Incorporation of exogenous lactic bacteria into the oral microflora
18 claims: 2 independent, 16 dependent
- 1Zastrzeżenia patentowe 1. Zastosowanie bakterii mlekowych, wybranych z grupy składającej się ze szczepów CNCM I-1984, CNCM I-1985, CNCM I-1986, CNCM I-1987 i LMG P-18997, które nie są częścią mikroflory stale występującej w ustach, są mniej zakwaszające niż patogeniczne szczepy, powodując że pH w jamie ustnej wynosi 5,5 - 7 i są zdolne do przywierania bezpośrednio do błonki zębów, to jest wykazują procent przywierania do złoża hydroksyapatytów pokrytych śliną co najmniej 1,96, po przywieraniu w ciągu 45 minut w temperaturze 37°C, do wytwarzania kompozycji przeznaczonej do profilaktyki lub leczenia próchnicy zębów, płytki nazębnej i zakażenia okołozębowego.
- 2Zastosowanie według zastrz. 1, znamienne tym, że bakterie mlekowe są wybrane z grupy składającej się z:- zakwaszających bakterii mlekowych, które przywierają do błonki zębów i które zostały genetycznie zmodyfikowane tak, że są słabo zakwaszające;- nieprzywierających bakterii mlekowych, które są słabo zakwaszające i które zostały genetycznie zmodyfikowane tak, że przywierają do błonki zębów;- nieprzywierających zakwaszających bakterii mlekowych, które zostały genetycznie zmodyfikowane tak, że przywierają do błonki zębów i genetycznie zmodyfikowane tak, że są słabo zakwaszające.
- 3Zastosowanie według zastrz. 1 albo 2, znamienne tym, że kompozycja jest przeznaczona do usuwania patogenów zębów lub do zapobiegania ich przyczepianiu się.
- 4Zastosowanie według zastrz. 1 albo 2, znamienne tym, że bakterie mlekowe są pochodzenia mleczarskiego.
- 5Zastosowanie według zastrz. 1 albo 2, znamienne tym, że bakterie mlekowe przywierają do błonki zębów za pomocą czynników adhezyjnych.
- 6Zastosowanie według zastrz. 1 albo 2, znamienne tym, że bakterie mlekowe zostały genetycznie zmodyfikowane dla zwiększenia ich przywierania do błonki zębów i/lub genetycznie zmodyfikowane tak, aby były jeszcze mniej zakwaszające.
- 7Zastosowanie według zastrz. 1 albo 2, znamienne tym, że kompozycja jest kompozycją jadalną zawierającą ilość bakterii mlekowych skuteczną dla profilaktyki lub leczenia próchnicy zębów, płytki nazębnej i zakażenia okołozębowego.
- 8Zastosowanie według zastrz. 7, znamienne tym, kompozycja zawiera co najmniej 10 4 - 10 9 cfu/g bakterii mlekowych.
- 9Zastosowanie według zastrz. 1 albo 2, znamienne tym, że bakterie mlekowe są połączone z mlekiem, fermentowanym mlekiem, pochodnymi mleka lub bakteriocyną.
- 10Zastosowanie według zastrz. 9, znamienne tym, że pochodne mleka są wybrane z dowolnych postaci kazeinoglikomakropeptydu, micelarnej kazeiny, fluorowanej micelarnej kazeiny lub podpuszczkowego mleka.
- 11Kompozycja zdrowotna do jamy ustnej, przeznaczona do profilaktyki lub leczenia próchnicy zębów, płytki nazębnej i zakażenia okołozębowego, zawierająca oprócz konwencjonalnych składników bakterie kwasu mlekowego, znamienna tym, że zawiera co najmniej jeden szczep bakterii mlekowych wybrany z grupy składającej się ze szczepów CNCM I-1984, CNCM I-1985, CNCM I-1986, CNCM I-1987 i LMG P-18997, które nie są częścią mikroflory stale występującej w ustach, są mniej zakwaszające niż PL 201 312 B1 patogeniczne szczepy, powodując, że pH w jamie ustnej wynosi 5,5 - 7 i są zdolne do przywierania bezpośrednio do błonki zębów.
- 12Kompozycja według zastrz.11, znamienna tym, że bakterie mlekowe zostały genetycznie zmodyfikowane dla zwiększenia ich przywierania do błonki zębów i/lub genetycznie zmodyfikowane tak, aby były jeszcze mniej zakwaszające.
- 13Kompozycja według zastrz.11 albo 12, znamienna tym, że bakterie mlekowe są wybrane z grupy skł adają cej się z:- zakwaszających bakterii mlekowych, które przywierają do błonki zębów i które zostały genetycznie zmodyfikowane tak, że są słabo zakwaszające;- nieprzywierających bakterii mlekowych, które są słabo zakwaszające i które zostały genetycznie zmodyfikowane tak, że przywierają do błonki zębów;- nieprzywierających zakwaszających bakterii mlekowych, które zostały genetycznie zmodyfikowane tak, że przywierają do błonki zębów i genetycznie zmodyfikowane tak, że są słabo zakwaszające.
- 14Kompozycja według zastrz. 11 albo 12, znamienna tym, że bakterie mlekowe zostały genetycznie zmodyfikowane tak, że przywierają do błonki zębów za pomocą czynników adhezyjnych i powodują ż e pH w jamie ustnej wynosi 5,5 - 7.
- 15Kompozycja według zastrz. 11 albo 12, znamienna tym, że zawiera ilość bakterii mlekowych skuteczną dla profilaktyki lub leczenia próchnicy zębów, płytki nazębnej i zakażenia okołozębowego.
- 16Kompozycja według zastrz.15, znamienna tym, że zawiera co najmniej 10 4 - 10 9 cfu/g bakterii mlekowych.
- 17Kompozycja według zastrz. 11 albo 12, znamienna tym, że bakterie mlekowe są połączone z mlekiem, fermentowanym mlekiem, pochodnymi mleka lub bakteriocyną .
- 18Kompozycja według zastrz.17, znamienna tym, że pochodne mleka są wybrane z dowolnych postaci kazeinoglikomakropeptydu, micelarnej kazeiny, fluorowanej micelarnej kazeiny lub podpuszczkowego mleka lub bakteriocyny.
Independent claims18
218 paragraphs in 10 sections, as filed
(12) PATENT DESCRIPTION (19) PL (11) 201312 (13) B1 (21) Application number: 345890 <sup>(13)</sup> (22) Filing date: 1997-07-26 (86) Date and number of the international application:
1999-07-26, PCT / EP99 / 05473 (87) Date and publication number of the international application:
February 24, 2000, WO00 / 09080 PCT Gazette No. 08/00 (51) Int.Cl.
A61K 35/74 (2006.01) A61P 1/02 (2006.01) A61K 8/99 (2006.01) A61Q 11/00 (2006.01) A23L 1/29 (2006.01) A23L 1/30 (2006.01)
Use of lactic bacteria for the preparation of an oral health composition and an oral health composition
<td></td><td>(73) The right holder of the patent:</td>
<td></td><td>SOCIETE DES PRODUITS NESTLE SA,</td>
<td>(30) Priority:</td><td>Vevey, CH</td>
<td>1998-08-12, EP 98202707.0</td><td>(72) Inventor (s):</td>
<td>(43) Application announced: 14.01.2002 BUP 02/02</td><td>Jean-Richard Neeser, Savigny, CH Bernhard Guggenheim, Erlenbach, CH Elena-Maria Comelli, Lausanne, CH Francesca Stingele, Lausanne, CH</td>
<td>(45) The grant of the patent was announced:</td><td>Pier Sandro Cocconcelli, Piacenza, IT</td>
<td>March 31, 2009 WUP 03/09</td><td>(74) Representative:</td>
<td></td><td>Agnieszka Jakobsche, PATPOL Sp. z o. o</td>
<sup>(57)</sup> 1. Application of lactic acid bacteria, selected from the group consisting of CNCM strains
I-1984, CNCM I-1985, CNCM I-1986, CNCM I-1987 and LMG P-18997, which are not part of the persistent oral microflora, are less acidifying than pathogenic strains, resulting in an oral pH of 5. 5 - 7 and are able to adhere directly to the pellicle of the teeth, that is, show a percentage of adhesion to the saliva-coated hydroxyapatite bed of at least 1.96, after adhesion for 45 minutes at 37 ° C, for the manufacture of a composition intended for the prophylaxis or treatment of dental caries, plaque and periodontal infection.
11. An oral health composition for the prophylaxis or treatment of dental caries, plaque and periodontal infection, containing, in addition to conventional ingredients, lactic acid bacteria, characterized in that it contains at least one strain of lactic bacteria selected from the group consisting of the strains CNCM I-1984 , CNCM I-1985, CNCM I-1986, CNCM I-1987 and LMG P-18997, which are not part of the persistent oral microflora, are less acidifying than pathogenic strains. causing the oral pH to be 5.5-7 and able to adhere directly to the pellicle of the teeth.
PL 201 312 B1
Description of the invention
The invention relates to the use of lactic bacteria for the preparation of an oral health composition intended for the prophylaxis or treatment of dental caries, plaque and periodontal infection, and an oral health composition.
The oral cavity contains constantly occurring and non-permanent microflora. The first includes microorganisms that are capable of more or less continuous occurrence on the surface of the oral cavity. These bacteria are mainly located on the tongue, buccal mucosa and teeth, while the gums, lips, cheeks, palate and the bottom of the mouth are only a substrate for very rarely disseminated microflora. On the tongue and buccal mucosa, the naturally occurring microflora includes microorganisms selected from Streptococcus, Veillonella, Bacteroides and Haemophilus. The teeth are dominated by Streptococci, Lactobacilli and Actynomyces, but different gram positive and negative cocci and rodents can be found.
For example, Frandsen et al. They showed that S. sanguis predominates on the buccal mucosa, but its main environment is the surface of the teeth, that S. oralis and S. gordonii grow in mature plaque above the gum, that S. mitis grows in the initial stage of plaque formation (Oral Microbiol Immunol., 6, 129-133, 1991).
Strains belonging to the mutant group are localized on the teeth (S. criscetus, S. downei, S. ferus, S. macacae, S. mutans, S. rattus, S. sobrinus). Strains belonging to the S. milleri group predominate in tooth abscesses (S. anginosus, S. constellatus, S. intermedius; Bentley et al. Int. J. System. Bacter. 1991, 41, 487-494; Wood et al., The Genera of Lactic Acid Bacteria, Blackie Academic and Professional, Chapman & Hall, WH Publishers, 1995).
Many of these microorganisms are harmless commensal microorganisms, but many have been recognized as the etiological agents of quite a number of diseases (Hill, MJ, and Marsh, PD Human Microbial Ecology, 1990, CRC Press, Boca Raton Florida, USA).
Dental plaque is the membrane that forms on the surface of the teeth made up of bacterial cells in a matrix of extracellular polysaccharides and salivary products. Immediately after eruption, the teeth are covered with an amorphous layer of saliva, an acquired enamel pellicle (AEP) approximately 1.3 μm thick, which cannot be removed by normal tooth brushing. The deposition of bacteria on the teeth occurs immediately after AEP is formed, and plaque becomes visible within 8-12 hours as a multilayer structure. The first layer consists of bacteria (earliest colonizers) that attach to the teeth mainly by recognizing specific adhesion receptors, they form the substrate for subsequent colonizers that stick to each other by analogous specific attachment or by simple juxtaposition. The compactness of the wafer is generally guaranteed by three mechanisms; the presence of a salivary pellicle on the outer layer of the bacteria, the specific coaggregation between different species of bacteria and the glucans synthesized by the bacteria, and so are retained in the plaque matrix (Skopek et al., Oral Microbiol. Immunol., 9, 19 - 24, 1994; Kolenbrander et al. ., Meth. Enzymol., 253, 385-397, 1995; Hiroi et al., FEMS Microbiol Lett., 96, 193-198, 1992; Gibbons et al., Infect. Immun., 52, 555-561, 1986 ).
Organic acids produced by bacteria that are constantly present in the flora of the mouth during fermentation processes directly cause tooth decay. These acids attack the hard tissue of the tooth and, as a consequence, release ions such as calcium, phosphate, carbonate, magnesium, fluoride and sodium. When the pH in the mouth rises to about neutral again, the saliva becomes saturated with calcium and therefore its release from the teeth is prevented.
Among the food residues present in the oral cavity, carbohydrates show the greatest effect on caries, being directly accessible to the bacterial fermentation processes occurring in the mouth.
Potentially all sugar-fermenting microorganisms are cariogenic, but the major etiological factors of tooth crown and root caries are the streptococci mutants as they are strong acid producers, but lactobacilli, which are highly acidic, may also be involved. In humans, S. mutants and S. sobrinus are the most cariogenic strains, they live on the teeth but do not colonize the entire dentition. A decline in numbers from molars to anterior teeth has been demonstrated (Lindquist et al., Dent. Res., 69, 1160-1166, 1990). Moreover, in humans in the average plaque, S. mutants and S. sobrinus preferentially colonize the most prone to caries apical sites to the contact surface of the teeth (Ahmady et al., Caries Res., 27, 135-139,
PL 201 312 B1
1993). The S. sobrinus strain has also been found to be more prevalent in the molars compared to S. mutans (Lindquist et al., Caries Res., 25, 146-152, 1991).
It has been shown that S. mutans and S sobrinus attack the pellicle of the teeth mainly via specific adhesion receptors. Gibbons et al. showed that S. mutans has an adhesin that binds to the salivary components of the pellicle, while S. sobrinus cells appear to have an adhesin that binds to the glucan of the pellicle (Infect. Immun., 52, 555 - 561, 1986).
The transient microflora contains exogenous bacteria which may be present in the oral cavity occasionally but are not permanent flora (even with repeated introductions of these bacteria). All food bacteria, and especially lactic acid bacteria, can be part of this transient microflora. These exogenous lactic acid bacteria have never been shown to be able to adhere directly to the pellicle of the teeth. The repeated administration of exogenous lactic bacteria, however, can lead to colonization of the oral cavity on all surfaces, such as the tongue, buccal mucosa, gums, lips, cheeks, palate, floor of the mouth and teeth. This colonization can result from the attachment of a persistent microflora by specific binding to bacteria (co-aggregation phenomenon), or by retention in a matrix of polysaccharides produced by persistent bacteria or by adherence to saliva proteins (especially glycoproteins).
Lactobacillus casei rhamnosus GG (ATCC53103) has been reported to colonize the oral cavity, most likely on the buccal mucosa epithelium. This strain also adheres to the epithelium of the intestinal tract (US Patent No. 5,032,399, Gorbach et al., Micr. Ecol. In Health and Dis., 7, 295-298, 1994). In contrast, L. rhamnosus does not stick to the teeth.
Japanese Patent JP-4021633 (Cyconmedix KK) also describes the colonization of the oral cavity by Lactobacillus acidophilus, most likely on the buccal mucosa epithelium. Many Lactobacillus acidophilus are known to adhere to the epithelium of the intestinal tract (EP-577904; EP199535, Perdigon et al., Medicina, 46, 751-754, 1986; Perdigon et al., Immunology, 63, 17-23, 1988).
Exogenous bacteria can also produce factors that inhibit the growth of the persistent microflora in the mouth. For example, EP-759469 (Societe des Produits Nestle) describes the use of bacteriocin, produced by Micrococcus varians, to inhibit the development of pathogens in the mouth of S. sobrinus, S. sanguis, S. mutans and A. viscosus.
Certain strategies have been used to minimize the development of persistent oral microflora, namely, administration of commensal bacteria of persistent microflora that are not cariogenic such as Streptococcus mutans (JP 59220191) or Streptococcus salivarius (JP 05004927) and / or Stomatococcus mucilaginosus and / or exogenous lactic bacteria such as L. casei, L. fer mentum, L. acidophilus, L. crispatus. L. gasseri. L. salivarius, L. bulgaricus and S. salivarius (Tanzer et al., Infec. and Immunity, 48, 44-50, 1985; WO 92/14475; EP-0524732).
The use of bacteriocins is also one of the investigated methods that have been proposed for reducing tooth decay. These molecules are attractive as potential anti-caries agents and as factors important in modulating oral colonization. The anticaries power of certain bacteriocins comes from their potent and broad antimicrobial activity against streptococcal mutants and plaque-associated bacteria and their natural occurrence in bacteria recognized as safe for humans (US Patent 5,368,845 to Colgate, and WO 94/12150) for SmithKline Beecham).
The use of milk derivatives is also interesting for oral health. US Pat. No. 5,427,769 (Nestec SA) describes another alternative where tooth decay is prevented by contacting the teeth with an edible composition containing sufficient micellar casein to inhibit colonization by Streptococcus sobrinus. EP 748591 (Societe des Produits Nestle SA) also describes the use of fluorinated micellar casein or its micellar subunits for the treatment of caries and dental plaque. U.S. Patent No. 4,992,420 (Nestec SA) describes the treatment of the oral cavity with a milk-derived kappa-caseinoglycomacropeptide to remove plaque and caries.
It has never been shown that lactic acid bacteria, which are not part of the persistent microflora in the mouth, are actually able to adhere directly to the pellicle of the teeth.
Thus, by colonizing the surface of the teeth, such lactic acid bacteria could have activity in inhibiting the growth of the persistent microflora, including oral pathogens.
PL 201 312 B1
The subject of the invention is the use of lactic acid bacteria, selected from the group consisting of the strains CNCM I-1984, CNCM I-1985, CNCM I-1986, CNCM I-1987 and LMG P-18997, which are not part of the constant microflora present in the oral cavity, are less acidifying than pathogenic strains, making the oral pH 5.5-7 and able to adhere directly to the pellicle of the teeth, i.e. show a percentage of adhesion to the saliva-coated hydroxyapatite deposit of at least 1.96, after adhering for 45 minutes at 37 ° C, for the preparation of a composition intended for the prophylaxis or treatment of dental caries, plaque and periodontal infection.
Preferably, the lactic acid bacteria are selected from the group consisting of:
- acidifying lactic bacteria which adhere to the pellicle of the teeth and which have been genetically modified to be low acidifying;
- non-stick lactic acid bacteria that are low acidifying and that have been genetically engineered to stick to the pellicle of the teeth;
- non-stick acidifying lactic acid bacteria that have been genetically modified to stick to the pellicle of the teeth and genetically modified to be low acidifying.
Preferably, the composition is for removing tooth pathogens or preventing them from sticking.
Preferably, the lactic acid bacteria are of dairy origin.
Preferably, the lactic acid bacteria adhere to the pellicle of the teeth by adhesive agents.
It is particularly preferred that the lactic acid bacteria have been genetically modified to increase their adherence to the pellicle of the teeth and / or genetically modified to be even less acidifying.
Most preferably, the composition is an edible composition containing an amount of lactic acid bacteria effective to prevent or treat dental caries, plaque and periodontal infection.
It is particularly preferred that the composition contains at least 10<sup>4</sup> - 10<sup>9</sup> cfu / g of lactic acid bacteria.
Moreover, preferably the lactic acid bacteria are combined with milk, fermented milk, milk derivatives or bacteriocin.
Most preferably, the milk derivatives are selected from any of the forms of caseinoglycomacropeptide, micellar casein, fluorinated micellar casein or rennet milk.
The invention further relates to an oral health composition intended for the prophylaxis or treatment of dental caries, plaque and periodontal infection, comprising, in addition to conventional ingredients, lactic acid bacteria, containing at least one strain of lactic bacteria selected from the group consisting of the strains CNCM I-1984 , CNCM I-1985, CNCM I-1986, CNCM I -1987 and LMG P-18997, which are not part of the microflora constantly present in the mouth, they are less acidifying than pathogenic strains, making the oral pH 5.5-7, and are able to adhere directly to the pellicle of the teeth.
In this composition, preferably the lactic acid bacteria have been genetically modified to increase their adherence to the pellicle of the teeth and / or genetically modified to be even less acidifying.
Preferably, the lactic acid bacteria are selected from the group consisting of:
- acidifying lactic acid bacteria which adhere to the pellicle of the teeth and which have been genetically modified to be low acidifying;
- non-stick lactic acid bacteria which are weakly acidifying and which have been genetically modified to stick to the pellicle of the teeth;
- non-stick acidifying lactic acid bacteria that have been genetically modified to adhere to the pellicle of the teeth and genetically modified to be weakly acidifying.
Preferably, the lactic acid bacteria have been genetically modified such that they adhere to the pellicle of the teeth with adhesive agents and cause the pH in the oral cavity to be 5.5-7.
Preferably, the composition of the invention comprises an amount of lactic acid bacteria effective to prevent or treat dental caries, plaque and periodontal infection.
Preferably, the composition according to the invention contains at least 10<sup>4</sup> - 10<sup>9</sup> cfu / g of lactic acid bacteria.
Preferably, in the composition according to the invention, the lactic acid bacteria are combined with milk, fermented milk, milk derivatives or bacteriocin.
PL 201 312 B1
It is particularly preferred that the milk derivatives are selected from any of the forms of caseinoglycomacropeptide, micellar casein, fluorinated micellar casein or rennet milk or bacteriocin.
In the following description, oral cavity means the oral cavity of humans or animals such as pets, consisting of the oral mucosa (gums, lips, cheeks, palate and bottom of the mouth), tongue and teeth (including artificial teeth).
The microflora that is constantly present in the mouth includes all the microorganisms that naturally live in the mouth, as they can still be found on the surfaces of the mouth. The persistent microflora in the mouth also includes bacteria that live in the area between the hard and soft tissues (tooth-gingiva junction), even when the gingival gap and periodontal pocket are not present in a healthy mouth. This microflora includes microorganisms selected from Streptococcus, Staphylococcus, Enterococcus, Micrococcus, Peptostreptococcus, Peptococcus, Lactobacillus, Corynebacterium, Actinomyces, Arachnia, Rothia, Alcaligenes, Eubacterium, Propionibacterium, Bifillidobacterium, Bifillidobacterium, Bifillidobacterae / Eikenella, Actinobacillus, Capnocytophga, Haemophilus, Simonsiella, Bacteroides, Fusobacterium, Porphyromonas, Prevotella, Leptotrichia, Wolinella / Selenomonas, Mycoplasma, Candida, Spirochaetes, Protozoa.
The transient microflora includes exogenous bacteria that may occasionally be present in the mouth but which are not continuous. This transient microflora may include all food microorganisms such as bifidobacteria (B. infantis, B. adolescentis, B. breve and B. longum), lactococci (Lactococcus lactis subsp. Lactis, lactococcus lactis subsp. Cremoris, and Lactococcus lactis subsp. lactic biovar diacetylactis); streptococci (Streptococcus thermophilus, S. lactis, S. lactis cremoris and S. lactis diacetylactis); lactobacilli (Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus Helvetius, Lactobacillus farciminis, Lactobacillus alimentarius, Lactobacillus casei subsp.
casei, Lactobacillus delbruckii subsp. lactis, Lactobacillus sake, Lactobacillus curvatus, Lactobacillus fermentum, and an acidophilic group including L. johnsonii, see Fujisawa et al., Int. J. Syst. Bact., 42, 487
- 491, 1992); pediococci (Pediococcus pentosaceus, Pediococcus acidilactici and Pediococcus halophilus); enterococci, staphylococci (Staphylococcus xylosus and Staphylococcus carnosus); micrococci (Mikrococcus varians); yeasts of the genera Debaromyces, Candida, Pichia, Torulopsis and Saccharomyces, and molds of the genera Aspergillus, Rhizopus, Mucor and Penicillium.
The lactic acid bacteria used in the invention adhere to the pellicle of the teeth by means of specific or non-specific interactions and / or adhesive factors. The specific adhesion factors are proteins or polysaccharides.
The lactic acid bacteria used are selected from the strains CNCM I -1984, CNCM I-1985, CNCM I-1986, CNCM 1-1987 and LMP P-18997.
These strains have been selected from lactic acid bacteria strains for their ability to adhere to the pellicle of the teeth and for optimal growth at about 37 ° C, that is, oral temperature. Moreover, they are capable of fermenting glucose and sucrose and do not synthesize glucans, which are pathogenicity factors in cariogenic strains.
As indicated above, genetically modified lactic acid bacteria are preferably used.
Modification of lactic bacteria can be achieved, for example, by inserting the genes X17390, X14490 or X53657 (GenBank designations). These genes are responsible in S. mutans for the expression of the I / II antigen which mediates adhesion to salivary glycoproteins.
You can also genetically modify the lactic acid bacteria so that they are low acidifying. For lactic acid bacteria that are already weakly acidifying, this modification aims to increase this effect by reducing their lactic acid production.
Modifications can be carried out in a number of ways, and preferably according to one of the protocols described in the following documents: Boumerdassi et al., Appl. Environ. Microbiol. 63, 2293
- 2299, 1997; Platteeuw et al., Appl. Environ. Microbiol., 61, 3967-3971,1995; Ito et al., Biosci. Biotechnol. Biochem. 58, 1569-1573, 1994.
As indicated above, at least one strain of genetically modified or unmodified lactic bacteria is used in an "effective amount" in the preparation of a composition for the prophylaxis or treatment of dental caries, plaque and periodontal infection in humans or animals such as pets. This amount is preferably between 10<sup>4</sup> up to 10<sup>9</sup> cfu / g.
It is also possible to use, as mentioned above, at least one strain of lactic bacteria in combination with milk derivatives such as milk or fermented milk or derivative thereof.
For example, milk is selected from the form of caseinoglycomacropeptides, micellar casein, fluorinated micellar casein, rennet milk or bacteriocins.
Biochemical characteristics of selected strains
Fermentation pattern: 49 simple sugars were tested in the api 50 CH bioMerieux strip test (bioMerieux SA, 69280 Marcyl'Etoile, France) and the results are shown in Table 1 below.
Acidification curves: The acidification curves were determined at 37 ° C under the following conditions;
S. sobrinus OMZ 176: FUM sucrose 1% and FUM glucose 1%
S. thermophilus CNCM 1-1985: sucrose Belliker 1% and glucose Belliker 1%
Inoculation was always 5%, pH was read every 20 minutes.
S. thermophilus CNC I-1985, during sucrose fermentation, lowers the pH to 4.5, while S. sobrinus OMZ 176 to 4.
Table 1
Sugar fermentation by strains L. lactis CNCM I-1987, L. lactis CNCM I-1986,
S. thermophilus CNCM I-1984, S. thermophilus CNCM I-1985 and S. thermophilus LMG P-18997
<td rowspan="2">Sugar</td><td>L. lactis</td><td>L. lactis</td><td>S. th.</td><td>S. th.</td><td>S. th.</td>
<td>CNCM I-1987</td><td>CNCM I-1986</td><td>CNCM I-1984</td><td>CNCM I-1985</td><td>LMG P-18997</td>
<td>Adonitol</td><td> +++</td><td></td><td></td><td></td><td></td>
<td>Aesculin</td><td> ++</td><td> ++++</td><td></td><td></td><td></td>
<td>Amygdalin</td><td> ++++</td><td></td><td></td><td></td><td></td>
<td>D-arabinose L-arabinose D-arabitol L-arabitol</td><td> +++</td><td></td><td></td><td></td><td></td>
<td>Arbutin</td><td> +++</td><td> +++</td><td></td><td></td><td></td>
<td>Cellobiosis Dulcitol</td><td> +++</td><td> ++++</td><td></td><td></td><td></td>
<td>Erythriol D-fructose</td><td> +</td><td> ++++</td><td></td><td></td><td></td>
<td>D-fucose L-fucose Galactose</td><td> ++</td><td> ++++</td><td></td><td></td><td></td>
<td>β-gentiobiosis Gluconate</td><td></td><td> +++</td><td></td><td></td><td></td>
<td>2-keto gluconate 5-keto gluconate GlcNAc</td><td> +</td><td> ++++</td><td></td><td></td><td></td>
<td>D-glucose Glycerol</td><td> +</td><td> ++++</td><td> +</td><td> ++</td><td> ++</td>
<td>Glycogen Inositol</td><td></td><td></td><td></td><td></td><td></td>
<td>Inulin Lactose</td><td> +</td><td> ++++</td><td> +++</td><td> ++++</td><td> ++++</td>
<td>D-lyxosis Maltose</td><td> ++</td><td></td><td></td><td></td><td></td>
<td>Mannitol</td><td> +++</td><td> ++</td><td></td><td></td><td></td>
<td>D-mannose Melezitosis Melibiosis</td><td> +</td><td> ++++</td><td></td><td></td><td></td>
<td>α-methyl-D-glucoside α-methyl-D-mannoside D-raffinose Rhamnosis Ribose</td><td> ++</td><td> ++</td><td></td><td></td><td></td>
<td>Salicin Sorbitol L-sorbose Starch Saccharose</td><td> +++</td><td> +++</td><td> +++</td><td> ++++</td><td> +++</td>
<td>D-tagatosis Trehalose</td><td> ++</td><td></td><td></td><td></td><td></td>
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<td>D-turanose Xylitol D-xylose L-xylose β-methyloxyloside</td><td> ++ +++</td><td></td><td></td><td></td><td></td>
+, ++, +++, ++++ show that fermentation started after 3, 6, 24 or 48 hours.
The compositions of the invention are of particular interest for the prophylaxis or treatment of dental caries and periodontal infection. The lactic acid bacteria strains contained in this composition can, for example, be incorporated into food, petfood, cosmetic or pharmaceutical compositions.
Accordingly, such compositions are preferably, for example, toothpaste, mouth rinse, gum, spray, drink, candy, baby formula, ice cream, frozen desserts, sweet salad dressings, milk preparations, cheese, cottage cheese. , yoghurt, acidified milk, coffee cream or whipped cream.
In these compositions, the strains of lactic bacteria may be introduced alone or in combination with milk derivatives, for example to obtain synergistic preparations. In that case, the oral health compositions contain:
- the above-described lactic acid bacteria, which are not part of the persistent microflora of the mouth, and which are able to adhere directly to the pellicle of the teeth;
- any form of dairy glycopeptides, rennet milk or bacteriocin, such as caseinoglycomacropeptides (CGMP), fluorinated or non-micellar casein (which can be obtained as described in EP-0604802 and EP-0748591) or rennet milk. Caseinoglycomacropeptides are added in a minimal amount of about 0.1%. It was also shown that caseinoglycomacropeptides did not prevent the adhesion of the above-mentioned lactic bacteria to the pellicle of the teeth (Figs. 2 and 3).
Synergistic compositions can also be prepared by adding at least one bacteriocin that is active against oral gram-positive bacteria. In this case, the oral hygiene compositions may contain 0.00001 to 50%, preferably 0.00001 to 15%, of purified bacteriocin by weight of the composition. The bacteriocin is preferably variacin (EP-0759469).
Oil-soluble antioxidants can also be added to protect the composition from degradation. Suitable antioxidants include "tocopherols", butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), and ascorbyl palmitate. The oil-soluble antioxidants are preferably present in an amount of from 0.005% to 0.5%, preferably 0.005% to 0.01% by weight of the composition.
Abrasives suitable for use in the dental 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 according to the invention have a pH which is acceptable in the mouth and at which the activity of said strains of lactic bacteria is not reduced. The pH may be in the range 3.0-9.5, preferably in the range 3.5 to 6.5.
These compositions can be prepared by conventional methods involving mixing the ingredients in the appropriate relative amounts and finally and, if necessary, adjusting the pH to the required value.
The method of screening lactic acid bacteria capable of adhering to teeth as described in the examples may include the steps of:
(1) producing monoclonal antibodies that specifically recognize the surface of the proteins of a strain of lactic bacteria capable of adhering to teeth, and (2) screening any strain of lactic bacteria with the monoclonal antibody of the strain capable of adhering to the teeth.
Monoclonal antibodies are used as a tool to detect the listed bacterial strains among other strains growing alongside.
The present invention is illustrated by the following examples, without limiting the scope thereof, which will be apparent to those skilled in the art from the light of the present description and accompanying drawings. Modifications falling within the scope of the claims are permissible.
Various publications are cited herein, the disclosures of which are incorporated by reference herein, to the extent necessary for an understanding of the invention. DNA manipulation, cloning and transformation of bacterial cells are carried out in accordance with the publication, unless otherwise stated
PL 201 312 B1
Sambrook et al. (Sambrook et al., Molecular Cloning, A Laboratory manual, Cold Spring Harbor Laboratory Press, USA, 1989). The following examples are intended to give a brief description of the plasmids, strains, and various media used, as well as the method of producing a monoclonal antibody.
Strains of S. termophilus S118 (NCC 1529), S123 (NCC 1561), L. lactis subsp. lactis 29 (NCC 2211), L. lactis subsp. lactis biovar dioacetylactis 69 (NCC 2225) has been deposited in accordance with the Budapest Treaty at the National Collection of Microbial Cultures (CNCM I-1984, CNCM I-1985, CNCM I-1986 and CNCM I-1987 respectively), INSTITUT PASTEUR, 25 Rue du docteur Roux , 75724, Paris, France on March 3, 1998. Strain S. thermophilus BF11116 (CNBL1177) has been deposited under the Budapest Treaty with the Belgian Coordinated Microbial Collection LMP P-18997, KL Ledeganckstraat 35, B-9000 Gean, Belgium, on 5 July 1999. All restrictions on the availability of these deposits will be removed upon the first publication of this application or other publications that claim priority from this application.
Description of the drawings
Figures 1a, 1b and 1c show the saturation of attachment curves for S sobrinus OMZ 176, L. lactis NCC2211 and S. thermophilus NCC1561 strains, respectively.
Fig. 2 shows the curves obtained for the three strains by recording the number of attached cells versus increasing amount of CGMP.
Fig. 3 shows the curves obtained for the three strains by recording the number of attached cells against the increasing amount of As-CGMP.
Example 1; Strains and breeding conditions
More than 100 strains (belonging to the Nestle culture collection) were screened for their ability to attack the saliva-covered hydroxyapatite deposit, and in particular the following 23 strains were tested: S. thermophilus YS4 (NCC 2284), S. thermophilus Sfi6 (NCC 1971), S. thermophilus Sfi13 (NCC 2008), S. thermophilus Sfi21 (NCC 2038), S. thermophilus Sfi39 (NCC 2130), S. thermophilus Sfi42 (NCC 2145), S. thermophilus Sfi47 (NCC 2172), S. thermophilus S118 (NCC 1529) , S. thermophilus S119 (NCC 1536), S. thermophilus S122 (NCC 1554), S. thermophilus S123 (NCC 1561), S. thermophilus S126 (NCC 1587), L. lactis subsp. cremoris 15 (NCC 92), L. lactis subsp. cremoris 25 (NCC 1932), L. lactis subsp. cremoris 136 (NCC 2419), L. lactis subsp. cliacetylactis 8 (NCC 1970), L. lactis subsp. diacetylactis 28 (NCC 2057), L. lactis subsp. diacetylactis 69 (NCC 2225), L. lactis subsp. diacetylactis 80 (NCC 2272), L. lactis subsp. lactis 29 (NCC 2211), L. lactis subsp. lactis 50 (NCC 2224), L. lactis subsp. lactis 54 (NCC 2228), S. macedonicus 216 (NCC 2484).
oral strains, S. sobrinus OMZ 176, S. oralis OMZ 607, A. naeslundii OMZ 745, V. dispar OMZ 493 and F. nucleatum OMZ 596 were obtained from the Institut far Orale Mikrobiologie und Allgemeine Immunologie, University of ZOrich, and were bred in a FUM environment under anaerobic conditions (GasPack-System, BBL) at 37 ° C.
All strains were stored in glycerol at -20 ° C and pre-cultured for 14 hours prior to use at their specific optimum temperature; S. sobrinus OMZ 176 grew in the FUM environment (Gmϋr et al.), Lactococcus and streptococcus in M17 (Difco) with the exception of
S. thermophilus NCC1529, S119, S122, NCC1561 and S126 which were grown in Belliker (prepared by dissolving water in 1 liter of 20 g of tryptone, 5 g of yeast extract, 2.5 g of gelatin, 5 g of dextrose, 5 g of sucrose, 5 g of lactose , 4 g of NaCl, 0.5 g of ascorbic acid, 10 g of beef extract).
Counting per plate: S. sorbinus OMZ 176 was grown in Mitis-Salivarius (Difco), S. thermophilus NCC1529, S119, S122, NCC1561, BF11116 and S126 agar in Belliker agar (prepared by adding 15 g of Bacto, Difco agar to liquid Belliker) and the remaining strains of lactic bacteria in M17 agar (Oxoid).
Example 2; Production of a monoclonal antibody
The monoclonal antibody can be used as a tool for the detection of L. lactis subsp. lactis NC2211 between 5 oral strains growing together on S-HA discs and forming a biofilm that mimics plaque. The monoclonal antibody was tested on these strains to verify that there were no cross reactions.
Finally, a monoclonal antibody is produced as described in Granato et al., "A mouse monoclonal IgE antibody antibovine milk lactoglobulin allows studies of allergy in gastrointestinal tract.", Glin. Exp. Immunol., 63, 703-710, 1986.
PL 201 312 B1
Example 3; Selection of sticky lactic bacteria
Attachment to the saliva-coated hydroxyapatite (S-HA) bed
The procedure previously described by Neeser et al. (1994) with minor modifications: the beds were rinsed with 150 μl by volume, and Hyamine hydroxide was replaced with benzothionium hydroxide (Sigma).
All strains were grown to the end of the log phase in FUM except for S. thermophilus NCC1529, S119, S122, NCC1561 and S126 which were grown in Belliker. S. sobrlnus OMZ 176, L. lactis subsp. lactis NCC2211, 50 and 54, S. thermophilus NCC1529, S119, S122, NCC1561 and S126 were grown at 37 ° C, the remaining lactococci at 30 ° C and the remaining streptococci at 42 ° C.
mg of hydroxyapatite resin (BDH Chemicals Ltd, Poole, England) was covered with 70 μl of clarified saliva obtained from volunteers in the laboratory and prepared as previously explained (Neeser et al., 1994). The saliva-covered beds were kept overnight at 4 ° C, then washed (first with distilled water and then with HEPES buffer), and finally 100 μl of metabolically labeled bacterial suspension was inoculated (bacteria growing in their environment, supplemented with 10 μθί / ml acetic acid)<sup>14</sup>C). Attachment occurred during 45 minutes at 37 ° C, then unbound bacteria were washed away and directly attached cells were counted with a LKB scintillation counter (1219 Rackbet type).
The percentage of adhesion is expressed as the amount of radioactive cells attached to the bed to the total amount of radioactive cells added to each well. All measurements were carried out in triplicate. Table 2 shows the percentages of adhesion to the saliva-coated hydroxyapatite bed obtained for a dozen or so tested strains and for S. sobrinus OMZ176 (strain to determine the reference level).
Table 2
Percentage of adhesion to the saliva-coated hydroxyapatite deposit obtained for a dozen or so studied strains
<td>Strain</td><td>% adhesion (+/- SD)</td>
<td>S. sobrinus OMZ 176</td><td> 2,23 +/- 0,49</td>
<td>S. thermophilus Sfi42 (NCC 2145)</td><td> 0,08 +/- 0, 02</td>
<td>S. thermophilus Sfi47 (NCC 2172)</td><td> 0,14 +/- 0,04</td>
<td>S. thermophilus NCC1529</td><td> 2,89 +/- 0,260</td>
<td>S. thermophilus S119 (NCC 1536)</td><td> 0,15 +/- 0,04</td>
<td>S. thermophilus S122 (NCC 1554)</td><td> 0,93 +/- 0,17</td>
<td>S. thermophilus NCC1561</td><td> 2,19 +/- 0,50</td>
<td>S. thermophilus S126 (NCC 1587)</td><td> 1,19 +/- 0,56</td>
<td>S. lactis subsp. diacetylactis S28 (NCC 2057)</td><td> 1,59 +/- 0,17</td>
<td>S. lactis subsp. diacetylactis NCC2225</td><td> 1,96 +/- 0,40</td>
<td>S. lactis subsp. diacetylactis 80 (NCC 2272)</td><td> 1,20 +/- 0,35</td>
<td>S. lactis subsp. lactis NCC2211</td><td> 2,85 +/- 0,85</td>
Four strains of S. thermophilus NCC1529 (CNCM I-1984), S. thermophilus NCC1561 (CNCM I-1985), L. lactis subsp. lactis NCC2211 (CNCM I-1986) (hereinafter referred to as L. lactis NCC2211) and L. lactis subsp. diacetylactis NCC2225 (CNCM I-1987) showed values close to that of S. sobrinus OMZ 176.
L. lactis NCC2211 and S. thermophilus NCC1561 were selected as the more promising strains because they grow very well at 37 ° C, that is, mouth temperature, while L. diacetylactis NCC2225 has an optimal growth temperature of 30 °. In particular, L. lactis NCC2211 cannot grow on sucrose, but can ferment a wide range of sugars, moreover, other orally present strains can supply glucose via their invertase.
Saturation sticking curves
The attachment curves of CFU versus CF inoculated per well were determined to check whether saturation of the bed could be obtained. The 50% saturation was derived directly from the point of attachment in the obtained curves. Saturation sticking curves were determined for S. sobrinus OMZ 176, L. lactis NCC2211 and S. thermophilus NCC1561. They are shown in Figure 1.
For the three strains, the CFU titer inoculated into the well to give 50% saturation of the bed and the corresponding CFU titer was read directly from the junction point of the curves, the results are given in Table 3.
PL 201 312 B1
Table 3
Titer of CFU inoculated per well to obtain 50% bed saturation
<td></td><td>cfu / well</td><td>related cfu</td><td>% adhesion</td>
<td>S. sobrinus OMZ 176</td><td>4.00E + 07</td><td>4.00E + 06</td><td> 10%</td>
<td>L. lactis NCC2211</td><td>1.00E + 07</td><td>9.00E + 05</td><td> 9%</td>
<td>S. thermophilus NCC1561</td><td>3.00E + 07</td><td>2.00E + 06</td><td> 7%</td>
Example 4; Effect of caseinoglycomacropeptides
The effect of CGMP on the adherence of L. lactis NCC2211 and S. thermophilus NCC1561 was investigated to test their suitability for the development of an advantage of one of the two strains over a pathogenic strain (specifically S. sorbrinus OMZ 176). Caseinoglycopeptide (CGMP) and a sialic acid derivative thereof (As-CGMP) were obtained from Nestec SA, Lausanne (for preparation see Neeser et al., 1994).
The dose-response effect on cell adhesion to the S-HA matrix was investigated by inoculating 100 μl of a bacterial suspension (CFU / ml corresponding to a previously calculated 50% bed saturation) per well, which suspension contained CGMP or As-CGMP at various concentrations, and then adhesion was assessed as usual. Concentrations ranging from 0.05-3 mg / ml were tested. There was no prior incubation of bacteria in the presence of CGMP or As-CGMP.
Figure 2 shows the curves obtained for the three strains by reading the amount of bound cells versus the increasing amount of CGMP, the amount of inoculated cells corresponding to 50% of the bed saturation was previously determined for each strain. The observed strong inhibition in S. sobrinus OMZ 176 confirms the previous results obtained by Neeser et al. (1994) and Schijpbach et al. (J. Dent. Res., 75, 1779-1788, 1996).
As clearly seen in Fig. 2, 0.25 mg / ml gives 50% inhibition of S. sobrinus 176 adhesion, while more than 2 mg / ml was necessary for S. thermophilus NCC1561 to achieve the same result. CGMP slightly increases the adherence of L. lactis NCC2211.
As in the case of CGMP, from which the sialic acid residue has been removed, the derivative inhibits the adhesion of S. sobrinus OMZ 176; only 0.05 mg / ml is sufficient to obtain a 50% reduction in percent stick. As-CGMP had no effect on the adherence of L. lactis NCC2211, it slightly nourished one of the S. thermophilus NCC1561 (Fig. 3).
Example 5; Toothpaste
Prepare toothpaste by adding 10<sup>5</sup> cfu / ml of at least one strain of lactic acid bacteria CNCM I-1984, CNCM I-1985, CNCM I-1986, CNCM I-1987 or LMG P-18997 in freeze-dried form, for the following mixture containing:
<td>cetylpyridinium chloride</td><td> 1,65%</td>
<td>sorbitol (70% solution)</td><td> 33,0%</td>
<td>glycerine</td><td> 25,0%</td>
<td>sodium carboxymethyl cellulose</td><td> 2,0%</td>
<td>sodium fluoride</td><td> 0,25%</td>
<td>silica (RP 93)</td><td> 26,3%</td>
<td>thickening silica (Sident 22)</td><td> 8,1%</td>
<td>sodium saccharin</td><td> 0,5%</td>
<td>poloxamer (Pluropnic F108)</td><td> 3,2%</td>
<td colspan="2">This toothpaste is intended for the prevention or treatment of tooth decay, plaque on-</td>
dental and periodontal infection.
Example 6; Ice cream
A cream was prepared containing 10.8% milk fat, 13.5% milk solids (non-fat), 0.3% Emulstab® SE30 in 0.3% Emulstab® foam (Grindsted, DK) which was then pasteurized at 105 ° C for 20 seconds, homogenized at 75 ° C at 300 bar, cooled to 38 ° C and inoculated with MRS precultures taken in exponential growth phase at a concentration of 10<sup>7</sup> - 10<sup>8</sup> cfu / ml of at least one lactic acid bacteria strain CNCM I-1984, CNCM I-1985, CNCM I-1986, CNCM I-1987 or LMG P-18997. The cream was then fermented for 10 hours at 38 ° C to a pH of about 4.5. At the end of fermentation, sucrose and glucose syrup were added. The composition of the compositions is shown in Table 4 below.
PL 201 312 B1
The mix was then whipped, cooled to 4 ° C, stored at 4 ° C, cooled to 95 ° C volumetric expansion.
Table 4
<td>Ingredients</td><td>Composition (kg)</td><td>Fats (%)</td><td>Solid non-fat ingredients (%)</td><td>Saccharose (%)</td><td>Content constants ingredients (%)</td>
<td>cream (35%)</td><td> 30,83</td><td> 10,79</td><td> 1,54</td><td></td><td> 12,33</td>
<td>skim milk powder</td><td> 12,45</td><td></td><td> 11,95</td><td></td><td> 11,95</td>
<td>Emulstab ® SE30</td><td> 0,41</td><td></td><td></td><td></td><td> 0,37</td>
<td>Emulstab ® foam</td><td> 0,41</td><td></td><td></td><td></td><td> 0,36</td>
<td>water</td><td> 55,91</td><td></td><td></td><td></td><td></td>
<td>whole: cream base</td><td> 100,00</td><td> 10,79</td><td> 13,49</td><td> -</td><td> 25,01</td>
<td>cream base</td><td> 74,14</td><td> 8,00</td><td> 10,00</td><td> -</td><td> 18,54</td>
<td>saccharose</td><td> 22,06</td><td></td><td></td><td> 15,00</td><td> 15,00</td>
<td>glucose syrup</td><td> 3,80</td><td></td><td></td><td></td><td> 3,00</td>
<td>fermented cream ice cream</td><td> 100,00</td><td> 8,00</td><td> 10,00</td><td> 15,00</td><td> 36,54</td>
Example 7; The yogurt 1 culture in MRS medium was sterilized for 15 minutes at 121 ° C and then inoculated with 5% by volume of an active culture of at least one S. Thermophilus strain CNCM I-1984, CNCM I-1985 or LMG P-18997 containing approximately 10<sup>9</sup> cfu / ml. After incubation for 8 hours at 41 ° C, a starter containing 4.5x10 was obtained<sup>8</sup> cfu / ml.
l 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% active commercial culture concentrates Streptococus thermophilus containing approximately 10<sup>9 </sup>cells / ml. After incubating for 4 hours at 41<sup>about</sup> a starter containing 4.5x10 was obtained<sup>8</sup> cells / ml.
One tank of whole milk containing 3.7% fat fortified with 2.5% skim milk powder, then pasteurized for 30 minutes at 90 ° C and then inoculated with 2% v / v of starter with at least one strain CNCM I-1984, CNCM I- 1985 or LMG P-18997 and 3% by volume of a thickening starter of Streptococcus thermophilus. The inoculated milk was mixed, poured into dishes and incubated for 4 hours at 41 ° C.
The yoghurt obtained had a good firm and smooth texture and is intended for oral health.
Example 8; Chewing gum
A chewing gum may be prepared to prevent or treat tooth decay, plaque or periodontal infection by adding an active culture of at least one S. Thermophilus strain CNCM I-1984, CNCM I-1985, or LMG P-18997 such that it contains approximately 10<sup>4</sup> up to 10<sup>9</sup> cfu / g, for the following common ingredients:
xylitol 67.5% rubber base 20% calcium carbonate 5% glycerin 3%
Pluronic F127 2% cellulose gum 1% ballast compounds 0.5% flavor compounds 1%
Example 9; Composition for feeding pets
The pet feed composition is prepared by preparing a starting mixture of corn, wheat gluten, poultry and fish meat, salt, vitamins, and minerals. The initial mixture is introduced into the pre-flotation mixer and humidified.
PL 201 312 B1
The wetted batch leaving the primary flotation mixer is then fed into the boil extruder and gelatinized. The gelled matrix exiting the extruder is passed through a die and extruded. The extruded product is cut into pieces suitable for feeding dogs, dried at about 100 ° C for about 20 minutes and cooled to form pellets which have a water activity of about 0.6.
The pellets are spray-coated with 3 coating mixtures. Each coating mixture contains an active culture of at least one strain of S. Thermophilus CNCM I-1984, CNCM I-1985 or LMG P-18997, but one coating mixture contains hardened soybean fat as the coating substrate, one coating mixture contains water as the coating substrate and one coating mixture the coating blend contains extracted protein as a coating substrate. The pellets contain approximately 10<sup>4</sup> up to 10<sup>9</sup> cfu / g of said strain.
Contents10
3 sheets
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Titles2
- English
- INCORPORATION OF EXOGENOUS LACTIC BACTERIA INTO THE ORAL MICROFLORA
- Polish
- Zastosowanie bakterii mlekowych do wytwarzania kompozycji zdrowotnej do jamy ustnej i kompozycja zdrowotna do jamy ustnej
Classification
- CPC, 6
- A23C9/123
- A61Q11/00
- A23C9/1236
- A61K8/99
- A61P1/02
- A61P43/00
- IPC, 17
- G01N33 569
- A23C9 123
- A23G4 00
- A23G9 04
- A23L1 28
- A23L1 30
- A23L33 00
- A61K8 00
- A61K8 99
- A61K35 74
- A61K35 744
- A61P1 02
- A61P43 00
- A61Q11 00
- C12N1 20
- C12R1 01
- G01N33 577
