Incorporation of exogenous lactic bacteria into the oral microflora

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Incorporation Of Exogenous Lactic Bacteria Into The Oral Microflora

Societe des Produits Nestle S.A.

INCORPORATION OF EXOGENOUS LACTIC BACTERIA INTO

THE ORAL MICROFLORA

The present invention relates to the incorporation of exogenous lactic 5 bacteria into the oral microflora intended for the prophylaxis or the treatment of dental caries, dental plaque and periodontal infection.

Background of the invention 10

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 15 and floor of the mouth only support a very sparse microflora.

On the tongue and the buccal mucosa, the natural resident microflora includes microorganisms selected from Streptococcus, Veillonella, Bacteroides and Haemophilus. On the teeth, Streptococci, Lactobacilli and Actynomyces 20 predominate but a variety of Gram positive and negative cocci and rods can be found.

For example, Frandsen et al. showed that 5. sanguis predominates on the buccal mucosa but its primary habitat is the surface of teeth, that S. gordonii 25 grows in the mature supragingival plaque, that S. oralis and 5. mitis grow in the initial dental plaque (Oral Microbiol. Immunol., 6, 129-133, 1991). Strains belonging to the mutans group are localised on teeth (0*. criscetus, S. dovmei, S. ferus, S. macacae, S. mutans, S. rattus, S. sobrinus). Strains belonging to the S. milleri group predominate in dental abscesses (5. anginosus, S. constellatus, S. 30 intermedius', Bentley et al., Int. J. System. Bacter. 1991, 41, 487-494; Wood et al., The Genera of Lactic Acid Bacteria, Blackie Academic and Professional, Chapman & Hall, W. H. eds., 1995).

Many of these microorganisms are innocuous commensal, but a lot of them 35 have been recognised 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

Florida, USA) NO 6202. -. 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 pm 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 adhesin-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 coaggregation among the different bacterial species and the glucans synthesised by the bacteria and 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., 52, 555-561, 1986).

The organic acids produced by oral bacteria during the fermentation process directly cause dental caries. These acids attack the hard tissue of teeth with consequent release of ions such as calcium, phosphate, carbonate, magnesium, fluoride, sodium. When the pH in the oral cavity increases again around the neutrality, saliva becomes saturated with calcium and therefore its liberation from the tooth is prevented.

Among all the food residues found in the mouth, carbohydrates show the highest caries promoting effect being directly available for oral bacteria fermentation.

Potentially all micro-organisms fermenting 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, can also be implicated. In humans, S. mutans and S. sobrinus are the more cariogenic strains, and live on teeth while not colonising the entire dentition. A decrease in their number from molar to anterior teeth was indeed demonstrated (Lindquist et al., Dent. Res., 62, 1160-1166, 1990). Moreover in human approximal plaque, 5. mutans and S. sobrinus preferentially colonise the most caries-prone site apical to the contact area (Ahmady et al., Caries Res., 22, 135-139, 1993). A higher prevalence of 5. sobrinus was also found in the molar regions compared with that of S. mutans (Lindquist et al., Caries Res., 25, 146-152, 1991). S. mutans and S. sobrinus have been shown to attach to the pellicle of teeth mainly via specific adhesin-receptor. Gibbons et al. showed that S. mutans carries an adhesin which binds to salivary components in the pellicle, while S. sobrinus cells appear to possess an adhesin which binds to glucan in the pellicle (Infect. Immun., 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 however lead to colonisation 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 colonisation may result from attachments via specific bindings to bacteria of the resident microflora (co-aggregation phenomena), or via entrapment in the matrix of polysaccharides produced by the resident bacteria, or via adhesion to saliva proteins (especially glycoproteins).

Lactobacillus casei rhamnosus GG (ATCC53103) has been reported to colonise the mouth, most probably on the epithelium of the buccal mucosa. This strain also adheres to the epithelium of the intestinal tract (US5032399, Gorbach et al.·, Mier. Ecol. In Health and Dis., 2, 295-298, 1994). By contrast L. rhamnosus does not adhere to teeth.

Japanese patent n°4021633 (Cyconmedix KK) also reported colonisation of the mouth by Lactobacillus acidophilus, most probably on the epithelium of the buccal mucosa. Many Lactobacillus acidophilus are known to adhere also to the epithelium of the intestinal tract (EP577904; EPI99535; Perdigon et al., Medicina, 46, 751-754, 1986; Perdigon et al., Immunology, 52, 17-23, 1988).

Exogenous bacteria can also produce factors that inhibit the growth of the resident microflora in the mouth. For example, EP759469 (Societe des Produits Nestle) described the use of a bacteriocin produced by Micrococcus varians for /"'••'η'·* 08-2000, inhibitingihe development of the oral pathogens £ sobrinus, mutans and A. viscosus. DE3CPAMD EPO-DU7 ' ' θ 1 08. 2000 S. s^^uis, S.

Some strategies have been used to minimise the development of the resident 5 microflora of the mouth, namely an administration of commensal bacteria of the resident microflora that are not cariogenic, such as Streptococcus mutans (JP 59220191) or Streptococcus salivarius (JP 05004927) and/or Stomatococcus mucilaginosus, and/or repeated administrations of exogenous lactic bacteria such as L. casei, L. fermentum, L. acidophilus, L. crispatus, L. gasseri, L. salivarius, L io bulgaricus and A salivarius (Tanzer et al., Infec. and Immunity, 48, 44-50, 1985; WO92/14475; EP 0524732).

The application of bacteriocins is also one of the investigated strategies which have been set up to reduce tooth caries. These molecules have attracted 15 interest as prospective anticaries agents and as factors important in modulating colonisation of the oral cavity. The anti-caries potential of applications of some 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 human safe (US5368845 of Colgate, and 20 WO94/12150 of Smithkline Beecham).

The application of milk derivatives is also of interest for the health of the mouth. Indeed, US5427769 (Nestec S.A) describes another alternative where dental caries are prevented by contacting teeth with an edible composition 25 containing micellar casein in amount sufficient to inhibit colonisation by Streptococcus 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. US4992420 (Nestec S.A.) describes treatment of the buccal cavity with kappa-caseino-glycomacropeptide derived from milk for 30 eradicating plaque and caries. :21 2000 11 08-2000 5

'.DESCPAMD _____________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. Thus, by colonising the surface of teeth, such lactic bacteria would exert an inhibitory activity against the growth of the resident microflora, including oral 5 pathogens.

Summary of the invention 10 The object of the present invention is to provide the use of 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, for the preparation of a composition intended for the prophylaxis or the treatment of dental caries, dental plaque and periodontal infection. 15

The lactic bacteria that may 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 5.5-7. 20

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; a non adherent lactic bacteria that is low acidifying and that has been 25 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. 30 Another object is to provide a composition for the health of the mouth comprising a lactic bacteria that is not part of the resident microflora, that is low acidifying and that is capable of adhering directly to the pellicle of the teeth.

21 20W '2 Ό8. FCT/EF99/05473; 4 , S \ z%s ' \ ' '·. 6 •DESCPAMf3';

In a prefered embodiment, the composition for the health of the mouth comprises at least one lactic bacteria strain selected from the group consisting of the strains CNCM 1-1984, CNCM 1-1985, CNCM 1-1986, CNCM 1-1987 and LMG P-18997. 5

The composition for the health of the mouth may comprise at least one lactic bacteria that has 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 and contributes to a pH in the oral cavity of about 5.5-7. io

The composition for the health of the mouth may comprise lactic bacteria 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; 15 - 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. 20

Detailed description of the invention 25 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). 30 The 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 1-2Ϊ £000' \ 'v * \ ν' tooth-gingiva), even thought the gingival crevice and the periodontal pocket are not present in a healthy mouth. This microflora includes microorganisms selected from Streptococcus, Staphylococcus, Enterococcus, Micrococcus, Peptostreptococcus, Peptococcus, Lactobacillus, Corynebacterium, Actinomyces, 5 Arachnia, Rothia, Alcaligenes, Eubacterium, Propionibacterium, Bifidobacterium, Bacillus, Clostridium, Neisseria/Branhamella, Veillonella, Enterobacteriaceae, Campylobacter, Eikenella, Actinobacillus, Capnocytophga, Haemophilus, Simonsiella, Bacteroides, Fusobacterium, Porphyromonas, Prevotella, Leptotrichia, Wolinella/Selenomonas, Mycoplasma, Candida, 10 Spirochaetes, Protozoa.

The transient microflora comprises exogenous bacteria that can be occasionally present in the mouth, but that do not establish a permanent residence. This transient microflora may comprise all the food micro-organisms, 15 such as the bifidobacteria (B. infantis, B. adolescentis, B. breve and B. longum); the lactococci (Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, and Lactococcus lactis subsp. lactic biovar diacetylactis}; the streptococci (Streptococcus thermophilus, S. lactis, S. lactis cremoris and S. lactis diacetylactis}; the lactobacilli (Lactobacillus delbrueckii subsp. bulgaricus, 20 Lactobacillus helveticus, Lactobacillus farciminis, Lactobacillus alimentarius, Lactobacillus casei subsp. casei, Lactobacillus delbruckii subsp. lactis, Lactobacillus sake, Lactobacillus curvatus, Lactobacillus fermentum and the acidophile group comprising L. johnsonii', see Fujisawa et al., Int. J. Syst. Bact., 42, 487-491, 1992); the pediococci (Pediococcus pentosaceus, Pediococcus 25 acidilactici and Pediococcus halophilus}; the enterococci; the staphilococci (Staphylococcus xylosus and Staphylococcus camosus}; the micrococci (Micrococcus varians}; yeast of the genus Debaromyces, Candida, Pichia, Torulopsis and Saccharomyces; and mold of the genus Aspergillus, Rhizopus, Mucor and Penicillium. 30

With respect to the first object of the present invention, the use of 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, for the preparation of a composition intended for the prophylaxis or the treatment of 35 dental caries, dental plaque and periodontal infection, is concerned.

The lactic bacteria according to the invention are low acidifying and they are capable of adhering directly to the pellicle of the teeth so that the compositions-prepared with these lactic bacteria are intended for deplacing pathogens of the teeth or preventing their attachment. 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 5 about 5.5-7. They are preferably from dairy origin.

The lactic bacteria according to the invention adhere to the pellicle of the teeth via specific or unspecific interaction and/or adhesion factors. The specific adhesion factors are proteins or polysaccharides. 10

At least one lactic bacteria is 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 CNCM 1-1984, CNCM 1-1985, CNCM 1-1986, CNCM 1-1987 and 15 LMG P-18997.

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. Moreover 20 they are capable of fermenting glucose and sucrose and do not synthesise glucans, which are factors of pathogenicity of the cariogenic strains.

According to another object of the present invention, it is considering modifying genetically lactic bacteria so that it adheres to the pellicle of the teeth 25 via adhesion factors. For lactic bacteria that already adheres to the pellicle of the teeth, this modification intends to make the strains more adherent to the surface of the teeth. In the same way, any non-adherent lactic acid bacteria (not Lactobacilli) can be genetically modified so that it adheres to the pellicle of the teeth. 30 This modification of the lactic bacteria can be achieved by insertion of the genes X17390, X14490 or X53657 (GenBank accession numbers), for example. These gene are responsible in S. mutans for the expression of the Antigen I/II that mediates adhesion to salivary glycoproteins. 35 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 is intended to increase the said effect by decreasing its lactic acid production. ΚΤΠ -----This modification can be "achieved “in many ways and preferably according to one the protocols described in the following documents : Boumerdassi et al., Appl. Environ. Microbiol., til, 2293-2299, 1997; Platteeuw et al., Appl. Environ. Microbiol, til, 3967-3971, 1995; Ito et al., Biosci. Biotechnol. Biochem.,ti£, 1569-1573, 1994.

According to the present invention, at least one lactic bacteria genetically modified or not, is used in an "effective quantity" 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 comprised between 104 to 109 cfu/g.

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

BIOCHEMICAL CHARACTERIZATION OF THE SELECTED STRAINS

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

Acidification curves: the acidification curves were determined at 37°C in the following conditions: - S. sobrinus OMZ 176: FUM sucrose 1% and FUM glucose 1% - S. thermophilus CNCM 1-1985: Belliker sucrose 1% and Belliker glucose 1% Inoculation was always 5%; pH was recorded every 20 min. S. thermophilus CNCM 1-1985, from sucrose fermentation, lowers the pH to 4.5, while S. sobrinus OMZ 176 to 4. 10

Sugar L. lactis CNCMI- 1987 L. lactis CNCM I- 1986 S. th. CNCM I- 1984 S. th. CNCM I- 1985 S. th. LMG P- 18997 Adonitol +++ Aesculin ++ ++++ Amygdalin D-Arabinose ++++ L-Arabinose D-Arabitol L-Arabitol +++ Arbutin +++ +++ Cellobiose +++ ++++ Dulcitol Erythritol D-Fructose D-Fucose L-Fucose + ++++ Galactose ++ ++++ β-Gentiobiose Gluconate 2-keto-Gluconate 5-keto-Gluconate +++ GlcNAc + ++++ D-Glucose Glycerol Glycogen Inositol Inulin + ++++ + ++ ++ Lactose + ++++ +++ ++++ ++++ D-Lyxose Maltose ++ Mannitol +++ ++ D-Mannose Melezitose Melibiose + ++++ a-Methyl-D-glucoside a-Methyl-D-mannoside Μ/Ί 11 D-Raffmose Rhamnose Ribose Salicin Sorbitol L-Sorbose Starch Sucrose +++ ++ +++ +++ ++++ +++ D-Tagatose Trehalose ++ D-Turanose ++ Xylitol +++ D-Xylose L-Xylose β-methil-xyloside +, ++, +++, ++++ show if the fermentation beguns after 3, 6, 24 or 48 hours. Table 1. Sugar fermentation of L. lactis CNCM I- 1987, L. lactis CNCM1-1986, S. thermophilus CNCM 1-1984, S. thermophilus CNCM 1-1985 and ,S. thermophilus LMG P-18997. 5

The second main object of the present invention concerns a composition for the health of the mouth comprising 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. 10

These compositions are in particular intended for the prophylaxis or the treatment of dental caries, dental plaque and periodontal infection.

The said lactic bacteria strain is selected from the group consisting of 15 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 1-1985, LMG P-18997, CNCM 1-1986 and CNCM 1-1987. 20 In such compositions for the health of the mouth, lactic bacteria strains may be genetically modified as described above.

X TO 12

The said 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.

In these compositions 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 not micellar casein (which can be obtained as described in the patents EP 0 604 802 and EP 0 748 591) or renneted milk may also be added. The caseino-glycomacopeptides are preferably added in a minimum amount of about 0.1%. It has also been shown that the caseino-glycomacropeptides do not prevent the said lactic bacteria from adhering to the teeth pellicle (fig. 2 and 3).

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 0.00001 to 15% of purified bacteriocin, by weight of the composition. The bacteriocin is preferably variacin (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 ΝΤΓ» Aim 2000 <κ<νχ·Λ·:&*:·χ·κ<·:·κ·:·<κ:^ χ·. · , ·.·.·, ·. 13 ;descpamd 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 5 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 10 admixing the ingredients together in the appropriate relative amounts and finally, and if necessary, adjusting the pH to desired value. A method for screening lactic bacteria capable of adhering to tooth may comprise the steps of: 15 (1) preparing monoclonal antibodies recognising 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. 20

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

The present invention is not to be limited in scope by the specific 25 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 and accompanying figures. 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 30 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). These examples are preceded by a brief 35 description of the plasmids, strains and the various media used, as well as the method for producing a monoclonal antibody. -:08-2000 14

The strains S. thermophilus SI 18 (NCC 1529), S123 (NCC 1561), L. lactis subsp. lactis 29 (NCC 2211), L. lactis subsp. lactis biovar dioacetylactis 69 (NCC 2225) were deposited under the Budapest Treaty, at the Collection Nationale de Culture de Microorganismes (CNCM 1-1984, CNCM 1-1985, 5 CNCM 1-1986 and CNCM 1-1987 respectively), 25 rue du docteur Roux, 75724 Paris, France, on March 3rd, 1998. The strain S. thermophilus BF11116 (CNBL1177) 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 July 5th , 1999. All restrictions as 10 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.

Figures 15 - Figures la, lb and lc represent respectively the adhesion saturation curves for S. sobrinus OMZ 176, L. lactis NCC2211 and S. thermophilus NCC 1561. - Figure 2 represents the curves obtained for the three strains by plotting the number of bound cells versus increasing amounts of CGMP. 20 - Figure 3 represents the curves obtained for the three strains by plotting the number of bound cells versus increasing amounts of As-CGMP.

Example 1: STRAINS AND CULTURE CONDITIONS 25 More than 100 strains (belonging to the Nestle culture collection) were screened for their ability to attach to saliva-coated hydroxyapatite beads, and in particular the following 23 strains: 5. thermophilus YS4 (NCC 2284), S. thermophilus Sfi6 (NCC 1971), S. thermophilus Sfil3 (NCC 2008), S. thermophilus Sfi21 (NCC 2038), S. thermophilus Sfi39 (NCC 2130), S. 30 thermophilus Sfi42 (NCC 2145), 5. thermophilus Sfi47 (NCC 2172), S. thermophilus SI 18 (NCC 1529), S. thermophilus SI 19 (NCC 1536), S. thermophilus S122 (NCC 1554), £ thermophilus S123 (NCC 1561), S. thermophilus S126 (NCC 1587), L. lactis subsp. cremoris 15 (NCC 92), L. lactis subsp. cremoris 25 (NCC 1932), L. lactis subsp. cremoris 136 (NCC 2419), L. 35 lactis subsp. diacetylactis 8 (NCC 1970), L. lactis subsp. diacetylactis 28 (NCC 2057), L. lactis subsp. diacetylactis 69 (NCC 2225), L. lactis subsp. diacetylactis 80 (NCC 2272), L. lactis subsp. lactis 29 (NCC 2211), L. lactis subsp. lactis 50 (NCC 2224), L. lactis subsp. lactis 54 (NCC 2228), S. macedonicus 216 (NCC 2484). no 6?n? 15

The 5 oral strains, S. sobrinus OMZ 176, S. oralis OMZ 607, A. naeslundii OMZ 745, V. dispar OMZ 493 and F. nucleatum OMZ 596 were obtained from the Institute fur Orale Mikrobiologie und Allgemeine Immunologie, University 5 of Zurich 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; 5. sobrinus OMZ 176 grew in 10 FUM medium (Gmiir et al.) lactococci and streptococci in Ml7 (Difco) except S. thermophilus NCC1529, SI 19, S122, NCC1561 and S126 that grew in Belliker (prepared by dissolution in 1 1 water 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, 10 g beef extract). 15

For the plate counting: S. sobrinus OMZ 176 was cultured in Mitis-Salivarius agar (Difco), S. thermophilus NCC1529, SI 19, S122, NCC1561, BF11116 and S126 in Belliker agar (prepared by adding to the liquid Belliker 15 g of Bacto agar, Difco), and the remaining lactic bacteria strains in Ml7 agar 20 (Oxoid).

Example 2: PRODUCTION OF A MONOCLONAL ANTIBODY A monoclonal antibody would be used as a tool to detect L. lactis subsp. 25 lactis NCC2211 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 30 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.

Example 3: SELECTION OF ADHERENT LACTIC BACTERIA 35

Attachment to saliva-coated hydroxyapatite beads (S-HA)

To select among the lactic bacteria dairy strains those able to attach to saliva-coated hydroxyapatite beads (S-HA), the procedure previously described by Neeser et al. (1994) was used with slight modifications: beads washings were wo done with 150 μΐ volumes and Hyamine hydroxide was substituted with

Benzethonium hydroxide (Sigma). 16

Briefly, all the strains were grown to the end of the log phase in FUM 5 except S. thermophilus NCC1529, SI 19, S122, NCC1561 and S126 that were cultured in Belliker. S. sobrinus OMZ 176, L. lactis subsp. lactis NCC2211, 50 and 54, S. thermophilus NCC1529, SI 19, SI22, NCC1561 and SI26 grew at 37°C, the remaining lactococci at 30°C and the remaining streptococci at 42°C. io 5 mg of hydroxyapatite beads (BDH Chemicals Ltd, Poole, England) were covered with 70 μΐ clarified saliva obtained from volunteers in the lab and prepared as previously explained (Neeser et al., 1994). 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 μΐ of the metabolically labelled 15 bacterial suspension (bacteria had been grown in their medium, supplemented with 10 pCi/ml 14C 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). 20 The adhesion percentages are expressed as radioactivity bound to the beads on 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 for 0*. sobrinus OMZ 176 (the reference strain). 25 STRAIN " % ADH)ESION(+/-SD) S. sobrinus OMZ 176 2.23 +/-0.49 5. thermophilus Sfi42 (NCC 2145) 0.08 +/-0.02 S. thermophilus Sfi47 (NCC 2172) 0.14+/-0.04 5. thermophilus NCC1529 2.89 +/-0.60 S. thermophilus SI 19 (NCC 1536) 0.15 +/-0.04 S. thermophilus SI22 (NCC 1554) 0.93 +/-0.17 S. thermophilus NCC1561 2.19 +/-0.50 S. thermophilus S126 (NCC 1587) 1.19+/-0.56 L. lactis subsp. diacetylactis 28 (NCC 2057) 1.59+/-0.17 L. lactis subsp. diacetylactis NCC2225 1.96 +/-0.40 L. lactis subsp. diacetylactis 80 (NCC 2272) 1.20+/-0.35 L. lactis subsp. lactis NCC2211 2.85 +/-0.85 17

Table 2: percentages of adhesion to saliva-coated hydroxyapatite beads obtained for several screened strains

Four strains, S. thermophilus NCC1529 (CNCM 1-1984), S. thermophilus 5 NCC1561 (CNCM 1-1985), L. lactis subsp. lactis NCC2211 (CNCM 1-1986) (hereinafter L. lactis NCC2211) and L. lactis subsp.diacetylactis NCC2225 (CNCM 1-1987) showed values close to S. sobrinus OMZ 176. L. lactis NCC2211 and S. thermophilus NCC1561 were chosen as the io more promising candidates since they grow very well at 37°C, which is the temperature in the mouth, while L. diacetylactis NCC2225 has an optimal growth temperature of 30°C. In particular, L. lactis NCC2211 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 beads saturation could be obtained. The 50% saturation was directly 20 drawn from the bending point of the curves obtained. The adhesion saturation curves for S. sobrinus OMZ 176, L. lactis NCC2211 and 5. thermophilus NCC1561 were determined. They are shown in the Figure 1.

For the three strains the CFU number to be inoculated in the well to get the 25 50% beads 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. cfu/well Bound cfu % adhesion 5. sobrinus OMZ 176 4.00E+07 4.00E+06 10% L. lactis NCC2211 1.00E+07 9.00E+05 9% 5. thermophilus NCC1561 3.00E+07 2.00E+06 7%

Table 3: Number of CFU to be inoculated per well to get the 50% beads saturation. 30

Example 4: EFFECT OF CASEINOGLYCOMACROPEPTIDES

I

The influence of CGMP on the adhesion of L. lactis NCC2211 and 5. thermophilus NCC1561 was studied to verify the possibility of using it to foster 35 the predominance of one of this two strains on the pathogenic ones (namely £. Sobrinus OMZ 176. Caseino-glycopeptide (CGMP) and its desialylated MO ΑΟΠΟ 18 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 μΐ of bacterial suspension (CFU/ml corresponding to the 50% beads saturation previously calculated) which contained CGMP or As-CGMP in different concentrations and then performing the adhesion assay as usual. Concentrations in the range 0.05-3 mg/ml were tested. No previous incubation of the bacteria in presence of CGMP or As-CGMP was done.

In the figure 2, the curves obtained for the three strains by plotting the number of bound cells versus increasing amounts of CGMP are presented, the number of inoculated cells being the one corresponding to the 50% beads saturation formerly calculated for each strain. The strong inhibition observed in the case of S. sobrinus OMZ 176 confirms the previous results obtained by Neeser et al. (1994) and Schiipbach et al. (J. Dent. Res., 25, 1779-1788, 1996).

As evidenced in the figure 2, 0.25 mg/ml produced 50% inhibition of the adhesion of S. sobrinus OMZ 176, while more than 2 mg/ml were necessary to have the same effect with S. thermophilus NCC1561. CGMP slightly enhances the adhesion of L. lactis NCC2211.

As in the case of CGMP, the desyalilated derivative inhibits the adhesion of S. sobrinus OMZ 176; only 0.05 mg/ml are needed to produce 50% decrease in the adhesion percentage. As-CGMP does not influence L. lactis NCC2211 adhesion, while it slightly fosters the one of S. thermophilus NCC1561 (Fig. 3).

Example 5: TOOTHPASTE

Toothpaste is prepared by adding 105 cfu/ml of at least one of the lactic bacteria strain CNCM 1-1984, CNCM 1-1985, CNCM 1-1986, CNCM 1-1987 or LMG P-18997 in a lyophilised form, to the following mixture containing:

Cetyl pyridinium chloride Sorbitol (70% soln) Glycerin 1.65% 33.0% 25.0%

Sodium carboxymethyl cellulose 2.0% no

Sodium fluoride 0.25%

Silica (RP 93) 26.3%

Thickening Silica (Sident 22) 8.1% 5 Sodium saccharine 0.5%

Poloxamer (Pluronic F108) 3.2%, 19

This toothpaste is intended for the prophylaxis or the treatment of dental caries, dental plaque and periodontal infection. 10

Example 6: ICE CREAM A cream comprising 10.8% lactic fats, 13.5% milk solids (non fat), 0.3% Emulstab® SE30 et 0.3% Emulstab® foam (Grindsted, DK) is prepared, it is is then pasteurised at 105°C for 20s, homogenised at 75°C and 300 bar, cooled to 38°C and inoculated with precultures in MRS medium, taken in exponential growth phase, at a rate of 107-108 cfu/ml of at least one of the lactic bacteria strain 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. 20 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, chilled to a degree of expansion of 95°C by volume.

Ingredients Composi tion (kg) Fats . (%) Non-fat solids (%) Sucrose (%) Solids content (%) Cream (35%) 30,83 10,79 1,54 12,33 Powdered skimmed milk 12,45 11,95 11,95 Emulstab® SE30 0,41 0,37 Emulstab® foam 0,41 0,36 Water 55,91 Total: cream base 100,00 10,79 13,49 - 25,01 Cream base 74,14 8,00 10,00 - 18,54 Sucrose 22,06 15,00 15,00 Glucose syrup 3,80 3,00 Fermented Ice cream 100,00 8,00 10,00 15,00 36,54 25 Table 4 20

Example 7: YOGHURT 5 1 MRS culture medium are sterilised for 15 min at 121 °C and then 5 inoculated with 5% by volume of an active culture of at least one of the S.Thermophilus strain CNCM 1-1984, CNCM 1-1985 or LMG P-18997 containing approximately 109 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 10 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 109 cells/ml. After incubation for 4 h at 41°C, a starter containing 4.5.108 cells/ml is obtained.

One batch of whole milk containing 3.7% fats strengthened with 2.5% 15 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 strain CNCM 1-1984, CNCM 1-1985 or LMG P-18997 and 3% by volume of the starter of thickening Streptococcus thermophilus. The inoculated milk is stirred, poured into pots and incubated for 4 h at 41 °C. 20

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

Example 8: CHEWING GUM 25 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 the S.Thermophilus strain CNCM 1-1984, CNCM 1-1985 or LMG P-18997 so that it contains approximately 104 to 109 cfu/g, to the following typical 30 ingredients:

Xylitol 67.5 % Gum base 20% Calcium carbonate 5% Glycerin 3% Pluronic FI27 2% Cellulose gum 1% Balast compounds 0.5% Flavor 1 % \rn Aim 21

Example 9: PET FOOD COMPOSITION A pet food for mouth health is obtained by preparing a feed mixture made

5 up of com, com 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 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 10 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 strain CNCM 1-1984, CNCM 1-1985 or LMG P-18997 but one coating mixture uses hydrogenated soy fat as a 15 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 104 to 109 cfu/g of said strains. ‘ , crrixan rwzn arn pnow pnszn irn nr “|»ob ,Ρ’ηη ηχΰ- πώοβπβ mzrna π^πιώώ np’ioz .zruwan ms rnp’nn pmi1? oxnm □ιηπη Pi? _____88°4003 01 J' ·aoi 0851 :H 40200 .(moiB nzrnn) cras&'an nwa