USE OF EXOGENOUS LACTIC BACTERIA STRAIN AGAINST i ACTINOMYCES NAESLUNDII /i RELATED DISEASES

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DESCRIPTION

Use of an exogenous lactic acid bacteria strain against Actinomyces naeslundii-related diseases.

The present invention is related to the incorporation into the oral microflora of exogenous lactic acid bacteria that are capable of modulating the colonization of A. naeslundii and of reducing the severity of diseases related to A. naeslundii.

Background of the invention

The mouth (oral cavity) contains a resident and a non-resident microflora. The first includes microorganisms that are capable of establishing a more or less permanent residence on oral surfaces. These bacteria are mainly located on the tongue, oral mucosa and teeth, while only a very dispersed microflora is found on the gums, lips, cheeks, palate and base of the mouth.

Dental plaque is a film that forms on the surface of the teeth that consists of bacterial cells enclosed in a matrix of extracellular polysaccharides and salivary products. Immediately upon appearance, teeth are covered with an amorphous layer of saliva, the Acquired Enamel Film (EAP) that is about 1.3 μm thick and cannot be removed by normal toothbrushing.

The deposition of bacteria on the teeth occurs immediately after the formation of the PAE and the plaque is evident in 8-12 hours as a multilayered structure. The first layer is made up of bacteria (early colonizers) that attack teeth mainly through the recognition of specific adhesin receptors; this forms a substrate for the second colonizers that adhere to each other through specific analogous junctions or by simple juxtaposition.

On the tongue and oral mucosa, the natural resident microflora includes microorganisms selected from Streptococcus, Veillonella, Bacteroides, and Haemophilus. Streptococci and Actinomyces predominate on teeth, but a number of Gram-positive and negative cocci and rods can be found.

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

In particular, Actinomyces naeslundii genospecies 1 (previously A. naeslundii) and 2 (previously A. viscosus) are common members of human dental plaque. These are among the strongest plaque-forming oral strains, because of their ability to firmly adhere to teeth and co-aggregate with many other species of bacteria, thus promoting their establishment in the mouth. In addition, in the elderly they are frequently isolated at root caries sites, and are believed to be the main etiological agent 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, 1800-1809).

Transient microflora includes exogenous bacteria that may occasionally be present in the mouth, but do not establish permanent residence (even with repeated oral administrations of these bacteria). All bacteria in food, and in particular lactic acid bacteria, can be part of this transient microflora.

Some of these exogenous lactic acid bacteria have been shown to be able to adhere to the film of the teeth. For example, WO 00/09080 (Société des Produits Nestlé) describes strains of lactic acid bacteria, which are not part of the resident microflora of the mouth, which produce reduced acidification and which are capable of adhering directly to the film of the teeth. . These bacteria are used specifically for the treatment or prevention of dental caries and periodontal infection caused by cariogenic microorganisms such as Streptococcus mutans and Streptococcus sobrinus.

Exogenous bacteria can also produce factors that inhibit the growth of the resident microflora in the mouth. For example, PE 759469 (Société des Produits Nestlé) describes the use of a bacteriocin produced by Micrococcus varians for the inhibition of the development of the oral pathogens S. sobrinus, S. sanguis, S. mutans and A. viscosus. The application of bacteriocins is also one of the investigated strategies that have been optimized for the reduction of dental caries. These molecules are interesting when considering the next anti-caries agents and as important factors in the modulation of the colonization of the oral cavity.

It should be noted that prior knowledge does not provide any information on the strains that can become established in the oral cavity by adhering directly to the tooth film and that also produce factors such as growth inhibition factors, which can modulate the colonization of A. naeslundii. as well as to reduce the severity of diseases related to A. naeslundii.

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Summary of the invention

Consequently, the present invention aims to provide the use of lactic bacteria that are exogenous to the oral microflora, selected for their ability to adhere to the surface of the teeth and produce a growth inhibition factor, for the preparation of a composition with the which is intended to treat or prevent Actinomyces naeslundii-related diseases in mammals.

Lactic acid bacteria can be selected from the group consisting of Streptococcus thermophilus, Lactococcus lactis subsp. lactis, and Lactococcus lactis subsp. lactis biovar diacetylactis, and in particular from the group consisting of the strains CNCM I-1984, CNCM I-1985, CNCM I-1986 and CNCM I-1987.

Thus, by colonizing the tooth surface and producing growth-inhibiting factors, such lactic acid bacteria can achieve a significant reduction in the spread of Actinomyces naeslundii, thus reducing dental plaque, root caries and other infections. related to Actinomyces naeslundii.

Another object is to provide a composition to maintain oral health by reducing the colonization of Actinomyces naeslundi, a composition comprising exogenous lactic acid bacteria that have been selected for their ability to adhere to the surface of the teeth and to produce an inhibiting factor. growth.

Such a composition can contain at least 10⁴-10⁹ CFU / g of lactic acid bacteria.

Detailed description of the invention

In the following description, the mouth is defined as the oral cavity of humans, or animals such as pets, which is made up of the oral mucosa (gums, lips, cheeks, roof of the mouth and base of the mouth), the tongue and the teeth ( which includes man-made structures).

The term "growth inhibiting factor" defines any extracellular substance produced by adherent exogenous lactic acid bacteria that allows them to inhibit the growth of A. naeslundii.

Regarding the first object of the present invention, this is related to the use of exogenous lactic acid bacteria that have been selected for their ability to adhere to the surface of the teeth and to produce a growth inhibition factor, for the preparation of a composition with which it is intended to treat or prevent diseases related to Actinomyces naeslundii in mammals.

Lactic acid bacteria can be selected from the group consisting of Streptococcus thermophilus, Lactococcus lactis subsp. lactis, and Lactococcus lactis subsp. lactis biovar diacetylactis, and in particular from the group consisting of the Streptococcus thermophilus strains (NCC 1529) (CNCM I-1984); Streptococcus thermophilus (nCc 1561) (CNCM I-1985), Lactococcus lactis subsp. lactis (NCC 2211) (CNCM I-1986), Lactococcus lactis subsp. lactis biovar dioacetylactis (NCC 2225) (CNCM I-1987).

Lactic acid bacteria are preferably of dairy origin (ie those obtained from milk or cheese, for example).

Lactic acid bacteria according to the invention result in "reduced acidification", which means that they are less acidifying than pathogenic strains. Therefore, they can contribute to a pH in the oral cavity of around 5.5-7.

These strains have been selected from among lactic bacteria strains for their ability to adhere to the film of the teeth and their optimal growth temperature of around 37 ° C, which is the temperature of the oral cavity. They are also capable of producing a growth inhibition factor, which, combined with their adhesion properties, allows them to significantly decrease the extent of colonization by A. naeslundii genospecies 1 and 2.

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

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

The lactic acid bacteria strains can be included in a food, pet food, cosmetic or pharmaceutical composition, for example. Accordingly, these compositions are preferably toothpastes, mouthwashes, chewing gums, sprays, drinks, candies, infant formulations, ice creams, frozen desserts, sweet salad dressings, dairy preparations, cheeses, quark, yogurt, acidified milks, cream. for coffee or whipped cream, for example.

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

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The effect of incorporating the aforementioned bacteria into the oral microflora was tested in a rat model. The CNCM I-1985 and CNCM-1986 strains were able to modulate oral microbial ecology, significantly reducing the number of total CFUs. More specifically, the strains were able to significantly reduce the extent of colonization of A. naeslundii genospecies 2, with which the rats had become infected (see examples).

Biochemical characterization of the selected strains

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

TABLE 1

Fermentation of sugars from L. lactis CNCM I-1987 (A), L. lactis CNCM I-1986 (B), S. thermophilus CNCM I-1984 (C), S. thermophilus CNCM I-1985 (D)

<td>Sugar</td><td>TO</td><td>B</td><td>C</td><td>D</td><td>Sugar</td><td>TO</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>Esculin</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-Lixosa</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>Sweet</td><td></td><td></td><td></td><td></td>

<td>Arbutin</td><td> +++</td><td> +++</td><td></td><td></td><td>Melibiosa</td><td></td><td></td><td></td><td></td>

<td>Cellobiose</td><td> +++</td><td> ++++</td><td></td><td></td><td>a-Methyl-D-</td><td></td><td></td><td></td><td></td>

<td>Dulcitol</td><td></td><td></td><td></td><td></td><td>glycoside</td><td></td><td></td><td></td><td></td>

<td>Erythritol</td><td></td><td></td><td></td><td></td><td>a-Methyl-D-</td><td></td><td></td><td></td><td></td>

<td>D-Fructose</td><td> +</td><td> ++++</td><td></td><td></td><td>mannoside</td><td></td><td></td><td></td><td></td>

<td>D-Fucosa</td><td></td><td></td><td></td><td></td><td>D-Raffinose</td><td></td><td></td><td></td><td></td>

<td>L-Fucosa</td><td></td><td></td><td></td><td></td><td>Ramnosa</td><td></td><td></td><td></td><td></td>

<td>Galactose</td><td> ++</td><td> ++++</td><td></td><td></td><td>Ribose</td><td> ++</td><td> ++</td><td></td><td></td>

<td>β-Gentiobiose</td><td> +++</td><td></td><td></td><td></td><td>Salicin</td><td> +++</td><td> +++</td><td></td><td></td>

<td>Gluconate</td><td></td><td></td><td></td><td></td><td>Sorbitol</td><td></td><td></td><td></td><td></td>

<td> 2-</td><td></td><td></td><td></td><td></td><td>L-Sorbose</td><td></td><td></td><td></td><td></td>

<td>Ketogluconate</td><td></td><td></td><td></td><td></td><td>Starch</td><td></td><td></td><td></td><td></td>

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<td>5- Ketogluconate GlcNAc D-Glucose</td><td> + +</td><td> ++++ ++++</td><td> +</td><td> ++</td><td>Saccharose D-Tagatose Trehalose 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-Methyl-</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td>xyloside</td><td></td><td></td><td></td><td></td>

+, ++, +++, ++++ shows if fermentation starts after 3, 6, 24 or 48 hours.

The strains of Streptococcus thermophilus (NCC 1529), Streptococcus thermophilus (NCC 1561), Lactococcus lactis subsp. lactis (NCC 2211), Lactococcus lactis subsp. lactis biovar dioacetylactis (NCC 2225) were deposited on March 3, 1998, under the Budapest Treaty, in the National Collection of Microorganism Cultures (CNCM I1984, CNCM I-1985, CNCM I-1986 and CNCM I-1987, respectively ), 25 rue du docteur Roux, 75724 Paris, France.

The second main object of the present invention is related to a composition for the maintenance of oral health by reducing the colonization of A. naeslundii in mammals, and said composition comprises exogenous lactic acid bacteria, which have been selected for their ability to adhere to the film of the teeth and to produce a growth inhibiting factor.

These compositions are particularly indicated in the prophylaxis or treatment of dental plaque and diseases related to infection by A. naeslundii, such as root caries, for example.

The lactic bacteria strain according to the present invention is selected from the group consisting of Streptococcus thermophilus, Lactococcus lactis subsp. lactis, and Lactococcus lactis subsp. lactis biovar diacetylactis, and preferably from the group consisting of the strains CNCM I-1984, CNCM I-1985, CNCM I-1986 and CNCM I1987.

Such a composition can contain at least 10⁴-10⁹ CFU / g of lactic acid bacteria.

Synergistic compositions can also be prepared by adding at least one bacteriocin, which is active against oral Gram positive bacteria. In that case, the oral hygiene compositions may comprise 0.00001 to 50%, and preferably 0.00001 to 15% of purified bacteriocin, by weight of the composition. The bacteriocin is preferably variacin (PE 0 759 469).

To protect the composition from degradation, an oil soluble antioxidant can also be included. Suitable antioxidants include "tocopherols", butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), and ascorbyl palmitate.

The oil soluble antioxidant is present in amounts of between 0.005% and 0.5%, preferably 0.005% to 0.01% by weight of the composition.

Suitable abrasives for use in the dentifrice compositions of the present invention include calcium carbonate, calcium aluminosilicate, alumina, hydrated alumina, zinc orthophosphate, plastic particles, and silica, of which silica is the preferred abrasive.

The compositions according to the invention will have a pH that is acceptable at the oral level and in which the activity of said lactic acid bacteria is not compromised. The pH can be in the range of 3.0-9.5, preferably in the range of 3.5 to 6.5.

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These compositions can be prepared by conventional processes, which comprises mixing the ingredients in the appropriate relative amounts and finally, and if necessary, adjusting the pH to the desired value.

Actinomyces naeslundii genospecies 1 (previously A. naeslundii) and 2 (previously A. viscosus) are among the strongest plaque-forming oral strains. These are commonly isolated from root caries sites, particularly in humans over 40 years of age, and are believed to be the main etiologic agent of this disease.

Example 1

In-vitro tests

The S. thermophilus strains NCC1561 (CNCM I-1985) and L. lactis subsp. lactis NCC2211 (CNCM I-1986) (hereinafter L. lactis NCC2211) were incorporated in vitro into a biofilm that mimics dental plaque in vitro.

The oral strains A. naeslundii genospecie 1 (previously A. naeslundii) OMZ745 and A. naeslundii genospecie 2 (previously A. viscosus) OMZ105 were obtained from the Institute für Orale Mikrobiologie und Allgemeine Immunologie, University of Zürich, and were cultured in FUM medium in anaerobiosis (GasPackSystem, BBL) at 37 ° C.

All strains were stored in glycerol at -20 ° C and precultured for 14 hours before use at their specific optimum temperature.

The two selected strains L. lactis NCC2211 and S. thermophilus NCC1561 were inoculated in an in vitro system in which a biofilm, composed of bacteria commonly found in the human mouth after 40 years, is generated on saliva-coated hydroxyapatite discs. . The universal fluid medium (FUM), the growth medium used, was specially formulated to buffer the acidity produced by the test strains and thus achieve continued growth (plaque development), as if they were in the mouth (Gmur and Guggenheim, 1983). The tests were carried out in triplicate and the mixtures, with and without the dairy strains, were tested in parallel. The strains listed in Table 2 were used.

TABLE 2

Bacterial strains used and culture conditions used in in vitro dental plaque experiments

<td>Strain</td><td>Properties relevant</td><td>Conditions of growth</td>

<td>S. thermophilus NCC1561</td><td>adherent to S-HA</td><td>FUM, Belliker;</td>

<td></td><td></td><td>37 ° C</td>

<td>L. lactis subsp. lactis</td><td>adherent to S-HA</td><td>FUM, M17-</td>

<td>NCC2211</td><td></td><td>lactose; 37 ° C</td>

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

<td>S. oralis OMZ607</td><td>plate former</td><td>FUM; 37 ° C</td>

<td>A. naeslundii OMZ745</td><td>plaque former, causative agent cavities in estate</td><td>FUM; 37 ° C</td>

<td>Dispar V. OMZ493</td><td>plate former</td><td>FUM; 37 ° C</td>

<td>F. nucleatum OMZ596</td><td>plate former</td><td>FUM; 37 ° C</td>

Process

- Formation of the saliva film: cover the 10 mm diameter synthetic hydroxyapatite discs (HYAPATITE®, Euro-Crystals, Landgraaf, The Netherlands) with 800 gl of human saliva and incubate for 4 h at room temperature while shaking (1 disc / well in a sterile 24-well Nunclon plate).

- Preparation of the bacterial association: cultivar S. thermophilus NCC1561, L. lactis subsp. lactis NCC2211, S. sobrinus OMZ176, S. oralis OMZ607, A. naeslundii OMZ745, V dispar OMZ493 and F. nucleatum OMZ596 overnight at 37 ° C in anaerobic in FUM-glucose (S. thermophilus NCC1561 in FUM-lactose) , adjust the DO_55oup to 1 with FUM and pool 2 ml of each of the oral bacteria suspensions with 2 ml of S. thermophilus NCC1561 or L. lactis subsp. lactis NCC2211. The control mix contains only the five oral strains.

- Biofilm formation and recovery: the procedure is as described in Guggenheim et al., 1998, Validation of a new biofilm model. J. Dent. Res., 77, (Spec Iss A): 110 (Abstract No. 38).

- Biofilm culture analysis: Spread the suspension on Columbia blood agar (5% sheep blood, Becton Dickinson, Meylan Cedex, France) for the total count and for the differentiation of A. naeslundii. Incubate the plates at 37 ° C in anaerobiosis for 48 h.

Growth antagonism between the oral and dairy strains of study

The strains and the culture conditions used are listed in Table 3.

<td>Strain</td><td>Properties relevant</td><td>Conditions of growth</td>

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

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

<td>S. thermophilus NCC1536</td><td>non-adherent</td><td>Belliker; 37 ° C</td>

<td>A. naeslundii OMZ745</td><td>plate former</td><td>BHI; 37 ° C, anaerobiosis</td>

<td>A. viscosus ΘΜΖ105</td><td>plate former</td><td>BHI; 37 ° C</td>

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Growth antagonism was tested between S. thermophilus NCC1561, S. thermophilus NCC1536 and L. lactis NCC2211 (killer strains) and A. naeslundii OMZ745 and A. viscosus OMZ105 (target strains).

Process

- Grow the killer strains overnight on agar plates in anaerobiosis and the target strains in BHI until reaching the stationary phase

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

- Solidify for 1 h at room temperature, then sow the killer strain from plate M17 in a cross shape. Seed in parallel only the killer and target strain as a control

- Incubate at 37 ° C in anaerobiosis for 24 hours.

Growth antagonism is detected by a halo of inhibition around the withers.

Statistics

Differences between controls and test associations were determined by Student's t-test. Results and Discussion

S. thermophilus NCC1561 and L. lactis NCC2211 can be incorporated and grown on the plate-like biofilm of S-HA discs, and their total CFU / disc after 40.5 h are provided in Table 4.

TABLE 4

Level of incorporation of the two dairy strains in the biofilm (CFU / disc). Values ​​are a mean of three experiments with their standard deviation

<td>Inoculation method</td><td>S. theuaophilus NCC1561 (xlO⁶)</td><td>i. lactis NCC2211 (xlO⁶)</td>

<td>Along with the strains oral</td><td> 4,08 +/- 1,78</td><td> 5,76 +/- 3,64</td>

<td>Before the strains oral</td><td> 5,03 +/- 2,21</td><td> 3,87 +/- 4,01</td>

The effect of the incorporation of the dairy strains in the biofilm on the oral species is indicated in Tables 5 and 6. When S. thermophilus NCC1561 (Table 5) is included, a general reduction of the total flora is observed, which is represented by the count in Columbia blood agar plates (ASC), and of 4 of the oral species.

When introducing L. lactis NCC2211 in the association of oral strains (Table 6), the total flora count decreased notably (CFU in ASC). The reduction was significant in the case of A. naeslundii OMZ745, which decreased significantly (p = 0.021). The reduction was even greater if the strain was inoculated on the discs before the oral bacteria.

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TABLE 5

Modulation of oral strain associations by S. thermophilus NCC1561 (CFU / disk)

<td>Treatment</td><td>ASC (x 10⁸)</td><td>A. naeslundii ΟΜΖΊ45 (x 10⁶)</td><td>MS (x 10⁸)</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 +/- 0,38°° 2,32 +/- 0,38°°</td><td> 4,78 +/- 2,29 4,78 +/- 2,29</td><td> 1,48 +/- 0,29 1,48 +/- 0,29</td>

<td colspan="4">N = 3. *: p-values ​​calculated with respect to the control (*: p &lt;0.05; **: p &lt;0.01); °: p-values ​​calculated with respect to treatment ”+ NCC1561 (°: p &lt;0.05; °°: p &lt;0.01).</td>

TABLE 6

Modulation of the associations of oral strains by L. lactis NCC2211 (CFU / disk)

<td>Treatment</td><td>ASC (x 10⁸)</td><td>A. naeslundii OMZ745 (x 10⁶)</td><td>MS (x 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 +/- 0,33</td>

<td>Pre-incubation with NCC2211</td><td> 0,27 +/- 0,11**°</td><td> 3,91 +/- 3,2*° 3,91 + /- 3,29*°°</td><td> 0,27 +/- 0,11**°°</td>

<td colspan="4">N = 3. *: p-values ​​calculated with respect to the control (*: p &lt;0.05; **: p &lt;0.01); p-values ​​calculated with respect to treatment + NCC2211 (°: p &lt;0.05; °°: p &lt;0.01).</td>

Some tests were carried out to verify if the reduction of the oral strains was due to a growth antagonism of the dairy strains towards them (Table 7). The strains A. viscosus OMZ105 and S. thermophilus NCC1536 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 lactic acid production. A. viscosus is capable of metabolizing lactate only under conditions

ES 2 275 697 T3 aerobic (van der Hoeven et al., (1990) Oral Microbiol. Immunol., 5, 223-225) and is highly aciduric. These findings have been confirmed by seeding A. viscosus in the presence of 1% lactic acid, and no inhibition was observed.

TABLE 7

Growth inhibition of oral strains by S. thermophilus NCC1561, S. thermophilus NCC 1536 and L. lactis NCC211

<td rowspan="2">Diana</td><td colspan="3">KILLER STRAINS</td><td rowspan="2">acid lactic 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 were able to be incorporated into a biofilm that mimics dental plaque and were able to modulate the oral microflora, significantly reducing the total number of CFUs, and more specifically, these strains were able to significantly reduce the spread of colonization of A. naeslundii genospecies 2. Furthermore, the strains were able to inhibit the growth of A. naeslundii genospecies 1 and 2 in cocultures.

Example 2

In vivo tests

An in vivo study was carried out in a rat model. In this study, the association of the selected strains continued on a daily basis during the entire experimental period, by supplying a cold dairy product.

The study took 58 days to complete. To carry out the experiment during the day, the active period of the animals had to be advanced 7 hours in total; this was done in three steps on day 16, 17 and 18 as described in more detail. The cariogenic strains were associated on days 21 and 22, while the association of dairy strains began on day 23 and was maintained until day 57. The animals were fed the dairy strains as a supplement in a yoghurt base that was included in their normal diet, as explained in the section. The rats' teeth were rubbed with a cotton swab at the end of the study, on day 58.

Animals and diet

Ten litters consisting of 4 Osborne-Mendel rat pups each were used in the experiment (animal production section of the Institute für Orale Mikrobiologie und Allgemeine Mikrobiologie, University of Zurich, Zurich, Switzerland). All animals were weighed at the beginning and at the end of the experimental period. When they were 13 days old, mothers and calves were transferred to stainless steel mesh-bottom cages without substrate and fed a low-fluoride powdered Nafag diet (0.2 jum) to avoid impacts on fissures (Rat Checkers n ° 184, NAFAG, Gossau SG, Switzerland), and tap water at will. The active phase during which the rats eat is during the night, that is, from 18:00 - 06:00.

In order to be able to refill the food containers during normal working hours, the circadian biorhythm was modified sequentially between days 16 and 18 by advancing the active phase of the rats each day on three occasions by an automatic adjustment of the light control. .

On 16/17, the start of the active period was advanced from 6:00 p.m. to 3:00 p.m., that is, it was at night from 3:00 p.m. to 3:00 a.m. and it was daytime from then on. On 17/18, the start of the active period was advanced from 3:00 p.m. to 12:00 p.m., that is, it was at night from 12:00 h - 00:00 h and during the day from 00:00 h. onwards.

Finally, on the 18/19, the beginning of the active period was advanced from 12:00 to 10:00, that is, it was at night from 10:00 a.m. to 10:00 p.m. and during the day from 10:00 p.m. onwards .

Therefore, on day 19 the change of the active phase of the rats from the hours of darkness to the normal working hours (10:00 - 22:00 h) had been completed.

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On day 20 the mothers were withdrawn, and the rats were fed ad libitum with the modified diet 2000a containing 40% sucrose, 28% skim milk substitute (soy protein extract SVPROPP 1611 to 39, 4%, 49.3% lactose, 0.6% L-methionine, 0.3% L-lysine, 0.1% HCl), 24% wheat flour, 5% brewer's yeast, 2% Gevral® instant protein (Whitehall-Robins SA, 6301 Zug, Switzerland) and 1% NaCl.

During the association period (days 21 and 22) the drinking water was supplemented with 2% glucose and 2% sucrose to support the implantation of the associated bacteria. On day 23 the litters were distributed in the 3 treatments, 1 animal per cage, in a programmed feeding equipment and they began to receive the test diet as indicated in Table 10. The test diet consisted of 18 yogurt meals containing the test strains, alternating with 18 previously described 2000a modified diet meals.

Drinking water was supplied ad libitum. Following the cotton swab rubbing procedure on day 58, animals were overdosed with thiopental sodium (100 mg / kg body weight) by intraperitoneal injection and decapitated when comatose.

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

TABLE 8

Bacterial strains that were used in this study

<td>Strain</td><td>Relevant properties</td><td>Conditions of growth</td>

<td>S. thermophilus NCC1561</td><td>adherent S-HA</td><td>Belliker; 42 ° C</td>

<td>S. thermophilus NCC1536</td><td>control no</td><td>Belliker; 42 ° C</td>

<td></td><td>adherent</td><td></td>

<td>L. lactis NCC2211</td><td>adherent S-HA</td><td>M17-lactose;</td>

<td></td><td></td><td>37 ° C</td>

<td>A. viscosus OMZ105</td><td>plate former,</td><td>BHI; 37 ° C</td>

<td></td><td>adherent S-HA</td><td>BHI, 37 ° C</td>

Preparation of the laboratory strains tested in the association

A preliminary study was carried out to assess the growth parameters, especially the hours required to reach the stationary phase under the specific conditions described. Thus, the cultivation of S. thermophilus strains was established for 7 h and that of L. lactis for 6 h. A study of the viability after freezing of the dairy strains was also carried out by seeding the same cell suspension before and after freezing. To associate them with the animals, the dairy strains were treated according to the following procedure.

Process

- Inoculate the 1% strains overnight in an appropriate medium from a glycerol reserve

- Inoculate them at 5% from that culture in 10 culture containers of 4 liters of appropriate medium pre-heated to the optimal growth temperature, and cultivate them until the end of the logarithmic phase / beginning of the stationary phase.

- Determine the final CFU / ml by sowing them on M17-lactose agar from two containers chosen at random for each of the 4 strains. Incubate the plates overnight in anaerobiosis.

- Centrifuge the cultures in each container at 6000 rpm for 10 min. and resuspend the buttons in 150 ml of fresh medium; keep overnight at 4 ° C.

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- Centrifuge again and resuspend in freezing medium (15% glycerol in Belliker or M17).

- Distribute in aliquots so that there are 2 x 10¹¹ viable cells / vial, taking into account the loss of viability due to freezing, and store at -20 ° C until necessary.

Association of animals with bacterial strains

The animals were distributed in the 3 treatments (Table 9). Each treatment was carried out on 10 pups that were distributed 1 per cage.

TABLE 9

Distribution of 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 initially infected on days 21 and 22 with A. viscosus OMZ105.

The tested laboratory strains were associated on a daily basis (since they were included in the yogurt-based meal), from day 23 on. 2 frozen vials were mixed, each with 2 x 10¹¹ viable cells of the test strain, in 200 ml of yogurt to obtain at least 10⁹ CFU / ml. S. thermophilus NCC1536, a strain that does not adhere to S-HA, was used as a negative control.

Yogurt and diet 2000a meals, 1 ml and 400 mg respectively, were offered alternatively 18 times a day at 20 min intervals. (Table 10). Therefore, each animal received a total of 18 ml of yogurt and 7.2 g of powdered diet.

The meals were dispensed into the feeding containers of a programmed feeding equipment that automatically offered the animals the correct food at the exact moment.

TABLE 10

Feeding guideline

<td>No. of food</td><td>Meals highly cariogenic</td><td>Yogurt meals</td><td>No. of food</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>

ES 2 275 697 T3

<img file="ES2275697T3_D0001.tif" />

Bacteriological evaluation

On day 58, five rats per treatment were swabbed with a cotton swab. Smear suspensions were seeded in Petri dishes for counting CFU (colony forming units) or immobilized on immunofluorescence slides for counting NCT (number of total cells).

Procedure for determining CFUs

- Rub the teeth of the rats with a sterile cotton swab and immediately place it in 5 ml of sterile 0.9% NaCl

- Vortex for 1 min. and sonicate for 5 s at 50 W.

- Seed in a spiral the suspensions diluted adequately in ASC, MS and HJL agar.

- Incubate the ASC and MS plates at 37 ° C and the HJL plates at 45 ° C.

Procedure for the determination of NCT

- Place 10 gl of the undiluted smear suspension prepared for CFU determination in each well of a 24-well glass slide (Dynatech Produkte AG, Embrach Embraport, Switzerland), and allow to air dry.

- Fix by immersion in methanol for 2 min. and let it air dry.

- Incubate with 10 gl of the appropriate antibody or serum diluted in ELISA buffer (section 4.2.2.5) and incubate at 37 ° C for 30 min.

- Aspirate each drop from the wall of the well and wash by immersing the slide, first in ELISA buffer and then in distilled water. Air dry.

- Apply 10 gl of goat anti-rabbit IgG (H + L) -FITC (Sigma) diluted 1: 400 and incubate at 37 ° C for 30 min.

- Wash as before and air dry.

- Apply 49 ml of mounting solution (section 4.2.2.5), cover with a coverslip and count the fluorescent cells with a fluorescence microscope.

Statistics

The data were treated with a bidirectional ANOVA (Snedecor and Cochran, 1980).

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Results of the continuous association of dairy strains by feeding a cold dairy product. Bacteriological evaluation (Table 11)

- Colonization of the strain. When the dairy product was supplemented with the non-adherent control S. thermophilus (NCC1536), 1.7 (+/- 1.1) x 10 were counted⁷ CFU of A. viscosus OMZ105 plaque former. Dairy strains could not be counted by microbiological methods. However, a qualitative evaluation was attempted by immunofluorescence. The three adherent milk strains could be recognized in the plaque samples from treatments 2 and 3. It was impossible to make a precise quantification, because they were co-aggregated with other oral bacteria and oral waste, thus generating large aggregates.

- Variations in the total flora (FT). The three treatments, containing the tested adherent strains S. thermophilus NCC1561 and L. lactis NCC2211, showed a significant reduction in the number of colony-forming units on ASC compared to the control group containing the non-adherent strain S. thermophilus NCC1536 (Table 11). Treatment 2 reduced the CFU count by a significant factor P_F&lt;0.01 and treatment 3 even more significantly (P_F&lt;0,001).

- Modulation of dental colonization by A. viscosus OMZ105 by the tested strains.

In the case of the plaque-forming bacterium A. viscosus OMZ105, an apparently less pronounced but more significant decrease in the number of colony-forming units was observed with the three treatments containing the adherent strains tested compared to treatment 1 (P_F&lt;0.01) (Table 11). In contrast, the percentages of A. viscosus on the total CFU count were not significantly different. Approximately 50% of the total CFUs in the four treatments were identified as colonies of A. viscosus.

TABLE 11

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

<td>Treatment</td><td>FT about ASC x 10⁶</td><td>OMZ105 about ASC xlO⁶</td><td>% OMZ105 about ASC</td><td>FT about MS x 10⁴</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> 8,0+/- 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 ★ go ★</td><td> 5,8+/- 2.07 **</td><td>43.4 +/- 18.16 ns</td><td>1.7 +/- 1.11 ns</td>

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

<td>Pf</td><td> 0,001</td><td> 0,01</td><td>ns</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>

<td colspan="5">Treatments 2-3 were compared with treatment 1.</td>

<td colspan="2">SEM = standard error of</td><td>average; ns</td><td colspan="2">. = not significant.</td>

<td colspan="5">ASC = Columbia Blood Agar; MS = Mitis-salivarius agar.</td>

<td colspan="3">OMZ105: Genospecies 2 of A.</td><td>naeslundii</td><td></td>

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In this in vivo test, the strains that were supplied daily showed a clear inhibitory effect on the total microflora, whose CFU decreased significantly. This decrease can be explained by the growth antagonism of the dairy strains against the oral species. For example, all of them, including S-HA non-adherent S. thermophilus NCC1536, can inhibit the growth of A. viscosus OMZ105 in vitro. However, in vivo such an effect is only present in strains adherent to S-HA, as was detected in treatments 2-4 compared to the first.

Specifically, as the animals were infected with A. viscosus OMZ105, quantification of these plaque-forming organisms was possible at the end of the experimental period, and the decrease in their CFU could be carefully monitored.

The percentages of A. viscosus OMZ105 on the total CFU do not decrease in parallel, so it can be deduced that the growth antagonism effect also occurs against other species, that is, Veillonellae, and consequently what is observed is a overall effect.

Thus, the CNCM I-1985 and CNCM-1986 strains are capable of modulating the oral microbial ecology, significantly reducing the colonization of A. naeslundii genospecies 2, with which the rats had been infected. Example 3

Production and initial analysis of the surfactant substances of S. thermophilus NCC1561 and S. thermophilus NCC1536

S. thermophilus NCC1561 and S. thermophilus NCC1536 were grown overnight in 1 L of Belliker at 42 ° C. For the production of biosurfactant, the procedure described in Busscher et al., (1997) Appl. Environ. Microbiol., 63, 3810-3817, (Busscher et al., 1997).

Preparation of surfactant substances

Process

- Wash the cells three times with PBS.

- Resuspend in 200 ml of distilled water or PBS.

- Produce the biosurfactant by gently shaking the suspension for 2 or 4 h at room temperature.

- Separate the bacteria by centrifugation at 10,000 rpm for 10 min.

- Centrifuge the supernatant twice at 10,000 rpm for 10 min.

- Freeze dry and weigh both the button and the surfactant substance solutions.

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

Procedure for SDS-PAGE

SDS-PAGE was carried out with a ready-to-use 12.5% ​​ExcelGel (Amersham Pharmacia Biotech). Silver staining was performed with the Plusone silver stainer kit (Amersham Pharmacia Biotech).

Procedure for measuring surface tension

The surface tension of the biosurfactant suspensions was measured with a TVT1 drop volume tensiometer (Lauda, ​​Lauda-Konigshofen, Germany), which is based on the one drop volume principle. In summary, the method consists of the exact determination of the volume of a drop of a suspension that separates from a capillary. This volume (critical volume) is proportional to the surface tension (s), whose value is calculated with the relation:

σ = V g Δρ F / 2 nr_chap in which:

- σ is the interfacial tension

- V is the volume of the drop

- G is the acceleration constant

ES 2 275 697 T3

- Δρ is the difference of the density is of both adjacent phases

- F is the correction factor

- r_chap is the radius of the capillary.

Measurements were made in duplicate at 37 ° C in air. Each measurement consisted of 10 cycles. A 6 mg / ml solution of released crude product reduced the surface tension of the water from 70 to 51 mN / m (Table 13). The SDS-pAge profile of the released bacterial products showed that there were many different substances of a protein nature in the solution.

TABLE 13

Surface tension values ​​of biosurfactant suspensions compared to water and PBS. Values ​​are the average of two experiments, each consisting of ten measurements

<td></td><td>Concentration of crude extract</td><td>Surface tension (mN / m)</td>

<td>Water</td><td> -</td><td> 69,07 +/- 0,01</td>

<td>PBS</td><td> -</td><td> 68,13 +/- 0,30</td>

<td>S. thermophilus NCC1561</td><td>6 mg / ml</td><td> 51,47 +/- 0,17</td>

<td>S. thermophilus NCC1536</td><td>6 mg / ml</td><td> 51,67 +/- 1,32</td>

Results

The cells of S. thermophilus NCC1561 and S. thermophilus NCC1536 are capable of releasing substances with surfactant activity. Therefore it is possible that the biosurfactant produced by S. thermophilus NCC1561 causes the bacteria itself and other nearby oral strains to separate from the tooth surface. On the contrary, this action is not observed in S. thermophilus NCC1536, since this strain does not adhere to the teeth.

Example 4

Toothpaste

Toothpaste is prepared by adding 10^{5 * *} cfu / ml of at least one of the lactic acid bacteria strains CNCM I1984, CNCM I-1985, CnCm I-1986 or CNCM I-1987 in lyophilized form, to the following mixture containing: 1.65% cetyl pyridinium chloride , 33.0% sorbitol (70% sol.), 25.0% glycerin, 2.0% sodium carboxymethylcellulose, 0.25% sodium fluoride, 26.3% silicon (RP 93), 8 , 1% thickener silicon (Sident 22), 0.5% sodium saccharin, 3.2% poloxamer (Pluronic F108).

This toothpaste is intended to be used for the prophylaxis or treatment of root caries, dental plaque, and other infections induced by A. naeslundii species.

Example 5

Yogurt µl of MRS culture medium were sterilized for 15 min. at 121 ° C and then inoculated with 5% by volume of an active culture of at least one of the S. thermophilus strains CNCM I-1984 or CNCM I-1985 containing approximately 10⁹ cfu / ml. After incubation for 8 h at 41 ° C, a starting culture of 4.5 x 10 was obtained.⁸ cfu / ml.

1 of reconstituted skim milk with a dry matter content of 10%, to which 0.1% yeast extract has been added, were sterilized for 15 min. at 121 ° C and inoculated with 2% of a commercial thickening Streptococcus thermophilus active culture containing approximately 10⁹ cells / ml. After incubation for 4 h at 41 ° C, a starter culture was obtained containing 4.5 x 10⁸ cells / ml.

ES 2 275 697 T3

A batch of whole milk containing 3.7% fat reinforced with 2.5% skim milk powder and then pasteurized for 30 min. at 90 ° C, it was inoculated with 2% by volume of the starter culture of at least one of the CNCM I-1984 or CNCM I-1985 strains, and 3% by volume of the thickening Streptococcus thermophilus starter culture. The inoculated milk was shaken, poured into containers and incubated for 4 h at 41 ° C.

The yogurt obtained has a good firmness and a smooth texture and is intended for oral hygiene.

Example 6

Chewing gum

A chewing gum can be prepared to prevent or treat root caries, dental plaque or other diseases related to A. naeslundii, by adding an active culture of at least one of the S. thermophilus strains CNCM I-1984 or CNCM I -1985, containing approximately 10⁴ to 10⁹ cfu / g, to the following typical ingredients: 67.5% xylitol, 20% gum base, 5% calcium carbonate, 3% glycerin, 2% Pluronic F127, 1% cellulose gum, 0.5 % Balast compounds and 1% flavoring.

Example 7

Composition for pet food

A food for the oral hygiene of domestic animals was obtained by preparing a food mixture made of corn, food based on corn gluten, chicken and fish, salts, vitamins and minerals. The food mixture is placed in a preconditioner and moistened. The moistened food coming out of the preconditioner is then placed in an extruder pot and gelatinized. The gelatinized matrix exiting the extruder is forced through a die and extruded. The extrudate is cut into pieces suitable for dog food, dried at about 110 ° C for about 20 minutes, and cooled to form buttons having a water activity of about 0.6.

Buttons are sprayed with 3 coating mixes. Each coating mix contains active culture of at least one of the S. thermophilus strains CNCM I-1984 or CNCM I-1985, but one of the coating mixes uses hydrogenated soybean fat as the coating substrate, another coating mix uses water as the coating substrate, and the other coating mixture uses digested protein as the coating substrate. The buttons contain approximately 10⁴ to 10⁹ cfu / g of said strains.

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<td>ro II has been proven the microorganism³It was viable³Π It was not feasible</td><td>March 3, 1998 ² On this date the</td>

1. Indicates the initial deposit date or, if a new deposit or transfer has been made, the most recent of the corresponding dates {date of new deposit or transfer date)

2. In the cases according to rule 10.2.a) ii) and iii), mention the most recent viability control

3. Choose the most convenient case.

ES 2 275 697 T3

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF DEPOSIT

OF MICROORGANISMS IN ORDER TO PROCEED YOUR PATENT

INTERNATIONAL FORMULA

DECLARATION ON FEASIBILITY, delivered in accordance with rule 10.2 by the INTERNATIONAL DEPOSIT AUTHORITY, identified on the next page

RECIPIENT: Monsieur F. GROS Patents Department

SOCIETE DES PRODUITS NESTLE SA Avenue Nestlé 55 CH-1800 VEVEY

<td>I. DEPOSITARY</td><td>II. IDENTIFICATION OF THE MICROORGANISM</td>

<td>NAME: SOCIETE DES PRODUITS NESTLE SA ADDRESS: Patents Department Avenue Nestlé 55 CH-1800 VEVEY</td><td>Order number assigned by the INTERNATIONAL DEPOSIT AUTHORITY 1-1985 Date of deposit or transfer¹: March 3, 1998</td>

<td colspan="2">III. DECLARATION OF FEASIBILITY</td>

<td colspan="2">The viability of the microorganism identified under number II has been verified on March 3, 1998 ² On this date the microorganism³It was viable³Ü It was not feasible</td>

1. Indicates the initial deposit date or, if a new deposit or transfer has been made, the most recent of the corresponding dates (new deposit date or transfer date)

2. In the cases according to rule 10.2.a) ii) and iii), mention the most recent viability control

3. Choose the most convenient case.

ES 2 275 697 T3

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF DEPOSIT

OF MICROORGANISMS IN ORDER TO PROCEED YOUR PATENT

INTERNATIONAL FORMULA

DECLARATION ON FEASIBILITY, delivered in accordance with rule 10.2 by the INTERNATIONAL DEPOSIT AUTHORITY, identified on the next page

RECIPIENT: Monsieur F. GROS Patents Department

SOCIETE DES PRODUITS NESTLE SA Avenue Nestlé 55 CH-1800 VEVEY

<td>I. DEPOSITARY</td><td>II. IDENTIFICATION OF THE MICROORGANISM</td>

<td>NAME: SOCIETE DES PRODUITS NESTLE SA ADDRESS: Patents Department Avenue Nestlé 55 CH-1800 VEVEY</td><td>Order number assigned by the INTERNATIONAL DEPOSIT AUTHORITY 1-1986 Date of deposit or transfer¹: March 3, 1998</td>

<td colspan="2">III. DECLARATION OF FEASIBILITY</td>

<td colspan="2">The viability of the microorganism identified under number II has been verified on March 3, 1998 ² On this date the microorganism³It was viable³Π It was not feasible</td>

1. Indicates the initial deposit date or, if a new deposit or transfer has been made, the most recent of the corresponding dates (new deposit date or transfer date)

2. In the cases according to rule 10.2.a) ii) and iii), mention the most recent viability control

3. Choose the most convenient case.

ES 2 275 697 T3

BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF DEPOSIT

OF MICROORGANISMS IN ORDER TO PROCEED YOUR PATENT

INTERNATIONAL FORMULA

DECLARATION ON FEASIBILITY, delivered in accordance with rule 10.2 by the INTERNATIONAL DEPOSIT AUTHORITY, identified on the next page

RECIPIENT: Monsieur F. GROS Patents Department

SOCIETE DES PRODUITS NESTLE SA Avenue Nestlé 55 CH-1800 VEVEY

<td>I. DEPOSITARY</td><td>II. IDENTIFICATION OF THE MICROORGANISM</td>

<td>NAME: SOCIETE DES PRODUITS NESTLE SA</td><td>Order number assigned by the INTERNATIONAL DEPOSIT AUTHORITY</td>

<td>ADDRESS: Patents Department Avenue Nestlé 55</td><td> 1-1987</td>

<td>CH-1800 VEVEY</td><td>Date of deposit or transfer¹: March 3, 1998</td>

<td colspan="2">III. DECLARATION OF FEASIBILITY</td>

<td colspan="2">The viability of the microorganism identified under the number</td>

<td>ro II has been proven the microorganism³It was viable³O It was not feasible</td><td>March 3, 1998 ² On this date the</td>

1. Indicates the initial deposit date or, if a new deposit or transfer has been made, the most recent of the corresponding dates (new deposit date or transfer date)

2. In the cases according to rule 10.2.a) ii) and iii), mention the most recent viability control

3. Choose the most convenient case.