Incorporation of exogenous lactic bacteria into the oral microflora

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DESCRIPTION

Incorporation of exogenous lactic acid bacteria into the oral microflora

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

Background of the present invention

The mouth (oral cavity) contains a resident microflora and a non-resident microflora. The first includes microorganisms capable of establishing themselves more or less permanently on oral surfaces. These bacteria are mainly located on the tongue, on the oral mucosa and on the teeth, while the gums, lips, cheeks, palate and floor of the mouth only support a very scarce microflora.

The natural microflora residing on the tongue and oral mucosa include microorganisms of the genera Streptococcus, Veillonella, Bacteroides, and Haemophilus. Streptococci, lactobacilli, and Actynomyces predominate on teeth, but many gram-positive and gram-negative cocci and rods can also be found.

For example, Frandsen et al. Showed that S. sanguis predominates on the buccal mucosa, although its main habitat is the tooth surface, that S. gordonii grows in mature supragingival plaque, that S. oralis and S. mitis grow in the initial dental plaque (Oral Microbiol. Immunol., 6, 129-133, 1991). Strains belonging to the group of mutants (S. cricetus, S. downei, S. ferus, S. macacae, S. mutans, S. rattus, S. sobrinus) are found in the teeth. The strains of the S. milleri predominates in dental abscesses (S. anginosus, S. constellatus, S. intermedius; Bentley et al., Int. J. System. Bacter. 1991, 41, 487-494; Wood et al., The Genera of Lactic Acid Bacteria [ Genera of lactic acid bacteria], Blackie Academic and Professional, Chapman & Hall, WH eds., 1995).

Many of these microorganisms are safe commensals, but they have also been identified as etiological agents of many diseases (Hill, MJ and Marsh, PD eds. Human Microbial Ecology, 1990, CRC Press, Boca Raton Florida, USA)

Dental plaque is a film that forms on the tooth surface and consists 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 Film (EAP), which is approximately 1.3 pm thick and cannot be removed by normal tooth brushing. The deposit of bacteria on the teeth occurs immediately after the formation of the PAE and is evident after 8-12 hours in the form of a multilayer structure. The first layer consists of bacteria (first colonizers) that adhere to teeth mainly by specific recognition of the adhesin receptor; it forms a substrate for the second colonizers that adhere to each other by analogous specific fixation or by simple juxtaposition. Plaque cohesion is ensured by three mechanisms: the presence of a salivary film on the outer layer of bacteria, the coaggregation between the different bacterial species and the glucans synthesized by the bacteria, and the fact that the bacteria are trapped in the plate matrix (Skopek et al. Oral Microbiol. Immunol., 9, 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).

Organic acids produced by oral bacteria during the fermentation process directly cause tooth decay. These acids attack the hard tissue of the teeth with the consequent release of ions such as calcium, phosphate, carbonate, magnesium, fluoride, sodium. When the pH of the oral cavity reaches neutrality again, the saliva becomes saturated with calcium and its detachment from the tooth is prevented.

Among all the food residues found in the mouth, carbohydrates show the greatest caries-promoting effect, as they are directly exposed to oral bacterial fermentation.

Potentially all sugar-fermenting microorganisms are cariogenic, but the main etiological agents of crown and root caries are mutant streptococci, because they are strong acid producers; lactobacilli, which are highly acidophilic, may also be involved. In humans the

S. mutans and S. sobrinus are the most cariogenic strains and live on teeth, without colonizing the entire dentition. A decrease in their number from the molars to the anterior teeth was demonstrated (Lindquist et al., Dent. Res., 69, 1160-1166, 1990). Furthermore, in the human proximal plate, S. mutans and S. sobrinus preferentially colonize the site most prone to caries, apical to the contact area (Ahmady et al., Caries Res., 27, 135139, 1993). A greater predominance of S. sobrinus was also found in the molar regions, compared to S. mutans (Lindquist et al., Caries Res., 25, 146-152, 1991).

S. mutans and S. sobrinus have been shown to adhere to the tooth film primarily by the specific adhesin-receptor pathway. Gibbons et al. Demonstrated that S. mutans carries an adhesin that binds to salivary components of the film, while S. sobrinus appears to possess an adhesin that binds to the glucan of the film (Infect. Immun., 52, 555- 561, 1986).

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The transient microflora includes exogenous bacteria that may occasionally be found in the mouth, but do not become permanently established (even with repeated oral administrations of these bacteria). All food bacteria, and in particular lactic acid bacteria, can be part of this transient microflora. These exogenous lactic acid bacteria have never been proven capable of adhering directly to dental film. However, the repeated administration of exogenous lactic acid bacteria leads to the colonization of all oral surfaces, such as the tongue, oral mucosa, gums, lips, cheeks, palate, floor and teeth. This colonization may be due to adhesions by specific bacteria attachment to the resident microflora (coaggregation phenomenon) or by entrapment in the matrix of the polysaccharides produced by the resident bacteria or by adhesion to salivary proteins (specifically glycoproteins).

Lactobacillus casei rhamnosus GG (ATCC53103) has been reported to colonize the mouth, most likely on the buccal mucosal epithelium. This strain also adheres to the epithelium of the intestinal tract (US 5032399, Gorbach et al; Micr. Ecol. In Health and Dis., 7, 295-298, 1994). In contrast, L. rhamnosus does not adhere to the teeth.

Japanese Patent No. 4021633 (Cyconmedix KK) also revealed colonization of the mouth by acidophilic lactobacilli, most likely on the epithelium of the buccal mucosa. Many acidophilic lactobacilli are also known to adhere to the epithelium of the intestinal tract (EP 577904; EP 199535; Perdigon et al., Medicine, 46.751754, 1986; Perdigon et al., Immunology, 63, 17-23, 1988).

Exogenous bacteria can also produce factors that inhibit the growth of the microflora resident in the mouth. For example, patent EP 759469 (Société des Produits Nestle) describes the use of a bacteriocin produced by Micrococcus varians to inhibit the development of the oral pathogens S. sobrinus, S. sanguis, S. mutans and A. viscosus.

Some strategies have been used to minimize the development of the resident microflora in the mouth, specifically the administration of commensal bacteria from 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 acid bacteria such as L. casei, L. fermentum, L. acidophilus, L. crispatus. L. gasserri, L. salivarius, L bulgaricus and S. salivarius (Tanzer et al., Infec. and Immunity, 48, 44-50, 1985; WO 92/14475; EP 0524732).

The application of bacteriocins is also one of the research strategies proposed to reduce dental caries. These molecules have aroused interest as presumed anticaries agents and as important modulating factors in the oral cavity. The anticaries potential of the applications of some bacteriocins comes from their energetic and broad antibacterial action against mutant streptococci and bacteria associated with dental plaque and from their natural existence in bacteria considered humanly safe (US 5368845 by Colgate, and WO 94/12150 by Smithkline Beecham).

The application of dairy products is also interesting for oral health. Indeed, patent US 5427769 (Nestec SA) describes an alternative consisting of preventing caries by contacting the teeth with an edible composition containing micellar casein in sufficient quantity to inhibit colonization by Streptococcus sobrinus. Patent EP 748591 (Société des Produits Nestlé SA) also indicates the use of fluorinated micellar casein or its micellar subunits to treat dental caries or plaque. US patent 4992420 (Nestec

SA) describes the treatment of the oral cavity with milk-derived kappa casein-glycomacropeptide to eradicate plaque and caries.

Patent EP 0 699 689 refers to soluble branched polysaccharides, formed by a main chain with repeating side chains of lactose units. It also reveals the branched polysaccharide-producing microorganism, which is a Lactobacillus helveticus strain, CNCM I-1449. The polypeptide and the microorganism can be used respectively in food or cosmetic compositions, eg. in a cosmetic product designed for oral hygiene, such as toothpastes or gels, mouthwashes, gums and / or chewing tablets.

It has never been proven that lactic acid bacteria foreign to the resident microflora of the mouth were actually capable of adhering directly to the dental film. Thus, by colonizing the tooth surface, these lactic acid bacteria would exert an inhibitory action against the growth of the resident microflora, including oral pathogens.

Summary of the present invention

The present invention aims to facilitate the use of lactic bacteria foreign to the resident microflora of the mouth, which are low acidifying and capable of adhering directly to the dental film, to prepare a composition intended for the prophylaxis or treatment of dental caries , dental plaque and periodontal infection.

According to the present invention, at least one lactic bacteria is chosen from the group consisting of CNCM 1-1984, CNCM 1-1985, CNCM 1-1986, CNCM I-1987 and LMG P-18997.

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According to one embodiment of the present invention, the composition is designed to displace pathogens from the teeth or to prevent their adherence.

Lactic acid bacteria can be less acidifying than pathogenic strains, thus helping to establish a pH of approximately 5.5-7 in the oral cavity.

Lactic acid bacteria can be of dairy origin.

In another embodiment, at least one of the lactic acid bacteria is chosen from the group consisting of Streptococcus thermophilus, Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. lactis biovar diacetylactis.

According to another embodiment, the lactic acid bacteria adhere to the dental film by means of adhesion factors.

In a preferred embodiment, the lactic acid bacteria have been genetically modified to increase its adhesion to the dental film and / or to be less acidifying; especially so that it adheres to the dental film through adhesion factors and helps to establish a pH of approximately 5.5-7 in the oral cavity.

In yet another embodiment of the present invention the composition is edible and comprises an effective amount of lactic acid bacteria for the prophylaxis or treatment of dental caries, dental plaque and periodontal infection.

The composition can contain at least 10⁴-10⁹ cfu / g of lactic bacteria.

In another embodiment, the lactic acid bacteria are combined with milk, fermented milk, dairy derivatives or bacteriocin; dairy derivatives are preferably chosen from any form of casein-glycomacropeptide, micellar casein, fluorinated micellar casein or curdled milk.

Another preferred embodiment of the present invention provides a composition for oral health comprising lactic bacteria foreign to the resident microflora of the mouth that are low acidifying and capable of adhering directly to the dental film, in which there is at least one strain of lactic acid bacteria chosen from the group formed by the strains CNCM I-1984, CNCM I-1985, CNCM I-1986, CNCM I-1987 and LMG P-18997.

Detailed description of the present invention

In the following description the mouth defines the oral cavity of humans or animals such as pets, composed of the oral mucosa (gums, lips, cheeks, palate and floor of the mouth), the tongue and the teeth (including artificial structures).

The resident microflora of the mouth includes all microorganisms that naturally live in the mouth because they are capable of permanently establishing themselves on oral surfaces. The resident microflora of the mouth also includes bacteria that live in the interface between hard and soft dental tissues (at the point of tooth-gum junction), although in a healthy mouth there is no gingival fissure or periodontal pocket. This microflora includes microorganisms of the genera Streptococcus, Staphylococcus, Enterococcus, Micrococcus, Peptostreptococcus, Peptococcus, Lactobacillus, Corynebacterium, Actinomyces, Arachnia, Rothia, Alcaligenes, Eubacterium, Propionilluslacterium, Bacterium / Bacteriaceae, Propionilluslacterium, Bacteriidifidobacteriaceae, Branmetriobacteriaceae, Branmelustriaceae, Branmetriobacterium , Eikenella, Actinobacillus, Capnocytophga, Haemophilus, Simonsiella, Bacteroides, Fusobacterium, Porphyromonas, Prevotella, Leptotrichia, Wolinella, Selenomonas, Mycoplasma, Candida, Spirochaetes, Protozoa.

The transient microflora includes exogenous bacteria that may occasionally be found in the mouth, but do not become permanently established in the mouth. This transient microflora can comprise all food microorganisms, such as bifidobacteria (B. infantis, B. adolescentis, B. breve, and B. longum); lactococci (Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris and Lactococcus lactis subsp. lactic biovar diacetylactis); streptococci (Streptococcus thermophilus, S. lactis, S. lactis cremoris and S. lactis diacetylactis); Lactobacilli (Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus helveticus, Lactobacillus farciminis, Lactobacillus Alimentarius, Lactobacillus casei subsp. casei, Lactobacillus delbruckii subsp. lactis, Lactobacillus acidus Lactobacillus curvillus, Lactobacillus curvillus, Lactobacillus curvillus group Lactobacillus, Lactobacillus curvillus, Lactobacillus acidus lactobacillus group et al., Int. J. Syst. Bact., 42, 487-491, 1992); pediococci (Pediococcus pentosaceus, Pediococcus acidilactici and Pediococcus halophilus); the enterococci; staphylococci (Staphylococcus xylosus and Staphylococcus carnosus); micrococci (Micrococcus varians); yeasts of the Debaromyces, Candida, Pichia, Torulopsis and Saccharomyces genera; and molds of the genera Aspergillus, Rhizopus, Mucory Penicillium.

The first object of the present invention is the use of lactic bacteria foreign to the resident microflora of the mouth, which are low acidifying and capable of adhering directly to the dental film, to prepare a composition for the prophylaxis or treatment of caries dental, dental plaque and periodontal infection.

The lactic acid bacteria according to the present invention are low acidifying and capable of adhering directly to the 4

ES 2 363 201 T3 dental film, so that the compositions prepared with these lactic bacteria are designed to displace pathogens from the teeth or to prevent their adherence. Lactic acid bacteria according to the present invention are "low acidifying", which means that they are less acidifying than pathogenic strains. Therefore they contribute to establish a pH of approximately 5.5-7 in the oral cavity. They are preferably of dairy origin.

The lactic acid bacteria according to the present invention adhere to the dental film through specific or non-specific interactions and / or adhesion factors. Specific adhesion factors are proteins or polysaccharides.

At least one lactic bacteria is chosen from the group consisting of Streptococcus thermophilus, Lactococcus lactis subsp. lactis and lactococcus lactis subsp. lactis biovar diacetylactis, and in particular from the group formed by the strains CNCM 1-1984, CNCM I-1985, CNCM 1-1986, CNCM 1-1987 and LMG P-18997.

These strains have been chosen among lactic bacteria for their ability to adhere to dental film, their optimal growth temperature is about 37 ° C, which is the temperature of the oral cavity. They are also capable of fermenting glucose and sucrose and do not synthesize glucans, which are the pathogenic factors of cariogenic strains.

The genetic modification of lactic acid bacteria is considered so that they adhere to the dental film through adhesion factors. For lactic bacteria that already adhere to the dental film, this modification aims to increase the adhesion of the strains to the teeth. Similarly, any non-adherent lactic acid bacteria (not lactobacilli) can be genetically modified to adhere to dental film.

This modification of lactic acid bacteria can be achieved by inserting the genes X17390, X14490 or X53657 (gene bank accession numbers), for example. In S. mutans these genes are responsible for the expression of antigen I / II that mediates adhesion to salivary glycoproteins.

It is also possible to genetically modify lactic acid bacteria so that they are not very acidifying. For lactic acid bacteria that are already slightly acidifying, this modification aims to increase this effect by reducing their production of lactic acid.

This modification can be achieved in many ways and preferably according to one of the protocols described in the following documents: Boumerdassi et al., Appl. Environ. Microbe., 63, 2293-2299, 1997; Platteeuw et al., Appl. Environ. Microbiol., 61, 3967-3971, 1995; Ito and others, Biosci. Biotechnol. Biochem., 58, 15691573, 1994.

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

It is also possible to use at least one lactic acid bacteria of the above strains in combination with dairy derivatives such as milk or fermented milk or with dairy derivatives chosen from any form of caseinoglycomacropeptide, micellar casein, fluorinated micellar casein, curdled milk or bacteriocin, for example.

BIOCHEMICAL CHARACTERIZATION OF THE SELECTED STRAINS

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

Acidification curves: the acidification curves were determined at 37 ° C under 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%; the inoculation was always at 5%; pH was recorded every 20 min.

S. thermophilus CNCM 1 -1985 from sucrose fermentation lowers the pH to 4.5 and S. sobrinus OMZ 176 to 4.

Table 1: Fermentation of sugars with L. lactis CNCM 1-1987, L. lactis CNCM I-1986,

S. thermophilus CNCM I-1984, S. thermophilus CNCM I-1985 and S. thermophilus LMG P-18997.

<td>Sugar</td><td>L. lactis CNCM 1-1987</td><td>L. lactis CNCM I-1986</td><td>S. th. CNCM I1984</td><td>S. th. CNCM I1985</td><td>S. th. LMG P18997</td>

<td>Adonitol</td><td> +++</td><td></td><td></td><td></td><td></td>

<td>Esculin</td><td> ++</td><td> ++++</td><td></td><td></td><td></td>

<td>Amygdalin D-Arabinose</td><td> +++</td><td></td><td></td><td></td><td></td>

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<td>L-Arabinose D-Arabitol L-Arabitol</td><td> +++</td><td></td><td></td><td></td><td></td>

(continuation)

<td>Sugar Arbutin Cellobiose Dulcitol Erythritol D-Fructose D-Fucose L-Fucose Galactose β-Genthiobiose Gluconate 2-keto-Gluconate 5-keto-Gluconate GlcNAc D-Glucose Glycerin Glycogen Inositol Inulin Lactose D-Lixose Maltose Mannitol D-Mannose Melezitosa Melibiosa α-Methyl-D-glucoside α-Methyl-D-mannoside D-Rafinose Ramnose Ribose Salicin Sorbitol L-Sorbose Starch Sucrose D-Tagatose Trehalose D-Turanose Xylitol D-Xylosa L-Xylosa β-methyl-xyloside</td><td>L. lactis CNCM 1-1987 +++ +++ + ++ + + + ++ +++ + ++ +++ ++ ++ +++</td><td>L. lactis CNCM I-1986 +++ ++++ ++++ ++++ +++ ++++ ++++ ++++ ++ ++++ ++ +++</td><td>S. th. CNCM I1984 + +++ +++</td><td>S. th. CNCM I1985 ++ ++++ ++++</td><td>S. th. LMG P18997 ++ ++++ +++</td>

<td colspan="6">+, ++, +++, ++++ indicate if fermentation starts after 3, 6, 24 or 48 hours.</td>

The second object of the present invention refers to a composition for oral health that comprises a lactic bacteria alien to the resident microflora of the mouth that is low acidifying and capable of adhering directly to the dental film.

These compositions are specifically designed for the prophylaxis or treatment of dental caries, dental plaque and periodontal infection.

Said strain of lactic bacteria is chosen from the group formed by Streptococcus thermophilus, Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. lactis biovar diacetylactis and preferably from the group consisting of the strains CNCM 1-1984, CNCM I-1985, LMG P-18997, CNCM 1-1986 and CNCM I-1987.

Said strains of lactic acid bacteria can be included in a meal, in a feed, in a cosmetic or pharmaceutical composition, for example.

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Therefore these compositions are preferably toothpastes, mouthwashes, chewing gums, sprays, beverages, sweets, infant formulas, ice creams, frozen desserts, sweet salad dressings, dairy preparations, cheeses, cottage cheese, yogurt, curd, coffee cream or whipped cream, for instance.

In these compositions the lactic acid bacteria strains can be included alone or in combination with dairy derivatives, for example, in order to obtain synergistic preparations. Thus, these oral health compositions comprise:

- a lactic bacteria alien to the resident flora of the mouth that is capable of adhering directly to the dental film;

- any form of lactic glycopeptides, curdled milk or bacteriocin.

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

Synergistic compositions can also be prepared by adding at least one bacteriocin that is active against gram-positive oral bacteria. In this case, the oral hygiene compositions can contain 0.00001 to 50% and preferably 0.00001 to 15% of bacteriocin relative to the weight of the composition. Bacteriocin is preferably variacin (EP 0 759 469).

To protect the composition against degradation, a fat-soluble antioxidant can also be included. Suitable antioxidants include "tocopherols", butyl hydroxyanisole (BHA), butyl hydroxytoluene (BHT) and ascorbyl palmitate. The amount of the fat-soluble antioxidant is 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, alumina hydrates, zinc orthophosphate, plastic particles and silica, among which silica is the preferred abrasive.

The compositions can have an orally acceptable pH that does not affect the activity of said lactic acid bacteria. The pH can be in the range 3.0-9.5, preferably between 3.5 and 6.5.

The compositions can be prepared by conventional processes that comprise mixing the ingredients together in the appropriate proportions and finally adjusting the pH to the desired value.

A method of selecting lactic acid bacteria capable of adhering to teeth may comprise the steps of:

(1) prepare monoclonal antibodies that recognize specific surface proteins of a strain of lactic acid bacteria capable of adhering to teeth, and (2) detect any strain of lactic acid bacteria by using the monoclonal antibody of the strain capable of adhering to teeth.

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

The scope of the present invention should not be limited to the specific embodiments described herein. In fact there are several modifications of the present invention, apart from those described herein, which will be apparent to those skilled in the art, from the foregoing description and the 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 entirety, to the extent necessary to understand the present invention. DNA manipulation, cloning and transformation of bacterial cells are carried out, if not indicated otherwise, in accordance with the manual by Sambrook et al. (Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, USA , 1989). These examples are preceded by a brief description of the plasmids, strains, and various media used, as well as the method for producing a monoclonal antibody.

The strains S. thermophilus S118 (NCC 1529), S123 (NCC 1561), L. lactis subsp. lactis 29 (NCC 2211), L. lactis subsp. lactis biovar dioacetylactis 69 (NCC 2225) were deposited according to the Budapest treaty in the Collection Nationale de Culture de Microorganismes (CNCM 1-1984, CNCM 1-1985, CNCM I-1986 and CNCM I-1987 respectively), 25 rue du docteur Roux, 75724 Paris, France, March 3, 1998. The S. thermophilus BF11116 (CNBL1177) was deposited according to the Budapest treaty in the Belgian Coordinated Collections of Microorganisms LMG P-18997, KL Ledeganckstraat 35, B-9000 Ghent, Belgium, on July 5, 1999. All restrictions on the availability of these Deposits will be raised when this patent application or another that claims the benefit of the priority of this application is published for the first time.

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Figures (edit)

- Figures 1a, 1b and 1c respectively represent the adherence saturation curves for S. sobrinus OMZ 176, L. lactis (NCC2211 and S. thermophilus NCC1561.

- Figure 2 shows the curves obtained for the three strains when representing the number of fixed cells against increasing amounts of CGMP.

- Figure 3 shows the curves obtained for the three strains when representing the number of fixed cells against increasing amounts of As-CGMP.

Example 1: STRAINS AND GROWING CONDITIONS

More than 100 strains (belonging to the Nestle culture collection) were tested for their ability to bind to saliva-coated hydroxyapatite beads, and in particular the following 23 strains: S. thermophilus YS4 (NCC 2284), S. thermophilus Sfi6 (NCC 1971), S. thermophilus Sfi13 (NCC 2008), S. thermophilus Sfi21 (NCC 2038), S. thermophilus Sfi39 (NCC 2130), S. thermophilus Sfi42 (NCC 2145), S. thermophilus Sfi47 (NCC 2172), S. thermophilus S118 (NCC 1529), S. thermophilus S119 (NCC 1536), S. thermophilus S122 (NCC 1554), S. 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. 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).

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 für Orale Mikrobiologie und Allgemeine Immunologie [Institutes of Oral Microbiology and general immunology] of the University of Zürich and anaerobically cultured in FUM medium (GasPackSystem, BBL) at 37 ° C.

All strains were stored in glycerin at -20 ° C and precultured for 14 hours before being used at their specific optimum temperature; S. sobrinus OMZ 176 grew in FUM medium (Gmür et al.), lactococci and streptococci in M17 (Difco), except S. thermophilus NCC 1529, S119, S122, NCC 1561 and S126 that grew in Belliker medium (prepared by dissolving in 1 l of water 20 g of tryptone, 5 g of yeast extract, 2.5 g of gelatin, 5 g of dextrose , 5 g of sucrose, 5 g of lactose, 4 g of NaCl, 0.5 g of ascorbic acid, 10 g of beef extract).

For the plate count: S. sobrinus OMZ 176 was grown on Mitis-Salivarius agar (Difco), S. thermophilus NCC1529, S119, S122, NCC1561, BF11116 and S126 on Belliker agar (prepared by adding 15 g of agar Bacto, Difco, to Belliker liquid) and the other strains of lactic acid bacteria on M17 agar (Oxoid).

Example 2: PRODUCTION OF A MONOCLONAL ANTIBODY

A monoclonal antibody would be used as a tool to detect L. lactis subsp. lactis NCC2211 among 5 oral strains growing together on S-HA discs and forming a dental plaque simulating biofilm. Therefore the monoclonal antibody was tested against these strains, to check that there was no cross reaction.

For this, the monoclonal antibody is produced in the manner described by Granato et al. "A monoclonal mouse IgE anti-bovine milk lactoglobulin antibody allows the study of allergy in the gastrointestinal tract, Clin. Exp. Immunol., 63, 703-710, 1986.

Example 3: SELECTION OF ADHERENT LACTIC BACTERIA

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

The procedure previously described by Neeser et al. (1994) was used with slight modifications to select among the lactic bacteria strains those that were capable of binding to saliva-coated hydroxyapatite beads (S-HA), with slight modifications: the beads were washed with volumes of 150 µl and the hyamine hydroxide was replaced by benzethonium hydroxide (Sigma).

In summary, all strains were grown to the end of the log phase in FUM, except S. thermophilus NCC 1529, S119, S122, NCC1561 and S126 which were grown in Belliker. S. sobrinus OMZ 176, L. lactis subsp. lactis NCC2211, 50 and 54, and S. thermophilus NCC1529, S119, S122, NCC1561 and S126 grew at 37 ° C, and the remaining streptococci at 42 ° C.

5 mg of hydroxyapatite beads (BDH Chemicals Ltd, Poole, England) were coated with 70 µl of clarified saliva obtained from volunteers in the laboratory and prepared as explained above (Neeser et al., 1994).

The saliva-coated beads were stored overnight at 4 ° C, then washed (first with distilled water and then with HEPES buffer) and finally inoculated with 100 µl of the labeled bacterial suspension.

ES 2 363 201 T3 metabolically (the bacteria had been cultured in their medium, supplemented with 10 pCi / ml of acetic acid C¹⁴). Adhesion took place for 45 minutes at 37 ° C, then the unfixed bacteria were washed away and the fixed cells were counted directly in an LKB scintillation counter (1219 Rackbeta type).

The adhesion percentages are expressed as the radioactivity bound to the beads, based on the total radioactivity added to each well. All measurements were made in triplicate. Table 2 shows the percentages of adhesion to the saliva-coated hydroxyapatite beads obtained for various strains tested and for S. sobrinus OMZ 176 (as reference strain).

Table 2: percentages of adhesion to saliva-coated hydroxyapatite beads, obtained for various strains analyzed.

<td>CEPA</td><td>% ADHESION (± SD)</td>

<td>S. sobrinus OMZ 176</td><td> 2,23 ± 0,49</td>

<td>S. thermophilus Sfi42 (NCC 2145)</td><td> 0,08 ± 0,02</td>

<td>S. thermophilus Sfi47 (NCC 2172)</td><td> 0,14 ± 0,04</td>

<td>S. thermophilus NCC1529</td><td> 2,89 ± 0,60</td>

<td>S. thermophilus S119 (NCC 1536)</td><td> 0,15 ± 0,04</td>

<td>S. thermophilus S122 (NCC 1554)</td><td> 0,93 ± 0,17</td>

<td>S. thermophilus NCC1561</td><td> 2,19 ± 0,50</td>

<td>S. thermophilus S126 (NCC 1587)</td><td> 1,19 ± 0,56</td>

<td>L. lactis subsp. diacetylactis 28 (NCC 2057)</td><td> 1,59 ± 0,17</td>

<td>L. lactis subsp. diacetylactis NCC2225</td><td> 1,96 ± 0,40</td>

<td>L. lactis subsp. diacetylactis 80 (NCC 2272)</td><td> 1,20 ± 0,35</td>

<td>L. lactis subsp. lactis NCC2211</td><td> 2,85 ± 0,85</td>

The four strains S. thermophilus NCC1529 (CNCM I-1984), S. thermophilus NCC1561 (CNCM I-1985), L. lactis subsp. lactis NCC2211 (CnCm I-1986) (hereinafter L. lactis NCC2211) and L. lactis subsp.diacetylactis NCC2225 (CNCM I-1987) showed values ​​close to S. sobrinus OMZ 176.

The strains L. lactis NCC2211 and S. thermophilus NCC1561 were chosen as the most promising because they grow well at 37 ° C, which is the temperature in the mouth, while L. diacetylactis NCC2225 has optimal growth at 30 ° C. L. lactis NCC2211, in particular, cannot grow on sucrose, but is capable of fermenting a wide variety of sugars. In addition, other oral strains can provide glucose through their invertase.

Adhesion saturation curves

Curves of fixed CFU versus CFU inoculated in the well were determined to check if the beads could be saturated. The 50% saturation was taken directly from the bending point of the curves obtained. Adhesion saturation curves were determined for S. sobrinus OMZ 176, L. lactis NCC2211 and S. thermophilus NCC 1561. They are shown in Figure 1.

The number of CFUs of the three strains that must be inoculated into the well to obtain 50% saturation of the beads and the corresponding number of fixed CFUs was deduced directly from the bending point of the curves and is indicated in Table 3.

Table 3: number of CFUs to be inoculated in the well to obtain 50% saturation of the beads.

<td></td><td>cfu / well</td><td>fixed cfu</td><td>% adhesion</td>

<td>S. sobrinus OMZ 176</td><td>4.00E + 07</td><td>4.00E + 06</td><td> 10%</td>

<td>L. lactis NCC2211</td><td>1.00E + 07</td><td>9.00E + 05</td><td> 9%</td>

<td>S. thermophilus NCC1561</td><td>3.00E + 07</td><td>2.00E + 06</td><td> 7%</td>

Example 4: EFFECT OF CASEINOGLYCOMACROPEPTIDES

The influence of CGMPs on the adhesion of L. lactis NCC2211 and S. thermophilus NCC 1561 was studied in order to verify the possibility of using them to promote the predominance of one of these two strains in the pathogenic ones (S. Sobrinus OMZ 176 , the caseino-glycopeptide (CGMP) and its desialylated derivative (As-CGMP) were obtained from Nestec

SA, Lausanne (for its preparation see Neeser et al., 1994).

The dose-response effect of adhesion to S-HA beads was studied, inoculating the well with 100 μl of bacterial suspension (CFU / ml corresponding to the 50% saturation of beads previously calculated) containing different concentrations of CGMP or As- CGMP and then performing the adhesion test in the usual way. Concentrations in the range of 0.05-3 mg / ml were tested. No previous incubation of the bacteria was carried out in the presence of CGMP or As-CGMP.

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Figure 2 shows the curves obtained for the three strains by representing the number of fixed cells against increasing amounts of CGMP, the number of inoculated cells corresponding to the 50% saturation of beads previously calculated for each strain. The strong inhibition observed in the case of S. sobrinus OMZ 176 confirms the previous results obtained by Neeser and others (1994) and by Schüpbach and others (J. Dent. Res., 75, 1779-1788, 1996).

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

As in the case of CGMP, the desialylated derivative inhibits the adhesion of S. sobrinus OMZ 176; only 0.05 mg / ml is needed to decrease the adhesion percentage by 50%. As-CGMP does not affect the adhesion of L. lactis NCC2211, but slightly favors that of S. thermophilus NCC1561 (Fig. 3).

Example 5: TOOTHPASTE

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

<td>Cetylpyridinium chloride</td><td> 1,65%</td>

<td>Sorbitol (sol. 70%)</td><td> 33,0%</td>

<td>Glycerin</td><td> 25,0%</td>

<td>Sodium carboxymethylcellulose</td><td> 2,0%</td>

<td>Sodium fluoride</td><td> 0,25%</td>

<td>Silica (RP 93)</td><td> 25,3%</td>

<td>Thickener Silica (Sident 22)</td><td> 8,1%</td>

<td>Sodium sacharine</td><td> 0,5%</td>

<td>Poloxamer (Pluronic F 108)</td><td> 3,2%</td>

This toothpaste is designed for the prophylaxis or treatment of tooth decay, dental plaque and periodontal infection.

Example 6: ICE CREAM

A cream is prepared consisting of 10.8% milk fat, 13.5% milk solids (non-fat), 0.3% Emulstab® SE30 and 0.3% Emulstab® foam (Grindsted, DK), then pasteurized at 105 ° C for 20 s, homogenized at 75 ° C and 300 bar, cooled to 38 ° C and inoculated with precultures in MRS medium taken in the exponential growth phase, at a concentration of 10⁷-10⁸ cfu / ml, of at least one of the lactic acid bacteria strains CNCM I-1984, CNCM I-1985, CNCM 1-1986, CNCM I-1987 or LMG P-18997. The cream is then fermented for 10 hours at 38 ° C until a pH of approx. 4.5. At the end of the fermentation, sucrose and glucose syrup are added. The composition of the cream is indicated in the following table 4.

The mixture is then beaten, cooled to 4 ° C and stored at 4 ° C, quenched to a degree of expansion of 95% by volume.

Table 4

<td>Ingredients</td><td>Composition (kg)</td><td>Fats (%)</td><td>Non-fatty solids (%)</td><td>Saccharose (%)</td><td>Solid content (%)</td>

<td>Cream (35%)</td><td> 30,83</td><td> 10,79</td><td> 1,54</td><td></td><td> 12,33</td>

<td>Skimmed milk powder</td><td> 12,45</td><td></td><td> 11,95</td><td></td><td> 11,95</td>

<td>Emulstab® SE30</td><td> 0,41</td><td></td><td></td><td></td><td> 0,37</td>

<td>Emulstab® foam</td><td> 0,41</td><td></td><td></td><td></td><td> 0,36</td>

<td>Water</td><td> 55,91</td><td></td><td></td><td></td><td></td>

<td>Total: cream base</td><td> 100,00</td><td> 10,79</td><td> 13,49</td><td> -</td><td> 25,01</td>

<td>Cream base</td><td> 74,14</td><td> 8,00</td><td> 10,00</td><td> -</td><td> 18,54</td>

<td>Saccharose</td><td> 22,06</td><td></td><td></td><td> 15,00</td><td> 15,00</td>

<td>Glucose syrup</td><td> 3,80</td><td></td><td></td><td></td><td> 3,00</td>

<td>Fermented ice cream</td><td> 100,00</td><td> 8,00</td><td> 10,00</td><td> 15,00</td><td> 36,54</td>

Example 7: YOGURT

5 μl of MRS culture medium are sterilized for 15 minutes at 121 ° C and then inoculated with 5% by volume of an active culture of at least one of the S. Thermophilus CNCM 1-1984, CNCM 1-1985 or LMG strains P-18997, which contains approximately 10⁹ cfu / ml. After incubation for 8 h at 41 ° C an initiator is obtained containing

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4,5·10⁸ cfu / ml.

5 l of reconstituted skim milk, whose dry matter content is 10% and to which 0.1% yeast extract has been added, are sterilized for 15 minutes at 121 ° C, and then inoculated with 2% of a commercial thickening Streptococcus thermophilus active culture containing approximately 10⁹ cells / ml. After incubation for 4 h at 41 ° C an initiator is obtained containing 4.5 · 10⁸ cells / ml.

A batch of whole milk containing 3.7% fat, fortified with 2.5% skim milk powder and then pasteurized for 30 minutes at 90 ° C, is inoculated with 2% by volume of the starter of at least one of the strains CNCM 1-1984, CNCM I-1985 or LMG P-18997 and 3% by volume of the initiator of Streptococcus thermophilus thickener. The inoculated milk is then shaken, poured into cans and incubated for 4 h at 41 ° C.

The yogurt obtained has a very firm and smooth texture and is designed for oral health.

Example 8: CHEWING GUM

A chewing gum to prevent or treat tooth decay, dental plaque or periodontal infection can be prepared by adding an active culture of at least one of the S.Thermophilus strains CNCM 1-1984, CNCM 11985 or LMG P-18997, so that it contains approximately 10⁴ to 10⁹ cfu / g, to the following usual ingredients:

<td>Xylitol</td><td> 67,5%</td>

<td>Rubber base</td><td> 20%</td>

<td>Calcium carbonate</td><td> 5%</td>

<td>Glycerin</td><td> 3%</td>

<td>Pluronic F 127</td><td> 2%</td>

<td>Cellulose rubber</td><td> 1%</td>

<td>Loads</td><td> 0,5%</td>

<td>Smell</td><td> 1%</td>

Example 9: FOOD COMPOSITION FOR PETS

A healthy pet food for the mouth is obtained, preparing a food mixture composed of corn, corn gluten, chicken and fish meal, salts, vitamins and minerals. The mixture is placed in a preconditioner and moistened. The wetted mix exiting the preconditioner is fed into a cook extruder and gelatinized. At the exit of the extruder the gelatinized matrix is ​​pushed through a die and extruded. The extrudate is cut into pieces suitable for feeding dogs, dried at about 110 ° C for 20 minutes and cooled, forming a granulate having a water activity of about 0.6.

The granules are sprayed with 3 coating mixtures. Each coating mix contains an active culture of at least one S.Thermophilus CNCM I-1984, CNCM 1-1985 or LMG P-18997 strain, but hydrogenated soybean fat is used as the coating substrate in one of the mixes. in another water and in another protein digest. The granulate contains approximately 10⁴ to 10⁹ cfu / g of said strains.