Treatment of actinomyces naeslundii-related diseases with exogenous lactic bacteria strains

A lactic bacteria strain that is exogenous to the oral microflora is selected for its ability to adhere the pellicle of the teeth and to produce a growth inhibition factor. This strain is used 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.

TECHNICAL FIELD

The present invention relates to the incorporation in the oral microflora of exogenous lactic bacteria that are able to modulate the colonization ofA. naeslundiiand to reduce the severity ofA. naeslundii-related diseases.

BACKGROUND ART

The mouth (oral cavity) contains a resident and a non-resident microflora. 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 teeth while the gingiva, 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 salivary products. Immediately after eruption, the teeth are covered with an amorphous layer of saliva, the acquired enamel pellicle (AEP) that is about 1.3 μm thick and cannot be removed by normal tooth brushing. The deposition of bacteria on teeth follows immediately 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. Plaque cohesion is essentially guaranteed by three mechanisms: the presence of a salivary pellicle on the outer bacteria layer, the specific co-aggregation among the different bacterial species, and the glucans synthesized by the bacteria that remain entrapped in the plaque matrix (Skopek et al., Oral Microbiol. Immunol., 2, 19-24, 1994; Kolenbrander et al., Meth. Enzymol., 253, 385-397, 1995; Hiroi et al., FEMS Microbiol Lett., 96, 193-198, 1992; Gibbons et al., Infect. Immun., 52555-561, 1986).

On the tongue and the buccal mucosa, the natural resident microflora includes microorganisms selected fromStreptococcus, Veillonella, BacteroidesandHaemophilus. On the teeth, Streptococci andActinomycespredominate but a variety of Gram positive and negative cocci and rods can be found.

Many of these microorganisms are innocuous commensally, but a lot of them have been recognized as the etiologic agent of quite a few diseases (Hill, M. J. and Marsh, P. D. eds. Human Microbial Ecology, 1990, CRC Press, Boca Raton Fla., USA).

In particular,Actinomyces naeslundiigenospecies 1 (formerlyA. naeslundii) and 2 (formerlyA. viscosus) 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 etiological agent of this disease (Bowden, G. H., et al. 1999, The diversity and distribution of the predominant ribotypes ofActinomyces naeslundiigenospecies 1 and 2 in samples from enamel and from healthy and carious root surfaces of teeth.J. Dent. Res.78, 1800-1809).

The organic acids produced by oral bacteria during the fermentation process directly cause dental caries. These acids attack the hard tissue of teeth with the consequent release of ions such as calcium, phosphate, carbonate, magnesium, fluoride, and sodium. When the pH in the oral cavity again increases to around neutrality, the saliva becomes saturated with calcium so that calcium liberation from the tooth is prevented. Among all the food residues found in the mouth, carbohydrates show the highest caries promoting effect since they are directly available for fermentation by oral bacteria.

Potentially all microorganisms that ferment sugars are cariogenic, but the primary etiological agents of coronal and root caries are the mutans streptococci because they are strong acid producers;Lactobacilli, that are highly aciduric, however, can also be implicated. In humans,S. mutansandS. sobrinusare the more cariogenic strains, and live on teeth while not colonizing the entire dentition. Their number is also less on anterior teeth than on molar teeth (Lindquist et al., Dent. Res., 69, 1160-1166, 1990). Moreover in human approximal plaque,S. mutansandS. sobrinuspreferentially colonize the most caries-prone site apical to the contact area (Ahmady et al., Caries Res., 27, 135-139, 1993). A higher prevalence ofS. sobrinuswas also found in the molar regions compared with that ofS. mutans(Lindquist et al., Caries Res., 25, 146-152, 1991).

S. mutansandS. sobrinushave been shown to attach to the pellicle of teeth mainly via specific adhesion-receptor interaction. Gibbons et al. showed thatS. mutanscarries an adhesion which binds to salivary components in the pellicle, whileS. sobrinuscells appear to possess an adhesion which binds to glucan in the pellicle (Infect. human., 52, 555-561, 1986).

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.

These exogenous lactic bacteria have never been shown to be capable of directly adhering to the pellicle of teeth. Repeated administration of exogenous lactic bacteria may lead to colonization of the mouth on all the oral surfaces, such as the tongue, the buccal mucosa, the gingiva, lips, cheeks, palate, floor, and the teeth. This colonization may result from attachments via specific bindings to bacteria of the resident microflora (co-aggregation phenomena), via entrapment in the matrix of polysaccharide produced by the resident bacteria, or via adhesion to saliva proteins (especially glycoproteins).

Lactobacillus casei rhamnosusGG (ATCC53103) has been reported to colonize the mouth, most probably on the epithelium of the buccal mucosa. This strain also adheres to the epithelium of the intestinal tract (U.S. Pat. No. 5,032,399, Gorbach et al.; Micr. Ecol. In Health and Dis., 2, 295-298, 1994). By contrastL. rhamnosusdoes not adhere to teeth.

Japanese patent no. 4021633 (Cyconmedix KK) also reported colonization of the mouth byLactobacillus acidophilus, most probably on the epithelium of the buccal mucosa. ManyLactobacillus acidophilusare known to also adhere to the epithelium of the intestinal tract (EP577904; EP199535; Perdigon et al., Medicina, 46, 751-754, 1986; Perdigon et al., Immunology, 63, 17-23, 1988).

Exogenous bacteria can also produce factors that inhibit the growth of the resident microflora in the mouth. For example, EP759469 (Sociétédes Produits Nestlé) described the use of a bacteriocin produced byMicrococcus variansfor inhibiting the development of the oral pathogensS. sobrinus, S. sanguis, S. mutansandA. viscosus.

There are several strategies to minimize the development of resident microflora of the mouth. For example, by administering commensal bacteria of the resident microflora that are not cariogenic, such asStreptococcus salivariusand/orStomatococcus mucilaginosus, and/or repeated administration of exogenous lactic bacteria such asL. casei, L. fermentum, L. acidophilus, L. crispatus, L. gasseri, L. salivarius, L bulgaricus, andS. salivarius(Tanzer et al., Infec. and Immunity, 48, 44-50, 1985; WO92/14475).

The application of bacteriocins is also one of the investigated strategies that have been set up to reduce tooth caries. These molecules have attracted interest as prospective anticaries agents and as factors important in modulating colonization of the oral cavity. The anti-carie potential of applying bacteriocins comes from their potent and broad antibacterial activity against mutans streptococci and bacteria associated with dental plaque and their natural occurrence in bacteria regarded as being safe to humans (U.S. Pat. No. 5,368,845 to Colgate, and WO 94/12150 to Smithkline Beecham).

The application of milk derivatives is also of interest for the health of the mouth. Indeed, U.S. Pat. No. 5,427,769 (Nestec S. A.) describes another alternative wherein dental caries are prevented by contacting teeth with an edible composition containing micellar casein in amount sufficient to inhibit colonization byStreptococcus sobrinus. EP748591 (Societe des Produits Nestle S. A.) also reports the use of fluoridated micellar casein or its micellar subunits for treating dental caries or plaque. U.S. Pat. No. 4,992,420 (Nestec S. A.) describes treatment of the buccal cavity with kappa-caseino-glycomacropeptide derived from milk for eradicating plaque and caries.

Lactic bacteria that are not part of the resident microflora of the mouth have never been shown to be really capable of directly adhering to the pellicle of teeth. By colonizing the surface of teeth, however, such lactic bacteria could exert an inhibitory activity against the growth of the resident microflora, including oral pathogens.

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 ofA. naeslundiiso as to reduce the severity ofA. naeslundii-related diseases.

SUMMARY OF THE INVENTION

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 aActinomyces naeslundiigrowth inhibition factor, and the preparation of a composition intended for reducing dental plaque and for treating or preventing root caries or other diseases or infections related to or caused byActinomyces naeslundiiin mammals.

The lactic bacteria may be selected from the group consisting ofStreptococcus thermophilis, Lactococcus lactissubsp.lactis, andLactococcus lactissubsp.lactis biovar diacetylactisand particularly from the group consisting of the strains CNCM I-1984, CNCM I-1985, CNCM I-1986, and CNCM I-1987. The lactic bacteria strain may be of dairy origin.

Thus, by colonizing the surface of teeth and producing growth inhibition factors, such lactic bacteria can exert a significant reduction of the extent ofActinomyces naeslundii, thus reducing dental plaque, root caries and otherActinomyces naeslundiirelated diseases and infections.

The lactic bacteria strain may be administered in an edible composition, and the composition may contain at least 104-109cfu/g of the lactic bacteria strain. The lactic bacteria may be administered in combination with a bacteriocin.

Another object is to provide a composition for maintaining the health of the mouth by reducing the colonization ofActinomyces 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 104-109cfu/g of lactic bacteria.

The present invention also relates to a composition for maintaining mammal mouth health by reducingActinomyces naeslundiicolonization therein. The composition comprises at least one lactic bacteria strain that is exogenous to oral microflora, where the strain is selected for its ability to adhere to teeth pellicle and to produce a growth inhibition factor and is present in an amount sufficient to reduce or inhibit the colonization ofActinomyces naeslundii.

The invention also provides a method of preventing or treatingActinomyces naeslundiirelated infections, particularly dental plaque extent and root caries in mammals.Actinomyces naeslundiirelated diseases may be prevented or treated in mammals according to the present invention by administering to a mammal a composition containing at least one lactic bacteria strain selected for its ability to adhere to the tooth surface and to produce aActinomyces naeslundiigrowth inhibition factor in an amount sufficient to reduce or inhibit the colonization ofActinomyces naeslundii. Further, such lactic bacteria strain may be provided in a dentifrice composition for maintaining mammal mouth health.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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 ofA. naeslundii.

Resident microflora of the mouth includes all microorganisms that naturally live in the mouth because they can establish a permanent residence on the oral surfaces. The resident microflora of the mouth also includes bacteria that live in the interfacial region between the dental hard and soft tissues (the junction tooth-gingiva), even thought the gingival crevice and the periodontal pocket are not present in a healthy mouth. This microflora includes microorganisms selected fromStreptococcus, Staphylococcus, Enterococcus, Micrococcus, Peptostreptococcus, Peptococcus, Lactobacillus, Corynebacterium, Actinomyces, Arachnia, Rothia, Alcaligenes, Eubacterium, Propionibacterium, Bifidobacterium, Bacillus, Clostridium, Neisseria/Branhamella, Veillonella, Enterobacteriaceae,Campylobacter, Eikenella, Actinobacillus, Capnocytophga, Haemophilus, Simonsiella, Bacteroides, Fusobacterium, Porphyromonas, Prevotella, Leptotrichia, Wohlinella/Selenomonas, Mycoplasma, Candida, Spirochaetes, Protozoa.

The object of the present invention is to use lactic bacteria that are not part of the resident microflora of the mouth, that is lactic bacteria that are low acidifying and that are capable of adhering directly to the pellicle of the teeth, to prepare a composition intended for the prophylaxis or the treatment of dental caries, dental plaque, and periodontal infection.

In one embodiment of the invention the lactic bacteria have been genetically modified to increase its adherence to the pellicle of the teeth via adhesion factors and/or genetically modified to be even less acidifying, contributing to a pH in the oral cavity of about 5.5 to 7.

The lactic bacteria may be selected from the group consisting of:

an acidifying lactic bacteria that adheres to the pellicle of the teeth and that has been genetically modified so that it is low acidifying compared to resident microflora;

a non adherent lactic bacteria that is low acidifying and that has been genetically modified so that it adheres to the pellicle of the teeth;

a non-adherent acidifying lactic bacteria that has been genetically modified so that it adheres to the pellicle of the teeth and genetically modified so that it is low acidifying compared to resident microflora.

In another embodiment the bacteria, that is not part of the resident microflora, is low acidifying compared to resident microflora and is capable of adhering directly to the pellicle of the teeth.

In another embodiment the composition for the health of the mouth comprises (1) at least a lactic bacteria that is not part of the resident microflora of the mouth, which is capable of adhering directly to the pellicle of the teeth and contributing to a pH in the oral cavity of above 5.5, and (2) any form of caseinoglycomacropeptide, micellar casein, fluorinated micellar casein, renneted milk, or bacteriocin.

The invention also provides a method for screening lactic bacteria capable of adhering to tooth. The method comprises the steps of: (1) preparing monoclonal antibody recognizing specific surface proteins of a lactic bacteria strain capable of adhering to the teeth, and (2) screening any lactic bacteria strain by use of the monoclonal antibody of strain capable of adhering to the teeth.

The lactic bacteria according to the invention that are low acidifying and capable of adhering directly to the pellicle of the teeth that are used to prepare compositions for the prophylaxis or the treatment of dental caries, dental plaque, and periodontal infection displace pathogenic bacteria from the teeth or prevent the attachment of the pathogenic bacteria. The lactic bacteria according to the invention are “low acidifying,” which means that they are less acidifying than pathogenic strains. Accordingly, they contribute to a pH in the oral cavity of about 5.5 to 7. Preferably, they are from dairy origin.

The lactic bacteria according to the invention adhere to the pellicle of the teeth via specific or unspecific interactions and/or adhesion factors. The specific adhesion factors are proteins or polysaccharides.

At least one lactic bacteria is selected from the group consisting ofStreptococcus thermophilus, Lactococcus lactissubsp.lactis, andLactococcus lactissubsp.Lactis biovar diacetylactisand particularly from the group consisting of the strains CNCM 1-1984, CNCM 1-1985, CNCM 1-1986, CNCM 1-1987, and LMG P-18997. These strains have been selected among lactic bacteria strains for their capacity to adhere to the pellicle of the teeth and their optimal growth temperature of about 37° C., which is the temperature in the oral cavity. Moreover they are capable of fermenting glucose and sucrose and do not synthesize glucans, which are factors leading to the pathogenicity of the cariogenic strains.

In one embodiment of the invention the lactic bacteria are genetically modifying so that they adhere to the pellicle of the teeth via adhesion factors. For lactic bacteria that already adhere to the pellicle of the teeth, this modification makes the strains more adherent to the surface of the teeth. In the same way, any non-adherent lactic acid bacteria (notLactobacilli) can be genetically modified so that it adheres to the pellicle of the teeth. This modification of the lactic bacteria can be achieved, for example, by insertion of the genes X17390, X14490 or X53657 (GenBank accession numbers). These gene are responsible inS. mutansfor the expression of the Antigen I/II that mediates adhesion to salivary glycoproteins.

According to the invention, it is also possible to genetically modify lactic bacteria so that they are low acidifying. For lactic bacteria that is already low acidifying this modification increases the effect by further decreasing lactic acid production. This modification can be achieved in many ways. Preferably, the modification is achieved according to one the protocols described in the following documents: Boumerdassi et al., Appl. Environ. Microbiol., 63, 2293-2299, 1997; Plattecuw et al., Appl. Environ. Microbiol, 61, 3967-3971, 1995; Ito et al., Biosci. Biotechnol. Biochem., 58, 1569-1573, 1994.

With respect to one 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 toActinomyces naeslundii, is concerned.

The lactic bacteria are preferably of 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 lactic bacteria strains for their capacity of adherence to the pellicle of the teeth, and 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 ofA. naeslundiigenospecies 1 and 2.

Moreover they are capable of fermenting glucose and sucrose and do not synthesize glucans, which are factors of pathogenicity of the cariogenic strains.

It is also possible to use at least one lactic bacteria strain in combination with a bacteriocin, for example.

According to the invention, at least one lactic bacteria, genetically modified or not, is used in an “effective amount” for the preparation of compositions intended for the prophylaxis or the treatment of dental caries, dental plaque, and periodontal infection in humans or animals such as pets. This quantity is preferably between 104-109cfu/g.

It is also possible to use the at least one lactic bacteria, in combination with milk derivatives, such as milk, fermented milk, or milk derivatives selected from any forms of caseino-glycomacropeptide, micellar casein, fluorinated micellar casein, renneted milk, or bacteriocin, for example.

The exogenous lactic bacteria may be used in an amount of at least 104-109cfu/g of lactic bacteria.

The effect of incorporating the above-mentioned bacteria in the oral microflora was tested in a rat model. The strains CNCM I-1985 and CNCM-1986 were able to modulate the oral microbial ecology, significantly reducing the number of total CFU. More specifically, the strains were able to significantly decrease the colonization extent ofA. naeslundiigenospecies 2, with which the rats had been infected (see examples).

Biochemical Characterization of the Selected Strains

Fermentation patterns: 49 simple sugars were tested with the api 50 CH bioMerieux strip test (bioMérieux SA, 69280 Marcy-l'Etoile, France) and the results are given in Table 1.

Acidification curves: Acidification curves were determined at 37° C. under the following conditions:

The strainsSreptococcus thermophilus(NCC 1529),Sreptococcus thermophilus(NCC 1561),Lactococcus lactissubsp.lactis(NCC 2211),Lactococcus lactissubsp.lactis biovar dioaetylactis(NCC 2225) were deposited under the Budapest Treaty, at the Collection Nationale de Culture de Microorganismes (CNCM I-1984, CNCM I-1985, CNCM I-1986 and CNCM I-1987 respectively), 25 rue du docteur Roux, 75724 Paris, France, on Mar. 3rd, 1998. The strainS. thermophilusBF11116 (CNBL 1177) was deposited under the Budapest Treaty at the Belgian Coordinated Collections of Microorganisms LMG P-18997, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium, on Jul. 5, 1999. All restrictions as to the availability of these deposits will be withdrawn upon first publication of this application or another application which claims benefit of priority to this application.

The invention is also directed to compositions for the health of the mouth that comprise a lactic bacteria that is not part of the resident microflora of the mouth, that is low acidifying, and that is capable of adhering directly to the pellicle of the teeth. The compositions are particularly intended for the prophylaxis or the treatment of dental caries, dental plaque, and periodontal infection. The lactic bacteria strain may be selected from the group consisting ofStreptococcus thermophilus, Lactococcus lactissubsp.lactis, andLactococcus lactissubsp.lactis biovar diacetylactisand preferably from the group consisting of the strains CNCM I-1984, CNCM I-1985, LMG P-18997, CNCM I-1986, and CNCM I-1987. In these compositions the lactic bacteria strains may be genetically modified as described above.

In the compositions of the invention, the lactic bacteria strains may be included alone or in combination with milk derivatives, for example, in order to obtain synergistic preparations. Accordingly, these compositions for the health of the mouth comprise:

a lactic bacteria that is not part of the resident microflora of the mouth, which is capable of adhering directly to the pellicle of the teeth;

any forms of lactic glycopeptides, renneted milk, or bacteriocin.

The lactic glycopeptides are preferably caseino-glycomacropeptides (CGMP), fluorinated or non-fluorinated micellar casein (which can be obtained as described in EP 0 604 802 and EP 0 748 591), or renneted milk. The caseino-glycomacropeptides are preferably added in a minimum amount of about 0.1%. It has also been shown that the caseino-glycomacropeptides do not prevent the lactic bacteria from adhering to the teeth pellicle (FIGS. 2 and 3).

Synergistic compositions may also be prepared by adding at least one bacteriocin which is active against Gram-positive oral bacteria. In this embodiment the oral hygiene compositions may comprise 0.00001 to 50%, and preferably from 0.00001 to 15% of purified bacteriocin, by weight of the composition. The bacteriocin is preferably variacin (EP 0759469).

To protect the composition from degradation, an oil-soluble antioxidant may also be included. Suitable antioxidants include the “tocopherols,” butyl-hydroxyanisole (BHA), butyl-hydroxytoluene (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 silica, of which silica is the preferred abrasive.

Compositions according to the invention will have a pH which is orally acceptable and within a range such that the activity of the lactic bacteria is not compromised. The pH may be in the range of 3.0 to 9.5, preferably in the range 3.5 to 6.5.

The compositions of the invention may be prepared by conventional processes that comprise admixing the ingredients together in the appropriate relative amounts and finally, if necessary, adjusting the pH to the desired value.

The present invention also relates to a composition for maintaining the health of the mouth by reducing the colonization ofA. naeslundiiin mammals, said composition comprises an exogenous lactic bacteria, which has been selected for its ability to adhere to the tooth surface and to produce a growth inhibition factor.

These compositions are particularly intended for the prophylaxis or the treatment of dental plaque and infection related toA. naeslundiidisease such as root caries, for example.

The lactic bacteria strain according to the present invention is selected from the group consisting ofStreptococcus thermophilus, Lactococcus lactissubsp.lactis, andLactococcus lactissubsp.lactis biovar diacetylactisand preferably from the group consisting of the strains CNCM I-1984, CNCM I-1985, CNCM I-1986 and CNCM I-1987.

Such a composition may contain at least 104-109cfu/g of lactic bacteria.

Actinomyces naeslundiigenospecies 1 (formerlyA. naeslundii) and 2 (formerlyA. 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 etiological agent of this disease.

Thus, the invention also provides a method for the prevention or the treatment ofActinomyces naeslundii-related 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 tooth surface and to produce a growth inhibition factor.

The amount to be administered may be of at least about 104-109cfu/g of lactic bacteria.

The invention is further directed to a method for screening lactic bacteria capable of adhering to tooth. This method comprises the steps of:

(1) preparing monoclonal antibodies that recognize specific surface proteins of a lactic bacteria strain capable of adhering to the teeth, and

(2) screening any lactic bacteria strain by using the monoclonal antibody of strain capable of adhering to the teeth.

The monoclonal antibodies are used as a tool to detect the said lactic bacteria strain among other strains growing nearby.

EXAMPLES

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. DNA manipulation, cloning and transformation of bacteria cells are, except where otherwise stated, carried out according to the textbook of Sambrook et al. (Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, U.S.A., 1989).

The examples are preceded by a brief description of the plasmids, strains, and the various media used, as well as the method for producing a monoclonal antibody.

Strains and Culture Conditions

All the strains were stored in glycerol at −20° C. and pre-cultured for 14 hours prior to use at their specific optimal temperature;S. sobrinusOMZ 176 grew in FUM medium lactococci and streptococci in M 17 (Difco) exceptS. thermophilusNCC1529, Si 19, S122, NCC1561 and S126 that grew in Belliker (prepared by dissolution of 20 g tryptone, 5 g yeast extract, 2.5 g gelatine, 5 g dextrose, 5 g sucrose, 5 g lactose, 4 g NaCl, 0.5 g Ascorbic acid, and 10 g beef extract in 1 L of water).

For plate counting,S. sobrinusOMZ 176 was cultured in Mitis-Salivarius agar (Difco),S. thermophilusNCC1529, S119, S122, NCC1561, BF11116, and S126 in Belliker agar (prepared by adding to liquid Belliker 15 g of Bacto agar, Difco), and the remaining lactic bacteria strains in M17 agar (Oxoid).

Production of Monoclonal Antibody

A monoclonal antibody would be used as a tool to detectL. lactissubsp.lactisNCC22111 among 5 oral strains growing together on S-HA discs and forming a biofilm that simulates dental plaque. Therefore the monoclonal antibody was tested against these strains to verify there was no cross-reaction. To this end, the monoclonal antibody is produced as described by Granato et al. “A mouse monoclonal IgE antibody anti-bovine milk lactoglobulin allows studies of allergy in the gastrointestinal tract., Clin. Exp. Immunol., 63, 703-710, 1986.

Selection of Adherent Lactic Bacteria

Attachment to Saliva-coated Hydroxyapatite Beads (S-HA)

To select among the lactic bacteria dairy strains those strains that are able to attach to saliva-coated hydroxyapatite beads (S-HA), the procedure previously described by Neeser et al. (1994) was used with slight modification in that the bead washings were done with 150 μl volumes and Hyamine hydroxide was substituted with Benzethonium hydroxide (Sigma).

Briefly, all the strains were grown to the end of the log phase in FUM exceptS. thermophilusNCC1529, S119, S122, NCC1561, and S126 that were cultured in Belliker.S. sobrinusOMZ 176,L. lactissubsp.lactisNCC2211, 50 and 54,S. thermophilusNCC1529, S119, S122, NCC1561, and S126 grew at 37 C°, the remaining lactococci at 30° C., and the remaining streptococci at 42° C.

5 mg of hydroxyapatite beads (BDH Chemicals Ltd, Poole, England) were covered with 70 μl clarified saliva obtained from volunteers in the lab and prepared as previously explained (Neeser et al, 1-994). Saliva coated beads were kept overnight at 4° C., then washed (first with distilled water and after with HEPES buffer) and finally inoculated with 100 μl of metabolically labeled bacterial suspension (bacteria had been grown in medium supplemented with 10 μCi/ml14C acetic acid). Adhesion took place during 45 min at 37° C., then unbound bacteria were washed away and the attached cells directly counted in a LKB scintillation counter (type 1219 Rackbeta).

Adhesion percentages are expressed as radioactivity bound to the beads relative to the total radioactivity added to each well. All measurements were done in triplicate. Table 2 reports the percentages of adhesion to saliva-coated hydroxyapatite beads obtained for several screened strains and forS. sobrinusOMZ 176 (the reference strain).

L. lactisNCC2211 andS. thermophilusNCC1561 were chosen as the more promising candidates since they grow very well at 37° C., which is the temperature in the mouth, whileL. diacetylactisNCC2225 has an optimal growth temperature of 30° C. In particular,L. lactisNCC2211 cannot grow on sucrose, but it can ferment a wide range of sugars, moreover other oral strain can provide glucose via their invertase.

Adhesion Saturation Curves

Curves of bound CFU versus CFU inoculated into the well were determined to verify if bead saturation could be obtained. The 50% saturation was obtained directly from the bending point of the curves. The adhesion saturation curves forS. sobrinusOMZ 176,L. lactisNCC2211, andS. thermophilusNCC 1561 were determined. They are shown inFIG. 1.

For each of the three strains the CFU number inoculated in the well to get 50% bead saturation and the corresponding number of bound CFU were directly deduced from the bending point of the curves and are given in the table 3.

Effect of Caseino-glycomacropeptides

The influence of CGMP on the adhesion ofL. lactisNCC2211 andS. thermophilusNCC 1561 was studied to verify the possibility of using CGMP to foster the predominance of one of these two strains over pathogenic strains, namelyS. SobrinusOMZ 176. Caseino-glycopeptide (CGMP) and its desialylated derivative (As-CGMP) were obtained from Nestec S. A., Lausanne (for their preparation see Neeser et al., 1994).

The dose-response effect was studied on the adhesion to S-HA beads by inoculating, in the well, 100 μl of bacterial suspension (CFU/ml corresponding to the 50% bead saturation previously calculated) which contained CGMP or AsCGMP in different concentrations and then performing the adhesion assay in the usual manner. Concentrations in the range 0.05 to 3 mg/ml were tested. No previous incubation of the bacteria in presence of CGMP or As-CGMP was done.

FIG. 2provides the curves obtained for the three strains by plotting the number of bound cells versus increasing amounts of CGMP, the number of inoculated cells corresponds to 50% bead saturation formerly calculated for each strain. The strong inhibition observed in the case ofS. sobrinusOMZ 176 confirms the previous results obtained by Neeser et al. (1994) and Schupbach et al. (J. Dent. Res., 75, 1779-1788, 1996).

FIG. 2shows that 0.25 mg/ml produced 50% inhibition of the adhesion ofS. sobrinusOMZ 176, while more than 2 mg/ml were necessary to have the same effect withS. thermophilusNCC1561. CGMP slightly enhances the adhesion ofL. lactisNCC2211.

As in the case of CGMP, the desyalilated derivative inhibits the adhesion ofS. sobrinusOMZ 176; only 0.05 mg/ml are needed to produce 50% decrease in the adhesion percentage. As-CGMP does not influenceL. lactisNCC2211 adhesion, while it slightly fosters the adhesion ofS. thermophilusNCC1561 (FIG. 3).

Toothpaste

The toothpaste is intended for the prophylaxis or the treatment of dental caries, dental plaque and periodontal infection.

Ice Cream

A cream comprising 10.8% lactic fats, 13.5% milk solids (non fat), 0.3% Emulstab® SE30 and 0.3% Emulstab® foam (Grindsted, DK) is prepared and then pasteurized at 105° C. for 20s, homogenized at 75° C. and 300 bar, cooled to 38° C., and inoculated with pre-cultures in MRS medium, taken in the exponential growth phase, at a rate of 107to 108cfu/ml of at least one of the lactic bacteria strain of CNCM 1-1984, CNCM 1-1985, CNCM 1-1986, CNCM 1-1987 or LMG P-18997. The cream is then fermented for 10 hours at 38° C. up to a pH of about 4.5. At the end of the fermentation, sucrose and glucose syrup is added thereto. The composition of the cream is presented in table 4 below. The mixture is then beaten, cooled to 4° C., stored at 4° C., and chilled to a degree of expansion of 95° C. by volume.

Yogurt

5 L MRS culture medium were sterilized for 15 min at 121° C. and then inoculated with 5% by volume of an active culture of at least one of theS. Thermophilusstrain CNCM I-1984, CNCM I-1985, or LMG P-18997 containing approximately 109cfu/ml. After incubation for 8 h at 41° C., a starter containing 4.5×108cfu/ml was obtained.

5 L reconstituted skimmed milk having a dry matter content of 10%, to which 0.1% yeast extract had been added, was sterilized for 15 min at 121° C. and inoculated with 2% of an active culture of commercial thickeningStreptococcus thermophiluscontaining approximately 109cells/ml. After incubation for 4 h at 41° C., a starter containing 4.5×108cells/ml was obtained.

One batch of whole milk containing 3.7% fats strengthened with 2.5% skimmed milk powder and then pasteurized for 30 min at 90° C. was then inoculated with 2% by volume of the starter of at least one of the strains CNCM I-1984, CNCM I-1985 or LMG P-18997 strains and 3% by volume of the starter of thickeningStreptococcus thermophilus. The inoculated milk is stirred, poured into pots, and incubated for 4 h at 41° C. The resulting yogurt obtained has a good firm and smooth texture and is intended for the health of the mouth.

Chewing Gum

A chewing gum for preventing or treating dental caries, dental plaque, or periodontal infection can be prepared adding an active culture of at least one of theS. Thermophilusstrains CNCM I-1984, CNCM I-1985, or LMG P-18997, so that it contains approximately 104to 109cfu/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.

Pet Food Composition

A pet food for mouth health is obtained by preparing a feed mixture made up of corn, corn gluten, chicken and fish meal, salts, vitamins, and minerals. The feed mixture is fed into a pre-conditioner and moistened. The moistened feed leaving the pre-conditioner 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 theS. Thermophilusstrains CNCM I-1984, CNCM I-1985, or LMG P-18997 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 104to 109cfu/g of the said strains.

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 strainsL. lactisNCC2211 andS. thermophilusNCC1561 were inoculated in an in vitro. system in which a biofilm, composed by bacteria commonly 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 5 were used.

TABLE 5Bacterial strains used and culture conditions appliedin the in vitro dental plaque experiments.StrainRelevant propertiesGrowth conditionsS. thermophilusNCC1561S-HA adherentFUM, Belliker; 37° C.L. lactissubsp.lactisS-HA adherentFUM, M17-lactose;NCC221137° C.S. sobrinusOMZ176CariogenicFUM; 37° C.S. oralisOMZ607Plaque formingFUM; 37° C.A. naeslundiiOMZ745Plaque formingFUM; 37° C.root cariescausative agentV. disparOMZ493Plaque formingFUM; 37° C.F. nucleatumOMZ596Plaque formingFUM; 37° C.
ProcedureSaliva pellicle formation: cover synthetic hydroxyapatite discs of 10 mm diameter (HY-APATITE®, Euro-Crystals, Landgraaf, The Netherlands) with 800 μl 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: growS. thermophilusNCC1561,L. lactissubsp.lactisNCC2211,S. sobrinusOMZ176,S. oralisOMZ607,A. naeslundiiOMZ745,V. disparOMZ493 andF. nucleatumOMZ596 overnight at 37° C. in anaerobiosis in FUM-glucose (S. thermophilusNCC1561 in FUM-lactose), adjust the OD550to 1 with FUM and pool 2 ml of each oral bacterial suspension with 2 ml of eitherS. thermophilusNCC1561 orL. lactissubsp.lactisNCC2211. The control mixture contains the five oral strains only.Biofilm formation and recovery: the procedure is as described in Guggenheim et al., 1998, Validation of a new biofilm model. J. Dent. 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 ofA. 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 6.

TABLE 6Bacterial strains used and culture conditionsapplied in the growth antagonism experiments.StrainRelevant propertiesGrowth conditionsS. thermophilusNCC1561S-HA adherentBelliker; 42° C.L. lactissubsp.lactisS-HA adherentM17-lactose; 37° C.NCC2211S. thermophilusNCC1536non-adherentBelliker; 42° C.A. naeslundiiOMZ745plaque formingBHI, 37° C.;anaerobiosisA. viscosusOMZ105plaque formingBHI; 37° C.S. thermophilusNCC1561,S. thermophilusNCC 1536 andL. lactisNCC2211 (killer strains) were tested for growth antagonism againstA. naeslundiiOMZ745 andA. viscosusOMZ105 (target strains).
ProcedureGrow the killer strains overnight on agar plates in anaerobiosis and the target strains in BHI until middle stationary phaseDilute 20 μl of the target strains suspension in 3 ml of BHI soft agar (agar 7 g/l) containing glucose and lactose, vortex and pour immediately on a BHI agar plateSolidify 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 controlIncubate at 37° C. in anaerobiosis for 24 hours
Growth antagonism is revealed by an inhibition halo around the cross.
Statistics
Differences between the control and test consortia were determined by Student's t test.
Results and Discussion

S. thermophilusNCC1561 andL. lactisNCC2211 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 7.

TABLE 7Level of incorporation of the two dairy strainsin the biofilm (CFU/disc). The values are the meanof three experiments with their standard deviations.S. thermophilusNCC1561L. lactisNCC2211Method of inoculation(×106)(×106)Together with the oral4.08 +/− 1.785.76 +/− 3.64strainsBefore the oral strains5.03 +/− 2.213.87 +/− 4.01

The effect of incorporating the dairy strains in the biofilm on the oral species is indicated in the Tables 8 and 9. WhenS. thermophilusNCC1561 was included (Table 8), 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.

WhenL. lactisNCC2211 was introduced in the oral strains consortium (Table 9) the total flora counts notably diminished (CFU on CBA). The decrease was significant in the case ofA. naeslundiiOMZ745 that significantly diminished (p=0.021) The decrease was even stronger if the strain was inoculated on the discs before the oral bacteria.

Some assays were carried out to verify if the reduction of the oral strains was due to growth antagonism of the dairy strains towards them (Table 10). The strainsA. viscosusOMZ105 andS. thermophilusNCC1536 were also included in the test since they are part of the in vivo model (Example 11)

All the four dairy strains inhibited the growth of the Gram-negative strainA. viscosusOMZ105. This inhibition cannot be attributed to lactic acid production.A. viscosusis 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 platingA. viscosusin presence of 1% lactic acid: no inhibition was observed.

CONCLUSIONS

S. thermophilusNCC1561 andL. lactisNCC2211 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 ofA. naeslundiigenospecies 2. In addition the strains could inhibit the growth ofA. naeslundiigenospecies 1 and 2 in co-cultures.

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 Osborne-Mendel rats pups each (animal production section of the Institute für 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 μm) Nafag diet to avoid fissure impaction (Rat Checkers No. 184, NAFAG, Gossau SG, Switzerland), and tap waterad 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.

During the association period (days 21 and 22) the drinking water was supplemented with 2% glucose and 2% sucrose to support the implantation of the associated bacteria. On day 23 the litters 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 13. 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 suppliedad 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.

Bacterial strains: The strains listed in the Table 11 were used.

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 theS. thermophilusstrains for 7 h and theL. lactisone for 6 h. Also a study of the viability 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 stockInoculate 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 phaseDetermine 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 Belliker or M17).Split in aliquots in order to have 2×1011viable 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 12). Each treatment consisted of 10 pups that were distributed in 1 per cage.

All rats were first infected on days 21 and 22 withA. viscosusOMZ105.

The tested LAB strains were associated daily (since contained in the yogurt base meal), starting on day 23. 2 frozen vials, each containing 2×1011viable cells of the test strain, were mixed in 200 ml of yogurt in order to have at least 109CFU/ml.S. thermophilusNCC 1536, 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 13). 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.

TABLE 13Eating times.N° of the mealHigh cariogenic mealsYogurt mealsN° of the meal110:0010:202310:4011:004511:2011:406712:0012:208912:4013:00101113:2013:40121314:0014:20141514:4015:00161715:2015:40181916:0016:20202116:4017:00222317:2017:40242518:0018:20262718:4019:00282919:2019:40303120:0020:20323320:4021:0034
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 immediately in 5 ml of sterile NaCl 0.9%Vortex for 1 min and sonicate for 5 s at 50 WSpirally plate the properly diluted suspensions on CBA, MS and HJL agarIncubate CBA and MS plates at 37° C. and HJL plates at 45° C.
Procedure for TCN DeterminationPut 10 μl 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 dryFix by soaking in methanol for 2 min and air dryIncubate with 10 μl of the proper antibody or serum diluted in ELISA buffer (section 4.2.2.5) and incubate at 37° C. for 30 minAspirate each drop from the side of the well and wash by soaking the slide first in ELISA buffer and then in distilled water. Air dryApply 10 μl of goat-anti-rabbit IgG (H+L)−FITC (Sigma) diluted 1:400 and incubate at 37° C. for 30 minWash as before and air dryApply 49 μl 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.

Colonization of the strain. 1.7 (+/−1.1)×107CFU were counted for the plaque formingA. viscosusOMZ105, when the dairy product was supplemented with the non-adherentS. thermophiluscontrol (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 strainsS. thermophilusNCC1561 andL. lactisNCC2211 displayed significant reduced numbers of colony forming units on CBA compared with the control group containing the non-adherent strainS. thermophilusNCC 1536 (Table 14). Treatment 2 reduced the CFU counts with a significant factor of PF<0.01 and treatment 3 even more significantly (PF<0.001).Modulation ofA. viscosusOMZ105 tooth colonization by the tested strains. In the case of the plaque forming bacteriumA. viscosusOMZ105, 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<0.01) (Table 14). By contrast the percentages ofA. viscosuson the total CFU counts were not significantly different. Approximately 50% of the total CFU for all four treatments were identified asA. viscosuscolonies.

In this in vivo assay, the strains that 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 adheringS. thermophilusNCC1536, can inhibit the growth ofA. viscosusOMZ105. 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 withA. viscosusOMZ105, 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 ofA. viscosusOMZ105 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.Veillonellae, and consequently it is a global effect that is observed.

Thus the strains CNCM I-1985 and CNCM-1986 are able to modulate the oral microbial ecology, significantly decreasing the colonization extent ofA. naeslundiigenospecies 2, with which the rats had been infected.

Production and Initial Analysis of Surfactant Substances fromS. thermophilusNCC1561 andS. thermophilusNCC1536

Preparation of the Surfactant Substances

Procedure

Wash cells three times in PBSResuspend in 200 ml of distilled water or PBSProduce the biosurfactant by gently stirring the suspension for 2 or 4 h at room temperatureSeparate bacteria by centrifugation at 10000 rpm for 10 minCentrifuge supernatant twice at 10000 rpm for 10 minFreeze-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 TVT1 Drop Volume Tensiometer (Lauda, Lauda-Königshofen, Germany), which is based on the drop volume principle. Briefly, the method consists in the exact determination of the volume of a suspension drop that detaches from a capillary. This volume (critical volume) is proportional to the surface tension (σ), whose value is calculated with the relation:
σ=V gΔρF/2πrcap
where:σ is the interfacial tensionV is the drop volumeG is the acceleration constantΔρ is the difference of the densities of both adjacent phasesF is the correction factorrcapis 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 15). SDS-PAGE profile of the bacteria released products, showed that there were many different substances of proteinaceous nature in the solution.

S. thermophilusNCC1561 andS. thermophilusNCC1536 cells are able to release substances with a surfactant activity. It is therefore possible that the biosurfactant produced byS. thermophilusNCC1561 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 byS. thermophilusNCC1536, since this strain does not adhere to the teeth.

Toothpaste

This toothpaste is intended for the prophylaxis or the treatment of root caries, dental plaque and other infections induced byA.naeslundiispecies.

5 L MRS culture medium are sterilized for 15 min at 121° C. and then inoculated with 5% by volume of an active culture of at least one of theS. thermophilusstrain CNCM I-1984, CNCM I-1985 containing approximately 109cfu/ml. After incubation for 8 h at 41° C., a starter containing 4.5·108cfu/ml is obtained.

5 L reconstituted skimmed milk having a dry matter content of 10%, to which 0.1% yeast extract has been added, are sterilized for 15 min at 121° C. and inoculated with 2% of an active culture of commercial thickeningStreptococcus thermophiluscontaining approximately 109cells/ml. After incubation for 4 h at 41° C., a starter containing 4.5·108cells/ml is obtained.

One batch of whole milk containing 3.7% fats strengthened with 2.5% skimmed milk powder and then pasteurized for 30 min at 90° C. is then inoculated with 2% by volume of the starter of at least one of the strains CNCM I-1984, CNCM I-1985 and 3% by volume of the starter of thickeningStreptococcus thermophilus. The inoculated milk is stirred, poured into pots and incubated for 4 h at 41° C.

The yogurt obtained has a good firm and smooth texture and is intended for the health of the mouth.

Chewing Gum

A chewing gum for preventing or treating root caries, dental plaque, or otherA.naeslundii-related diseases can be prepared adding an active culture of at least one of theS. thermophilusstrain CNCM I-1984, CNCM I-1985, so that it contains approximately 104to 109cfu/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.

Pet Food Composition

A pet food for mouth health is obtained by preparing a feed mixture made up of corn, corn gluten, chicken and fish meal, salts, vitamins and minerals. The feed mixture is fed into a preconditioner and moistened. The moistened feed leaving the preconditioner is then fed into an extruder-cooker and gelatinized. The gelatinized 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 that 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 theS. thermophilusstrains CNCM I-1984, CNCM I-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 104to 109cfu/g of said strains.