U.S. patent application number 11/087523 was filed with the patent office on 2005-08-25 for incorporation of exogenous lactic bacteria into the oral microflora.
Invention is credited to Cocconcelli, Pier Sandro, Comelli, Elena-Maria, Guggenheim, Bernhard, Neeser, Jean-Richard, Stingele, Francesca.
Application Number | 20050186148 11/087523 |
Document ID | / |
Family ID | 8234030 |
Filed Date | 2005-08-25 |
United States Patent
Application |
20050186148 |
Kind Code |
A1 |
Neeser, Jean-Richard ; et
al. |
August 25, 2005 |
Incorporation of exogenous lactic bacteria into the oral
microflora
Abstract
Compositions for the prophylaxis or treatment of dental caries,
dental plaque, and periodontal infection that include lactic
bacteria that are not part of the resident microflora of the mouth,
that are low acidifying, and that are capable of adhering directly
to the pellicle of the teeth. The compositions are used in methods
of treating or preventing dental caries, dental plaque, and
periodontal infection.
Inventors: |
Neeser, Jean-Richard;
(Savigny, CH) ; Guggenheim, Bernhard; (Erlenbach,
CH) ; Comelli, Elena-Maria; (Lausanne, CH) ;
Stingele, Francesca; (Lausanne, CH) ; Cocconcelli,
Pier Sandro; (Piacenza, IT) |
Correspondence
Address: |
BELL, BOYD & LLOYD LLC
P. O. BOX 1135
CHICAGO
IL
60690-1135
US
|
Family ID: |
8234030 |
Appl. No.: |
11/087523 |
Filed: |
March 23, 2005 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11087523 |
Mar 23, 2005 |
|
|
|
09779596 |
Feb 9, 2001 |
|
|
|
09779596 |
Feb 9, 2001 |
|
|
|
PCT/EP99/05473 |
Jul 26, 1999 |
|
|
|
Current U.S.
Class: |
424/50 ;
435/252.3; 435/252.9 |
Current CPC
Class: |
A61K 8/99 20130101; A61Q
11/00 20130101; A61P 1/02 20180101; A23C 9/1236 20130101; A61P
43/00 20180101; A23C 9/123 20130101 |
Class at
Publication: |
424/050 ;
435/252.3; 435/252.9 |
International
Class: |
A61K 007/16; A61K
007/28; C12N 001/20 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 12, 1998 |
EP |
98202707.0 |
Claims
The invention is claimed as follows:
1. A method of treating or preventing dental caries, dental plaque,
and periodontal infection in humans or animals comprising
administering to the oral cavity of a human or animal one or more
lactic bacteria that are not part of the resident microflora of the
human mouth, that are low acidifying in that when administered they
provide a pH in the oral cavity of 5.5 to 7, and that are capable
of adhering directly to the pellicle of the teeth to displace from
the teeth or prevent attachment to the teeth of cariogenic strains
of bacteria that are resident microflora of the mouth.
2. The method of claim 1, wherein the lactic bacteria are
originally derived from a diary.
3. The method of claim 1, wherein the lactic bacteria comprise one
or more of Streptococcus thermophilus,llactococcus lactis subsp.
lactis, or Lactococcus lactis subsp. lactis biovar
diacetylactis.
4. The method of clam 1, wherein the lactic bacteria have optimal
growth at a temperature of 30.degree. C. to 42.degree. C.
5. The method of claim 1, wherein the lactic bacteria have been
genetically modified to have improved adherence to the pellicle of
the teeth by insertion of the X17390 gene, the X14490 gene, or the
X53657 gene.
6. The method of claim 1, further comprising administering the
lactic bacteria in combination with one or more of milk, fermented
milk, milk derivatives, or bacteriocin.
7. The method of claim 6, wherein the milk derivative comprises one
or more of a caseino-glycomacropeptide, micellar casein,
fluorinated micellar casein, or renneted milk.
8. The method of claim 1, wherein the lactic bacteria that are used
for treating or preventing dental caries are administered by way of
a composition that contains the lactic bacteria in an amount of
10.sup.4 to 10.sup.9 cfu/g in order to provide the pH of at least
5.5 when the composition is administered to the oral cavity of a
human or animal.
9. The method of claim 8, wherein the composition further contains
one or more of milk, fermented milk, or a milk derivative.
10. The method of claim 9, which further comprises a bacteriocin in
an amount of 0.00001 to 50 percent by weight of the
composition.
11. The method of claim 10, wherein the composition includes a milk
derivative comprising one or more of a caseino-glycomacropeptide,
micellar casein, fluorinated micellar casein, or renneted milk in
an amount of at least 0.1 percent by weight of the composition.
12. The method of claim 8, wherein the composition further
comprises one or more of an oil soluble antioxidant or an
abrasive.
13. The method of claim 8, wherein the composition is in the form
of a toothpaste, mouth rinse, gum, spray, beverage, candy, infant
formula, ice cream, frozen dessert, sweet salad dressing, milk
preparation, cheese, quark, yogurt, acidified milk, coffee cream or
whipped cream.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of the U.S. national
phase designation of PCT application no. PCT/EP99/05473, filed Jul.
26, 1999, the entire contents of which are incorporated herein by
reference thereto.
FIELD OF THE INVENTION
[0002] The present invention relates to the incorporation of
exogenous lactic bacteria into the oral microflora for the
prophylaxis or the treatment of dental caries, dental plaque, and
periodontal infection.
BACKGROUND OF THE INVENTION
[0003] The mouth (oral cavity) contains resident and non-resident
microflora. The resident microflora includes microorganisms that
are able to establish a more or less permanent residence on the
oral surfaces. These bacteria are mainly localized on the tongue,
the buccal mucosa, and the teeth while the gingiva, lips, checks,
palate, and floor of the mouth only support a very sparse
microflora.
[0004] On the tongue and the buccal mucosa, the natural resident
microflora includes microorganisms selected from Streptococcus,
Veillonella, Bacteroides, and Haemophilus. On the teeth,
Streptococci, Lactobacilli and Actynomyces predominate but a
variety of Gram positive and negative cocci and rods can be also
found.
[0005] For example, Frandsen et al. showed that S. sanguis
predominates on the buccal mucosa but its primary habitat is the
surface of teeth, that S. gordonji grows in the mature
supragingival plaque, and that S. oralis and S. mitis grow in the
initial dental plaque (Oral Microbiol. Immunol., 6, 129-133, 1991).
Strains belonging to the mutans group are localized on teeth (S.
criscetus, S. downei, S. ferus, S. macacae, S. mutans, S. rattus,
S. sobrinus). Strains belonging to the S. milleri group predominate
in dental abscesses (S. anginosus, S. constellatus, S. intermedius)
(Bentley et al., Int. J. System. Bacter. 1991, 41, 487-494; Wood et
al., The Genera of Lactic Acid Bacteria, Blackie Academic and
Professional, Chapman & Hall, W. H. eds., 1995).
[0006] Many of these microorganisms are innocuous commensal
microorganisms, but a lot of them have been recognized as being the
etiologic agent responsible for several diseases (Hill, M. J. and
Marsh, P. D. eds. Human Microbial Ecology, 1990, CRC Press, Boca
Raton Fla., USA).
[0007] Dental plaque is a film that forms on the surface of teeth
consisting of bacterial cells in a matrix of extracellular
polysaccharide and salivary products. Immediately after eruption,
the teeth are covered with an amorphous layer of saliva, the
acquired enamel pellicle (AEP), that is about 1.3 .mu.m thick and
cannot be removed by normal tooth brushing. The deposition of
bacteria on teeth immediately follows the formation of the AEP and
plaque becomes evident in 8-12 hours as a multi-layered structure.
The first layer consists of bacteria (earliest colonizers) that
attach to teeth, mainly via specific adhesion-receptor recognition,
and forms a substratum for the second colonizers that adhere one to
the other by analogous specific binding or by simple juxtaposition.
Plaque cohesion is essentially guaranteed by three mechanisms: the
presence of a salivary pellicle on the outer bacteria layer, the
specific co-aggregation among the different bacterial species, and
the glucans synthesized by the bacteria that remain entrapped in
the plaque matrix (Skopek et al., Oral Microbiol. Immunol., 2,
19-24, 1994; Kolenbrander et al., Meth. Enzymol., 253, 385-397,
1995; Hiroi et al., FEMS Microbiol Lett., 96, 193-198, 1992;
Gibbons et al., Infect. Immun., 52, 555-561, 1986).
[0008] The organic acids produced by oral bacteria during the
fermentation process directly cause dental caries. These acids
attack the hard tissue of teeth with the consequent release of ions
such as calcium, phosphate, carbonate, magnesium, fluoride, and
sodium. When the pH in the oral cavity again increases to around
neutrality, the saliva becomes saturated with calcium so that
calcium liberation from the tooth is prevented. Among all the food
residues found in the mouth, carbohydrates show the highest caries
promoting effect since they are directly available for fermentation
by oral bacteria.
[0009] Potentially all microorganisms that ferment sugars are
cariogenic, but the primary etiological agents of coronal and root
caries are the mutans streptococci because they are strong acid
producers; Lactobacilli, that are highly aciduric, however, can
also be implicated. In humans, S. mutans and S. sobrinus are the
more cariogenic strains, and live on teeth while not colonizing the
entire dentition. Their number is also less on anterior teeth than
on molar teeth (Lindquist et al., Dent. Res., 69, 1160-1166, 1990).
Moreover in human approximal plaque, S. mutans and S. sobrinus
preferentially colonize the most caries-prone site apical to the
contact area (Ahmady et al., Caries Res., 27, 135-139, 1993). A
higher prevalence of S. sobrinus was also found in the molar
regions compared with that of S. mutans (Lindquist et al., Caries
Res., 25, 146-152, 1991).
[0010] S. mutans and S. sobrinus have been shown to attach to the
pellicle of teeth mainly via specific adhesion-receptor
interaction. Gibbons et al. showed that S. mutans carries an
adhesion which binds to salivary components in the pellicle, while
S. sobrinus cells appear to possess an adhesion which binds to
glucan in the pellicle (Infect. Immun., 52, 555-561, 1986).
[0011] The transient microflora comprise exogenous bacteria that
are occasionally present in the mouth, but that do not establish a
permanent residence therein (even if repeated oral administrations
of these bacteria are carried out). All the food bacteria, and in
particular lactic acid bacteria, can be part of this transient
microflora. These exogenous lactic bacteria have never been shown
to be capable of directly adhering to the pellicle of teeth.
Repeated administration of exogenous lactic bacteria may, however,
lead to colonization of the mouth on all the oral surfaces, such as
the tongue, the buccal mucosa, the gingiva, lips, cheeks, palate,
floor, and the teeth. This colonization may result from attachments
via specific bindings to bacteria of the resident microflora
(co-aggregation phenomena), via entrapment in the matrix of
polysaccharide produced by the resident bacteria, or via adhesion
to saliva proteins (especially glycoproteins).
[0012] Lactobacillus casei rhamnosus GG (ATCC53103) has been
reported to colonize the mouth, most probably on the epithelium of
the buccal mucosa. This strain also adheres to the epithelium of
the intestinal tract (U.S. Pat. No. 5,032,399, Gorbach et al.;
Micr. Ecol. In Health and Dis., 2, 295-298, 1994). By contrast L.
rhamnosus does not adhere to teeth.
[0013] Japanese patent no. 4021633 (Cyconmedix KK) also reported
colonization of the mouth by Lactobacillus acidophilus, most
probably on the epithelium of the buccal mucosa. Many Lactobacillus
acidophilus are known to also adhere to the epithelium of the
intestinal tract (EP577904; EP199535; Perdigon et al., Medicina,
46, 751-754, 1986; Perdigon et al., Immunology, 63, 17-23,
1988).
[0014] Exogenous bacteria can also produce factors that inhibit the
growth of the resident microflora in the mouth. For example,
EP759469 (Socit des Produits Nestle) described the use of a
bacteriocin produced by Micrococcus varians for inhibiting the
development of the oral pathogens S. sobrinus, S. sanguis, S.
mutans, and A. viscosus.
[0015] There are several strategies to minimize the development of
resident microflora of the mouth. For example, by administering
commensal bacteria of the resident microflora that are not
cariogenic, such as Streptococcus salivarius and/or Stomatococcus
mucilaginosus, and/or repeated administration of exogenous lactic
bacteria such as L. casei, L. fermentum, L. acidophilus, L.
crispatus, L. gasseri, L. salivarius, L. bulgaricus, and S.
salivarius (Tanzer et al., Infec. and Immunity, 48,44-50, 1985;
WO92/14475).
[0016] The application of bacteriocins is another investigated
strategy which has been used to reduce tooth caries. These
molecules have attracted interest as prospective anti-carie agents
and as factors important in modulating colonization of the oral
cavity. The anti-carie potential of applying bacteriocins comes
from their potent and broad antibacterial activity against mutans
streptococci and bacteria associated with dental plaque and their
natural occurrence in bacteria regarded as being safe to humans
(U.S. Pat. No. 5,368,845 to Colgate, and WO 94/12150 to Smithkline
Beecham).
[0017] The application of milk derivatives is also of interest for
the health of the mouth. Indeed, U.S. Pat. No. 5,427,769
(Nest{umlaut over (e )}S. A.) describes another alternative wherein
dental caries are prevented by contacting teeth with an edible
composition containing micellar casein in amount sufficient to
inhibit colonization by Streptococcus sobrinus. EP748591 (Societe
des Produits Nestle S. A.) also reports the use of fluoridated
micellar casein or its micellar subunits for treating dental caries
or plaque. U.S. Pat. No. 4,992,420 (Nestec S. A.) describes
treatment of the buccal cavity with kappa-caseino-glycomacr-
opeptide derived from milk for eradicating plaque and caries.
[0018] Lactic bacteria that are not part of the resident microflora
of the mouth have never been shown to be really capable of directly
adhering to the pellicle of teeth. By colonizing the surface of
teeth, however, such lactic bacteria could exert an inhibitory
activity against the growth of the resident microflora, including
oral pathogens.
SUMMARY OF THE INVENTION
[0019] The present invention is directed to a method of treating or
preventing dental caries, dental plaque, and periodontal infection
in a humans or animals comprising administering to the oral cavity
of a human or animal one or more lactic bacteria that are not part
of the resident microflora of the mouth, that are low acidifying,
and that are capable of adhering directly to the pellicle of the
teeth to displace from the teeth or prevent attachment to the teeth
of cariogenic strains of bacteria that are resident microflora of
the mouth. In one embodiment the lactic bacteria to be administered
provides a pH in the oral cavity of about 5.5 to 5.7.
Advantageously, the lactic bacteria may be of dairy origin.
[0020] The lactic bacteria is preferably one or more of
Streptococcus thermophilus, Lactococcus lactis subsp. lactis, or
Lactococcus lactis subsp. lactis biovar diacetylactis. In
particular the lactic bacteria is one of the strains CNCM I-1984,
CNCM I-1985, CNCM I-1986, CNCM I-1987, and LMG P-18997.
[0021] Preferably, the lactic bacteria has optimal growth at a
temperature of about 37.degree. C., i.e., the temperature of the
mouth. The lactic bacteria may have been genetically modified to
have improved adherence to the pellicle of the teeth or to be less
acidifying than resident microflora found in the mouth. The lactic
bacteria may be genetically modified to have improved adherence to
the pellicle of the teeth by insertion of the X17390 gene, the
X14490 gene, or the X53657 gene.
[0022] In another embodiment the method of the invention further
involves administering the lactic bacteria in combination with one
or more of milk, fermented milk, milk derivatives, or bacteriocin.
The milk derivative may be one or more of a
caseino-glycomacropeptide, micellar casein, fluorinated micellar
casein, or renneted milk.
[0023] The invention also relates to dental compositions for use in
the methods of the invention. The lactic bacteria may be present in
these compositions in an amount of 10.sup.4 to 10.sup.9 cfu/g in
order to provide a pH of at least 5.5 when the composition is
administered to the mouth of a human or animal. When bacteriocin is
present in an the composition, it is typically present in an mount
of 0.00001 to 50 percent by weight of the composition. When the
milk derivative is one or more of a caseino-glycomacropeptide,
micellar casein, fluorinated micellar caesin, or renneted milk it
may be present in an amount of at least about 0.1 percent by weight
of the composition. The composition may further include one or more
of an oil soluble antioxidant in an amount of about 0.005 to 0.5
percent by weight of the composition and an abrasive. The
composition may be in the form of a toothpaste, mouth rinse, gum,
spray, beverage, candy, infant formula, ice cream, frozen dessert,
sweet salad dressing, milk preparation, cheese, quark, yogurt,
acidified milk, coffee cream, or whipped cream.
[0024] The invention also relates to a method for screening lactic
bacteria capable of adhering to teeth. The method involves the
steps of preparing monoclonal antibodies that recognize specific
surface proteins of lactic bacteria strains that are capable of
adhering to the teeth and screening lactic bacteria strains with
the monoclonal antibody to identify the strains of lactic bacteria
that adhere to teeth.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 represents the adhesion saturation curves for S.
sobrinus OMZ 176 (1a), L. lactis NCC2211 (1b), and S. thermophilus
NCC1561 (1c);
[0026] FIG. 2 represents the effect of CGMP on the adhesion to S-HA
beads of S. sobrinus OMZ 176, L. lactis NCC2211, and S.
thermophilus NCC1561;
[0027] FIG. 3 represents the effect of As-CGMP on the adhesion to
S-HA beads of S. sobrinus OMZ 176, L. lactis NCC2211, and S.
thermophilus NCC1561.
DETAILED DESCRIPTION OF THE INVENTION
[0028] The object of the present invention is to use lactic
bacteria that are not part of the resident microflora of the mouth,
that is lactic bacteria that are low acidifying and that are
capable of adhering directly to the pellicle of the teeth, to
prepare a composition intended for the prophylaxis or the treatment
of dental caries, dental plaque, and periodontal infection.
[0029] In one embodiment of the invention the lactic bacteria have
been genetically modified to increase its adherence to the pellicle
of the teeth via adhesion factors and/or genetically modified to be
even less acidifying, contributing to a pH in the oral cavity of
about 5.5 to 7.
[0030] The lactic bacteria may be selected from the group
consisting of:
[0031] an acidifying lactic bacteria that adheres to the pellicle
of the teeth and that has been genetically modified so that it is
low acidifying compared to resident microflora;
[0032] a non adherent lactic bacteria that is low acidifying and
that has been genetically modified so that it adheres to the
pellicle of the teeth;
[0033] a non-adherent acidifying lactic bacteria that has been
genetically modified so that it adheres to the pellicle of the
teeth and genetically modified so that it is low acidifying
compared to resident microflora.
[0034] In another embodiment the bacteria, that is not part of the
resident microflora, is low acidifying compared to resident
microflora and is capable of adhering directly to the pellicle of
the teeth.
[0035] In another embodiment the composition for the health of the
mouth comprises (1) at least a lactic bacteria that is not part of
the resident microflora of the mouth, which is capable of adhering
directly to the pellicle of the teeth and contributing to a pH in
the oral cavity of above 5.5, and (2) any form of
caseinoglycomacropeptide, micellar casein, fluorinated micellar
casein, renneted milk, or bacteriocin.
[0036] The invention also provides a method for screening lactic
bacteria capable of adhering to tooth. The method comprises the
steps of: (1) preparing monoclonal antibody recognizing specific
surface proteins of a lactic bacteria strain capable of adhering to
the teeth, and (2) screening any lactic bacteria strain by use of
the monoclonal antibody of strain capable of adhering to the
teeth.
[0037] The term "mouth," as used herein defines the oral cavity of
humans or animals such as pets, composed by the oral mucosa (gums,
lips, cheeks, palate, and floor of the mouth), the tongue, and the
teeth (including artificial structures).
[0038] Resident microflora of the mouth includes all microorganisms
that naturally live in the mouth because they can establish a
permanent residence on the oral surfaces. The resident microflora
of the mouth also includes bacteria that live in the interfacial
region between the dental hard and soft tissues (the junction
tooth-gingiva), even thought the gingival crevice and the
periodontal pocket are not present in a healthy mouth. This
microflora includes microorganisms selected from Streptococcus,
Staphylococcus, Enterococcus, Micrococcus, Peptostreptococcus,
Peptococcus, Lactobacillus, Corynebacterium, Actinomyces, Arachnia,
Rothia, Alcaligenes, Eubacterium, Propionibacterium,
Bifidobacterium, Bacillus, Clostridium, Neisseria/Branhamella,
Veillonella, Enterobacteriaceae, Campylobacter, Eikenella,
Actinobacillus, Capnocytophga, Haemophilus, Simonsiella,
Bacteroides, Fusobacterium, Porphyromonas, Prevotella,
Leptotrichia, Wohlinella/Selenomonas, Mycoplasma, Candida,
Spirochaetes, Protozoa.
[0039] Transient microflora comprises exogenous bacteria that can
be occasionally present in the mouth, but that do not establish a
permanent residence. This transient microflora may comprise all the
food micro-organisms, such as the bifidobacteria (B. infantis, B.
adolescentis, B. breve and B. longum); the lactococci (Lactococcus
lactis subsp. lactis, Lactococcus lactis subsp. cremoris, and
Lactococcus lactis subsp. lactic biovar diacetylactis); the
streptococci (Streptococcus thermophilus, S. lactis, S. lactis
cremoris and S. lactis diacetylactis); the Lactobacilli
(Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus
helveticus, Lactobacillus farciminis, Lactobacillus alimentarius,
Lactobacillus casei subsp. casei, Lactobacillus delbruckii subsp.
lactis, Lactobacillus sake, Lactobacillus curvatus, Lactobacillus
fermentum; and the acidophile group comprising L. johnsonii; (see
Fujisawa et al., Int. J. Syst. Bact., 42, 487-491, 1992); the
pediococci (Pediococcus pentosaceus, Pediococcus acidilactici, and
Pediococcus halophilus); the enterococci; the staphilococci
(Staphylococcus xylosus and Staphylococcus carnosus); the
micrococci (Micrococcus varians); yeast of the genus Debaromyces,
Candida, Pichia, Torulopsis and Saccharomyces; and mold of the
genus Aspergillus, Rhizopus, Mucor and Penicillium.
[0040] The lactic bacteria according to the invention that are low
acidifying and capable of adhering directly to the pellicle of the
teeth that are used to prepare compositions for the prophylaxis or
the treatment of dental caries, dental plaque, and periodontal
infection displace pathogenic bacteria from the teeth or prevent
the attachment of the pathogenic bacteria. The lactic bacteria
according to the invention are "low acidifying," which means that
they are less acidifying than pathogenic strains. Accordingly, they
contribute to a pH in the oral cavity of about 5.5 to 7.
Preferably, they are from dairy origin.
[0041] The lactic bacteria according to the invention adhere to the
pellicle of the teeth via specific or unspecific interactions
and/or adhesion factors. The specific adhesion factors are proteins
or polysaccharides.
[0042] At least one lactic bacteria is selected from the group
consisting of Streptococcus thermophilus, Lactococcus lactis subsp.
lactis, and Lactococcus lactis subsp. Lactis biovar diacetylactis
and particularly from the group consisting of the strains CNCM
1-1984, CNCM 1-1985, CNCM 1-1986, CNCM 1-1987, and LMG P-18997.
These strains have been selected among lactic bacteria strains for
their capacity to adhere to the pellicle of the teeth and their
optimal growth temperature of about 37.degree. C., which is the
temperature in the oral cavity. Moreover they are capable of
fermenting glucose and sucrose and do not synthesize glucans, which
are factors leading to the pathogenicity of the cariogenic
strains.
[0043] In one embodiment of the invention the lactic bacteria are
genetically modifying so that they adhere to the pellicle of the
teeth via adhesion factors. For lactic bacteria that already adhere
to the pellicle of the teeth, this modification makes the strains
more adherent to the surface of the teeth. In the same way, any
non-adherent lactic acid bacteria (not Lactobacilli) can be
genetically modified so that it adheres to the pellicle of the
teeth. This modification of the lactic bacteria can be achieved,
for example, by insertion of the genes X17390, X14490 or X53657
(GenBank accession numbers). These gene are responsible in S.
mutans for the expression of the Antigen I/II that mediates
adhesion to salivary glycoproteins.
[0044] According to the invention, it is also possible to
genetically modify lactic bacteria so that they are low acidifying.
For lactic bacteria that is already low acidifying this
modification increases the effect by further decreasing lactic acid
production. This modification can be achieved in many ways.
Preferably, the modification is achieved according to one the
protocols described in the following documents: Boumerdassi et al.,
Appl. Environ. Microbiol., 63, 2293-2299, 1997; Plattecuw et al.,
Appl. Environ. Microbiol, 61, 3967-3971, 1995; Ito et al., Biosci.
Biotechnol. Biochem., 58, 1569-1573, 1994.
[0045] According to the invention, at least one lactic bacteria,
genetically modified or not, is used in an "effective amount" for
the preparation of compositions intended for the prophylaxis or the
treatment of dental caries, dental plaque, and periodontal
infection in humans or animals such as pets. This quantity is
preferably between 10.sup.4 to 10.sup.9 cfu/g.
[0046] It is also possible to use the at least one lactic bacteria,
in combination with milk derivatives, such as milk, fermented milk,
or milk derivatives selected from any forms of
caseino-glycomacropeptide, micellar casein, fluorinated micellar
casein, renneted milk, or bacteriocin, for example.
Biochemical Characterization of the Selected Strains
[0047] Fermentation patterns: 49 simple sugars were tested with the
api 50 CH bioMerieux strip test (bioMorieux SA, 69280
Marcy-l'Etoile, France). The results are given in the Table 1.
[0048] Acidification curves: Acidification curves were determined
at 37.degree. C. under the following conditions:
[0049] S. sobrinus OMZ 176: FUM sucrose 1% and FUM glucose 1%
[0050] S. thermophilus CNCM 1-1985: Belliker sucrose 1% and
Belliker glucose 1% Inoculation was always 5%. The pH was recorded
every 20 min.
[0051] S. thermophilus CNCM 1-1985, from sucrose fermentation,
lowers the pH to 4.5, while S. sobrinus OMZ 176 lowers the pH to
4.
1TABLE I Sugar fermentation of 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. L. lactis L. lactis S. th.
S. th. S. th. Sugar CNCM I-1987 CNCM I-1986 CNCM I-1984 CNCM I-1985
LMG P-18997 Adonitol +++ Aesculin ++ ++++ Amygdalin ++++
D-Arabinose L-Arabinose D-Arabitol L-Arabitol +++ Arbutin +++ +++
Cellobiose +++ +++ Dulcitol Erythritol D-Fructose + ++++ D-Fucose
L-Fucose Galactose ++ ++++ .beta.-Gentiobiose +++ Gluconate
2-keto-Gluconate 5-keto-Gluconate GlcNAc + ++++ D-Glucose + ++++ +
++ ++ Glycerol Glycogen Inositol Inulin Lactose + ++++ +++ ++++
++++ D-Lyxose Maltose ++ Mannitol +++ ++ D-Mannose + ++++
Melezitose Melibiose .alpha.-Methyl-D-glucoside
.alpha.-Methyl-D-mannoside D-Raffinose Rhamnose Ribose ++ ++
Salicin +++ +++ Sorbitol L-Sorbose Starch Sucrose +++ ++++ +++
D-Tagatose Trehalose ++ D-Turanose ++ Xylitol +++ D-Xylose L-Xylose
.beta.-methil-xyloside +, ++, +++, ++++ show if the fermentation
begins after 3, 6, 24, or 48 hours, respectively.
[0052] The invention is also directed to compositions for the
health of the mouth that comprise a lactic bacteria that is not
part of the resident microflora of the mouth, that is low
acidifying, and that is capable of adhering directly to the
pellicle of the teeth. The compositions are particularly intended
for the prophylaxis or the treatment of dental caries, dental
plaque, and periodontal infection. The lactic bacteria strain may
be 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, LMG
P-18997, CNCM I-1986, and CNCM I-1987. In these compositions the
lactic bacteria strains may be genetically modified as described
above.
[0053] The lactic bacteria strains may be included in a food, pet
food, cosmetic, or pharmaceutical composition, for example.
Accordingly, the compositions are preferably a toothpaste, mouth
rinse, gum, spray, beverage, candy, infant formula, ice cream,
frozen dessert, sweet salad dressing, milk preparation, cheese,
quark, yogurt, acidified milk, coffee cream, or whipped cream, for
example.
[0054] In the compositions of the invention, the lactic bacteria
strains may be included alone or in combination with milk
derivatives, for example, in order to obtain synergistic
preparations. Accordingly, these compositions for the health of the
mouth comprise:
[0055] a lactic bacteria that is not part of the resident
microflora of the mouth, which is capable of adhering directly to
the pellicle of the teeth;
[0056] any forms of lactic glycopeptides, renneted milk, or
bacteriocin.
[0057] The lactic glycopeptides are preferably
caseino-glycomacropeptides (CGMP), fluorinated or non-fluorinated
micellar casein (which can be obtained as described in EP 0 604 802
and EP 0 748 591), or renneted milk. The caseino-glycomacropeptides
are preferably added in a minimum amount of about 0.1%. It has also
been shown that the caseino-glycomacropeptides do not prevent the
lactic bacteria from adhering to the teeth pellicle (FIGS. 2 and
3).
[0058] Synergistic compositions may also be prepared by adding at
least one bacteriocin which is active against Gram-positive oral
bacteria. In this embodiment the oral hygiene compositions may
comprise 0.00001 to 50%, and preferably from 0.00001 to 15% of
purified bacteriocin, by weight of the composition. The bacteriocin
is preferably variacin (EP 0759469).
[0059] To protect the composition from degradation, an oil-soluble
antioxidant may also be included. Suitable antioxidants include the
"tocopherols," butyl-hydroxyanisole (BHA), butyl-hydroxytoluene
(BHT), and ascorbyl palmitate. The oil soluble antioxidant is
present in amounts of from 0.005% to 0.5%, preferably 0.005% to
0.01% by weight of the composition.
[0060] Suitable abrasives for use in dentifrice compositions of the
present invention include calcium carbonate, calcium
aluminosilicate, alumina hydrates, alumina, zinc orthophosphate,
plastic particles, and silica, of which silica is the preferred
abrasive.
[0061] Compositions according to the invention will have a pH which
is orally acceptable and within a range such that the activity of
the lactic bacteria is not compromised. The pH may be in the range
of 3.0 to 9.5, preferably in the range 3.5 to 6.5.
[0062] The compositions of the invention may be prepared by
conventional processes that comprise admixing the ingredients
together in the appropriate relative amounts and finally, if
necessary, adjusting the pH to the desired value.
[0063] The invention is further directed to a method for screening
lactic bacteria capable of adhering to tooth. This method comprises
the steps of:
[0064] (1) preparing monoclonal antibodies that recognize specific
surface proteins of a lactic bacteria strain capable of adhering to
the teeth, and
[0065] (2) screening any lactic bacteria strain by using the
monoclonal antibody of strain capable of adhering to the teeth.
[0066] The monoclonal antibodies are used as a tool to detect the
said lactic bacteria strain among other strains growing nearby.
[0067] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention, in addition to those described
herein, will become apparent to those skilled in the art from the
foregoing description and accompanying figures. Such modifications
are intended to fall within the scope of the claims. Various
publications are cited herein, the disclosures of which are
incorporated by reference in their entireties to the extent
necessary for understanding the present invention. DNA
manipulation, cloning and transformation of bacteria cells are,
except where otherwise stated, carried out according to the
textbook of Sambrook et al. (Sambrook et al., Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Laboratory Press, U.S.A.,
1989).
EXAMPLES
[0068] The examples are preceded by a brief description of the
plasmids, strains, and the various media used, as well as the
method for producing a monoclonal antibody.
[0069] The strains S. thermophilus S118 (NCC 1529), S123 (NCC
1561), L. lactis subsp. Lactis 29 (NCC 2211), L. lactis subsp.
lactis biovar dioacetylactis 69 (NCC 2225) were deposited under the
Budapest Treaty at the Collection Nationale de Culture de
Microorganismes (CNCM 1-1984, CNCM 1-1985, CNCM 1-1986 and CNCM
1-1987, respectively), 25 rue du docteur Roux, 75724 Paris, France,
on Mar. 3, 1998. The strain S. thermophilus BFI 1116 (CNBL 1177)
was deposited under the Budapest Treaty at the Belgian Coordinated
Collections of Microorganisms LMG P-18997, K. L. Ledeganckstraat
35, B-9000 Gent, Belgium, on Jul. 5, 1999. All restrictions as to
the availability of these deposits will be withdrawn upon first
publication of this application or another application which claims
benefit of priority to this application.
Example 1
Strains and Culture Conditions
[0070] More than 100 strains (belonging to the Nestle culture
collection) were screened for their ability to attach to
saliva-coated hydroxyapatite beads, and in particular the following
23 strains: S. thermophilus Y54 (NCC 2284), S. thermophilus 5fi6
(NCC 1971), S. thermophilus Sfi13 (NCC 2008), S. thermophilus Sfi21
(NCC 2038), S. thermophilus Sfi39 (NCC 2130), S. thermophilus Sfi42
(NCC 2145), S. thermophilus Sfi47 (NCC 2172), S. thermophilus S118
(NCC 1529), S. thermophilus S119 (NCC 1536), S. thermophilus S122
(NCC 1554), S. thermophilus S123 (NCC 1561), S. thermophilus S126
(NCC 1587), L. lactis subsp. cremoris 15 (NCC 92), L. lactis subsp.
cremoris 25 (NCC 1932), L. lactis subsp. cremoris 136 (NCC 2419),
L. lactis subsp. diacetylactis 8 (NCC 1970), L. lactis subsp.
diacetylactis 28 (NCC 2057), L. lactis subsp. diacetylactis 69 (NCC
2225), L. lactis subsp. diacetylactis 80 (NCC 2272), L. lactis
subsp. lactis 29 (NCC 2211), L. lactis subsp. lactis 50 (NCC 2224),
L. lactis subsp. lactis 54 (NCC 2228), S. macedonicus 216 (NCC
2484).
[0071] The 5 oral strains, S. sobrinus OMZ 176, S. oralis OMZ 607,
A. naeslundii OMZ 745, V. dispar OMZ 493 and F. nucleatum OMZ 596
were obtained from the Institute fur Orale Mikrobiologie und
Aligemeine Immunologie, University of Zurich and were cultured in
FUM medium in anaerobiosis (GasPackSystem, BBL) at 37.degree.
C.
[0072] All the strains were stored in glycerol at -20.degree. C.
and pre-cultured for 14 hours prior to use at their specific
optimal temperature; S. sobrinus OMZ 176 grew in FUM medium
lactococci and streptococci in M17 (Difco) except S. thermophilus
NCC1529, S119, S122, NCC1561 and S126 that grew in Belliker
(prepared by dissolution of 20 g tryptone, 5 g yeast extract, 2.5 g
gelatine, 5 g dextrose, 5 g sucrose, 5 g lactose, 4 g NaCl, 0.5 g
Ascorbic acid, and 10 g beef extract in 1 L of water).
[0073] For plate counting, S. sobrinus OMZ 176 was cultured in
Mitis-Salivarius agar (Difco), S. thermophilus NCC1529, S119, S122,
NCC1561, BFI 1116, and S126 in Belliker agar (prepared by adding to
liquid Belliker 15 g of Bacto agar, Difco), and the remaining
lactic bacteria strains in M17 agar (Oxoid).
Example 2
Production of Monoclonal Antibody
[0074] A monoclonal antibody would be used as a tool to detect L.
lactis subsp. lactis NCC2211 among 5 oral strains growing together
on S-HA discs and forming a biofilm that simulates dental plaque.
Therefore the monoclonal antibody was tested against these strains
to verify there was no cross-reaction. To this end, the monoclonal
antibody is produced as described by Granato et al. "A mouse
monoclonal IgE antibody anti-bovine milk lactoglobulin allows
studies of allergy in the gastrointestinal tract., Clin. Exp.
Immunol., 63, 703-710, 1986.
Example 3
Selection of Adherent Lactic Bacteria
[0075] Attachment to Saliva-Coated Hydroxyapatite Beads (S-HA)
[0076] To select among the lactic bacteria dairy strains those
strains that are able to attach to saliva-coated hydroxyapatite
beads (S-HA), the procedure previously described by Neeser et al.
(1994) was used with slight modification in that the bead washings
were done with 150 .mu.l volumes and Hyamine hydroxide was
substituted with Benzethonium hydroxide (Sigma).
[0077] Briefly, all the strains were grown to the end of the log
phase in FUM except S. thermophilus NCC1529, S119, S122, NCC1561,
and S126 that were cultured in Belliker. S. sobrinus OMZ 176, L.
lactis subsp. lactis NCC2211, 50 and 54, S. thermophilus NCC1529,
S119, S122, NCC1561, and S126 grew at 37.degree. C., the remaining
lactococci at 30.degree. C., and the remaining streptococci at
42.degree. C.
[0078] 5 mg of hydroxyapatite beads (BDH Chemicals Ltd, Poole,
England) were covered with 70 .mu.l clarified saliva obtained from
volunteers in the lab and prepared as previously explained (Neeser
et al, 1994). Saliva coated beads were kept overnight at 4.degree.
C., then washed (first with distilled water and after with HEPES
buffer) and finally inoculated with 100 .mu.l of metabolically
labeled bacterial suspension (bacteria had been grown in medium
supplemented with 10 .mu.Ci/ml .sup.14C acetic acid). Adhesion took
place during 45 min at 37.degree. C., then unbound bacteria were
washed away and the attached cells directly counted in a LKB
scintillation counter (type 1219 Rackbeta).
[0079] Adhesion percentages are expressed as radioactivity bound to
the beads relative to the total radioactivity added to each well.
All measurements were done in triplicate. Table 2 reports the
percentages of adhesion to saliva-coated hydroxyapatite beads
obtained for several screened strains and for S. sobrinus OMZ 176
(the reference strain).
2TABLE 2 Percentages of Adhesion to Saliva-coated Hydroxyapute
Beads for Several Screened Strains. STRAIN % ADHESION (.+-.SD) S.
sobrinus OMZ 176 2.23 .+-. 0.49 S. thermophilus Sfi42 (NCC 2145)
0.08 .+-. 0.02 S. thermophilus Sfi47 (NCC 2172) 0.14 .+-. 0.04 S.
thermophilus NCC1529 2.89 .+-. 0.60 S. thermophilus S119 (NCC 1536)
0.15 .+-. 0.04 S. thermophilus S122 (NCC 1554) 0.93 .+-. 0.17 S.
thermophilus NCC1561 2.19 .+-. 0.50 S. thermophilus S126 (NCC 1587)
1.19 .+-. 0.56 L. lactis subsp. diacetylactis 28 (NCC 2057) 1.59
.+-. 0.17 L lactis subsp. diacetylactis NCC2225 1.96 .+-. 0.40 L.
lactis subsp. diacetylactis 80 (NCC 2272) 1.20 .+-. 0.35 L lactis
subsp. lactis NCC2211 2.85 .+-. 0.85
[0080] Four strains, S. thermophilus NCC 1529 (CNCM 1-1984), S.
thermophilus NCC1561 (CNCM 1-1985), L. lactis subsp. lactis NCC2211
(CNCM 1-1986) (hereinafter L. lactis NCC2211) and L. lactis subsp.
diacetylactis NCC2225 (CNCM 1-1987) showed adhesion values close to
S. sobrinus OMZ 176.
[0081] L. lactis NCC2211 and S. thermophilus NCC1561 were chosen as
the more promising candidates since they grow very well at
37.degree. C., which is the temperature in the mouth, while L.
diacetylactis NCC2225 has an optimal growth temperature of
30.degree. C. In particular, L. lactis NCC2211 cannot grow on
sucrose, but it can ferment a wide range of sugars, moreover other
oral strain can provide glucose via their invertase.
[0082] Adhesion Saturation Curves
[0083] Curves of bound CFU versus CFU inoculated into the well were
determined to verify if bead saturation could be obtained. The 50%
saturation was obtained directly from the bending point of the
curves. The adhesion saturation curves for S. sobrinus OMZ 176, L.
lactis NCC2211, and S. thermophilus NCC 1561 were determined. They
are shown in FIG. 1.
[0084] For each of the three strains the CFU number inoculated in
the well to get 50% bead saturation and the corresponding number of
bound CFU were directly deduced from the bending point of the
curves and are given in the table 3.
3TABLE 3 Number of CPU Inoculated Per Well to get 50% Bead
Saturation. cfu/well Bound cfu % adhesion S. sobrinus OMZ 176
4.00E+07 4.00E+06 10% L. lactis NCC2211 1.00E+07 9.00E-f-05 9% S.
thermophilus NCC156I 3.00E+07 2.00E+06 7%
Example 4
Effect of Caseino-Glycomacropeptides
[0085] The influence of CGMP on the adhesion of L. lactis NCC2211
and S. thermophilus NCC 1561 was studied to verify the possibility
of using CGMP to foster the predominance of one of these two
strains over pathogenic strains, namely S. Sobrinus OMZ 176.
Caseino-glycopeptide (CGMP) and its desialylated derivative
(As-CGMP) were obtained from Nestec S. A., Lausanne (for their
preparation see Neeser et al., 1994).
[0086] The dose-response effect was studied on the adhesion to S-HA
beads by inoculating, in the well, 100 .mu.l of bacterial
suspension (CFU/ml corresponding to the 50% bead saturation
previously calculated) which contained CGMP or AsCGMP in different
concentrations and then performing the adhesion assay in the usual
manner. Concentrations in the range 0.05 to 3 mg/ml were tested. No
previous incubation of the bacteria in presence of CGMP or As-CGMP
was done.
[0087] FIG. 2 provides the curves obtained for the three strains by
plotting the number of bound cells versus increasing amounts of
CGMP, the number of inoculated cells corresponds to 50% bead
saturation formerly calculated for each strain. The strong
inhibition observed in the case of S. sobrinus OMZ 176 confirms the
previous results obtained by Neeser et al. (1994) and Schupbach et
al. (J. Dent. Res., 75, 1779-1788, 1996).
[0088] FIG. 2 shows that 0.25 mg/ml produced 50% inhibition of the
adhesion of S. sobrinus OMZ 176, while more than 2 mg/ml were
necessary to have the same effect with S. thermophilus NCC1561.
CGMP slightly enhances the adhesion of L. lactis NCC2211.
[0089] As in the case of CGMP, the desyalilated derivative inhibits
the adhesion of S. sobrinus OMZ 176; only 0.05 mg/ml are needed to
produce 50% decrease in the adhesion percentage. As-CGMP does not
influence L. lactis NCC2211 adhesion, while it slightly fosters the
adhesion of S. thermophilus NCC1561 (FIG. 3).
Example 5
Toothpaste
[0090] Toothpaste is prepared by adding 10.sup.5 cfu/ml of at least
one of the lactic bacteria strain CNCM 1-1984, CNCM 1-1985, CNCM
1-1986, CNCM 1-1987 or LMG P-18997 in a lyophilized form, to a
mixture containing:
4 Cetyl pyridinum chloride 1.65% Sorbitol (70% soln) 33.0% Glycerin
25.0% Sodium carboxymethyl cellulose 2.0% Sodium fluoride 0.25%
Silica (RP 93) 26.3% Thickening Silica (Sident 22) 8.1% Sodium
saccharine 0.5% Poloxamer (Pluronic F 108) 3.2%
[0091] The toothpaste is intended for the prophylaxis or the
treatment of dental caries, dental plaque, and periodontal
infection.
Example 6
Ice Cream
[0092] A cream comprising 10.8% lactic fats, 13.5% milk solids (non
fat), 0.3% Emulstab.RTM. SE30 and 0.3% Emulstab.RTM. foam
(Grindsted, DK) is prepared and then pasteurized at 105.degree. C.
for 20s, homogenized at 75.degree. C. and 300 bar, cooled to
38.degree. C., and inoculated with pre-cultures in MRS medium,
taken in the exponential growth phase, at a rate of 10.sup.7 to
10.sup.8 cfu/ml of at least one of the lactic bacteria strain of
CNCM 1-1984, CNCM 1-1985, CNCM 1-1986, CNCM 1-1987 or LMG P-18997.
The cream is then fermented for 10 hours at 38.degree. C. up to a
pH of about 4.5. At the end of the fermentation, sucrose and
glucose syrup is added thereto. The composition of the cream is
presented in table 4 below. The mixture is then beaten, cooled to
4.degree. C., stored at 4.degree. C., and chilled to a degree of
expansion of 95.degree. C. by volume.
5TABLE 4 Ice Cream Composition Non-fat Solids Composition Fats
solids Sucrose content Ingredients (kg) (%) (%) (%) (%) Cream (35%)
30.83 10.79 1.54 12.33 Powdered 12.45 11.95 11.95 skimmed milk
Emulstab .RTM. 5E30 0.41 0.37 Emulstab .RTM. foam 0.41 0.36 Water
55.91 Total: cream base 100.00 10.79 13.49 -- 25.01 Cream base
74.14 8.00 10.00 -- 18.54 Sucrose 22.06 15.00 15.00 Glucose syrup
3.80 3.00 Fermented 100.00 8.00 10.00 15.00 36.54 Ice cream
Example 7
Yogurt
[0093] 5 L MRS culture medium were sterilized for 15 min at
121.degree. C. and then inoculated with 5% by volume of an active
culture of at least one of the S. Thermophilus strains CNCM 1-1984,
CNCM 1-1985, or LMG P-18997 containing approximately 10.sup.9
cfU/ml. After incubation for 8 h at 41.degree. C., a starter
containing 4.5.times.10.sup.8 cfu/ml was obtained.
[0094] 5 L of reconstituted skimmed milk having a dry matter
content of 10%, to which 0.1% yeast extract had been added, was
sterilized for 15 min at 121.degree. C. and inoculated with 2% of
an active culture of commercial thickening Streptococcus
thermophilus containing approximately 10.sup.9 cells/ml. After
incubation for 4 h at 41.degree. C., a starter containing
4.5.times.10.sup.8 cells/ml was obtained.
[0095] One batch of whole milk containing 3.7% fats strengthened
with 2.5% skimmed milk powder and then pasteurized for 30 min at
90.degree. C. was then inoculated with 2% by volume of the starter
of at least one of the CNCM 1-1984, CNCM 1-1985 or LMG P-18997
strains and 3% by volume of the starter of thickening Streptococcus
thermophilus. The inoculated milk is stirred, poured into pots, and
incubated for 4 h at 41.degree. C. The resulting yogurt obtained
has a good firm and smooth texture and is intended for the health
of the mouth.
Example 8
Chewing Gum
[0096] A chewing gum for preventing or treating dental caries,
dental plaque, or periodontal infection can be prepared adding an
active culture of at least one of the S. Thermophilus strains CNCM
1-1984, CNCM 1-1985, or LMG P-18997 so that it contains
approximately 10.sup.4 to 10.sup.9 cfu/g, to the following typical
ingredients:
6 Xylitol 67.5% Gum base 20% Calcium carbonate 5% Glycerin 3%
PluronicFl27 2% Cellulose gum 1% Ballast compounds 0.5% Flavor
1%
Example 9
Pet Food Composition
[0097] A pet food for mouth health is obtained by preparing a feed
mixture made up of corn, corn gluten, chicken and fish meal, salts,
vitamins, and minerals. The feed mixture is fed into a
pre-conditioner and moistened. The moistened feed leaving the
pre-conditioner is then fed into an extruder-cooker and
gelatinised. The gelatinised matrix leaving the extruder is forced
through a die and extruded. The extrudate is cut into pieces
suitable for feeding to dogs, dried at about 110.degree. C. for
about 20 minutes, and cooled to form pellets which have a water
activity of about 0.6. The pellets are sprayed with 3 coating
mixtures. Each coating mixture contains an active culture of at
least one of the S. Themmophilus strains CNCM 1-1984, CNCM 1-1985,
or LMG P-18997 but one coating mixture uses hydrogenated soy fat as
a coating substrate, one coating mixture uses water as a coating
substrate, and one coating mixture uses protein digest as a coating
substrate. The pellets contain approximately 10.sup.4 to 10.sup.9
cfu/g of the strains.
* * * * *