Indian Patents. 209522:A PROCESS FOR PREPARATION OF A COMPOSITION CONTAINING EXOGENOUS LACTIC BACTERIA STRAINS AGAINST ACTINOMYCES NAESLUNDII RELATED DISEASE

Title of Invention	A PROCESS FOR PREPARATION OF A COMPOSITION CONTAINING EXOGENOUS LACTIC BACTERIA STRAINS AGAINST ACTINOMYCES NAESLUNDII RELATED DISEASE
Abstract	The use of a lactic bacteria strain that is exogenous to the oral microflora, which has been selected for its ability to adhere the pellicle of the teeth and to produce a growth inhibition factor, for the preparation of a composition intended for reducing dental plaque and for treating or preventing root caries and other diseases related to Actinomyces naeslundii in mammals.

Full Text	Use of exogenous lactic bacteria strain against Actinomyces naeslundiU related diseases The present invention relates to the incorporation in the oral micro flora of exogenous lactic bacteria which are able to modulate the colonization of A. naeslundii and to reduce the severity of A -related diseases. Background of the invention The mouth (oral cavity) contains a resident and a non-resident micro flora. The first includes microorganisms that are able to establish a more or less permanent residence on the oral surfaces. These bacteria are mainly localised on the tongue, the buccal mucosa and the teed while the gingival, lips, cheeks, palate and floor of the mouth only support a very sparse microflora. The dental plaque is a film that forms on the surface of teeth consisting of bacterial cells in a matrix of extracellular polysaccharides and savory products. Immediately after eruption, the teeth are covered with an amorphous layer of saliva, the acquired enamel pellicle (AEP) that is about 13 ‘im thick and cannot be removed by normal tooth brushing. The deposition of bacteria on teeth follows iihmediately the formation of the AEP and plaque becomes evident in 8-12 hours as a multi-layered structure. The first layer consists of bacteria (earliest colonisers) that attach to teeth mainly via specific adhesion-receptor recognition; it forms a substratum for the second colonisers that adhere one to the other via analogous specific binding or via simple juxtaposition. On the tongue and the buccal mucosa, the natural resident microflora includes microorganisms selected from Streptococcus, Veillonella, Bacteroides and Haemophilus* On the teeth, Streptococci and Actinomyces predominate but a variety of Gram positive and negative coccid and rods can be found. Many of these microorganisms are innocuous commensally, but a lot of them have been recognised as the etiologic agent of quite a few diseases (Hill, M. J. and Amish, P. D. eds. Human Microbial Ecology, 1990, CRC Press, Boca Raton Florida, USA). In particular, Actinomyces naeslundii genospecies 1 (formerly A. naeslundii) and 2 (formerly A. viscous) are common members of human dental plaque. They are among the strongest plaque forming oral strains, because of their capacity to firmly adhere to the teeth and to coaggregate with many other bacterial species, thus fostering their establishment in the mouth. Moreover, in the elderly, they are commonly isolated at root caries sites, and they are believed to be the major aetiological agent of this disease (Bowden, G.H., et aL 1999, The diversity and distribution of the predominant biotypes of Actinomyces naeslundii genospecies 1 and 2 in samples from enamel and from healthy and carious root surfaces of teeth. J.DenuRes.1%, 1800-1809). The transient microflora comprises exogenous bacteria that can be occasionally present in the mouth, but that do not establish a permanent residence (even if repeated oral administrations of these bacteria are carried out). All the food bacteria, and in particular lactic acid bacteria, can be part of this transient microflora. Some of these exogenous lactic bacteria have been shown to be capable of adhering to the pellicle of teeth. For example, WO 00/09080 (Soci6t6 des Products Nestl6) discloses lactic bacteria strains, that are not part of the resident microflora of the mouth, that are low acidifying and that are capable of adhering directly to the pellicle of the teeth. These bacteria are particularly used for treating or preventing dental caries and periodontal infection that are caused by cryogenic microorganism such as Streptococcus mutants and Streptococcus sopranos. Exogenous bacteria can also produce factors that inhibit the growth of the resident microflora in the mouth. For example, EP759469 (Soci6t6 des Produits Nestl6) described the use of a bacteriocin produced by Micrococcus varians for inhibiting the development of the oral pathogens 5. sorbents, S, anguish, 5. mutants and A. viscosus The application of bacteriocins is also one of the investigated strategies which have been set up to reduce tooth caries. These molecules have attracted interest as prospective antiquaries agents and as factors important in modulating colonization of the oral cavity. It is to note that the prior art does not provide any information concerning strains that can establish in the oral cavity by directly adhering to the pellicle of the teeth and also produce factors such as growth inhibition factors, which can modulate the colonization of A, naeslundii so as to reduce the severity of A. naeslundii'- diseases. Summary of the invention Consequently the present invention aims to provide the use of lactic bacteria that are exogenous to the oral microflora, which have been selected for their ability to adhere to the tooth surface and to produce a growth inhibition factor, for the preparation of a composition intended for reducing dental plaque and for treating or preventing root caries or other disease related to Actinomyces naeslundii in mammals. The lactic bacteria may be selected from the group consisting of Streptococcus Thus, by colonizing the surface of teeth and producing growth inhibition factors, such lactic bacteria can exert an significant reduction of the extent of Actinomyces iaeslundii, thus reducing dental plaque,,root caries and other Actinomyces naeslundii related infections. Another object is to provide a composition for maintaining the health of the mouth by reediting the colonization of Actinomyces naeslundii said composition comprises an exogenous lactic bacteria that has been selected for its ability to adhere to the tooth surface and to produce a growth inhibition factor. Such a composition may contain at least 10' -10’ cfu /g of lactic bacteria. The invention also provides a method for the prevention or the treatment of Actinomyces naeslundi related infections, particularly dental plaque extent and root caries in a mammal, comprising the step of feeding a mammal a composition containing at least one lactic bacteria strain selected for its ability to adhere to the tooth surface and to produce a growth inhibition factor, said composition reduces the colonization of Actinomyces naeslundi. Detailed description of the invention Within the following description, the mouth defines the oral cavity of humans or animals such as pets, composed by the oral mucosa (gums, lips, cheeks, palate and floor of the mouth), the tongue and the teeth (including artificial structures). The terms "inhibition growth factor" defines any extracellular substance produced by the adherent exogenous lactic bacteria that enables it to inhibit the growth of A.naeslundL With respect to the first object of the present invention, the use of an exogenous lactic bacteria that has been selected for its ability to adhere to the tooth surface and to produce a growth inhibition factor, for the preparation of a composition intended for reducing dental plaque and for treating or preventing root caries or other disease related to Actinomyces naeslundU is concerned. The lactic bacteria may be selected from the group consisting of Streptococcus thermophilus’ Lactococcus lactis subsp, lactis, and Lactococcus lactis subsp. lactis biovar diacetylactis and particularly from the group consisting of the strains Sreptococcus thermophilus (NCC 1529) (CNCM 1-1984), Sreptococcus thermophilus (NCC 1561) (CNCM 1-1985), Lactococcus. lactis subsp. lactis (NCC 2211) (CNCM 1-1986), Lactococcus, lactis subsp. lactis biovar dioacetylactis (NCC 2225) (CNCM 1-1987). The lactic bacteria is preferably from dairy origin (i.e. originating from milk or cheese, for example)* The lactic bacteria according to the invention is "low acidifying", which means that it is less acidifying than pathogenic strains- Accordingly, it can contribute to a pH in the oral cavity of about 5,5-7. These strains have been selected among latic bacteria strains for their capacity of adherence to the pellicle of the teeth, their optimal growth temperature is about 37°C, which is the temperature in the oral cavity. They are also capable of producing a growth inhibition factor, which combined to their adhesion properties allow them to significantly decrease the colonization extent of A. naeslundii genospecies 1 and 2. Moreover they are capable of fermenting glucose and sucrose and do not synthesised gleans, which are factors of pathogen city of the cariogenic strains. It is also possible to use at least one lactic bacteria strain in combination with a bacteriocin, for example. The lactic bacteria strains may be included in a food, pet food, cosmetic or pharmaceutical composition, for example. Accordingly, these compositions are preferably toothpaste, mouth rinse, gum, spray, beverage, candies, infant formula, ice cream, frozen dessert, sweet salad dressing, milk preparations, cheese, quark, yogurt, acidified milk, coffee cream or whipped cream, for example. The exogenous lactic bacteria may be used in an amount of at least 10"‘-10’ cfu /g of lactic bacteria. The effect of incorporating the above-mentioned bacteria in the oral microflora wa$ tested m a rat model. The strains CNCM1-1985 and CNCM-1986 were able to modulate the oral microbial ecology, significatively reducing the number of total CFU* More specifically, the strains were able to significantly decrease the colonization extent of A. naeslundii genospecies 2, with which the rats had been infected (see examples). BIOCHEMICAL CHARACTERIZATION OF THE SELECTED STRAINS Fermentatioii patterns: 49 simple sugars were tested with the ape 50 CH bioMerieux strip test (bioM6rieux SA, 69280 Marcy-lEtoile, France) and the results are given in Table 1, Table 1. Sugar fermentation of L lactis CNCM I- 1987 (A), L lactis CNCM I-1986 (B), 5. thenvophilus CNCM 1-1984 (C), S, thermophilus CNCM M985 (D). The strains Sreptococcus thermophilus (NCC 1529), Sreptococcus thermophilus (NCC 1561), Lactococcus, lactis subsp. lactis (NCC 2211), Lactococcus. lactis subsp. lactis biovar dioacetylactis (NCC 2225) were deposited under the Budapest Treaty, at the Collection Nationale de Culture de Microorganismes (CNCM M984, CNCM M985, CNCM M986 and CNCM I-1987 respectively), 25 rue du docteur Roux, 75724 Paris, France, on March 3’’, 1998. The second main object of the present invention relates to a composition for maintaining the health of the mouth by reducing the colonization of A. naeslundii in mammals, said composition comprises an exogenous lactic bacteria, which has been selected for its ability to adhere to the tooh stxrface and to produce a growth inhibition factor. These compositions are particularly intended for the prophylaxis or the treatment of dental plaque and infection related to A. naeslundii disease such as root caries, for example. The lactic bacteria strain according to the present invention is selected from the group consisting of Streptococcus thermophilus, Lactococcus lactis subsp. lactis, and Lactococcus lactis subsp. lactis biovar diacetylactis and preferably from the group consisting of the strains CNCM 1-1984, CNCM I-1985, CNCM 1-1986 and CNCM 1-1987, Such ajcomposition may contain at least lO'‘-lO’ cfu /gof lactic bacteria. Synergistic compositions may also be prepared, adding at least one bacteriocin, which is active against Gram-positive oral bacteria. In that case, the oral hygiene compositions may comprise 0.00001 to 50%, and preferably from O.OOOOI to 15% of purified bacteriocin, by weight of the composition. The bacteriocin is'preferably vaiiacin (EP 0 759 469). In order to protect the composition from degradation, an oil-soluble antioxidant may also be included. Suitable antioxidants include the "tocopherols", butyl-hydroxyanisole (BHA), butyl-hydrxytoluene (BHT), and ascorbyl palmitate. The oil soluble antioxidant is present in amounts of from 0.005% to 0.5%, preferably 0.005% to 0.01% by weight of the composition. Suitable abrasives for use in dentifrice compositions of the present invention include calcium carbonate, calcium aluminosilicate, alumina, hydrates alumina, zinc orthophosphate, plastic particles, and siUca, of which silica is the prefeixed abrasive. Compositions according to the invention will have a pH which is orally acceptable and within which the activity of the said lactic bacteria is not compromised. The pH may be in the range 3.0-9,5, preferably in the range 3,5 to 6.5. These compositions may be prepared by conventional processes comprising admixing the ingredients together in the appropriate relative amounts and finally, and if necessary, adjusting the pH to desired value. Actinomyces naeslundii genospecies 1 (formerly A. naeslundii) and 2 (formerly A, viscosus) are among the strongest plaque forming oral strains. They are commonly isolated at root caries sites, in particular in humans over 40 years, and they are believed to be the major aetiological agent of this disease. Thus, the invention also provides a method for the prevention- or the treatment of Actinomyces naeslundi-xtldXtd. infections in mammals, particularly dental plaque extent and root caries, comprising the step of feeding the mammal a composition containing at least one lactic bacteria strain selected for its ability to adhere to the tooh surface and to produce a growth inhibition factor. The amount to be administred may be of at least about 10"‘-10’ cfu /g of lactic bacteria. The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the claims. Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties to the extent necessary for understanding the present invention. Example 1: in’vitro trials The strains 5, thermophilus NCC 1561 (CNCM M985) and L lactis subsp. lactis NCC2211 (CNCM M986) (hereinafter L. lactis NCC2211) were incorporated in vitro in a biofilm mimicking dental plaque in vitro. The oral strain A, naeslundii genospecies 1 (formerly A, naeslundii) OMZ745 and A. naeslundii genospecies 2 (formerly A. viscosus) OMZ105 were obtained from the Institute fiir Orale Mikrobiologie und AUgemeine Immunologie, University of Ziiricii and they were cultured in FUM medium in anaerobiosis (GasPackSystem, BBL) at 37°C. All the strains were stored in glycerol at -20°C and precultured for 14 hours prior to use at their specific optimal temperature; The two selected strains L. lactis NCC2211 and S. thermophilus NCC1561 were inoculated in an in vitro system in which a biofilra, composed by bacteria conmionly found in the human mouth after 40 years, developed on saliva coated-hydroxyapatite discs. Fluid Universal Medium (FUM), the growth medium used, was especially formulated to buffer the acidity produced by the test strains and to have therefore a continued growth (plaque development), like it is in the mouth (Gmur and Guggenheim, 1983). The assays were done in triplicate and the mixtures with and without the dairy strains were tested in parallel The strains listed in the Table 2 were used. ; ■ Procedure - Saliva pellicle formation: cover synthetic hydroxyapatite discs of 10 mm diameter (HY-APATITE®, Euro-Crystals, Landgraaf, The Netheriands) with 800 \|il of human saliva and incubate for 4 h at room temperature under shaking (1 disc/well in a 24 holes sterile Nunclon plate). - Bacterial consortium preparation: grow S, thermophilus NCC1561, L. lactis subsp. lactis NCC2211, S. sobrinus OMZ176, S. oralis OMZ607, A. naeslundii OMZ745, V. dispar OMZ493 and F. nucleatum OMZ596 overnight at 3TC in anaerobiosis in FUM-glucose (S. thermophilus NCC1561 in FUM-lactose), adjust the OD550 to 1 witli FUM and pool 2 ml of each oral bacterial suspension with 2 mi of either 5. thermophilus NCC1561 or L. lactis subsp, lactis NCC221L The control mixture contains the five oral strains only. - Biofilm fonnation and recovery: the procedure is as described in Guggenheim et al., 1998, Validation of a new biofilm model. LDent.Res. 77, (Spec Iss A): 110 (Abstract #38), - Cultural analysis of the biofilm: spiral plate the suspension on Columbia Blood Agar (5% sheep blood, Becton Dickinson, Meylan Cedex, France) for the total count and for the differentiation of A. naeslundii. Incubate the plates at 37°C in anaerobiosis for 48 h. Growth antagonism between the oral and the dairy strains under study Strains and culture conditions used are listed in the Table 3. 5. thermophilus NCC1561, 5. thermophilus NCC 1536 and L lactis NCC2211 (killer strains) were tested for growth antagonism against A. naeslundii OMZ745 and A. viscosus OMZ105 (target strains). Procedure - Grow the killer strains overnight on agar plates in anaerobiosis and the target strains in BHI until middle stationary phase - Dilute 20 fil of the target strains suspension in 3 ml of BHI soft agar (agar 7 g/1) containing glucose and lactose, vortex and pour immediately on a BHI agar plate - Solidify for 1 h at room temperature, then streak the killer strain from the M17 plate in the form of a cross. Streak in parallel the killer and the target strains alone as a control - Incubate at 37 ’C in aiiaerobiosis for 24 hours Growth antagonism is revealed by an inhibition halo around the cross. Statisticals Differences between the control and test consortia were determined by Student's t test. RESULTS AND DISCUSSION S. thermophilus NCC1561 and L. lactis NCC2211 could be incorporated and grown in the plaque-like biofilm on the S-HA discs, and their total CFU/disc after 40.5 h are given in the Table 4. Table 4. Level of incorporation of the two dairy strains in the biofilm (CFU/disc). The values are the mean of three experiments with their standard The effect of incorporating the dairy strains in the biofilm on the oral species is indicated in the Tables 5 and 6. When S. thermophilus NCC1561 was included (Table 5), a general decrease of the total flora, that is represented by the counts on Columbia blood agar plates (CBA), and of 4 of the oral species was noticed. All the four dairy strains inhibited the growth of the Gram-negative strain A, viscosus OMZ105 This inhibition cannot be attributed to lactic acid production. A. viscosus is able to metabolize lactate only under aerobic conditions (van der Hoeven et al, (1990) Oral Microbiol Immunol 5, 223-225) and it is very aciduric. These findings have been confirmed by plating A, viscosus in presence of 1% lactic acid: no inhibition was observed. CONCLUSIONS S, thermophilus NCC1561 and L. lactis NCC22U could be incorporated in a biofilm mimicking dental plaque and were able to modulate the oral microflora, significantly reducing the number total of cfu, and more specifically, these strains were able to significantly decrease the colonization extent of A. naeslundii genospecies 2, In addition the strains could inhibit the growth of A, naeslundii genospecies 1 and 2 in co-cultures. Example 2; in-vivo trials. An in-vivo study was performed on a rat model. In this study, the association of the selected strains was continued during the whole experimental period on a daily basis, by the way of a chilled dairy product feeding. The study was carried out in 58 days. In order to perform the experiment during the day, the active period of the animals had to be advanced of 7 hours totally; this was done in three steps on day 16, 17 and 18 as further described in detail. The cariogenic strains were associated on days 21 and 22, while ■ association of the dairy strains started on day 23 and lasted until day 57. The animals were fed the dairy strains as supplement in a yogurt base that was included in the normal diet, as explained in the section. The rats teeth were swabbed at the end of the study, on day 58. Animals and diet 10 litters consisting of 4 Osbome-Mendel rats pups each (animal production section of the Institute fiir Orale Mikrobiologie und Allgemeine Mikrobiologie, University of Zurich, Zurich, Switzerland) were used in the experiment* All animals were weighed at the beginning and at the end of the experimental period. When 13 days old, the dams and the pups were transferred to screen-bottom stainless-steel cages without bedding and nourished with low-fluoride powdered (0,2 \|j.m) Nafag diet to avoid fissure impaction (Rat Checkers No. 184, NAFAG, Gossau SO, Switzerland), and tap water ad libitum. The active phase during which the rats eat is during the night, i. e. 18:00 - 06:00. In order to allow refilling of the food cups during normal working hours, the circadian biorhythm was stepwise reversed between days 16 and 18 by advancing the active phase of the rats each day on three occasions by adjusting the automatic light controls. On day 16/17 the beginning of the active period was brought forward from 18:00 h to 15:00 i. .e. it was night from 15:00 - 03:00 and it was day onwards. On day 17/18 the beginning of the active period was brought forward from 15:00 to 12:00 h, i. e, it was night from 12:00 h - 00:00 h and day from 00:00 h onwards. Finally on day 18/19 the beginning of the active period was brought forward from 12:00 to 10:00 i. e. it was night from 10:00 h to 22:00 h and day from 22:00 h onwards. Therefore by day 19 the shift of the active phase for the rats from the hours of darkness to normal working hours (10:00 - 22:00 h) was completed. On day 20 Uie dams were removed, and the rats started to be fed ad libitum with the modified diet 2000a containing 40% sucrose, 28% substitute for skim milk (soya protein extract SVPRO-PP 1611 39.4%, lactose 49.3%, 0.6%, L-Methionine 0.3%, L-Lysine HCl 0.1%), 24% wheat flour, 5% brewer's yeast, 2% Gevral® Instant Protein (Whitehall-Robins SA, 6301 Zug, Switzeriand) and 1% NaCL During the association period (days 21 and 22) die drinking water was supplemented with 2% glucose and 2% sucrose to support the implantation of the associated bacteria. On day 23 the Utters were distributed among the 3 treatments, 1 animal per cage, in a programmed feeder machine and began to receive the test diet as indicated in the table 10. The test diet consisted of 18 yogurt meals containing the test strains alternating with 18 meals of the modified diet 2000a previously described. Drinking water was supplied ad libitum. Following the swabbing procedure on day 58 the animals were overdosed with Thiopental sodium (100 mg/Kg of body weight) given by intra-peritoneal injection and decapitated when comatose. Preparation of the tested LAB strains for the association. A preliminary study was done to assess the growth parameters, especially the hours required to reach the stationary phase in the specific conditions further described. It was therefore established to grow the S. themiophilus strains for 7 h and die L lactis one for 6 h. Also a study of the viabihty after freezing of the dairy strains was performed by plating the same cell suspension before and after freezing. In order to be associated to the animals, the dairy strains were treated according to the following procedure. Procedure - Inoculate the strains 1% overnight in their proper medium from a glycerol stock - Inoculate them 5% from this culture into 10 batches of 4 liters of their proper medium pre-heated at the optimal growth temperature, and grow them until the end of the log phase/beginning stationary phase - Determine the final CFU/ml by plating on M17-lactose agar from two randomly chosen batches for each of the 4 strains. Incubate the plates overnight in anaerobiosis. - Centrifuge the cultures from each batch at 6000 rpm for 10 min and re- suspend the pellets in 150 ml of fresh medium; keep overnight at 4°C - Centrifuge again and re-suspend in the freezing medium (15% glycerol in BellikerorMl?); - Split in aliquots in order to have 2x10’* viable cells/vial, taking into account the viability loss due to freezing, and store at-20°C until needed. Association of the animals with the bacterial strains The animals were arranged in 3 treatments (Table 9). Each treatment consisted of 10 pups that were distributed in 1 per cage. All the rats were first infected on days 21 and 22 with A, viscosus OMZ105. The tested LAB strains were associated daily (since contained in the yogurt base meal), starting on day 23. 2 frozen vials, each containing 2x10’* viable cells of the test strain, were mixed in 200 mJ of yogurt in order to have at least 10’ CFU/ml 5. thermophilus NCC1536, a non S-HA adhering strain, was used as a negative control The yogurt and diet 2000a meals, of 1 ml and 400 mg respectively, were offered alternatively 18 times per day at 20-min intervals (Table 10). Therefore, each animal received in total 18 ml of yogurt and 7.2 g of powdered diet. The meals were dispensed in the food cups of a programmed feeder machine that automatically offered to the animals the right meal at the exact time. Bacteriological evaluation Five rats per treatment were swabbed on day 58. The swab suspensions were either plated on Petri dishes for CFU (colony forming units) counts or immobilized on glass slides for immunofluorescence for TCN (total cells number) counts. Procedure for CFU determination - Swab rats' teeth with a sterile cotton-tipped stick and place it iramediately in 5 ml of sterile NaCl 0.9% - Vortex for 1 min and sonicate for 5 s at 50 W - Spirally plate the properly diluted suspensions on CBA, MS and HJL agar - Incubate CBA and MS plates at 37 °C and HJL plates at 45 °C. Procedure for TCN determination - Put 10 ‘il of the undiluted swab suspension prepared for CFU determination per well on a 24 wells glass slide (Dynatech Produkte AG, Embrach Embraport, Switzerland), and air dry - Fix by soaking in methanol for 2 min and air dry - Incubate with 10 \x\ of the proper antibody or serum diluted in ELISA buffer (section 4.2.2.5) and incubate at 37 'C for 30 min T Aspirate each drop from the side of the well and wash by soaking the slide first in ELISA buffer and then in distilled water. Air dry - Apply 10 \xl of goat-anti-rabbit IgG (H + L) - FITC (Sigma) diluted 1:400 and incubate at 37 °C for 30 min - Wash as before and air dry - Apply 49 \|il of mounting fluid (section 4.2.2.5), cover with a glass slip and count the fluorescent cells with a fluorescence microscope Statistics Data were treated with two-way ANOVA ((Snedecor and Cochran, 1980)). RESULTS from the continuous association of the dairy strains by the way of a chilled dairy product feeding. Bacteriological evaluation (Table 11) - Colonization of the strain. 1.7 (+/- 1.1) xlO’ CFU were counted for the plaque forming A. viscosus OMZ105, when the dairy product was supplemented with the non-adherent S. thermophilus control (NCC1536). The dairy strains could not be counted by microbiological methods. By immunofluorescence, however, a qualitative evaluation was tempted. The three adherent dairy strains could be recognized in the plaque samples from treatments 2 and 3. Since they were co-aggregated with other oral bacteria and mouth debris, thus generating big clusters, a precise quantification was impossible. - Variations in the total flora (TF), The three treatments containing the adherent tested strains 5, thermophilus NCC1561 and L lactis NCC2211 displayed significant reduced numbers of colony forming units on CBA compared with the control group containing the non-adherent strain S. thermophilus NCC1536 (Table 11). Treatment 2 reduced the CFU counts with a significant factor of PF - Modulation of A. viscosus OMZ105 tooth colonization by the tested strains. In the case of the plaque forming bacterium A. viscosus OMZ105, an apparently less pronounced but more significant decrease in the number of colony forming units was observed for the three treatments containing the tested adherent strains with respect to treatment 1 (PF In this in vivo assay, the strains Uiat were supplied daily, displayed clear inhibitory effect on the total microflora, whose CFU significantly diminished. This diminution can be explained by the growth antagonism of the dairy strains versus oral species. For instance in vitro, all of them, including the non S-HA adhering S, thermophilus NCC1536, can inhibit the growth of A, viscosus OMZ105., However, in vivo such an effect can only be displayed by the S-HA adhering strains, since it was detected in the treatments 2-4 compared to the first. in particular, since the animals had been infected with A. viscosus OMZ105, the quantification of this plaque-forming organism was possible at the end of the experimental period, and the decrease of its CFU could be closely monitored. The percentages of A. viscosus OMZ105 on the total CFU did not decrease in parallel, therefore one can deduce that the growth antagonism effect was also displayed versus other species, ie. VeiUonellae, and consequently it is a global effect which is observed. Thus the strains CNCM H985 and CNCM-1986 are able to modulate the oral microbial ecology, significatively decreasing the colonization extent of A. naeslundii genospecies 2, with which the rats had been infected. Example 3: Production and initial analysis of surfactant substances from S. thermophilus NCC1561 andS. thermophilus NCC1536. S. thermophilus NCC1561 and S. thermophilus NCC1536 were grown overnight in 1 1 of Belliker at 42 C, For biosurfactant production, the procedure described in Busscher et al. (1997) Appl Environ. Microbiol 63, 3810-3817, (Busscher et ai, 1997)was used. Preparation of the surfactant substances Procedure - Wash cells three times in PBS - Resuspend in 200 ml of distilled water or PBS - Produce the biosurfactant by gently stirring the suspension for 2 or 4 h at room temperature - Separate bacteria by centrifugation at 10000 rpm for 10 min - Centrifuge supernatant twice at 10000 rpm for 10 min - Freeze-dry and weigh both the pellet and the surfactant substances solutions. The crude biosurfactant suspension was first analyzed by SDS-PAGE and then submitted to surface tension measurements. Procedure for SDS-PAGE SDS-PAGE was carried out with a precast 12.5% ExcelGel (Amersham Pharmacia Biotech) Silver staining was performed with the Plusone Silver Staining Kit (Amersham Pharmacia Biotech). Procedure for surface tension measurement The surface tension of the biosurfactant suspensions was measured with a TVTl Drop Volume Tensiometer (Lauda, Lauda-Konigshofen, Germany), which is based on the drop volume principle. Briefly, the method ccsnsists in the exact determination of the volume of a suspension drop ttiat detaches from a capillary. This volume (critical volume) is proportional to the surface tension (a), whose value is calculated with the relation; - a is the interfacial tension - V is the drop volume - G is the acceleration constant - Ap is the difference of the densities of both adjacent phases - F is the correction factor " reap Is the radius of the capillary The measurements were done in duplicate at 37 ‘‘C against air. Each measurement consisted of 10 cycles. A solution of 6 mg/ml of crude product released, made the water surface tension decrease from 70 to 51 mN/m (Table 13). SDS-PAGE profile of the bacteria released products, showed that there were many different substances of proteinaceous nature in the. solution. Table 13. Surface tension values of the biosurfactant suspensions compared to water and PBS. The values are the mean of two experiments, each one consisting of ten measurements. RESULT 5. thermophilus NCC1561 and S. thermophilus NCC1536 cells are able to release substances with a surfactant activity. It is therefore possible that the ' biosurfactant produced by 5. thermophilus NCC1561 makes the bacterium itself and the other oral strains established close to it detach from the tooth surface. By contrast, this action would not be displayed by S. thermophilus NCC1536, since this strain does not adhere to the teeth. Example 4: TOOTHPASTE Toothpaste is prepared by adding 10’ cfu/ml of at least one of the lactic bacteria strain CNCM1-1984, CNCM M985, CNCM M986, CNCM M987 in a lyophilised form, to the following mixture containing: 1,65% Cetyl pyridinium chloride. 33.0% Sorbitol (70% soln), 25.0% Glycerin, 2.0% Sodium carboxymethyl cellulose, 0.25% Sodium fluoride. 26.3% Silica (RP 93), 8.1% Thickening Silica (Sident 22), 05% Sodium saccharine, 3.2% Poloxamer (Pluronic F108), This toothpaste is intended for the prophylaxis or the treatment of root caries, dental plaque and other infections induced by A.naeslundii species. Example 5: YOGHURT 5 1 MRS culture medium.are steriUsed for 15 min at lll'‘C and then inoculated with 5% by volume of an active culture of at least one of the SJhermophilus strain CNCM 1-1984, CNCM M985 containing approximately 10’ cfu/ml. After incubation for 8 h at 41 °C, a starter containing 4.5.10’ cfu/ml is obtained, 5 1 reconstituted skimmed milk having a dry matter content of 10%, to which 0.1% yeast extract has been added, are sterilised for 15 min at 121’C and inoculated with 2% of an active culture of commercial thickening Streptococcus thermophilus containing approximately 10’ cells/ml. After incubation for 4 h at 4PC, a starter containing 4.5.10’ cells/ml is obtained One batch of whole milk containing 3 J% fats strengthened with 2,5% skimmed milk powder and then pasteurised for 30 min at 90°C is then inoculated with 2% by volume of the starter of at least one of the strains CNCM 1-1984, CNCM 1-1985 and 3% by volume of the starter of thickening Streptococcus thermophilus. The inoculated milk is stirred, poured into pots and incubated for 4 hat4rC. The yoghurt obtained has a good firm and smooth texture and is intended for the health of the mouth. Example 6: CHEWING GUM A chewing gum for preventing or treating root caries, dental plaque or other A.nae’/wnrfw-related deseases can be prepared adding an active culture of at least one of the SJhermophilus strain CNCM 1-1984, CNCM H985 so that it contains approximately lO'‘ to 10’ cfu/g, to the following typical ingredients: 67.5 % Xylitol, 20 % Gum base, 5 % Calcium carbonate, 3 % Glycerin, 2 % Pluronic F127, 1 % Cellulose gum, 0.5 % Balast compounds and 1 % Flavor, Example 7: PET FOOD COMPOSITION A pet food for mouth health is obtained by preparing a feed mixture made up of com, com gluten chicken and fish meal, salts, vitamins and minerals. Tlie feed mixture is fed into a preconditioner and moistened. The moistened feed leaving the preconditioner is then fed into an extruder-cooker and gelatinised. The gelatinised matrix leaving the extruder is forced through a die and extruded. The extrudate is cut into pieces suitable for feeding to dogs, dried at about 110°C for about 20 minutes and cooled to form pellets which have a water activity of about 0.6* The pellets are sprayed with 3 coating mixtures. Each coating mixture contains active culture of at least one of the S.thermophilus strains CNCM I-1984, CNCM 1-1985 but one coating mixture uses hydrogenated soy fat as a coating substrate, one coating mixture uses water as a coating substrate and one coating mixture uses protein digest as a coating substrate* The pellets contain approximately 10’ to 10’ cfu/g of said strains. We Claim 1. A process for preparation of a composition intended for reducing dental plaque and for preventing root caries, said process characterized by using lactic acid bacteria, wherein lactic acid bacteria is selected from the group consisting of Streptococcus thermophilus, lactococcus lactis subsp. Lactis, and lactococcus lactis subsp biovar diacetylactis, which is exogenous to the oral micro flora, which has been selected for its ability to adhere to the pellicle of the teeth and produce a growth inhibition factor. 2. The process as claimed in claim 1 wherein the lactic acid bacteria are from dairy origin. 3. The process as claimed in claims 1 to 2 wherein the lactic acid bacteria strain is selected from the group consisting of the strains CNCM 1-1984, CNCM 1-1985, CNCM1-1986, CNCM 1-1987. 4. The process claimed in claims 1 to 3 wherein the composition is an edible composition comprising an effective quantity of lactic acid bacteria for reducing dental plaque and for preventing root caries and other disease related to Actinomyces naeslundi in mammals. 5. The process as claimed in claims 1 to 4, wherein the composition contains at least cfu/g of the lactic acid bacteria strain. 6. The process as claimed in claims 1 to 5, wherein the lactic acid bacteria strain is combined with a bacteriocin. 7. The process as claimed in claim 1 wherein the said composition is helpful in maintaining the health of the mouth by reducing the colonization of Actinomyces naeslundi in mammals, said composition containing at least one lactic acid bacteria strain that is exogenous to the oral micro flora, which has been selected from for its ability to adhere the pellicle of the teeth and to produce a growth inhibition factor. 8. The process as claimed in claim 7 wherein the composition maintaining the health of the mouth by reducing the colonization of Actinomyces naeslundi in mammals, said composition comprises: - a lactic acid bacteria strain which has been selected from its ability to adhere to the pellicle of the teeth and to produce a growth inhibition factor, and - a bacteriocin, 9. The process as claimed in claim 7 or 8 wherein the said composition comprises at least one lactic acid bacteria selected from the group consisting of Streptococcus thermophilus, lactococcus lactis subsp. Lactis, and lactococcus lactis subsp lactis biovar diacetylactis. 10. The process as claimed in claims 7 to 9, wherein the said composition comprises at least one lactic bacteria selected from the group consisting of the strains CNCM 1-1984, CNCM 1-1985, CNCM 1-1986, CNCM 1-1987. 11. The process as claimed in claims 7 to 10, wherein the said composition comprises at least 10"*-10’ cfu/g of the lactic acid bacteria strain.

Full Text

Use of exogenous lactic bacteria strain against Actinomyces naeslundiU related diseases
The present invention relates to the incorporation in the oral micro flora of exogenous lactic bacteria which are able to modulate the colonization of A. naeslundii and to reduce the severity of A -related diseases.
Background of the invention
The mouth (oral cavity) contains a resident and a non-resident micro flora. The first includes microorganisms that are able to establish a more or less permanent residence on the oral surfaces. These bacteria are mainly localised on the tongue, the buccal mucosa and the teed while the gingival, lips, cheeks, palate and floor of the mouth only support a very sparse microflora.
The dental plaque is a film that forms on the surface of teeth consisting of bacterial cells in a matrix of extracellular polysaccharides and savory products. Immediately after eruption, the teeth are covered with an amorphous layer of saliva, the acquired enamel pellicle (AEP) that is about 13 ‘im thick and cannot be removed by normal tooth brushing. The deposition of bacteria on teeth follows iihmediately the formation of the AEP and plaque becomes evident in 8-12 hours as a multi-layered structure. The first layer consists of bacteria (earliest colonisers) that attach to teeth mainly via specific adhesion-receptor recognition; it forms a substratum for the second colonisers that adhere one to the other via analogous specific binding or via simple juxtaposition.
On the tongue and the buccal mucosa, the natural resident microflora includes microorganisms selected from Streptococcus, Veillonella, Bacteroides and Haemophilus* On the teeth, Streptococci and Actinomyces predominate but a variety of Gram positive and negative coccid and rods can be found.
Many of these microorganisms are innocuous commensally, but a lot of them have been recognised as the etiologic agent of quite a few diseases (Hill, M. J. and Amish, P. D. eds. Human Microbial Ecology, 1990, CRC Press, Boca Raton Florida, USA).

In particular, Actinomyces naeslundii genospecies 1 (formerly A. naeslundii) and 2 (formerly A. viscous) are common members of human dental plaque. They are among the strongest plaque forming oral strains, because of their capacity to firmly adhere to the teeth and to coaggregate with many other bacterial species, thus fostering their establishment in the mouth. Moreover, in the elderly, they are commonly isolated at root caries sites, and they are believed to be the major aetiological agent of this disease (Bowden, G.H., et aL 1999, The diversity and distribution of the predominant biotypes of Actinomyces naeslundii genospecies 1 and 2 in samples from enamel and from healthy and carious root surfaces of teeth. J.DenuRes.1%, 1800-1809).
The transient microflora comprises exogenous bacteria that can be occasionally present in the mouth, but that do not establish a permanent residence (even if repeated oral administrations of these bacteria are carried out). All the food bacteria, and in particular lactic acid bacteria, can be part of this transient microflora.
Some of these exogenous lactic bacteria have been shown to be capable of adhering to the pellicle of teeth. For example, WO 00/09080 (Soci6t6 des Products Nestl6) discloses lactic bacteria strains, that are not part of the resident microflora of the mouth, that are low acidifying and that are capable of adhering directly to the pellicle of the teeth. These bacteria are particularly used for treating or preventing dental caries and periodontal infection that are caused by cryogenic microorganism such as Streptococcus mutants and Streptococcus sopranos.
Exogenous bacteria can also produce factors that inhibit the growth of the resident microflora in the mouth. For example, EP759469 (Soci6t6 des Produits Nestl6) described the use of a bacteriocin produced by Micrococcus varians for inhibiting the development of the oral pathogens 5. sorbents, S, anguish, 5. mutants and A. viscosus The application of bacteriocins is also one of the investigated strategies which have been set up to reduce tooth caries. These molecules have attracted interest as prospective antiquaries agents and as factors important in modulating colonization of the oral cavity.
It is to note that the prior art does not provide any information concerning strains that can establish in the oral cavity by directly adhering to the pellicle of

the teeth and also produce factors such as growth inhibition factors, which can modulate the colonization of A, naeslundii so as to reduce the severity of A. naeslundii'- diseases.
Summary of the invention
Consequently the present invention aims to provide the use of lactic bacteria that are exogenous to the oral microflora, which have been selected for their ability to adhere to the tooth surface and to produce a growth inhibition factor, for the preparation of a composition intended for reducing dental plaque and for treating or preventing root caries or other disease related to Actinomyces naeslundii in mammals.
The lactic bacteria may be selected from the group consisting of Streptococcus
Thus, by colonizing the surface of teeth and producing growth inhibition factors, such lactic bacteria can exert an significant reduction of the extent of Actinomyces iaeslundii, thus reducing dental plaque,,root caries and other Actinomyces naeslundii related infections.
Another object is to provide a composition for maintaining the health of the mouth by reediting the colonization of Actinomyces naeslundii said composition comprises an exogenous lactic bacteria that has been selected for its ability to adhere to the tooth surface and to produce a growth inhibition factor.
Such a composition may contain at least 10' -10’ cfu /g of lactic bacteria.
The invention also provides a method for the prevention or the treatment of Actinomyces naeslundi related infections, particularly dental plaque extent and root caries in a mammal, comprising the step of feeding a mammal a composition containing at least one lactic bacteria strain selected for its ability to adhere to the tooth surface and to produce a growth inhibition factor, said composition reduces the colonization of Actinomyces naeslundi.

Detailed description of the invention
Within the following description, the mouth defines the oral cavity of humans or animals such as pets, composed by the oral mucosa (gums, lips, cheeks, palate and floor of the mouth), the tongue and the teeth (including artificial structures).
The terms "inhibition growth factor" defines any extracellular substance produced by the adherent exogenous lactic bacteria that enables it to inhibit the growth of A.naeslundL
With respect to the first object of the present invention, the use of an exogenous lactic bacteria that has been selected for its ability to adhere to the tooth surface and to produce a growth inhibition factor, for the preparation of a composition intended for reducing dental plaque and for treating or preventing root caries or other disease related to Actinomyces naeslundU is concerned.
The lactic bacteria may be selected from the group consisting of Streptococcus thermophilus’ Lactococcus lactis subsp, lactis, and Lactococcus lactis subsp. lactis biovar diacetylactis and particularly from the group consisting of the strains Sreptococcus thermophilus (NCC 1529) (CNCM 1-1984), Sreptococcus thermophilus (NCC 1561) (CNCM 1-1985), Lactococcus. lactis subsp. lactis (NCC 2211) (CNCM 1-1986), Lactococcus, lactis subsp. lactis biovar dioacetylactis (NCC 2225) (CNCM 1-1987).
The lactic bacteria is preferably from dairy origin (i.e. originating from milk or cheese, for example)*
The lactic bacteria according to the invention is "low acidifying", which means that it is less acidifying than pathogenic strains- Accordingly, it can contribute to a pH in the oral cavity of about 5,5-7.
These strains have been selected among latic bacteria strains for their capacity of adherence to the pellicle of the teeth, their optimal growth temperature is about 37°C, which is the temperature in the oral cavity. They are also capable of producing a growth inhibition factor, which combined to their

adhesion properties allow them to significantly decrease the colonization extent of A. naeslundii genospecies 1 and 2.
Moreover they are capable of fermenting glucose and sucrose and do not synthesised gleans, which are factors of pathogen city of the cariogenic strains.
It is also possible to use at least one lactic bacteria strain in combination with a bacteriocin, for example.
The lactic bacteria strains may be included in a food, pet food, cosmetic or pharmaceutical composition, for example. Accordingly, these compositions are preferably toothpaste, mouth rinse, gum, spray, beverage, candies, infant formula, ice cream, frozen dessert, sweet salad dressing, milk preparations, cheese, quark, yogurt, acidified milk, coffee cream or whipped cream, for example.
The exogenous lactic bacteria may be used in an amount of at least 10"‘-10’ cfu /g of lactic bacteria.
The effect of incorporating the above-mentioned bacteria in the oral microflora wa$ tested m a rat model. The strains CNCM1-1985 and CNCM-1986 were able to modulate the oral microbial ecology, significatively reducing the number of total CFU* More specifically, the strains were able to significantly decrease the colonization extent of A. naeslundii genospecies 2, with which the rats had been infected (see examples).
BIOCHEMICAL CHARACTERIZATION OF THE SELECTED STRAINS
Fermentatioii patterns: 49 simple sugars were tested with the ape 50 CH bioMerieux strip test (bioM6rieux SA, 69280 Marcy-lEtoile, France) and the results are given in Table 1,
Table 1. Sugar fermentation of L lactis CNCM I- 1987 (A), L lactis CNCM I-1986 (B), 5. thenvophilus CNCM 1-1984 (C), S, thermophilus CNCM M985 (D).

The strains Sreptococcus thermophilus (NCC 1529), Sreptococcus thermophilus (NCC 1561), Lactococcus, lactis subsp. lactis (NCC 2211), Lactococcus. lactis subsp. lactis biovar dioacetylactis (NCC 2225) were deposited under the Budapest Treaty, at the Collection Nationale de Culture de Microorganismes (CNCM M984, CNCM M985, CNCM M986 and CNCM I-1987 respectively), 25 rue du docteur Roux, 75724 Paris, France, on March 3’’, 1998.
The second main object of the present invention relates to a composition for maintaining the health of the mouth by reducing the colonization of A.

naeslundii in mammals, said composition comprises an exogenous lactic bacteria, which has been selected for its ability to adhere to the tooh stxrface and to produce a growth inhibition factor.
These compositions are particularly intended for the prophylaxis or the treatment of dental plaque and infection related to A. naeslundii disease such as root caries, for example.
The lactic bacteria strain according to the present invention is selected from the group consisting of Streptococcus thermophilus, Lactococcus lactis subsp. lactis, and Lactococcus lactis subsp. lactis biovar diacetylactis and preferably from the group consisting of the strains CNCM 1-1984, CNCM I-1985, CNCM 1-1986 and CNCM 1-1987,
Such ajcomposition may contain at least lO'‘-lO’ cfu /gof lactic bacteria.
Synergistic compositions may also be prepared, adding at least one bacteriocin, which is active against Gram-positive oral bacteria. In that case, the oral hygiene compositions may comprise 0.00001 to 50%, and preferably from O.OOOOI to 15% of purified bacteriocin, by weight of the composition. The bacteriocin is'preferably vaiiacin (EP 0 759 469).
In order to protect the composition from degradation, an oil-soluble antioxidant may also be included. Suitable antioxidants include the "tocopherols", butyl-hydroxyanisole (BHA), butyl-hydrxytoluene (BHT), and ascorbyl palmitate. The oil soluble antioxidant is present in amounts of from 0.005% to 0.5%, preferably 0.005% to 0.01% by weight of the composition.
Suitable abrasives for use in dentifrice compositions of the present invention include calcium carbonate, calcium aluminosilicate, alumina, hydrates alumina, zinc orthophosphate, plastic particles, and siUca, of which silica is the prefeixed abrasive.
Compositions according to the invention will have a pH which is orally acceptable and within which the activity of the said lactic bacteria is not compromised. The pH may be in the range 3.0-9,5, preferably in the range 3,5 to 6.5.

These compositions may be prepared by conventional processes comprising admixing the ingredients together in the appropriate relative amounts and finally, and if necessary, adjusting the pH to desired value.
Actinomyces naeslundii genospecies 1 (formerly A. naeslundii) and 2 (formerly A, viscosus) are among the strongest plaque forming oral strains. They are commonly isolated at root caries sites, in particular in humans over 40 years, and they are believed to be the major aetiological agent of this disease.
Thus, the invention also provides a method for the prevention- or the treatment of Actinomyces naeslundi-xtldXtd. infections in mammals, particularly dental plaque extent and root caries, comprising the step of feeding the mammal a composition containing at least one lactic bacteria strain selected for its ability to adhere to the tooh surface and to produce a growth inhibition factor.
The amount to be administred may be of at least about 10"‘-10’ cfu /g of lactic bacteria.
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the claims. Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties to the extent necessary for understanding the present invention.
Example 1: in’vitro trials
The strains 5, thermophilus NCC 1561 (CNCM M985) and L lactis subsp. lactis NCC2211 (CNCM M986) (hereinafter L. lactis NCC2211) were incorporated in vitro in a biofilm mimicking dental plaque in vitro.
The oral strain A, naeslundii genospecies 1 (formerly A, naeslundii) OMZ745 and A. naeslundii genospecies 2 (formerly A. viscosus) OMZ105 were obtained from the Institute fiir Orale Mikrobiologie und AUgemeine

Immunologie, University of Ziiricii and they were cultured in FUM medium in anaerobiosis (GasPackSystem, BBL) at 37°C.
All the strains were stored in glycerol at -20°C and precultured for 14 hours prior to use at their specific optimal temperature;
The two selected strains L. lactis NCC2211 and S. thermophilus NCC1561 were inoculated in an in vitro system in which a biofilra, composed by bacteria conmionly found in the human mouth after 40 years, developed on saliva coated-hydroxyapatite discs. Fluid Universal Medium (FUM), the growth medium used, was especially formulated to buffer the acidity produced by the test strains and to have therefore a continued growth (plaque development), like it is in the mouth (Gmur and Guggenheim, 1983). The assays were done in triplicate and the mixtures with and without the dairy strains were tested in parallel The strains listed in the Table 2 were used. ; ■

Procedure
- Saliva pellicle formation: cover synthetic hydroxyapatite discs of 10 mm diameter (HY-APATITE®, Euro-Crystals, Landgraaf, The Netheriands) with 800 |il of human saliva and incubate for 4 h at room temperature under shaking (1 disc/well in a 24 holes sterile Nunclon plate).

- Bacterial consortium preparation: grow S, thermophilus NCC1561, L. lactis
subsp. lactis NCC2211, S. sobrinus OMZ176, S. oralis OMZ607, A. naeslundii
OMZ745, V. dispar OMZ493 and F. nucleatum OMZ596 overnight at 3TC in
anaerobiosis in FUM-glucose (S. thermophilus NCC1561 in FUM-lactose),
adjust the OD550 to 1 witli FUM and pool 2 ml of each oral bacterial suspension
with 2 mi of either 5. thermophilus NCC1561 or L. lactis subsp, lactis NCC221L
The control mixture contains the five oral strains only.
- Biofilm fonnation and recovery: the procedure is as described in Guggenheim
et al., 1998, Validation of a new biofilm model. LDent.Res. 77, (Spec Iss A): 110
(Abstract #38),
- Cultural analysis of the biofilm: spiral plate the suspension on Columbia Blood
Agar (5% sheep blood, Becton Dickinson, Meylan Cedex, France) for the total
count and for the differentiation of A. naeslundii. Incubate the plates at 37°C in
anaerobiosis for 48 h.
Growth antagonism between the oral and the dairy strains under study
Strains and culture conditions used are listed in the Table 3.

5. thermophilus NCC1561, 5. thermophilus NCC 1536 and L lactis NCC2211 (killer strains) were tested for growth antagonism against A. naeslundii OMZ745 and A. viscosus OMZ105 (target strains).

Procedure
- Grow the killer strains overnight on agar plates in anaerobiosis and the target
strains in BHI until middle stationary phase
- Dilute 20 fil of the target strains suspension in 3 ml of BHI soft agar (agar 7 g/1)
containing glucose and lactose, vortex and pour immediately on a BHI agar plate
- Solidify for 1 h at room temperature, then streak the killer strain from the M17
plate in the form of a cross. Streak in parallel the killer and the target strains
alone as a control
- Incubate at 37 *’C in aiiaerobiosis for 24 hours
Growth antagonism is revealed by an inhibition halo around the cross.
Statisticals
Differences between the control and test consortia were determined by Student's t test.
RESULTS AND DISCUSSION
S. thermophilus NCC1561 and L. lactis NCC2211 could be incorporated and grown in the plaque-like biofilm on the S-HA discs, and their total CFU/disc after 40.5 h are given in the Table 4.
Table 4. Level of incorporation of the two dairy strains in the biofilm (CFU/disc). The values are the mean of three experiments with their standard

The effect of incorporating the dairy strains in the biofilm on the oral species is indicated in the Tables 5 and 6. When S. thermophilus NCC1561 was included (Table 5), a general decrease of the total flora, that is represented by the counts on Columbia blood agar plates (CBA), and of 4 of the oral species was noticed.

All the four dairy strains inhibited the growth of the Gram-negative strain A, viscosus OMZ105* This inhibition cannot be attributed to lactic acid production. A. viscosus is able to metabolize lactate only under aerobic conditions (van der Hoeven et al, (1990) Oral Microbiol Immunol 5, 223-225) and it is very aciduric. These findings have been confirmed by plating A, viscosus in presence of 1% lactic acid: no inhibition was observed.

CONCLUSIONS
S, thermophilus NCC1561 and L. lactis NCC22U could be incorporated in a biofilm mimicking dental plaque and were able to modulate the oral microflora, significantly reducing the number total of cfu, and more specifically, these strains were able to significantly decrease the colonization extent of A. naeslundii genospecies 2, In addition the strains could inhibit the growth of A, naeslundii genospecies 1 and 2 in co-cultures.
Example 2; in-vivo trials.
An in-vivo study was performed on a rat model. In this study, the association of the selected strains was continued during the whole experimental period on a daily basis, by the way of a chilled dairy product feeding.
The study was carried out in 58 days. In order to perform the experiment during the day, the active period of the animals had to be advanced of 7 hours totally; this was done in three steps on day 16, 17 and 18 as further described in detail. The cariogenic strains were associated on days 21 and 22, while ■ association of the dairy strains started on day 23 and lasted until day 57. The animals were fed the dairy strains as supplement in a yogurt base that was

included in the normal diet, as explained in the section. The rats teeth were swabbed at the end of the study, on day 58.
Animals and diet
10 litters consisting of 4 Osbome-Mendel rats pups each (animal production section of the Institute fiir Orale Mikrobiologie und Allgemeine Mikrobiologie, University of Zurich, Zurich, Switzerland) were used in the experiment* All animals were weighed at the beginning and at the end of the experimental period. When 13 days old, the dams and the pups were transferred to screen-bottom stainless-steel cages without bedding and nourished with low-fluoride powdered (0,2 |j.m) Nafag diet to avoid fissure impaction (Rat Checkers No. 184, NAFAG, Gossau SO, Switzerland), and tap water ad libitum. The active phase during which the rats eat is during the night, i. e. 18:00 - 06:00.
In order to allow refilling of the food cups during normal working hours, the circadian biorhythm was stepwise reversed between days 16 and 18 by advancing the active phase of the rats each day on three occasions by adjusting the automatic light controls.
On day 16/17 the beginning of the active period was brought forward from 18:00 h to 15:00 i. .e. it was night from 15:00 - 03:00 and it was day onwards. On day 17/18 the beginning of the active period was brought forward from 15:00 to 12:00 h, i. e, it was night from 12:00 h - 00:00 h and day from 00:00 h onwards.
Finally on day 18/19 the beginning of the active period was brought forward from 12:00 to 10:00 i. e. it was night from 10:00 h to 22:00 h and day from 22:00 h onwards.
Therefore by day 19 the shift of the active phase for the rats from the hours of darkness to normal working hours (10:00 - 22:00 h) was completed.
On day 20 Uie dams were removed, and the rats started to be fed ad libitum with the modified diet 2000a containing 40% sucrose, 28% substitute for skim milk (soya protein extract SVPRO-PP 1611 39.4%, lactose 49.3%, 0.6%, L-Methionine 0.3%, L-Lysine HCl 0.1%), 24% wheat flour, 5% brewer's yeast, 2% Gevral® Instant Protein (Whitehall-Robins SA, 6301 Zug, Switzeriand) and 1% NaCL
During the association period (days 21 and 22) die drinking water was supplemented with 2% glucose and 2% sucrose to support the implantation of the associated bacteria. On day 23 the Utters were distributed among the 3

treatments, 1 animal per cage, in a programmed feeder machine and began to receive the test diet as indicated in the table 10. The test diet consisted of 18 yogurt meals containing the test strains alternating with 18 meals of the modified diet 2000a previously described.
Drinking water was supplied ad libitum. Following the swabbing procedure on day 58 the animals were overdosed with Thiopental sodium (100 mg/Kg of body weight) given by intra-peritoneal injection and decapitated when comatose.

Preparation of the tested LAB strains for the association.
A preliminary study was done to assess the growth parameters, especially the hours required to reach the stationary phase in the specific conditions further described. It was therefore established to grow the S. themiophilus strains for 7 h and die L lactis one for 6 h. Also a study of the viabihty after freezing of the dairy strains was performed by plating the same cell suspension before and after freezing. In order to be associated to the animals, the dairy strains were treated according to the following procedure.
Procedure
- Inoculate the strains 1% overnight in their proper medium from a glycerol
stock
- Inoculate them 5% from this culture into 10 batches of 4 liters of their proper
medium pre-heated at the optimal growth temperature, and grow them until the
end of the log phase/beginning stationary phase

- Determine the final CFU/ml by plating on M17-lactose agar from two
randomly chosen batches for each of the 4 strains. Incubate the plates overnight
in anaerobiosis.
- Centrifuge the cultures from each batch at 6000 rpm for 10 min and re-
suspend the pellets in 150 ml of fresh medium; keep overnight at 4°C
- Centrifuge again and re-suspend in the freezing medium (15% glycerol in BellikerorMl?);
- Split in aliquots in order to have 2x10’* viable cells/vial, taking into account the viability loss due to freezing, and store at-20°C until needed.
Association of the animals with the bacterial strains
The animals were arranged in 3 treatments (Table 9). Each treatment consisted of 10 pups that were distributed in 1 per cage.

All the rats were first infected on days 21 and 22 with A, viscosus OMZ105.
The tested LAB strains were associated daily (since contained in the yogurt base meal), starting on day 23. 2 frozen vials, each containing 2x10’* viable cells of the test strain, were mixed in 200 mJ of yogurt in order to have at least 10’ CFU/ml 5. thermophilus NCC1536, a non S-HA adhering strain, was used as a negative control
The yogurt and diet 2000a meals, of 1 ml and 400 mg respectively, were offered alternatively 18 times per day at 20-min intervals (Table 10). Therefore, each animal received in total 18 ml of yogurt and 7.2 g of powdered diet.

The meals were dispensed in the food cups of a programmed feeder machine that automatically offered to the animals the right meal at the exact time.

Bacteriological evaluation
Five rats per treatment were swabbed on day 58. The swab suspensions were either plated on Petri dishes for CFU (colony forming units) counts or immobilized on glass slides for immunofluorescence for TCN (total cells number) counts.
Procedure for CFU determination
- Swab rats' teeth with a sterile cotton-tipped stick and place it iramediately in
5 ml of sterile NaCl 0.9%
- Vortex for 1 min and sonicate for 5 s at 50 W
- Spirally plate the properly diluted suspensions on CBA, MS and HJL agar
- Incubate CBA and MS plates at 37 °C and HJL plates at 45 °C.
Procedure for TCN determination

- Put 10 ‘il of the undiluted swab suspension prepared for CFU determination per well on a 24 wells glass slide (Dynatech Produkte AG, Embrach Embraport, Switzerland), and air dry
- Fix by soaking in methanol for 2 min and air dry
- Incubate with 10 \x\ of the proper antibody or serum diluted in ELISA buffer (section 4.2.2.5) and incubate at 37 'C for 30 min
T Aspirate each drop from the side of the well and wash by soaking the slide first in ELISA buffer and then in distilled water. Air dry
- Apply 10 \xl of goat-anti-rabbit IgG (H + L) - FITC (Sigma) diluted 1:400 and incubate at 37 °C for 30 min
- Wash as before and air dry
- Apply 49 |il of mounting fluid (section 4.2.2.5), cover with a glass slip and count the fluorescent cells with a fluorescence microscope
Statistics
Data were treated with two-way ANOVA ((Snedecor and Cochran, 1980)).
RESULTS from the continuous association of the dairy strains by the way of a chilled dairy product feeding.
Bacteriological evaluation (Table 11)
- Colonization of the strain. 1.7 (+/- 1.1) xlO’ CFU were counted for the plaque forming A. viscosus OMZ105, when the dairy product was supplemented with the non-adherent S. thermophilus control (NCC1536). The dairy strains could not be counted by microbiological methods. By immunofluorescence, however, a qualitative evaluation was tempted. The three adherent dairy strains could be recognized in the plaque samples from treatments 2 and 3. Since they were co-aggregated with other oral bacteria and mouth debris, thus generating big clusters, a precise quantification was impossible.
- Variations in the total flora (TF), The three treatments containing the adherent tested strains 5, thermophilus NCC1561 and L lactis NCC2211 displayed significant reduced numbers of colony forming units on CBA compared with the control group containing the non-adherent strain S.

thermophilus NCC1536 (Table 11). Treatment 2 reduced the CFU counts with a significant factor of PF - Modulation of A. viscosus OMZ105 tooth colonization by the tested strains.
In the case of the plaque forming bacterium A. viscosus OMZ105, an apparently less pronounced but more significant decrease in the number of colony forming units was observed for the three treatments containing the tested adherent strains with respect to treatment 1 (PF
In this in vivo assay, the strains Uiat were supplied daily, displayed clear inhibitory effect on the total microflora, whose CFU significantly diminished. This diminution can be explained by the growth antagonism of the dairy strains versus oral species. For instance in vitro, all of them, including the non S-HA

adhering S, thermophilus NCC1536, can inhibit the growth of A, viscosus OMZ105., However, in vivo such an effect can only be displayed by the S-HA adhering strains, since it was detected in the treatments 2-4 compared to the first.
in particular, since the animals had been infected with A. viscosus OMZ105, the quantification of this plaque-forming organism was possible at the end of the experimental period, and the decrease of its CFU could be closely monitored.
The percentages of A. viscosus OMZ105 on the total CFU did not decrease in parallel, therefore one can deduce that the growth antagonism effect was also displayed versus other species, i*e. VeiUonellae, and consequently it is a global effect which is observed.
Thus the strains CNCM H985 and CNCM-1986 are able to modulate the oral microbial ecology, significatively decreasing the colonization extent of A. naeslundii genospecies 2, with which the rats had been infected.
Example 3: Production and initial analysis of surfactant substances from S. thermophilus NCC1561 andS. thermophilus NCC1536.
S. thermophilus NCC1561 and S. thermophilus NCC1536 were grown overnight in 1 1 of Belliker at 42 **C, For biosurfactant production, the procedure described in Busscher et al. (1997) Appl Environ. Microbiol 63, 3810-3817, (Busscher et ai, 1997)was used.
Preparation of the surfactant substances
Procedure
- Wash cells three times in PBS
- Resuspend in 200 ml of distilled water or PBS
- Produce the biosurfactant by gently stirring the suspension for 2 or 4 h at room temperature
- Separate bacteria by centrifugation at 10000 rpm for 10 min
- Centrifuge supernatant twice at 10000 rpm for 10 min
- Freeze-dry and weigh both the pellet and the surfactant substances solutions.

The crude biosurfactant suspension was first analyzed by SDS-PAGE and then submitted to surface tension measurements.
Procedure for SDS-PAGE
SDS-PAGE was carried out with a precast 12.5% ExcelGel (Amersham Pharmacia Biotech)* Silver staining was performed with the Plusone Silver Staining Kit (Amersham Pharmacia Biotech).
Procedure for surface tension measurement
The surface tension of the biosurfactant suspensions was measured with a TVTl Drop Volume Tensiometer (Lauda, Lauda-Konigshofen, Germany), which is based on the drop volume principle. Briefly, the method ccsnsists in the exact determination of the volume of a suspension drop ttiat detaches from a capillary. This volume (critical volume) is proportional to the surface tension (a), whose value is calculated with the relation;

- a is the interfacial tension
- V is the drop volume
- G is the acceleration constant
- Ap is the difference of the densities of both adjacent phases
- F is the correction factor
" reap Is the radius of the capillary
The measurements were done in duplicate at 37 ‘‘C against air. Each measurement consisted of 10 cycles. A solution of 6 mg/ml of crude product released, made the water surface tension decrease from 70 to 51 mN/m (Table 13). SDS-PAGE profile of the bacteria released products, showed that there were many different substances of proteinaceous nature in the. solution.

Table 13. Surface tension values of the biosurfactant suspensions compared to water and PBS. The values are the mean of two experiments, each one consisting of ten measurements.

RESULT
5. thermophilus NCC1561 and S. thermophilus NCC1536 cells are able to release substances with a surfactant activity. It is therefore possible that the ' biosurfactant produced by 5. thermophilus NCC1561 makes the bacterium itself and the other oral strains established close to it detach from the tooth surface. By contrast, this action would not be displayed by S. thermophilus NCC1536, since this strain does not adhere to the teeth.
Example 4: TOOTHPASTE
Toothpaste is prepared by adding 10’ cfu/ml of at least one of the lactic bacteria strain CNCM1-1984, CNCM M985, CNCM M986, CNCM M987 in a lyophilised form, to the following mixture containing: 1,65% Cetyl pyridinium chloride. 33.0% Sorbitol (70% soln), 25.0% Glycerin, 2.0% Sodium carboxymethyl cellulose, 0.25% Sodium fluoride. 26.3% Silica (RP 93), 8.1% Thickening Silica (Sident 22), 0*5% Sodium saccharine, 3.2% Poloxamer (Pluronic F108),
This toothpaste is intended for the prophylaxis or the treatment of root caries, dental plaque and other infections induced by A.naeslundii species.
Example 5: YOGHURT
5 1 MRS culture medium.are steriUsed for 15 min at lll'‘C and then inoculated with 5% by volume of an active culture of at least one of the

SJhermophilus strain CNCM 1-1984, CNCM M985 containing approximately 10’ cfu/ml. After incubation for 8 h at 41 °C, a starter containing 4.5.10’ cfu/ml is obtained,
5 1 reconstituted skimmed milk having a dry matter content of 10%, to which 0.1% yeast extract has been added, are sterilised for 15 min at 121’C and inoculated with 2% of an active culture of commercial thickening Streptococcus thermophilus containing approximately 10’ cells/ml. After incubation for 4 h at 4PC, a starter containing 4.5.10’ cells/ml is obtained*
One batch of whole milk containing 3 J% fats strengthened with 2,5% skimmed milk powder and then pasteurised for 30 min at 90°C is then inoculated with 2% by volume of the starter of at least one of the strains CNCM 1-1984, CNCM 1-1985 and 3% by volume of the starter of thickening Streptococcus thermophilus. The inoculated milk is stirred, poured into pots and incubated for 4 hat4rC.
The yoghurt obtained has a good firm and smooth texture and is intended for the health of the mouth.
Example 6: CHEWING GUM
A chewing gum for preventing or treating root caries, dental plaque or other A.nae’/wnrfw-related deseases can be prepared adding an active culture of at least one of the SJhermophilus strain CNCM 1-1984, CNCM H985 so that it contains approximately lO'‘ to 10’ cfu/g, to the following typical ingredients: 67.5 % Xylitol, 20 % Gum base, 5 % Calcium carbonate, 3 % Glycerin, 2 % Pluronic F127, 1 % Cellulose gum, 0.5 % Balast compounds and 1 % Flavor,
Example 7: PET FOOD COMPOSITION
A pet food for mouth health is obtained by preparing a feed mixture made up of com, com gluten chicken and fish meal, salts, vitamins and minerals. Tlie feed mixture is fed into a preconditioner and moistened. The moistened feed leaving the preconditioner is then fed into an extruder-cooker and gelatinised. The gelatinised matrix leaving the extruder is forced through a die and extruded. The extrudate is cut into pieces suitable for feeding to dogs, dried at about 110°C for about 20 minutes and cooled to form pellets which have a water activity of about 0.6*

The pellets are sprayed with 3 coating mixtures. Each coating mixture contains active culture of at least one of the S.thermophilus strains CNCM I-1984, CNCM 1-1985 but one coating mixture uses hydrogenated soy fat as a coating substrate, one coating mixture uses water as a coating substrate and one coating mixture uses protein digest as a coating substrate* The pellets contain approximately 10’ to 10’ cfu/g of said strains.

We Claim
1. A process for preparation of a composition intended for reducing dental plaque and for preventing root caries, said process characterized by using lactic acid bacteria, wherein lactic acid bacteria is selected from the group consisting of Streptococcus thermophilus, lactococcus lactis subsp. Lactis, and lactococcus lactis subsp biovar diacetylactis, which is exogenous to the oral micro flora, which has been selected for its ability to adhere to the pellicle of the teeth and produce a growth inhibition factor.
2. The process as claimed in claim 1 wherein the lactic acid bacteria are from dairy origin.
3. The process as claimed in claims 1 to 2 wherein the lactic acid bacteria strain is selected from the group consisting of the strains CNCM 1-1984, CNCM 1-1985, CNCM1-1986, CNCM 1-1987.
4. The process claimed in claims 1 to 3 wherein the composition is an edible composition comprising an effective quantity of lactic acid bacteria for reducing dental plaque and for preventing root caries and other disease related to Actinomyces naeslundi in mammals.
5. The process as claimed in claims 1 to 4, wherein the composition contains at least cfu/g of the lactic acid bacteria strain.
6. The process as claimed in claims 1 to 5, wherein the lactic acid bacteria strain is combined with a bacteriocin.
7. The process as claimed in claim 1 wherein the said composition is helpful in maintaining the health of the mouth by reducing the colonization of Actinomyces naeslundi in mammals, said composition containing at least one lactic acid bacteria strain that is exogenous to the oral micro flora, which has been selected from for its ability to adhere the pellicle of the teeth and to produce a growth inhibition factor.

8. The process as claimed in claim 7 wherein the composition maintaining the health
of the mouth by reducing the colonization of Actinomyces naeslundi in mammals,
said composition comprises:
- a lactic acid bacteria strain which has been selected from its ability to adhere to the pellicle of the teeth and to produce a growth inhibition factor, and
- a bacteriocin,
9. The process as claimed in claim 7 or 8 wherein the said composition comprises at
least one lactic acid bacteria selected from the group consisting of Streptococcus
thermophilus, lactococcus lactis subsp. Lactis, and lactococcus lactis subsp lactis
biovar diacetylactis.
10. The process as claimed in claims 7 to 9, wherein the said composition comprises
at least one lactic bacteria selected from the group consisting of the strains CNCM
1-1984, CNCM 1-1985, CNCM 1-1986, CNCM 1-1987.
11. The process as claimed in claims 7 to 10, wherein the said composition comprises
at least 10"*-10’ cfu/g of the lactic acid bacteria strain.

Documents:

in-pct-2002-1972-che-abstract.pdf

in-pct-2002-1972-che-claims filed.pdf

in-pct-2002-1972-che-claims granted.pdf

in-pct-2002-1972-che-correspondnece-others.pdf

in-pct-2002-1972-che-correspondnece-po.pdf

in-pct-2002-1972-che-description(complete)filed.pdf

in-pct-2002-1972-che-description(complete)granted.pdf

in-pct-2002-1972-che-form 1.pdf

in-pct-2002-1972-che-form 26.pdf

in-pct-2002-1972-che-form 3.pdf

in-pct-2002-1972-che-form 5.pdf

in-pct-2002-1972-che-other document.pdf

in-pct-2002-1972-che-pct.pdf

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Patent Number

209522

Indian Patent Application Number

IN/PCT/2002/1972/CHE

PG Journal Number

50/2007

Publication Date

14-Dec-2007

Grant Date

04-Sep-2007

Date of Filing

29-Nov-2002

Name of Patentee

M/S. SOCIETE DES PRODUITS NESTLE S.A

Applicant Address

P.O. Box 353 CH-1800 Vevey

Inventors:

#	Inventor's Name	Inventor's Address
1	COMELLI, Elena-Maria	c/o Paulson, James, C. Scripps Research Institute Department of Molecular Biology 10550 N. Torrey Pines Road, CVN-24 La Jolla, CA 92037

PCT International Classification Number

A23C 9/123

PCT International Application Number

PCT/EP2001/006268

PCT International Filing date

2001-05-30

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	00201948.7	2000-06-02	EUROPEAN UNION