strains in the oral cavity was studied by evaluating their tolerance to different concentrations agents known to compromise bacterial survival such as hydrogen peroxide (HP) and lysozyme. A total number of 50 lactic acid bacteria candidates were evaluated and compared with commercial probiotics strains namely Streptococcus salivarius K12 (Blis Technologies, New Zeland) and Lactobacillus reuteri DSM17938 (Biogaia, Sweden) which were used as controls.

(12) Probiotic candidates and L. reuteri DSM17938 were grown in Man Rogosa Sharpe agar medium (MRSa) for 18-24 hours at 37° C. and microaerophilic conditions (5% of CO.sub.2). S. salivarius K12 was grown under the same conditions but using Brain Hearth Infusion medium (BHI) instead of MRS. Isolated colonies were used for preparing a bacterial suspension in 0.1 M Phosphate Buffered Saline (PBS) with an optical density corresponding to a McFarland standard 0.5 (approximately 1E+08 CFU/mL). Bacterial suspensions were subsequently 2-fold diluted in MRS or BHI liquid media. Microplates of 96 wells were inoculated with two-hundred microliters of the resulting dilution to which 50 μl of a solution containing either lysozyme or HP in PBS were added. The concentrations of lysozyme tested were 1×10.sup.6 and 5×10.sup.6 U/mL (final concentration in the well of 2×10.sup.5 and 1×10.sup.6 U/mL, respectively) and the concentrations of HP were 5 mM and 25 mM (final concentration in the well of 1 mM and 5 mM, respectively). Microwell plates were incubated for 6 h at 37° C. in microaerophilic conditions (5% of CO.sub.2). Bacterial growth was monitored by determining the absorbance at 625 nm. Percentage of growth was calculated comparing the increment observed in the presence of lysozyme or HP compared to the growth of the bacterial strain in the absence of these agents (positive control) using the following formula:

(13) $\mathrm{Growth}\left(\%\right)=\frac{{\mathrm{OD}}_{\mathrm{LH}}-{\mathrm{OD}}_{C-}}{{\mathrm{OD}}_{C+}-{\mathrm{OD}}_{C-}}*100$
wherein OD.sub.LH was the optical density of the well containing microorganism and either lysozyme or HP,
OD.sub.C− was the average optical density of three wells with the same amount of lysozyme without microorganism,
OD.sub.C+ was the average optical density of the three wells inoculated with bacteria but not lysozyme nor HP (positive control).

(14) Results:

(15) Seven of the fifty probiotic candidates did not grow even in control MRS media (not supplemented). Thus, these strains were discarded as potential candidates. The remaining 43 were ranked according to their capacity to grow in the presence of the highest concentration of lysozyme and HP tested. Results are shown in TABLE 1 and are expressed as means of survival in percentage compared to the growth of the same strain in media not supplemented with lysozyme nor HP.

(16) TABLE-US-00001 TABLE 1 Tolerance of bacteria to lysozyme and hydrogen peroxide concentrations. Lysozyme Lysozyme (2E+5 (1E+6 HP HP U/mL) U/mL) (1 mM) (5 mM) PERI3 n.i. n.i. n.i. n.i. F2043 n.i. n.i. n.i. n.i. F2002A n.i. n.i. n.i. n.i. PERI1 n.i. n.i. n.i. n.i. I1003 n.i. n.i. n.i. n.i. I3153 92.1 n.i. n.i. 90.8 L. reuteri DSM17938 n.i. n.i. 87.9 53.4 I3145 n.i. n.i. n.i. n.i. F3163 98.7 n.i. n.i. n.i. F1031 84.5 n.i. 91.7 92.3 F2003A n.i. n.i. n.i. n.i. PERI4 n.i. n.i. n.i. n.i. I3028 93.0 n.i. 86.0 86.0 I1005 n.i. n.i. n.i. n.i. F2008A 99.8 n.i. n.i. n.i. F2006 n.i. n.i. n.i. n.i. I3118 89.6 99.1 92.5 90.5 F3166 n.i. 97.8 n.i. n.i. PERI2 89.2 96.9 n.i. 92.4 I3143 93.5 96.9 90.9 84.9 I3030 91.3 92.0 92.0 92.8 I3061 92.8 91.6 93.4 90.0 I3149 n.i. 90.6 n.i. n.i. I3142 93.8 90.3 98.1 96.8 I3140 89.8 87.0 93.3 93.8 L. salivarius K12 99.9 86.7 92.9 80.5 I3142A n.i. 86.4 n.i. 90.4 I3130 91   84.1 90.2 88.3 I1004 90.9 83.3 106.0  96.5 F2002B 80.7 81.0 n.i. n.i. F2008B 72.4 76.7 n.i. n.i. F3162 86.0 63.2 95.7 n.i. F2009 85.4 62.3 n.i. n.i. I3142B 94.7 59.1 n.i. n.i. F2005 85.2 58.2 n.i. n.i. F3164 94.5 46.3 n.i. n.i. I1002 31.5 40.4 n.i. 95.7 I3096 41.9 38.3 90.4 87.9 F2041 36.9 29.3 n.i. n.i. F2044 34.8 25.1 n.i. n.i. F2003B 31.7 13.4 n.i. n.i. F3165 n.i. 11.8 n.i. n.i. I3086 87.1 10.9 87.2 87.0 F3159B n.i.  5.0 n.i. n.i. Abbreviations: n.i. = no inhibition (growth 100%); HP = hydrogen peroxide.

(17) The 25 first bacteria showing the highest tolerance to lysozyme and HP were considered the best candidates and were selected for subsequent in vitro test for evaluating their probiotic properties. As can be observed all the strains showed good tolerance to high concentrations of lysozyme showing a survival ratio not lower than 86%, which was similar to that of the commercial controls. The LAB candidates showed also good tolerance to HP with values higher than 84% at a concentration of 5 mM of HP. These results compared well with the survival ratio of the commercial controls L. reuteri DSM17938 and L. salivarius K12 (53.4 and 80.5% respectively).

Example 5: Use of Guar Gum as a Gelifier with Prebiotic Effect

(18) The capacity of the strains to use guar gum and increase their growth was studied in vitro. For this purpose, the growth of probiotic candidates in artificial saliva supplemented with guar gum was compared to their respective growth in artificial saliva that was not supplemented. Artificial saliva contained 1 g/L ‘Lab-lemco’ powder (Oxoid, Basingstoke, UK), 2 g/L yeast extract (Oxoid), 5 g/L proteose peptone (Oxoid), 2.5 g/L hog gastric mucin (Sigma Chemical Co., Poole, UK), 35 g/L sodium chloride (BDH Chemicals Ltd, Poole, UK), 0.2 g/L calcium chloride (BDH), 0.2 g/L potassium chloride (BDH) in distilled water. Artificial saliva was supplemented with guar gum (Genox Pharma, Barcelona, Spain) to a final concentration of 0.5% (w/v). Artificial saliva without gel ingredient was also prepared to compare the effect of guar gum in bacterial growth with a non-supplemented medium. After autoclaving, 1.25 mL of 40% urea per liter of artificial salivary medium were added. Two hundred microliters of the different media prepared were pipetted in 96-well plates. Immediately after 20 microliters of a suspension of probiotic candidates standardized to 1E+07 CFU/mL in PBS were added. The same amount of PBS without bacterial inoculum was used as a negative control. Plates were incubated for 24 h at 37° C. in anaerobiosis and bacterial growth monitored by determining the optical density at 625 nm. The capacity of probiotic candidates to use guar gum for growing was calculated according to the following formula:
ΔGrowth=(ΔDO.sub.gp−ΔDO.sub.g0)−(ΔDO.sub.sp−ΔDO.sub.s0)
wherein ΔDO.sub.gp is the difference between the optical density at 625 nm after 24 h compared with 0 h in the wells supplemented with guar gum and inoculated with probiotic candidates;
ΔDO.sub.g0 is the difference between the optical density at 625 nm at 24 h compared with 0 h in the wells supplemented with guar gum but containing PBS instead of LAB;
ΔDO.sub.sp is the difference between the optical density at 625 nm at 24 h compared with 0 h in the wells not supplemented with guar gum and inoculated with probiotic candidates; and
ΔDO.sub.s0 is the difference between the optical density at 625 nm at 24 h compared with 0 h in the wells not supplemented with guar gum and containing PBS instead of LAB.

(19) Results were compared with those obtained with the commercial probiotics L. reuteri DSM 17938, L. brevis CD2, Streptococcus salivarius K12 and with the pathogens Fusobacterium nucleatum and Porphyromonas gingivalis. The experiment was performed in duplicate.

(20) Results

(21) The capacity to use guar gum as a nutrient and potentiate their growth compared to non-supplemented artificial saliva is depicted in FIG. 1. The effect of guar gum on LAB growth was highly dependent on the strain tested. Whereas guar gum potentiated the growth of some strains, it had a detrimental effect in the growth of others compared to non-supplemented saliva. The strains PERI1; PERI2; PERI3; PERI4; F3163; I1003; I1005; I3028; I3030; I3140; I3142A; I3145 and I3153 benefit from the addition of guar gum. PERI1 was the strain showing the highest performance. Among the control strains tested, L. brevis CD2 and S. salivarius K12 benefit from the addition of guar gum, although the effect of guar gum on the growth of this strains was lower than other LAB candidates such as PERI1, PERI2, PERI3, F3163; I1003 and 3142A. Notably, the effect of guar gum was negligible in the case of Fusobacterium nucleatum and Porphyromonas gingivalis which is of interest for avoiding the undesirable growth of pathogens.

Example 6: Use of Hydroxyethylcellulose (HEC) as an Adhesive Agent with Prebiotic Effect

(22) The capacity of the strains to use HEC to potentiate their growth was assayed as explained above in Example 5 for guar gum.

(23) Results

(24) The effect of HEC on probiotic growth compared to non-artificial saliva is shown in FIG. 2. The growth of few strains was potentiated by the addition of HEC. Strains namely PERI4, I1005 and I3142A were significantly benefited by the use of HEC in the gel. Other strains, including the commercial strains had low capacity to use this ingredient.

Example 7: Use of Other Gelifier Agents as Ingredient with Prebiotic Effect

(25) The potential use of other gelifier agents to increase probiotic growth was also studied by using the same methodology explained above. Particularly, sodium alginate (SA) and methylcellulose (MC) were used as potential gelifier agents with prebiotic effect.

(26) Results

(27) The effect of SA supplementation on bacterial growth compared to non-supplemented artificial saliva is depicted in FIG. 3. Different strains were able to use SA as nutrient for increasing their growth including PERI; PERI2; PERI3; PERI4; I1003; I1005; I3028; I3030; I3130; I3142A; I3145 and I3153. In contrast, Fusobacterium nucleatum and Porphyromonas gingivalis had low capacity to use SA for growing. Results for MC are present in FIG. 4. None of the pathogen and control strains were able to use MC for increasing growth. In contrast, different probiotics strains benefit from the supplementation of MC, including F2008A; PERI2; F2043; PERI4; I1003; I1005; I3028; I3061; I3118; I3140 and I3142A.

Example 8: Antagonism Against Porphyromonas gingivalis, Fusobacterium nucleatum and Prevotella intermedia

(28) The antagonistic activity of probiotic candidates was assessed against bacteria abnormally abundant in patients presenting peri-implantitis. In particular, the pathogen strains were Porphyromonas gingivalis DSM-20709, Fusobacterium nucleatum DSM 20482 and Prevotella intermedia DSM-20706 DSM 8324. L. reuteri DSM 17938 from Biogaia (Sweden), L. brevis CD2 (Inersan®, VSL Pharmaceuticals, Inc., USA) and Streptococcus salivarius K12 (BLIS Technologies, New Zealand) were used as commercial controls. The capacity of the LAB candidates to inhibit pathogen growth was determined by using the Campbell protocol. Briefly, probiotic candidates and Lactobacillus controls were uniformly seeded in MRS agar plates and allowed to grow to confluence for 24 h at 37° C. and 5% of CO.sub.2. Streptococcus salivarius K12 was grown under the same conditions but using BHI medium.

(29) Pathogen strains were cultured overnight. Isolated colonies of these pathogens were used to prepare suspensions in phosphate buffered saline (PBS) medium and swabbed uniformly in appropriate solid medium for their growth: F. nucleatum and P. intermedia were seeded in blood agar and Porphyromonas gingivalis in Schaeder Anaerobe Sheep Blood Agar. Immediately, cylindrical sections of 6 mm in diameter of the confluent agar plate of the tested LAB candidates were placed lane-to-lane on the pathogen seeded plate, confronting the pathogen seeded plate with the grown-agar side of one of the cylinder sections and with the non-grown side of the other cylinder section. Plates were incubated 48 h at 37° C. in anaerobic conditions. Then, inhibition zones were measured by placing the agar plate over a flat rule and measuring the halos where pathogen growth was inhibited (either partially or completely). Growth inhibitory activity (GI) was then calculated by subtracting the cylinder diameter (CD) from the inhibition zone diameter (IZD) measured in millimeters. The final inhibitory activity was calculated as a mean value of the GI values for the two above-mentioned cylinder sections for each probiotic strain, i.e. averaging the duplicates. All experiments were performed in duplicate.

(30) Results

(31) The antagonistic activity of the different probiotic candidates is detailed in TABLE 2. The strains PERI3, PERI4 and F3166 were the three strains showing the greatest activity against F. nucleatum, and showed a higher activity than L. brevis CD2, L. reuteri DSM17938 and S. salivarius K12. The candidates F1031 and PERI2 were the candidates showing the highest activity against P. intermedia. Several strains were also efficient inhibiting P. gingivalis. Among them PERI1, F2006, PERI2, F3163, I1003, I3143, I3145 and I3153 showed higher activity than the commercial controls used for comparison purposes.

(32) TABLE-US-00002 TABLE 2 Inhibitory activity against F. nucleatum, P. intermedia and P. gingivalis (results expressed as means ± SD in mm) Fusobacterium Prevotella Porphyromonas Strain nucleatum intermedia gingivalis F1031 n.i 4.5 ± 0.7  3.5 ± 0.7 PERI1 1.5 ± 0.7 2.0 ± 1.4 15.0 ± 1.4 F2002A 3.0 ± 0.0 1.0 ± 0.0 n.i F2003A 1.5 ± 0.7 n.i n.i F2006 2.0 ± 1.4 n.i 17.5 ± 0.7 F2008A 3.5 ± 0.7 1.0 ± 0.0 11.5 ± 0.7 PERI2 2.0 ± 0.0 5.0 ± 0.0 13.5 ± 0.7 F2043 3.5 ± 0.7 n.i n.i PERI3 4.0 ± 1.4 n.i 14.0 ± 0.0 PERI4 4.0 ± 0.0 n.i 11.0 ± 1.4 F3163 2.0 ± 0.0 2.0 ± 2.8 13.0 ± 1.4 F3166 4.0 ± 0.0 n.i n.i I1003 0.5 ± 0.7 0.5 ± 0.7 16.0 ± 0.0 I1005 1.0 ± 0.0 n.i n.i I3028 3.0 ± 0.0 n.i n.i I3030 2.5 ± 0.7 n.i n.i I3061 n.i n.i  8.0 ± 0.0 I3118 2.0 ± 0.0 n.i n.i I3140 1.5 ± 0.7 n.i  9.0 ± 1.4 I3142 1.5 ± 0.7 1.0 ± 0.0  8.0 ± 1.4 I3142A n.i 0.5 ± 0.7 n.i I3143 2.5 ± 0.7 n.i 13.0 ± 1.4 I3145 3.0 ± 0.0 n.i 13.5 ± 0.7 I3149 2.0 ± 0.0 n.i n.i I3153 2.5 ± 0.7 1.0 ± 0.0 14.0 ± 0.0 L. brevis CD2 3.0 ± 0.0 2.5 ± 0.7 12.0 ± 0.0 L. reuteri DSM17938 n.i 1.0 ± 0.0  9.0 ± 0.0 S. salivarius K12 n.i 6.0 ± 2.8 10.0 ± 1.4

Example 9: Antagonism Against Aggregatibacter actinomycetemcomitans

(33) The activity of probiotic candidates to antagonize Aggregatibacter actinomycetemcomitans was studied in liquid medium. Probiotic candidates and Lactobacillus sp. controls were grown overnight at 37° C. in microaerophilic conditions (5% CO.sub.2) in MRS liquid medium. Staphylococcus salivarius K12 was grown in same conditions but using BHI medium. Cultures were centrifuged and supernatant filtered through 0.22 micrometers. Twenty microliters of the filtered supernatants were added to 96-well microplates containing 160 ml of BHI medium. Finally, 20 ml of a suspension of A. actinomycetemcomitans in PBS standardized to 1E+05 CFU/mL were added to the wells and incubated for 24 h in microaerophilic conditions (5% CO.sub.2) at 37° C. A. actinomycetemcomitans was monitored by determining the absorbance at 625 nm. The inhibitory capacity of probiotic supernatants was determined by comparing the growth of A. actinomycetemcomitans supplemented with probiotic supernatant and its growth without being supplemented (negative control) by using the following formula:

(34) $\mathrm{Inhibition}\left(\%\right)=\frac{\left({\mathrm{DO}}_C-{\mathrm{DO}}_B\right)-\left({\mathrm{DO}}_C-{\mathrm{DO}}_P\right)}{\left({\mathrm{DO}}_C-{\mathrm{DO}}_B\right)}\times 100$
wherein,
DO.sub.c corresponded to the negative control and was the optical density at 625 nm of wells containing 160 μl of BHI medium+20 μl of A. actinomycetemcomitans suspension+20 μl of MRS or BHI,
DO.sub.B corresponded to the blank and was the optical density at 625 nm of wells containing 160 μl of BHI medium+40 μl of MRS or BHI, and
DO.sub.P corresponded to probiotic candidates and was the optical density at 625 nm of wells containing 160 μl of BHI medium+20 μl of A. actinomycetemcomitans suspension+20 μl of probiotic supernatant.

(35) All experiments were performed in duplicate.

(36) Results

(37) The inhibitory activity of the probiotic candidates against A. actinomycetemcomitans is depicted in FIG. 5. Among the different strains, PERI3 showed the greatest activity, being able to reduce by 77.9% the growth of A. actinomycetemcomitans. This activity was significantly higher than commercial controls such as L. brevis CD2, L. reuteri DSM17938 and especially S. salivarius K12 (45.6, 38.8 and 2.42%, respectively).

Example 10: Capacity to Form Aggregates

(38) The capacity of bacteria to auto-aggregate is considered the first step necessary for forming a biofilm and can be used as a characteristic for assessing the potential biofilm-forming capacity of the strains. Biofilm formation allows to create a protective barrier that can reduce the attachment of pathogen to the oral surfaces. The capacity to form aggregates was evaluated for probiotic candidates PERI, PERI2, PERI3, PERI4, I3142A, I1005, I3030 and I3145. L. brevis CD2 and S. salivarius K12 were used as controls. Strains were grown overnight in MRS medium (or BHI for S. salivarius K12) at 37° C. and microaerophilic conditions (5% CO.sub.2). After this period, cultures were centrifuged at 1000 g for 5 minutes, supernatant discarded and pellet washed twice with PBS. Finally, PBS was added until obtaining a probiotic suspension having an optical density equivalent to a McFarland standard 1 (approximately. 3E+08 CFU/mL). Three mL of the suspension were transferred to spectrophotometer cuvettes and optical density monitored at 620 nm for 3 and 6 hours. The auto-aggregation capacity at this time intervals were determined by using the following formula:

(39) $\mathrm{Aggregation}\left(\%\right)=\frac{{\mathrm{DO}}_0-{\mathrm{DO}}_t}{{\mathrm{DO}}_0}\times 100$
wherein DO.sub.0 is the net absorbance at 620 nm of the bacterial suspension at the beginning of the test (time 0), and
DO.sub.t is the net absorbance at 620 nm of the bacterial suspension at either 3 or 6 hours.

(40) Results

(41) Percentage of aggregation at 3 and 6 h is summarized in TABLE 3. The probiotic candidate PERI4 was the strain showing the highest auto-aggregation capacity whereas the candidate I3030 and S. salivarius K12 showed the lowest activity.

(42) TABLE-US-00003 TABLE 3 Percentage of aggregation of probiotic candidates Strain 3 h 6 h I1005 11.2 13.3 I3030 6.9 6.9 I3142A 5.7 24.5 I3145 5.2 21.1 PERI1 8.3 17.2 PERI2 6.7 19.7 PERI3 4.3 14.7 PERI4 3.9 27.8 L. brevis CD2 11.1 26.9 S. salivarius K12 0.0 7.9

Example 11: Preparation of a Reconstitutable Probiotic Gel in Powder Form for Implant Application

(43) 500 g of a freeze-dried powder containing Pediococcus CECT 8904, Pediococcus CECT 8905 and Pediococcus CECT 8906 at 4E+10 cfu/g, 200 g of guar gum and 300 g of hydroxyethylcellulose were mixed and homogenized. 0.5 g of this powder blend were introduced into a glass vial provided with a screw cap. Upon the addition of 2.5 ml of water, preferably deionized or distilled water, and manual shaking, the reconstituted gel was formed. The final dose of probiotic was 1E+10 cfus/vial. After reconstitution of gel, the concentration of guar gum in gel was 4% and hydroxyethylcellulose 6%.

Example 12: Preparation of a Reconstitutable Probiotic Gel in Powder Form for Implant Application

(44) 535 g of a freeze-dried powder containing Pediococcus CECT 8904 and Pediococcus CECT 8905 at 3.75E+10 cfu/g, 150 g of sodium alginate, 15 g of calcium acetate and 300 g of hydroxyethylcellulose were mixed and homogenized. 0.5 g of this powder blend were introduced into a glass vial provided with a septum and aluminum capsule. Upon the addition of 2.5 mL of water with a syringe, preferably deionized or distilled water, and manual shaking, the reconstituted gel was formed. The final dose of probiotic was 1E+10 CFU/vial. After reconstitution of gel, the concentration of alginate in gel was 3% and hydroxyethylcellulose 6%.

Example 13: Preparation of a Reconstitutable Probiotic Gel in Powder Form for Implant Application

(45) 700 g of a freeze-dried powder containing Pediococcus CECT 8904, Pediococcus CECT 8905 and Pediococcus CECT 8906 at 2.9E+10 cfu/g, 200 g of guar gum and 100 g of polyvinylpyrrolidone were mixed and homogenized. 0.5 g of this powder blend were introduced into a glass vial provided with a septum and aluminum capsule. Upon the addition of 2.5 mL of water with a syringe, preferably deionized or distilled water, and manual shaking, the reconstituted gel was formed. The final dose of probiotic was 1E+10 cfu/vial. After reconstitution of gel, the concentration of guar gum in gel was 4% and polyvinylpyrrolidone 2%.

Example 14: Preparation of a Reconstitutable Probiotic Gel in Powder Form for Implant Application

(46) 500 g of a freeze-dried powder containing Pediococcus CECT 8904, Pediococcus CECT 8905 and Pediococcus CECT 8906 at 4E+10 cfu/g, and 500 g of guar gum were mixed and homogenized. 0.5 g of this powder blend were introduced into a glass vial provided with a screw cap. Upon the addition of 6 ml of water, preferably deionized or distilled water, and manual shaking, the reconstituted gel was formed. The final dose of probiotic was 1E+10 cfus/vial. After reconstitution of gel, the concentration of guar gum in gel was 4%.

Example 15: Preparation of a Reconstitutable Probiotic Gel in Powder Form for Teeth Application

(47) 600 g of a freeze-dried powder containing Pediococcus CECT 8903 at 3.35E+10 cfu/g, 100 g of guar gum and 300 g of hydroxyethylcellulose were mixed and homogenized. 0.5 g of this powder blend were introduced into a glass vial provided with a screw cap. Upon the addition of 2.5 ml of water, preferably deionized or distilled water, and manual shaking, the reconstituted gel was formed. The final dose of probiotic was 1E+10 cfu/vial. After reconstitution of gel, the concentration of guar gum in gel was 2% and hydroxyethylcellulose 6%.

Example 16: Preparation of a Reconstitutable Probiotic Gel in Powder Form for Teeth Application

(48) 590 g of a freeze-dried powder containing Pediococcus CECT 8903 at 3.4E+10 cfu/g, 100 g of sodium alginate, 10 g of calcium acetate, and 300 g of hydroxyethylcellulose were mixed and homogenized. 0.5 g of this powder blend were introduced into a glass vial provided with a screw cap. Upon the addition of 2.5 mL of water, preferably deionized or distilled water, and manual shaking, the reconstituted gel was formed. The final dose of probiotic was 1E+10 cfu/vial. After reconstitution of gel, the concentration of alginate in gel was 2% and hydroxyethylcellulose 6%.

Example 17: Preparation of a Reconstitutable Probiotic Gel in Powder Form for Teeth Application

(49) 280 g of a freeze-dried powder containing Pediococcus CECT 8906 at 7E+10 cfu/g, and 720 g of hydroxyethylcellulose were mixed and homogenized. 0.5 g of this powder blend were introduced into a glass vial provided with a screw cap. Upon the addition of 6 ml of water, preferably deionized or distilled water, and manual shaking, the reconstituted gel was formed. The final dose of probiotic was 1E+10 cfus/vial. After reconstitution of gel, the concentration of hydroxyethylcellulose in gel was 6%.

Example 18: Preparation of a Reconstitutable Probiotic Gel in Powder Form for Teeth Application

(50) 600 g of a freeze-dried powder containing Lactobacillus brevis CD2 at 4E+10 cfu/g, 100 g of guar gum and 300 g of hydroxyethylcellulose were mixed and homogenized. 0.5 g of this powder blend were introduced into a glass vial provided with a screw cap. Upon the addition of 2.5 ml of water, preferably deionized or distilled water, and manual shaking, the reconstituted gel was formed. The final dose of probiotic was 1E+10 cfu/vial. After reconstitution of gel, the concentration of guar gum in gel was 2% and hydroxyethylcellulose 6%.

Example 19: Preparation of a Reconstitutable Probiotic Gel in Powder Form for Teeth Application

(51) 590 g of a freeze-dried powder containing Lactobacillus brevis CD2 at 4E+10 cfu/g, 100 g of sodium alginate, 10 g of calcium acetate, and 300 g of hydroxyethylcellulose were mixed and homogenized. 0.5 g of this powder blend were introduced into a glass vial provided with a screw cap. Upon the addition of 2.5 mL of water, preferably deionized or distilled water, and manual shaking, the reconstituted gel was formed. The final dose of probiotic was 1E+10 cfu/vial. After reconstitution of gel, the concentration of alginate in gel was 2% and hydroxyethylcellulose 6%.

Example 20: Preparation of a Reconstitutable Probiotic Gel in Powder Form for Teeth Application

(52) 700 g of a freeze-dried powder containing Streptococcus salivarius K12 at 2.9E+10 cfu/g, 200 g of guar gum and 100 g of polyvinylpyrrolidone were mixed and homogenized. 0.5 g of this powder blend were introduced into a glass vial provided with a septum and aluminum capsule. Upon the addition of 2.5 mL of water with a syringe, preferably deionized or distilled water, and manual shaking, the reconstituted gel was formed. The final dose of probiotic was 1E+10 cfu/vial. After reconstitution of gel, the concentration of guar gum in gel was 4% and polyvinylpyrrolidone 2%.

Example 21. Application of the Reconstitutable Probiotic Gel in a Patient with Peri-Implantitis

(53) The crown was removed and local anesthesia was administered to the patient. The zone was cleaned and the subgingival plaque mechanically removed. Chlorhexidine 0.12% was administered and after that, saline solution. The reconstituted gel of Example 11 was obtained by adding 2.5 mL of sterile water to the freeze-dried powder containing Pediococcus CECT 8904, Pediococcus CECT 8905 and Pediococcus CECT 8906 at 4E+10 cfu/g, 200 g of guar gum and 300 g of hydroxyethylcellulose and vigorously mixing for 1 minute. The mixture was allowed to stand at room temperature for a period between 1 and 10 minutes and administered to the peri-implant pocket with a syringe and needle with blunt tip, positioning the tip of the blunt needle close to the base of the pocket and injecting the product until the solution reaches the upper edge of the gum. Then, after drawing the needle out of the pocket, saline solution washings and an air jet (during ca. 10 sec.) were applied on the treated zone. Immediately after, the crown was put in place. The patient was instructed not to brush the teeth within 6 hours post-treatment.

Example 22. Application of the Reconstitutable Probiotic Gel in a Patient for the Prevention of Caries

(54) Teeth were cleaned with a toothbrush and cleared of heavy plaque or debris. The teeth to be treated were lightly dried with air and isolated with cotton rolls to prevent recontamination with saliva. A small amount of gel (0.5 ml) made following Example 15 was dispensed by means of a brush to the teeth. The patient was instructed to avoid brushing for the rest of the day.

Example 23. Application of the Reconstitutable Probiotic Gel in a Patient for the Prevention of Caries

(55) Teeth were cleaned with a toothbrush and cleared of heavy plaque or debris. A self-film-forming composition comprising 50 mg of probiotic strain, 120 mg of guar gum and 360 mg of hydroxyethylcellulose was reconstituted with 6 mL of water and immediately aspired with a syringe. The gel was allowed to stand for 1 minute in the syringe and then uniformly distributed in a mouth splint. Subsequently, the splint was immediately applied in the mouth and removed after 5 minutes. The patient was provided with more vials containing the self-film-forming composition and instructed to follow the same procedure for self-administrating the reconstituted gel each 48 hours, preferably at night after brushing their teeth, just before going to sleep. Patients were instructed not to bush their teeth, eat or drink after applying the gel.

Example 24: Efficacy Study of the Reconstitutable Probiotic Gel in Animal Model

(56) The efficacy of the probiotic gel on the prevention of mucositis and peri-implantitis was studied in Beagle dog as animal model. All procedures were conducted under the supervision of a veterinary surgeon. Animals were pre-anesthetized with acepromazine (0.12%-0.25 mg/kg), buprenorphine (0.01 mg/kg) and medetomidine (35 lg/kg) by intramuscular injection in the femoral quadriceps. An intravenous catheter was inserted (diameter 22 or 20 gauge) into the cephalic vein, and propofol infused at the rate of 0.4 mg/kg/min at a slow constant infusion rate. Conventional dental infiltration anaesthesia (articaine 40 mg, 1% epinephrine) was administered at the surgical sites. Both quadrants of the lower jaws, second premolars (PM2) and first molars (M1) were used as experimental sites. Teeth were sectioned with a carbide tungsten drill and roots removed with forceps, without damaging the remaining bony walls. Sulcular marginal incisions were made along the vestibular and lingual areas adjoining the alveoli, separating tissues to make crestal hard tissue walls visible. After two months of site healing 8 implants were crestally placed and allowed to heal for another two more months with healing cups. After the two months of healing, silk ligatures were placed around each abutment. Oral gels were also administered around the implants. Five dogs were treated with a reconstituted liquid gel containing 4% of guar gum, 6% hydroxyethylcellulose and 4 CFU per mL of a probiotic mixture composed by Pediococcus CECT 8904, Pediococcus CECT 8905 and Pediococcus CECT 8906 (1:1:1). One of the animals was treated with the same gel, but not containing probiotic. The animals were then fed a soft diet to induce plaque accumulation and to provoke peri-implant inflammation and loss of bone. Additional ligatures were placed over the previous ones and around the implants every two weeks.

(57) Healing was uneventful after all surgeries, no exposure or secondary wound healing was observed. The experimental peri-implantitis resulted in signs of inflammation and bone loss. Generally, animals treated with probiotic gel showed less pronounced tissue loss, inflammatory response, probing depth, mucosal recession, and bleeding on probing, compared to the animal treated with gel not containing probiotic. Therefore, probiotic treatment ameliorated clinical signs associated with peri-implantitis.

Example 25: Study of Rheological Properties of Gelifier and Bioadhesive Agents

(58) The viscosity and adhesiveness of different agents was studied. The following compositions were studied: Sodium alginate at concentrations ranging from 2 to 8% in water (w/v), with or without calcium acetate at concentrations (0.02-0.2%). Guar gum at concentrations ranging from 1 to 5% in water (w/v). Methylcellulose at concentrations ranging from 1 to 5% in water (w/v). Hydroxyethylcellulose at concentrations ranging from 1 to 6% in water (w/v). Sodium carboxymethylcellulose at concentrations ranging from 1 to 3% in water (w/v).

(59) TABLE-US-00004 TABLE 4 Viscosity and adhesiveness capacity: Adhe- Agent Viscosity siveness Observations Sodium alginate High Very low Lump formation at high concentrations Guar gum Very high Very low Good solubility Methylcellulose Low Very low Foam formation under agitation Hydroxyethylcellulose Very low Very high Good solubility Carboxymethylcellulose Very low Low Good solubility

(60) The viscosity and adhesiveness conferred to the film-forming compositions was dependent on the agent used offering different possibilities depending on the clinical application of the gel. Combinations with sodium alginate and, especially, guar gum with hydroxyethylcellulose were considered good candidates to form gels combining properties such as high viscosity and adhesiveness.

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