Use of exogenous lactic bacteria strain against actinomyces naeslundii

Description of the invention

The invention relates to the use of a lactic acid bacterial strain and a composition containing the same.

The invention relates to the introduction into the oral microflora of exogenous lactic acid bacteria which have the ability to modulate the colonization of A. naeslundii bacteria and to alleviate A. naeslundii diseases.

The mouth (oral cavity) contains microflora that is constantly present and microflora that is only temporary. The first group includes microorganisms capable of being more or less permanently present on the surfaces of the oral cavity. These bacteria are found mainly on the tongue, cheek mucosa and teeth, while the gums, lips, cheeks, palate and the bottom of the mouth are home to only a few microflora.

Dental plaque is the membrane that forms on the surface of the teeth and is made up of bacterial cells in the matrix of extracellular polysaccharides and saliva products. Immediately after eruption, the teeth are covered with an amorphous layer of saliva, the acquired enamel pellicle (AEP), which is approximately 1.3 µm thick and cannot be removed by ordinary tooth brushing. Deposition of bacteria on the teeth occurs immediately after the formation of the AEP layer, and the plaque becomes clearly visible within 8-12 hours as a multilayer structure. The first layer consists of bacteria (the earliest colonizers) that adhere to the teeth mainly through recognition of a specific adhesin receptor. It creates a base for successive colonizers that adhere to each other through an analogous, specific bond or by simply placing themselves next to each other.

On the tongue and on the cheek mucosa, microorganisms selected from Streptococcus, Veillonella, Bacteroides and Haemophilus belong to the naturally occurring microflora. The teeth are dominated by Streptococcus and Actinomyces, but a variety of Gram-positive bacteria can be found there.

Gram-negative cocci and sticks.

Many of these microorganisms are harmless commensals, but many have also been recognized as an etiological agent in many diseases (Hill, MJ and Marsh, PD eds. Human Microbal Ecology, 1990, CRC Press, Boca Raton Florida, USA).

In particular, genomic species 1 Actinomyces naeslundii (formerly A. naeslundii) and (formerly A. viscosus) are common constituents of human plaque. They are among the oral cavity strains most strongly forming plaque because of their ability to adhere firmly to the teeth and to co-aggregate with many other species of bacteria to promote their persistent presence in the mouth. Moreover, in older people, they are commonly isolated at sites of root caries and are believed to be the main etiological factor of this disease (Bowden, GH et al. 1999, The diversity and distribution of the predominant ribotypes of Actinomyces naeslundii genospecies 1 and 2 in samples. from enamel and from healthy and carious root surfaces of teeth. J. Dent. Res 78, 1880-1809).

Transitional microflora includes exogenous bacteria that may occasionally be present in the mouth but are not permanently present (even when repeated oral administration of these bacteria is performed). All food bacteria, and in particular lactic acid bacteria, can be part of this transient microflora.

Some of these exogenous lactic acid bacteria have been shown to adhere to the pellicle of teeth. For example, WO 00/09080 (Societe des Produits Nestle) discloses strains of lactic acid bacteria which are not part of the constantly present oral microflora, have low acidifying properties and are able to adhere directly to the pellicle of the teeth. These bacteria are used particularly in the treatment and prevention of dental caries and periodontal infections which are caused by cariogenic microorganisms such as Streptococcus mutans and Streptococcus sobrinus.

Exogenous bacteria can also produce factors that inhibit the growth of specific microflora constantly present in the mouth. EP 759649 (Societe des Produits Nestle) describes, for example, the use of bacteriocins produced by Micrococcus varians to inhibit the development of the oral pathogens S. sobrinus, S. sanguis, S. mutans and A. viscosus. The use of bacteriocins is also one of the investigated strategies that have been developed to reduce tooth decay. These molecules have gained attention as promising anti-caries agents and as important factors in modulating oral colonization.

It should be noted that the prior art does not provide any information on strains that can grow in the oral cavity by direct adherence to the pellicle of the tooth and also produce

Factors such as growth inhibitory factors that can modulate A. naeslundii colonization to reduce the risk of A. naeslundii related diseases.

The invention relates to the use of a lactic acid bacterial strain selected from the group of Streptococcus thermophilus, Lactococcus lactis subs. lactis and Lactococcus lactis subs. lactis biovar diacetylactis, which is exogenous to the oral microflora and capable of adhering to the pellicle of the teeth and producing an Actinomyces naeslundii bacterium growth inhibitory agent, for the preparation of a composition intended for the treatment or prevention of diseases associated with Actinomyces naeslundii in mammals.

Preferably the lactic acid bacterial strain is derived from a dairy product, more preferably a strain selected from the group consisting of CNCM 1-1984, CNCM 1-1985, CNCM 1-1986 and CNCM 1-1987 strains.

Preferably the composition is an edible composition.

Preferably the composition comprises at least 10⁴-10⁹ cfu / g of lactic acid bacteria.

Preferably the lactic acid bacterial strain is in combination with bacteriocin.

Thus, by colonizing the tooth surfaces and producing growth inhibitory factors, such lactic acid bacteria can significantly reduce the proliferation of Actinomyces naeslundii, reducing plaque, root caries and other Actinomyces naeslundii infections.

Also within the scope of the invention is a composition intended to maintain oral health by reducing colonization of Actinomyces naeslundii in mammals, which comprises at least one lactic acid bacterial strain selected from the group consisting of Streptococcus thermophilus strains, Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. lactis biovar diacetylactis, exogenous to the oral microflora and able to adhere to the pellicle of the teeth and to produce the factor inhibiting the growth of Actinomyces naeslundii bacteria.

Preferably the composition further comprises bacteriocin.

Preferably the composition comprises at least one lactic acid bacterial strain selected from the group consisting of CNCM 1-1984, CNCM 1-1985, CNCM 1-1986, CNCM 1-1987 strains.

Preferably such a composition comprises at least 10⁴-10⁹ cfu / g (colony forming units) of lactic acid bacteria.

As used herein, the term "mouth" means the oral cavity of humans or animals such as pets, composed of the mucosa (gums, lips, cheeks, palate and floor of the mouth), tongue and teeth (including artificial structures).

The term "growth inhibitory factor" defines any extracellular substance, produced by adherent exogenous lactic acid bacteria, that allows the inhibition of the growth of A. naeslundii.

The lactic acid bacteria are selected from the group consisting of Streptococcus thermophilus, Lactococcus lactis subsp. lactis as well as Lactococcus lactis subsp. lactis biovar diacetylactis, and in particular from the group consisting of the strains Streptococcus thermophilus (NCC 1529) (CNCM 1-1984), Streptococcus thermophilus (NCC1561) (CNCM 1-1985), Lactococcus lactis subsp. lactis (NCC2211) (CNCM 1-1986), Lactococcus lactis subsp. lactis biovar dioacetylactis (NCC 2225) (CNCM 1-1987).

The lactic acid bacteria are preferably derived from dairy products, i.e., for example, milk or cheese. The lactic acid bacteria used according to the invention are "low acidifying", which means that they are less acidifying than the pathogenic strains. Accordingly, they may contribute to the maintenance of an oral pH of around 5.5-7.

These strains were selected from lactic acid bacteria strains because of their ability to adhere to the pellicle of the teeth and their optimal growth temperature of about 37 ° C, which is the temperature of the oral cavity. They also have the ability to produce an inhibitory factor for the growth of Actinomyces naeslundii bacteria, which, combined with their adherence properties, allows them to significantly reduce the degree of colonization by genomic species 1 and 2 of A. naeslundii. Moreover, they are capable of fermenting glucose and sucrose and do not synthesize glucans, which are pathogenicity factors in cariogenic strains.

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

The lactic acid bacterial strains can be incorporated into, for example, food, animal feed, cosmetic or pharmaceutical compositions. Accordingly, these compositions are

Preferably, for example, toothpastes, mouthwashes, chewing gums, sprays, drinks, sweets, infant formula, ice cream, frozen desserts, sweet salad dressings, dairy products, cheese, cottage cheese, yoghurt, sour milk, coffee cream or whipped cream.

Exogenous lactic acid bacteria can be used in an amount of at least 10⁴-10⁹ cfu / g of lactic acid bacteria.

The effect of introducing the above-mentioned bacteria into the microflora of the oral cavity was tested in a rat model. The CNCM 1-1985 and CNCM-1986 strains were able to modulate the oral microbial ecosystem, significantly reducing the total CFU value. More specifically, these strains were able to significantly reduce the degree of colonization of A. naeslundii genomic species 2 with which rats had been infected (see examples).

Biochemical characteristics of selected strains

Fermentation patterns: 49 simple sugars were tested using the api strip test

CH bioMerieux (bioMerieux SA, 69280 Marcy-l'Etoile, France) and the results are summarized in Table 1.

Table 1

Sugar fermentation by L. lactis CNCM 1-1987 (A) bacteria,

L. lactis CNCM 1-1986 (B), S. thermophilus CNCM 1-1984 (C), S. thermophilus CNCM 1-1985 (D)

<td>Sugar</td><td>AND</td><td>B</td><td>C.</td><td>D</td><td>Sugar</td><td>AND</td><td>B</td><td>C.</td><td>D</td>

<td>adonitol</td><td> +++</td><td></td><td></td><td></td><td>inulin</td><td></td><td></td><td></td><td></td>

<td>esculina</td><td> ++</td><td> ++++</td><td></td><td></td><td>lactose</td><td> +</td><td> ++++</td><td> +++</td><td> ++++</td>

<td>amygdalin</td><td> ++++</td><td></td><td></td><td></td><td>D-lixosis</td><td></td><td></td><td></td><td></td>

<td>D-arabinose</td><td></td><td></td><td></td><td></td><td>maltose</td><td> ++</td><td></td><td></td><td></td>

<td>L-arabinose</td><td></td><td></td><td></td><td></td><td>mannitol</td><td> +++</td><td> ++</td><td></td><td></td>

<td>D-arabitol</td><td></td><td></td><td></td><td></td><td>D-mannose</td><td> +</td><td> ++++</td><td></td><td></td>

<td>L-arabitol</td><td> +++</td><td></td><td></td><td></td><td>melezitosis</td><td></td><td></td><td></td><td></td>

<td>arbutin</td><td> +++</td><td> +++</td><td></td><td></td><td>melibiosis</td><td></td><td></td><td></td><td></td>

<td>cellobiosis</td><td> +++</td><td> ++++</td><td></td><td></td><td>α-mety l oDg lu goat d</td><td></td><td></td><td></td><td></td>

<td>dulcitol</td><td></td><td></td><td></td><td></td><td>α-methyl-D-mannoside</td><td></td><td></td><td></td><td></td>

<td>e rite l</td><td></td><td></td><td></td><td></td><td>D-raffinose</td><td></td><td></td><td></td><td></td>

<td>D-fructose</td><td> +</td><td> ++++</td><td></td><td></td><td>rhamnosis</td><td></td><td></td><td></td><td></td>

<td>D-fucose</td><td></td><td></td><td></td><td></td><td>ribose</td><td> ++</td><td> ++</td><td></td><td></td>

<td>L-fucose</td><td></td><td></td><td></td><td></td><td>salicin</td><td> +++</td><td> +++</td><td></td><td></td>

<td>galactose</td><td> ++</td><td> ++++</td><td></td><td></td><td>sorbitol</td><td></td><td></td><td></td><td></td>

<td>β-gentobiosis</td><td> +++</td><td></td><td></td><td></td><td>L-sorbose</td><td></td><td></td><td></td><td></td>

<td>gluconate</td><td></td><td></td><td></td><td></td><td>starch</td><td></td><td></td><td></td><td></td>

<td>2-ketogluconate</td><td></td><td></td><td></td><td></td><td>saccharose</td><td></td><td></td><td> +++</td><td> ++++</td>

<td>5-keto gluconate</td><td></td><td></td><td></td><td></td><td>D-tagatosis</td><td></td><td></td><td></td><td></td>

<td>GlcNAc (N-acetylgalacto- zoamine)</td><td> +</td><td> ++++</td><td></td><td></td><td>trehalose</td><td> ++</td><td></td><td></td><td></td>

<td>D-glucose</td><td> +</td><td> ++++</td><td> +</td><td> ++</td><td>D-turanose</td><td> ++</td><td></td><td></td><td></td>

<td>glycerol</td><td></td><td></td><td></td><td></td><td>xylitol</td><td> +++</td><td></td><td></td><td></td>

<td>glycogen</td><td></td><td></td><td></td><td></td><td>D-xylose</td><td></td><td></td><td></td><td></td>

<td>inositol</td><td></td><td></td><td></td><td></td><td>L-xylose</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td>β-methyloxyloside</td><td></td><td></td><td></td><td></td>

+, ++, +++, ++++ show whether fermentation started after 3, 6, 24 or 48 hours

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Strains Streptococcus thermophilus (NCC 1529), Streptococcus thermophilus (NCC 1561),

Lactococcus. lactis subsp lactis (NCC 2211), Lactococcus. lactis subsp. lactis biovar dioacetylactis (NCC 2225) has been deposited under the Budapest Treaty at Collection Nationale de Culture de Microorganismes (CNCM 1-1984, CNCM 1-1985, CNCM 1-1986 and CNCM 1-1987 respectively), 25 rue du docteur Roux, 75724 Paris , France, March 3, 1998.

A second subject matter of the invention is a composition for maintaining oral health by reducing the colonization of A. naeslundii in mammals, the composition comprising exogenous strains of lactic acid bacteria that have been selected for their ability to adhere to the tooth surface and produce a growth inhibitory agent.

These compositions are particularly intended for the prevention or treatment of plaque and infection related to A. naeslundii disease such as root caries.

The lactic acid bacterial strain in the composition of the present invention is selected from the group consisting of Streptococcus thermophilus, Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. lactis biovar diacetylactis and preferably from the group consisting of the strains CNCM 1-1984, CNCM 1-1985, CNCM 1-1986 and CNCM 1-1987.

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

It is also possible to prepare a synergistic composition by adding at least one bacteriocin that is active against gram-positive oral bacteria. In this case, the oral care compositions may contain from 0.00001 to 50%, preferably from 0.00001 to 15% by weight of the purified bacteriocin. The bacteriocin may advantageously be variacin (EP 0759 469).

An oil-soluble antioxidant may also be added to protect the composition from degradation. Suitable antioxidants include "tocopherols", butyl hydroxyanisole (BHA), butyl hydroxytoluene (BHT), and ascorbyl palmitate. The oil-soluble antioxidant is present in an amount from 0.005% to 0.5%, preferably 0.005% to 0.01% by weight based on the weight of the composition. Suitable abrasives for use in the dentifrice compositions of the present invention include calcium carbonate, calcium aluminosilicate, alumina, alumina hydrates, zinc orthophosphate, plastic particles, and silica, of which silica is the preferred abrasive.

The compositions of the invention will have a pH that is orally acceptable and within which the activity of the lactic acid bacteria is not limited. The pH may be in the range of 3.0-9.5, preferably in the range of 3.5 to 6.5. These compositions can be prepared by conventional methods which involve mixing the ingredients together in the correct proportions and ultimately adjusting the pH to the desired value if necessary.

The genomic species Actinomyces naeslundii 1 (formerly A. naeslundii) and 2 (formerly A. viscosus) are among the most plaque forming oral strains. They are commonly isolated in the places of tooth root caries, especially in people over 40 years of age, and are considered the main etiological factor of this disease.

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

Example 1. In vitro trials

Strains of S. thermophilus NCC1561 (CNCM 1-1985) and L. lactis subsp. lactis NCC 2211 (CNCM 1-1986) (here 1. lactis NCC2211).

Oral strain A. naeslundii genomic species 1 (formerly A. naeslundii) OMZ745 and

A. naeslundii genomic species 2 (formerly A. viscosus) OMZ105 was obtained from the Institute far Orale Mikrobiologie und Allgemeine Immunologie, University of Zurich and cultured in FUM (Fluid Universal Medium) under anaerobic conditions (GasPackSystem, BBL) at 37 ° C.

All strains were stored in glycerol at -20 ° C and pre-cultured for 14 hours at their optimal temperature before use.

Both L. lactis NCC 2211 and S. thermophilus NCC 1561 selected strains were inoculated with an in vitro system in which biofilm, composed of bacteria commonly found in the oral cavity of a person over 40 years of age, developed on saliva-coated hydroxyapatite discs. The growth medium used, Fluid Universal Medium (FUM), was specially formulated to buffer the acidity produced by the test strains and to give continuous growth (plaque development) similar to that of the mouth (Gmur and Guggenheim, 1983). The determinations were carried out in triplicate, and the mixtures with and without the content of milk strains were tested in parallel. The strains listed in Table 2 were used.

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Table 2

Bacterial strains used and culture conditions in in vitro plaque experiments

<td>Strain</td><td>Relevant properties</td><td>Growth conditions</td>

<td>S. thermophilus NCC1561</td><td>adheres to synthetic S-HA hydroxyapatite</td><td>FUM, Belliker; 37 ° C</td>

<td>L. lactis subsp. lactis NCC2211</td><td>adjacent to the S-HA</td><td>FUM, M17-lactose; 37 ° C</td>

<td>S. sobrinus OMZ176</td><td>cariogenic</td><td>FUM; 37 ° C</td>

<td>S. oralis OMZ607</td><td>forms plaque</td><td>FUM; 37 ° C</td>

<td>A. naeslundii OMZ74 5</td><td>forms plaque; the causative agent of tooth root caries</td><td>FUM; 37 ° C</td>

<td>V. dispar OMZ493</td><td>forms plaque</td><td>FUM; 37 ° C</td>

<td>F. nucleatum OMZ596</td><td>forms plaque</td><td>FUM; 37 ° C</td>

Procedure

- Formation of saliva pellicle: coating of 10 mm synthetic hydroxyapatite (S-HA) discs (Hy-APATITE®, Euro-Crystals, Landgraaf, The Netherlands) with 800 μl of human saliva and incubation for 4 hours at room temperature with shaking (1 disc / a well (in a sterile 24-hole Nunclon plate).

- Bacterial consortium production: growth of S. thermophilus NCC1561, L. lactis subsp. lactis NCC2211, S. sobrinus OMZ176, S. oralis OMZ607, A. naeslundii OMZ745, V. dispar OMZ493 and also F. nucleatum OMZ596 overnight at 37 ° C in FUM-glucose anaerobic medium (S. thermophilus NCC1561 in FUM medium -lactose), adjusting the OD550 to 1 from FUM, and pooling 2 ml of each oral bacterial suspension with 2 ml of either S. thermophilus NCC1561 or L. lactis subsp. lactis NCC2211. The control mixture contains only five oral strains.

- Biofilm formation and bacterial recovery: procedure as described in Guggenheim et al., 1998, Validation of a new biofilm model. J. Dent. Res. 77, (Spec Iss A): 110 (Abstract # 38).

- Biofilm culture analysis: tube plating the suspension onto Columbia Blood Agar (5% Sheep Blood, Becton Dickinson, Meylan Cedex, France) to determine the total amount and for differentiation of A. naeslundii. Incubation of the plates at 37 ° C in anaerobic conditions for 48 hours.

Growth antagonism between oral strains and the tested dairy strains

The strains and growing conditions are listed in Table 3.

Table 3

Bacterial strains used and culture conditions used in growth antagonism studies

<td>Strain</td><td>Relevant properties</td><td>Growth conditions</td>

<td>S. thermophilus NCC1561</td><td>adjacent to the S-HA</td><td>Belliker; 42 ° C</td>

<td>L. lactis subsp. lactis NCC2211</td><td>adjacent to the S-HA</td><td>M17-lactose; 37 ° C</td>

<td>S. thermophilus NCC 1536</td><td>does not stick</td><td>Belliker; 42 ° C</td>

<td>A. naeslundii OMZ745</td><td>forms plaque</td><td>BHI; 37 ° C; anaerobic conditions</td>

<td>A. viscosus OMZ105</td><td>forms plaque</td><td>BHI; 37 ° C</td>

S. thermophilus NCC 1561, S. thermophilus NCC 1536 and L. lactis NCC 2211 (killer strains) were tested for growth antagonism against A. naeslundii OMZ745 and A. viscosus OMZ105 (target strains).

Procedure

- Growth of "killer" strains overnight on agar plates under anaerobic conditions and growth of "target" strains in BHI up to the mean stationary phase.

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- Dilute 20 µl of the suspension of the "target" strains in 3 ml of soft BHI agar (7 g / l agar) containing glucose and lactose, mix on a Hortex mixer and immediately pour onto a BHI agar plate.

- Solidification for 1 hour at room temperature, then spreading the killer strains and the M17 plate as a cross. Parallel distribution of killer "and" target "strains separately as controls.

- Incubation at 37 ° C in anaerobic conditions for 24 hours.

Growth antagonism is revealed by the inhibition halo around the cross.

Statistics

The differences between the control and test community were determined using the Student's t-test.

Results and discussion

The S. thermophilus NCC1561 and L. lactis NCC 2211 strains can be introduced and grown on a plaque-like biofilm on S-HA discs and their total CFU / disc values ​​at 40.5 hours are given in Table 4.

Table 4

The level of introduction of the two dairy strains into the biofilm (CFU / disk). Values ​​are the mean values ​​of three standard deviation experiments

<td>Method of inoculation</td><td>S. thermophilus NCC561 (x 10⁶)</td><td>L. lactis NCC2211 (x 10⁶)</td>

<td>together with oral strains</td><td> 4,08 ± 1,78</td><td> 5,76 ± 3,64</td>

<td>against mouth strains</td><td> 5,03 ± 2,21</td><td> 3,87 ± 4,01</td>

The effect of introducing dairy strains into the biofilm on oral species is shown in Tables 5 and 6. When S. thermophilus NCC1561 (Table 5) was included, there was an overall decrease in total flora represented by the Columbia Blood Agar (CBA) plate counts and 4 species. from the mouth.

When the L. lactis NCC2211 strain was introduced into the oral strain consortium (Table 6) the total flora counts decreased noticeably (CFU per CBA). This decrease was significant in the case of A. naeslundii OMZ745, which significantly decreased (p = 0.021). The decrease was even greater when the strain was inoculated onto the discs in front of the oral bacteria.

Table 5

Oral strain consortium modulation by S. thermophilus NCC1561 (CFU / disc)

<td>Treatment</td><td>CBA (x10⁸)</td><td>A. naeslundii OMZ745 (x10⁶)</td><td>MS (x10⁸)</td>

<td>Control</td><td> 2,86 ± 2,14</td><td> 5,29 ± 2,58</td><td> 2,02 ± 1,68</td>

<td>+ NCC1561</td><td> 1,63 ± 0,55</td><td> 4,75 ± 1,45</td><td> 1,21 ± 0,81</td>

<td>Pre-incubation with NCC1561</td><td> 2,32 ± 10,38°°</td><td> 4,78 ± 2,29</td><td> 1,48 ± 0,29</td>

N = 3. *: p values ​​are calculated relative to the control (*: p &lt;0.05; **: p &lt;0.01); °: p values ​​are calculated from the test "+ NCC1561" (°: p &lt;0.05; °°: p &lt;0.01)

Table 6

Modulation of the oral strain consortium by L. lactis NCC2211 (CFU / disc)

<td>Treatment</td><td>CBA (· 10⁸)</td><td>A. naeslundii OMZ745 (· 10⁶)</td><td>MS (10⁸)</td>

<td>Control</td><td> 2,77 ± 2,16</td><td> 6,07 ± 2,70</td><td> 3,04 ± 2,88</td>

<td>+ NCC2211</td><td> 0,65 ± 0,33</td><td> 4,59 ± 2,81</td><td> 0,65 ± 10,33 *</td>

<td>Pre-incubation with NCC2211</td><td> 0,27 ± 10,11 **°</td><td> 3,91 ± 13,29 *°</td><td> 0,27 ± 10,11 **°°</td>

N = 3. *: p values ​​are calculated relative to the control sample (*: p &lt;0.05; **: p &lt;0.01); °: p values ​​are calculated from the test "+ NCC2211" (°: p &lt;0.05; °°: p &lt;0.01)

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Some trials have been carried out to determine whether the reduction of the oral strains was due to the growth antagonism of the dairy strains against them (Table 7). The A. viscosus OMZ105 and S. thermophilus NCC 1536 strains were also included in the test as they are part of the in vivo model (Example 2).

All four dairy strains inhibited the growth of the Gram negative strain A. viscosus OMZ105. This inhibition cannot be attributed to the production of lactic acid. A. viscosus is only capable of metabolizing lactate under aerobic conditions (van der Hoeven et al. (1990) Oral Microbiol. Immunol. 5, 223-225) and grows in an acid environment. These findings were confirmed by plating on an A. viscosus plate in the presence of a 1% lactic acid solution: no inhibition was observed.

Table 7

Growth inhibition of oral strains caused by S. thermophilus NCC 1561, S. thermophilus NCC 1536 and L. lactis NCC2211

<td rowspan="2">Target</td><td colspan="3">Killer Strains NCC1561 NCC1536 NCC2211</td><td rowspan="2">Lactic acid 1%</td>

<td>NCC1561</td><td>NCC1536</td><td>NCC2211</td>

<td>A. naeslundii OMZ745</td><td> +</td><td> +</td><td> +</td><td> -</td>

<td>A. viscosus OMZ105</td><td> +</td><td> +</td><td> +</td><td> -</td>

Conclusions

S. thermophilus NCC1561 and L. lactis NCC2211 could be incorporated into a plaque mimicking biofilm and were able to modulate the oral microflora, significantly reducing the total cfu value, and more specifically these strains were able to significantly reduce the degree of colonization of the genomic species A. naeslundii 2 Moreover, the strains were able to inhibit the growth of A. naeslundii genomic species 1 and 2 in co-cultures.

Example 2. In vivo trials

The studies were carried out in vivo on a rat model. In this study, the mating of selected strains was continued throughout the research period, in a 24-hour cycle, by feeding with a chilled dairy product.

This study was conducted over 58 days. In order to perform the daytime experiment, the period of activity of the animals had to be shifted by a total of 7 hours. This was done in three steps on days 16, 17 and 18 as further detailed below. The cariogenic strains were paired on day 21 and 22, while the mating of the dairy strains began on day 23 and continued until day 57. The animals were fed the dairy strains as a yoghurt additive, which was incorporated into the normal diet as explained later. At the end of the experiment, on day 58, a swab was taken from the rats' teeth.

Animals and diet

The experiment used 10 litters consisting of 4 young Osborne-Mendel rats (animal production department of the Institute far Orale Mikrobiologie und Allgemeine Mikrobiologie, University of Zurich, Switzerland). All animals were weighed at the beginning and end of the test period. After 13 days, mothers and pups were transferred to stainless steel cages with a clear bottom without lining and fed ad libitum Nafag powdered (0.2 μm) low-fluoride food to avoid the effect of scratching (Rat Checkers No. 184, NAFAG, Gossau SG, Switzerland) and tap water without restrictions. The active phase during which rats eat is overnight, i.e. 18:00 - 06:00.

In order to allow the feed cups to be replenished during normal working hours, the circadian rhythm of activity in the rats was progressively reversed between days 16 and 18 by shifting the activity period of the rats each day at three times by adjusting the automatic lighting control.

On 16/17, the start of the period of activity was shifted from 6:00 p.m. to 3:00 p.m., i.e. the night was moved from 3:00 p.m. to 3:00 p.m., followed by day. On 17/18 the beginning of the active period was moved from 3 p.m. to 12 p.m., that is, the night lasted from 12 p.m. until 00:00 and the day from 00:00.

Finally, on 18/19 the beginning of the period of activity was accelerated from 12:00 to 10:00, that is, the night was from 10:00 to 22:00 and the day was from 22:00.

PL 202 576 B1

Therefore, by day 19, the period of activity of the rats was completed shifting from dark hours to normal working hours (10:00 - 22:00).

on the day the mothers were removed, and the rats were fed ad libitum modified food 2000a, containing 40% sucrose, 28% skim milk substitute (SVPRO-PP 1611 soy protein extract 39.4%, lactose - 49.3%, 0.6%, L -methionine - 0.3%, L-lysine HCl 0.1%), 24% wheat flour, 5% brewer's yeast, 2% Gevral^® Instant Protein (Whitehall-Robins SA, 6301 Zug, Switzerland) and 1% NaCl.

During the mating period (days 21 and 22), the drinking water was supplemented with 2% glucose and 2% sucrose to maintain implantation of the associated bacteria. On day 23, the litters were divided into 3 treatment groups, one animal per cage on the programmed feeding machine, and began to receive a test diet as indicated in Table 10.

The test diet consisted of 18 meals of yoghurt containing the test strains alternating with 18 meals of the modified diet 2000a previously described.

Drinking water was given ad libitum. After swab collection on day 58, animals were overdosed with sodium thiopental (100 mg / kg body weight) by intra-peritoneal injection and decapitated during coma.

Bacterial strains: the strains listed in Table 8 were used.

Table 8

Bacterial strains used in the study

<td>Strain</td><td>Relevant properties</td><td>Growth conditions</td>

<td>S. thermophilus NCC1561</td><td>adjacent to the S-HA</td><td>Belliker; 42 ° C</td>

<td>S. thermophilus NCC1536</td><td>non-contiguous control</td><td>Belliker; 42 ° C</td>

<td>L. lactis NCC2211</td><td>adjacent to the S-HA</td><td>M17-lactose; 37 ° C</td>

<td>A. viscosus OMZ105</td><td>forms plaque; adjacent to the S-HA</td><td>BHI; 37 ° C</td>

Preparation of tested LAB strains for mating

A preliminary study was performed to evaluate growth parameters, especially the hours required to reach steady state under the specific conditions described later. Therefore, the growth time for S. thermophilus strains was set at 7 hours, and for L. lactis strain - 6 hours. A post-freezing viability study of the dairy strains was also performed by plating the same cell suspension before and after freezing. For mating with the animals, the dairy strains were treated according to the following procedure.

Procedure

- Strains inoculate 1% overnight in their appropriate medium with museum strain of glycerol.

- Inoculation of 5% strains of this culture into 10 4 liter aliquots of the appropriate medium warmed up to the optimal growth temperature, followed by growth until the end of the log phase / beginning of stationary phase.

- Determination of the final value of CFU / ml by plating on an agar plate with M17 - lactose from two randomly selected lots for each of the 4 strains. Incubation of the plates overnight under anaerobic conditions.

- Centrifugation of the culture from each batch at 6,000 rpm. for 10 minutes and suspending the pellets in 150 ml of fresh medium, keeping overnight at 4 ° C.

- Re-centrifugation and re-suspension in the freezing medium (15% glycerol in Belliker or M17).

- Split into samples to obtain 2-10¹¹ viable cells / vial considering loss of viability upon freezing and storage at -20 ° C until use.

Mating animals with bacterial strains

The animals were divided into three treatment groups (Table 9). Each group consisted of 10 cubs which were placed one in a cage.

PL 202 576 B1

Table 9 Setting the treatments

<td>Treatment</td><td>Associated bacteria</td>

<td> 1</td><td>A. viscosus OMZ105; S. thermophilus NCC1536</td>

<td> 2</td><td>A. viscosus OMZ105; S. thermophilus NCC1561</td>

<td> 3</td><td>A. viscosus OMZ105; L. lactis NCC2211</td>

All rats were first infected on days 21 and 22 with A. viscosus OMZ105 strain.

The test LAB strains were matched daily (as they were included in a basic yoghurt meal), starting on day 23. Two frozen vials, each containing 2 × 10¹¹ viable cells of the test strain, were mixed in 200 ml of yoghurt to obtain at least 10⁹ CFU / ml.

S. thermophilus NCC 1536, a S-HA non-adherent strain, was used as negative control.

1 ml and 400 mg yoghurt and feed 2000a, respectively, were administered alternately 18 times a day at 20-minute intervals (Table 10). Thus, each animal received a total of 18 ml of yogurt and 7.2 g of powdered food.

The meals were dispensed in food bowls from a programmed feed dispenser, which automatically fed the animals the right meal at the appointed time.

TABLE 10 Hours of feeding

<td>No meal</td><td>Highly cariogenic meals</td><td>Yoghurt meals</td><td>No meal</td>

<td> 1</td><td> 10:00</td><td> 10:20</td><td> 2</td>

<td> 3</td><td> 10:40</td><td> 11:00</td><td> 4</td>

<td> 5</td><td> 11:20</td><td> 11:40</td><td> 6</td>

<td> 7</td><td> 12:00</td><td> 12:20</td><td> 8</td>

<td> 9</td><td> 12:40</td><td> 13:00</td><td> 10</td>

<td> 11</td><td> 13:20</td><td> 13:40</td><td> 12</td>

<td> 13</td><td> 14:00</td><td> 14:20</td><td> 14</td>

<td> 15</td><td> 14:40</td><td> 15:00</td><td> 16</td>

<td> 17</td><td> 15:20</td><td> 15:40</td><td> 18</td>

<td> 19</td><td> 16:00</td><td> 16:20</td><td> 20</td>

<td> 21</td><td> 16:40</td><td> 17:00</td><td> 22</td>

<td> 23</td><td> 17:20</td><td> 17:40</td><td> 24</td>

<td> 25</td><td> 18:00</td><td> 18:20</td><td> 26</td>

<td> 27</td><td> 18:40</td><td> 19:00</td><td> 28</td>

<td> 29</td><td> 19:20</td><td> 19:40</td><td> 30</td>

<td> 31</td><td> 20:00</td><td> 20:20</td><td> 32</td>

<td> 33</td><td> 20:40</td><td> 21:00</td><td> 34</td>

Bacteriological evaluation

On day 58, five rats from each group were swabbed. Swab suspensions were either plated in Petri dishes for CFU (colony forming units) assessment or immobilized on immunofluorescence slides for TCN (total cell count) count.

Procedure for determining the CFU value

- Dental swab removal of rats using sterile cotton-tipped sticks and immediately placing them in 5 ml of sterile 0.9% NaCl solution.

PL 202 576 B1

- Mixing in a Hortex mixer for 1 minute and sonication for 5 s at 50 W.

- Tubing properly diluted suspensions onto CBA, MS and HJL agar media.

- Incubation of the CBA and MS plates at 37 ° C and the HJL plates at 45 ° C.

Procedure for determining the TCN

- Place 10 μl of undiluted swab suspension prepared for determination of CFU per well into a 24-well glass plate (Dynatech Produkte AG, Embrach Embraport, Switzerland) and air dry.

- Fix by soaking in methanol for 2 minutes and air drying.

- Incubation with 10 μl of the appropriate antibody or serum diluted in ELISA buffer (section 4.2.2.5) and incubation at 37 ° C for 30 minutes.

- Aspirating each drop on the side of the well and washing by soaking the slide first in ELISA buffer and then in distilled water.

Air drying

- Application of 10 μl rabbit anti-goat IgG (H + L) - FITC (Sigma) diluted 1: 400 and incubation at 37 ° C for 30 minutes.

- Washing as before and air drying.

- Use 49 μl of mounting fluid (section 4.2.2.5), cover with a coverslip and count the fluorescent cells using a fluorescent microscope.

Statistics

Data were subjected to a two-way ANOVA (Snedecor and Cochran, 1980).

Results of continuous mating of the dairy strains as a result of feeding chilled dairy products.

Bacteriological assessment (Table 11)

- Strain colonization. When the dairy product was supplemented with a non-adherent control strain of S. thermophilus (NCC1536), 1.7 (± 1.1) 10⁷ CFU for plate forming strain A. viscosus OMZ105. The dairy strains could not be counted by microbiological methods. However, attempts have been made to make a qualitative evaluation by immunofluorescence. Three adherent dairy strains could be recognized in the plaque samples for treatment groups 2 and 3. As they were aggregated with other oral bacteria and debris in the mouth to form large clusters, precise quantification was impossible.

- Variation in total flora (TF). The three treatment groups containing the adherent S. thermophilus NCC1561 and L. lactis NCC 2211 test strains showed a significant reduction in the number of colony forming units on the CBA medium compared to the control group containing the non-adherent S. thermophilus strain NCC 1536 (Table 11). In treatment group 2, CFU values ​​decreased with a significance factor PF &lt;0.01, and in group 3 even more significantly (PF &lt;0.001).

- Modulation of A. viscosus OMZ105 colonization on teeth by the tested strains. For the plaque forming A. viscosus OMZ105 bacteria, a clearly less visible but more significant decrease in the number of colony forming units was observed for the three treatments containing the adherent strains tested relative to Treatment 1 (PF &lt;0.01) (Table 11). In contrast, the percentages of A. viscosus with respect to the total CFU value were not significantly different. Approximately 50% of the total CFU value for all four treatments was identified as A. viscosus colonies.

Table 11

Mean values ​​per rat of colony forming units for total flora (TF) and strain A. viscosus OMZ105 and their respective percentages (N = 5)

<td>Treatment</td><td>TF to CBA x 10⁶</td><td>OMZ105 on CBA x 10⁶</td><td>% OMZ105 on CBA</td><td>TF to MS x 10⁴</td>

<td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td>

<td>1 (NCC1536)</td><td> 32,5 ± 13,87</td><td> 17,3 ± 11,01</td><td> 52,8 ± 23,09</td><td>41.9 ± 41 ns</td>

<td>2 (NCC1561)</td><td> 17,6 ± 6,57 **</td><td> 8,01 ± 1,96 **</td><td>47.2 ± 9.37 ns</td><td>5.6 ± 3.8 ns</td>

<td>3 (NCC2211)</td><td> 13,9 ± 4,85 ***</td><td> 5,81 ± 2,07</td><td>43.4 ± 18.16 ns</td><td>1.7 ± 1.11 ns</td>

<td>SEM</td><td> 2,99</td><td> 2,17</td><td> 5,65</td><td> 10,33</td>

PL 202 576 B1 cont. table 11

<td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td>

<td>Pf</td><td> 0, 001</td><td> 0,01</td><td>n. s.</td><td>ns</td>

<td>LSD 0.05 *</td><td> 9,21</td><td> 6,69</td><td> —</td><td> —</td>

<td>LSD 0.01 **</td><td> 12,92</td><td> 9,37</td><td> —</td><td> —</td>

<td>LSD 0.001 ***</td><td> 18,25</td><td> —</td><td> —</td><td> —</td>

Treatments 2-3 were compared to Treatment 1. SEM = standard error of the mean; ns = insignificant; CBA = Columbia Blond Agar; MS = Mitis-salivarius agar. OMZ105: A. naeslundii genomic species 2

In this in vivo test, the strains that were delivered daily showed a clear inhibitory effect on all microflora, the CFU of which had significantly decreased. This decrease can be explained by the growth antagonism of the dairy strains versus the oral strains. For example, all of these, including the non-S-HA adherent S. thermophilus NCC1536 strain, can inhibit the growth of A. viscosus OMZ105 in vitro. However, in vivo, such an effect can only be demonstrated for the strains adhering to the S-HA substrate, as it was detected in treatments 2-4 as opposed to the first.

In particular, since the animals were infected with A. viscous OMZ105 bacteria, it was possible, at the end of the experiment, to quantify this plaque-forming organism, and the decrease in its CFU value could be closely monitored.

The percentages of A. viscous OMZ105 strain in relation to the total value of CFU did not decrease in parallel, therefore it can be concluded that the effect of growth antagonism was also manifested against other species, ie Veillonellae, and consequently an effect of global scope is observed.

Thus, the CNCM 1-1985 and CNCM 1-1986 strains are able to modulate the microbial ecosystem of the oral cavity, significantly reducing A. naeslundii colonization of genomic species 2 in which rats have been infected.

Example 3. Preparation and preliminary analysis of surfactants from bacteria S. thermophilus NCC1561 and S. thermophilus NCC 1536

S. thermophilus NCC1561 and S. thermophilus NCC1536 strains were grown overnight in 1 liter Belliker medium at 42 ° C. To prepare the biosurfactant, the procedure described in Busscher et al. (1997), Appl. Environ. Microbiol. 63, 3810-3817, (Busscher et al., 1997).

Manufacture of surfactants

Procedure

- Washing cells three times in PBS.

- Suspension in 200 ml of distilled water or PBS.

- Preparation of a biosurfactant by gently stirring the suspension for 2 or 4 hours at room temperature.

- Separation of bacteria by centrifugation at 10,000 rpm. for 10 minutes.

- Centrifugation of the supernatant twice at 10,000 rpm. for 10 minutes.

- Freeze drying and weighing of both the sludge and surfactant solutions.

The crude surfactate suspension was first analyzed by SDS-PAGE and then subjected to surface tension measurements.

SDS-PAGE procedure

SDS-PAGE study was performed with ready-to-use 12.5% ​​ExcelGel (Amersham Pharmacia Biotech). Silver staining was performed using Plusone Silver Staining Kit (Amersham Pharmacia Biotech).

Surface tension measurement procedure

The surface tension of the biosurfactant suspensions was measured using a TVT1 Drop Volume Tensiometer (Lauda, ​​Lauda-Konigdshofen, Germany) which is based on the principle of the drop volume. In short, this method relies on the precise determination of the volume of a droplet of suspension that detaches from the capillary. This volume (critical volume) is proportional to the surface tension (σ), the value of which is calculated from the relationship:

σ = Vg Δρ F / 2π r_kap

PL 202 576 B1 where:

- σ - interfacial tension

- V - drop volume

- g - gravity constant

- Δμ - difference in density of both adjacent phases

- F - correction factor

- rkap - capillary radius

Measurements were made twice at 37 ° C in air. Each measurement consisted of 10 cycles. A solution of 6 mg / ml of the released crude product produced a decrease in the surface tension of water from 70 to 51 mN / m (Table 13). The SDS-PAGE profile of the bacteria released products indicated that many different substances of proteinaceous nature were present in the solution.

Table 13

Surface tension values ​​of biopsurfactant suspensions compared to water and PBS These values ​​are the average of two experiments with 10 measurements each

<td></td><td>Crude extract concentration</td><td>Surface tension (mN / m)</td>

<td>Water</td><td> —</td><td> 69,07 ± 0,001</td>

<td>PBS</td><td> —</td><td> 68,13 ± 0,300</td>

<td>S. thermophilus NCC1561</td><td>6 mg / ml</td><td> 51,47 ± 0,170</td>

<td>S. thermophilus NCC1536</td><td>6 mg / ml</td><td> 51,67 ± 1,320</td>

Score

Cells of S. thermophilus NCC1561 and S. thermophilus NCC 1536 strains are capable of releasing substances with surfactant activity. Therefore, it is possible that the biosurfactant produced by S. thermophilus NCC1561 easily detaches this bacterium or other oral strain from the tooth surface. On the other hand, S. thermophilus NCC1536 does not show such an effect, because this strain does not stick to the teeth.

Example 4. Toothpaste ₅

Toothpaste is made by adding 10⁵ cfu / ml of at least one strain of lactic acid bacteria CNCM 1-1984, CNCM 1-1985, CNCM 1-1986, CNCM 1-1987 in freeze-dried form, for the following mixture containing: 1.65% cetylpyridinium chloride, 33.0% sorbitol ( 70% solution), 25.0% glycerin, 2.0% sodium carboxymethylcellulose, 0.25% sodium fluoride, 26.3% silica (RP 93), 8.1% thickening silica (Sident 22), 0.5% Saccharin Sodium, 3.2% Poloxamer (Pluronic F108).

This toothpaste is intended for the prevention or treatment of tooth decay, plaque and other infections caused by A. naeslundii species.

Example 5 Yoghurt 1 of MRS medium is sterilized for 15 minutes at 121 ° C, and then inoculated with 5% by volume of an active culture of at least one strain of S. thermophilus CNCM 1-1984, CNCM 1-1985, containing approximately 10⁹ cfu / ml. After incubation for 8 hours at 41 ° C, a primer containing 4.5-10 was obtained⁸ cfu / ml.

liters of reconstituted skim milk with a dry matter content of 10%, to which 0.1% yeast extract was added, sterilized for 15 minutes at 121 ° C and inoculated with 2% of an active commercial thickening culture of Streptococcus thermophilus, containing approximately 10⁹ cells per 1 ml. After incubation for 4 hours at 41 ° C, a primer containing 4.5-10 was obtained⁸ cells / ml.

One portion of whole milk containing 3.7% fats was fortified with 2.5% skim milk powder, then pasteurized for 30 minutes at 90 ° C, then inoculated with 2% v / v starter from at least one strain CNCM 1-1984, CNCM 1-1985 and 3% by volume of a thickening starter of Streptococcus thermophilus strain. The inoculated milk is mixed, poured into dishes and incubated for 4 hours at 41 ° C.

The obtained yoghurt has a good, compact and smooth texture and is intended for the health prophylaxis of the oral cavity.

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Example 6. Chewing gum

A chewing gum intended for the prevention or treatment of dental caries, plaque or other diseases related to the activity of A. naeslundii can be prepared by adding an active culture of at least one strain of S. thermophilus CNCM 1-1984, CNCM 1-1985 in an amount approximately from 10⁴ up to 10⁹ cfu / g, for the following common ingredients: 67.5% xylitol, 20% gum base, 5% calcium carbonate, 3% glycerin, 2% Pluronic F127, 1% cellulose gum, 0.5% ballast compounds and 1% agent flavor.

Example 7. An animal feed composition

Animal feed, intended for oral prophylaxis, is obtained by preparing a mixture consisting of grain, corn gluten, poultry and fish meal, salt, vitamins and minerals. The feed mixture is placed in the pre-mixer and moistened. The moistened feed leaving the pre-mixer is then fed into the cooking extruder and gelatinized. The gelatinized matrix exiting the extruder is passed through a die and extruded. The extrudate is cut into pieces suitable for feeding dogs, dried at about 110 ° C for about 20 minutes, and cooled to form granules with a water activity of about 0.6.

The granules are sprayed with 3 layers of coating mixtures. Each coating blend contains an active culture of at least one of the strains of S. thermophilus CNCM 1-1984, CNCM 1-1985, but one coating blend uses hydrated soybean fat as a coating substrate, one coating blend uses water as a coating substrate, and one blend uses water as the coating substrate. the coating was used as a substrate coating the protein extract. The granules contain approximately 10⁴ up to 10⁹ cfu / g of said strains.