U.S. patent application number 15/602475 was filed with the patent office on 2017-09-14 for means and methods for preventing and/or treating caries.
The applicant listed for this patent is BASF SE. Invention is credited to Mewes Boettner, Eckhard Budde, Bruno KAESLER, Rolf Knoll, Christine Lang, Martin Ryser, Markus Veen.
Application Number | 20170258708 15/602475 |
Document ID | / |
Family ID | 34926496 |
Filed Date | 2017-09-14 |
United States Patent
Application |
20170258708 |
Kind Code |
A1 |
KAESLER; Bruno ; et
al. |
September 14, 2017 |
MEANS AND METHODS FOR PREVENTING AND/OR TREATING CARIES
Abstract
The present invention relates to a microorganism belonging to
the group of lactic acid bacteria characterized in that it is
capable of specifically binding to Streptococcus mutans, wherein
the specific binding is (i) resistant to heat treatment; and/or
(ii) resistant to protease treatment; and/or (iii)
calcium-dependent; and/or (iv) formed within a pH range between 4.5
and 8.5; and/or (v) formed in the presence of saliva. Preferably,
the specific binding can be assayed as follows: (a) growing said
microorganism to stationary phase; (b) mixing said microorganism
with Streptococcus mutans which has been grown to stationary phase;
(c) incubating the mixture obtained in step (b) under conditions
allowing the formation of aggregates of said microorganism and
Streptococcus mutans and (d) detecting aggregates by the occurrence
of a pellet. Another aspect of the present invention is an analog
or fragment of said microorganism which is thermally inactivated or
lyophilized, wherein said analog or fragment retains the capability
of specifically binding to Streptococcus mutans. In addition, the
present invention encompasses compositions and additives for food,
feed or drinks comprising the microorganism belonging to the group
of lactic acid bacteria which specifically bind to Streptococcus
mutans or an analog or fragment thereof. Moreover, uses of said
microorganism or said analog or fragment thereof for the
preparation of an anticariogenic or pharmaceutical composition or
anticariogenic food or feedstuff as well as methods for producing
said compositions or food or feedstuff are provided by the present
invention.
Inventors: |
KAESLER; Bruno; (Cuxhaven,
DE) ; Knoll; Rolf; (Laudenbach, DE) ;
Boettner; Mewes; (Berlin, DE) ; Budde; Eckhard;
(Koln, DE) ; Lang; Christine; (Berlin, DE)
; Ryser; Martin; (Dresden, DE) ; Veen; Markus;
(Altmuhldorf, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BASF SE |
Ludwigshafen |
|
DE |
|
|
Family ID: |
34926496 |
Appl. No.: |
15/602475 |
Filed: |
May 23, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13462171 |
May 2, 2012 |
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15602475 |
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11662347 |
Feb 14, 2008 |
8192978 |
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PCT/EP2005/009724 |
Sep 9, 2005 |
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13462171 |
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60608381 |
Sep 10, 2004 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A23K 50/10 20160501;
A23L 33/135 20160801; A23Y 2220/73 20130101; A23K 50/40 20160501;
A23K 50/70 20160501; A23V 2002/00 20130101; A23K 50/75 20160501;
A23V 2002/00 20130101; A23G 3/366 20130101; A23Y 2220/63 20130101;
A61K 8/99 20130101; A61P 1/02 20180101; A61K 8/60 20130101; A23G
4/123 20130101; A23Y 2240/49 20130101; A23K 50/80 20160501; A23V
2200/312 20130101; A61K 35/747 20130101; A23V 2200/3204 20130101;
C12N 1/20 20130101; A61K 8/042 20130101; A23K 50/30 20160501; A61P
19/00 20180101; C12R 1/225 20130101; A61Q 11/00 20130101; A23K
50/50 20160501; A61P 43/00 20180101; A61K 8/0216 20130101; A23L
7/10 20160801 |
International
Class: |
A61K 8/99 20060101
A61K008/99; A61K 8/60 20060101 A61K008/60; A61K 8/02 20060101
A61K008/02; A61K 8/04 20060101 A61K008/04; C12N 1/20 20060101
C12N001/20; C12R 1/225 20060101 C12R001/225; A23G 4/12 20060101
A23G004/12; A23L 33/135 20060101 A23L033/135; A23L 7/10 20060101
A23L007/10; A23K 50/40 20060101 A23K050/40; A23K 50/50 20060101
A23K050/50; A23K 50/80 20060101 A23K050/80; A23K 50/30 20060101
A23K050/30; A23K 50/10 20060101 A23K050/10; A23K 50/75 20060101
A23K050/75; A23K 50/70 20060101 A23K050/70; A61Q 11/00 20060101
A61Q011/00 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 10, 2004 |
EP |
04 02 1591.5 |
Claims
1-33. (canceled)
34. A liquid or solid composition comprising cells of at least one
microorganism belonging to Lactobacillus paracasei which is
thermally inactivated by heating at more than 95.degree. C. for at
least 20 minutes, the cells consisting of dead or inactivated
cells, wherein said cells bind to Streptococcus mutans in the
presence of saliva or at a pH range between 4.5 and 8.5, wherein
the binding between said cells and Streptococcus mutans is
calcium-dependent, resistant to heat treatment at more than
95.degree. C. for at least 20 minutes, and resistant to protease
treatment, and wherein the composition comprises the cells in an
amount of at least 0.001% by weight based on the weight of the
composition; wherein said composition optionally comprises less
than 1% (w/w) lactose or more than 6% (w/w) lactose based on the
composition; wherein the composition is an animal feed which
further comprises at least one orally acceptable carrier or
excipient.
35. The composition of claim 34, wherein the feed composition is a
pet feed for dogs, cats, and rats, a cattle feed for cows and pigs,
chicken feed for chicken and turkeys, or a fish cultivation feed
for porgy and yellowtail.
36. The composition of claim 34, wherein the at least one orally
acceptable carrier or excipient comprises a raw feed material
selected from the group consisting of cereals, brans, oil-seed
meals, animal-derived raw feed materials, and combinations
thereof.
37. The composition of claim 34, wherein the at least one orally
acceptable carrier or excipient comprises a cereal selected from
the group consisting of mile, wheat, barley, oats, rye, brown rice,
buckwheat, fox-tail millet, Chinese millet, Deccan grass, corn,
soybean, and combinations thereof.
38. The composition of claim 34, wherein the at least one orally
acceptable carrier or excipient comprises a bran selected from the
group consisting of rice bran, defatted rice bran, bran,
lowest-grade flour, wheat germ, barley bran, screening pellet, corn
bran, corn germ, and combinations thereof.
39. The composition of claim 34, wherein the at least one orally
acceptable carrier or excipient comprises an oil-seed meal selected
from the group consisting of soybean meal, soybean powder, linseed
meal, cottonseed meal, peanut meal, safflower meal, coconut meal,
palm meal, sesame meal, sunflower meal, rapeseed meal, kapok seed
meal, mustard meal, and combinations thereof.
40. The composition of claim 34, wherein the at least one orally
acceptable carrier or excipient comprises an animal-derived raw
feed material selected from the group consisting of fish powders,
import meal, whole meal, coast meal, meat powder, meat and bone
powder, blood powder, decomposed hair, bone powder, byproducts from
butchery, feather meal, silkworm pupa, skim milk, casein, dry whey,
krill, and combinations thereof.
41. The composition of claim 34, wherein the at least one orally
acceptable carrier or excipient comprises a raw feed material
selected from the group consisting of plant stems and leaves,
byproducts from corn processing industries, starch, sugar, yeast,
byproducts from fermentation, agricultural byproducts, and
combinations thereof.
42. The composition of claim 34, wherein the composition has
anticariogenic activity.
43. The composition of claim 34, wherein the formation of
aggregates of the microorganism and Streptococcus mutans occurs in
the presence of saliva or at a pH range between 4.5 and 8.5.
44. The composition of claim 34, wherein the composition comprises
at least 0.01% by weight of the cells.
45. The composition of claim 34, wherein the composition comprises
at least 0.1% by weight of the cells.
46. The composition of claim 34, wherein the microorganism binds to
Streptococcus mutans serotype c (DSMZ 20523) and/or serotype e
(NCTC 10923) and/or serotype f (NCTC 11060).
47. The composition according to claim 34, wherein the
microorganism is selected from the group consisting of L. paracasei
DSM 16667, L. paracasei DSM 16668, L. paracasei DSM 16669, L.
paracasei DSM 16670, and L. paracasei DSM 16671.
48. The composition according to claim 45, wherein the
microorganism is selected from the group consisting of L. paracasei
DSM 16667, L. paracasei DSM 16668, L. paracasei DSM 16669, L.
paracasei DSM 16670, and L. paracasei DSM 16671.
49. A method for the production of the composition according to
claim 34 comprising obtaining the cells of a microorganism
belonging to the group of lactic acid bacteria, and adding said
cells to a raw or cooked feed material, molding, and granulating to
form the composition.
50. A liquid or solid_composition comprising cells of at least one
microorganism belonging to Lactobacillus paracasei which is
thermally inactivated by heating at more than 95.degree. C. for at
least 20 minutes, the cells consisting of dead or inactivated
cells, wherein said cells bind to Streptococcus mutans in the
presence of saliva or at a pH range between 4.5 and 8.5, wherein
the binding between said cells and Streptococcus mutans is
calcium-dependent, resistant to heat treatment at more than
95.degree. C. for at least 20 minutes, and resistant to protease
treatment, and wherein the composition comprises the cells in an
amount of at least 0.001% by weight based on the weight of the
composition; wherein said composition optionally comprises less
than 1% (w/w) lactose or more than 6% (w/w) lactose based on the
composition; wherein the composition is a food or drink which
further comprises at least one orally acceptable carrier or
excipient.
51. The composition of claim 50, wherein the food or drink is
selected from the group consisting of juices, refreshing drinks,
soups, teas, sour milk beverages, dairy products, ices, butter,
cheese, processed milk, skim milk, meat products, fish meat cake
products, egg products, confectioneries, breads, noodles, pickles,
smoked products, dried fishes, and seasonings.
52. The composition of claim 50, wherein the composition is a
powder food, sheet-like food, bottled food, canned food, retort
food, capsule food, tablet food, or fluid food.
53. The composition of claim 50, wherein the composition comprises
at least 0.01% by weight of the cells.
54. The composition of claim 50, wherein the composition comprises
at least 0.1% by weight of the cells.
55. The composition according to claim 50, wherein the
microorganism is selected from the group consisting of L. paracasei
DSM 16667, L. paracasei DSM 16668, L. paracasei DSM 16669, L.
paracasei DSM 16670, and L. paracasei DSM 16671.
56. The composition of claim 50, wherein the microorganism binds to
Streptococcus mutans serotype c (DSMZ 20523) and/or serotype e
(NCTC 10923) and/or serotype f (NCTC 11060).
57. A method for the production of the composition according to
claim 50 comprising obtaining the cells of a microorganism
belonging to the group of lactic acid bacteria, and adding said
cells to a raw or cooked feed material, molding, and granulating to
form the composition.
Description
[0001] The present invention relates to a microorganism belonging
to the group of lactic acid bacteria characterized in that it is
capable of specifically binding to Streptococcus mutans, wherein
the specific binding is (i) resistant to heat treatment; and/or
(ii) resistant to protease treatment; and/or (iii)
calcium-dependent; and/or (iv) formed within a pH range between 4.5
and 8.5; and/or (v) formed in the presence of saliva. Preferably,
the specific binding can be assayed as follows: [0002] (a) growing
said microorganism to stationary phase; [0003] (b) mixing said
microorganism with Streptococcus mutans which has been grown to
stationary phase; [0004] (c) incubating the mixture obtained in
step (b) under conditions allowing the formation of aggregates of
said microorganism and Streptococcus mutans and [0005] (d)
detecting aggregates by the occurrence of a pellet.
[0006] Another aspect of the present invention is an analog or
fragment of said microorganism which is thermally inactivated or
lyophilized, wherein said analog or fragment retains the capability
of specifically binding to Streptococcus mutans. In addition, the
present invention encompasses compositions and additives for food,
feed or drinks comprising the microorganism belonging to the group
of lactic acid bacteria which specifically bind to Streptococcus
mutans or an analog or fragment thereof. Moreover, uses of said
microorganism or said analog or fragment thereof for the
preparation of an anticariogenic or pharmaceutical composition or
anticariogenic food or feedstuff as well as methods for producing
said compositions or food or feedstuff are provided by the present
invention.
[0007] Streptococcus mutans .mu.lays a central role in the
development of caries. S. mutans metabolises sucrose to organic
acids thereby developing an acidic micro environment. On the one
hand, this provides an advantage for the less acidophilic,
non-cariogenic oral plaque bacteria. On the other hand, the organic
acids demineralise dental enamel, leading to cariotic lesions.
Furthermore, S. mutans synthesises a non-water soluble glucan
matrix which enforces the plaque and adds to the adherence of S.
mutans to the tooth surface. The role of further bacterial species
that are connected to caries development like lactic acid bacteria
or actinomycetes is not conclusive. These bacteria are often found
in cariotic lesions, but only in association with S. mutans. To
present knowledge, the presence of S. mutans is an indispensable
condition of cariogenesis.
[0008] The initial binding of S. mutans to the surface of the teeth
occurs via two mechanisms. The first mechanism is binding of S.
mutans via the streptococcal antigen I/II (SA I/11)--a surface
protein also known by the synonyms B, IF, P1, SR, MSL-1 or PAc--to
the pellicle, a layer of saliva proteins on the teeth surface.
Antibodies against this protein have been shown to prevent the
adhesion of S. mutans in vitro.
[0009] Accordingly, the streptococcal antigen I/II (SA I/II) is a
target for vaccination. In different recombinant combinations--the
complete antigen, the saliva binding region, the protein coupled to
cholera toxin or expressed on the surface of an avirulent
Salmonella strain--a successful immunization of animals has been
shown. This resulted in high IgA titers and a reduction of S.
mutans colonization (Huang et al., Infect. Immun. 69 (2001),
2154-2161). Comparable results have been achieved using a
DNA-vaccine coding for SA I/II (Fan et al., J. Dent. Res. 81
(2002), 784-787).
[0010] Passive immunity has been achieved by recombinant expression
of anti-SA I/II antibodies on the surface of lactic acid bacteria.
These lactobacilli aggregate S. mutans and administration of the
bacteria to rats led to a reduction of caries development (Krueger
et al., Nature Biotechnology 20 (2002), 702-706).
[0011] The most important binding partner of the streptococcal
antigen is the salivary agglutinin, a protein similar to the lung
glycoprotein gp-340 from the scavenger receptor cysteine-rich
superfamily (Prakobphol et al., J. Biol. Chem. 275 (2000)
39860-39866). The role of agglutinin in cariogenesis is not
entirely understood so far. It can lead to the adhesion of S.
mutans when present bound to surfaces, and it can lead to an
aggregation of S. mutans when present in a soluble state. The
latter might result in a removal of aggregated S. mutans from the
mouth by saliva flow. A high agglutinin concentration in saliva
leads in vitro to an increase in the adhesion of S. mutans ,
whereas in vivo there is no clear correlation between the
agglutinin concentration in saliva and the risk for caries (Stenudd
et al., J. Dent. Res. 80 (2001), 2005-2010).
[0012] Monoclonal antibodies against agglutinin completely block
the binding of S. mutans to saliva-coated hydroxyapatite in vitro
and prevent the agglutinin dependent aggregation (Carlen and
Olsson, J. Dent. Res. 74 (1995), 1040-1047; Carlen et al., J. Dent.
Res. 77 (1998), 81-90). Brady et al., Infect. Immun. 60 (1992),
1008-1017 showed that the surface adhesion and the aggregation can
be independently inhibited by different antibodies. This indicates
that different epitopes of agglutinin are responsible for these two
effects.
[0013] Other saliva proteins frequently connected to the
development of caries are proline-rich proteins (PRPs). However,
the role of these proteins in the adhesion of cariogenic bacteria
is discussed controversially. These proteins are coded by two gene
loci (PRH-1 and PRH-2) and occur in different variants that differ
in only a few amino acids (PRP-1, PRP-2, PIF. Db-double band).
These variants can be cleaved proteolytically, resulting in the
so-called small PRPs (PRP-3, PRP-4. PIF-fund Db-f).
[0014] PRPs mediate a strong binding of commensales like
Actinomyces naeslundii or non-mutans streptococci. Interestingly,
this binding takes place only after adhesion of the protein to the
tooth surface, resulting in a conformational shift making the
binding sites accessible. S. mutans is only weakly bound. The
PRP-variant Db is of relevance for the effective binding of S.
mutans. A high concentration of Db correlates with a high adhesion
of S. mutans and a strong development of caries. A reduced part of
PRP-Db of a high total PRP concentration correlates with a low
development of caries (Stenudd et al., J. Dent. Res. 80 (2001),
2005-2010). It is unknown, if S. mutans binds directly to PRPs.
[0015] The second way of S. mutans to adhere to the tooth surface
is via a sucrose dependent adhesion. S. mutans expresses three
different glycosyltransferases (GTFs) that are capable of
synthesizing the sugar polymer glucan. Glucans exist in a water
soluble form (1-6 glycosidic linkage) and a non-soluble form called
mutan (1-3 glycosidic linkage). Mutan cannot be degraded either by
oral bacteria or by enzymes in saliva. It forms a sticky matrix
within the dental plaque that is the basis for the sucrose
dependent adhesion of S. mutans. The glycosyltransferases GTFB and
GTFC, the prevalent enzymes responsible for mutan formation, are
located on the cell surface of S. mutans. In contrast, the
glycosyltransferase GTFD synthesises the soluble glucan and is
secreted by S. mutans. Experiments using GTF deficient mutants of
S. mutans show that an interaction of all three enzymes is
necessary for a sucrose dependent adhesion (Ooshima et al., J.
Dent. Res. 80 (2001), 1672-1677).
[0016] Glycosyltransferases have an N-terminal sucrose binding site
and a C-terminal glucan binding site. Antibodies against the enzyme
or against the glucan binding site lead to an inhibition of the
sucrose dependent adhesion of S. mutans. It has not been possible
to block the N-terminal sucrose binding site using antibodies (Yu
et al., Infect. Immun. 65 (1997), 2292-2298).
[0017] An inhibition of glycosyltransferases followed by a reduced
adhesion of S. mutans can also be achieved by some flavonoids or
terpenoids (US 2004/0057908) or propolis extracts (Duarte et al.,
Biol. Pharmacol. Bull. 26 (2003), 527-531).
[0018] Lactic acid bacteria named S11 have been found, that reduce
mutan formation and, therefore, adherence of S. mutans in vitro. As
described above, mutan formation is essential for S. mutans to
adhere to the tooth surface. Accordingly, Chung et al. (Oral
Microbiol. Immunol. 19 (2004), 214-216) have found detached S.
mutans cells when they have been incubated with the lactic acid
bacteria of strain S11 which are said to reduce mutan formation.
The binding of S. mutans to mutan occurs via bacterial binding
proteins (glucan binding protein). The exact mechanism of this
binding has to be determined (Sato et al., Infect. Immun. 65
(1997), 668-675).
[0019] The fungi Trichoderma harzianum and Penicillium purpurogenum
produce homologous alpha-1,3-glucanases (Fuglsang et al., J. Biol.
Chem. 275 (2000), 2009-2018). The use of Enterococcus,
Lactobacillus and Lactococcus species effective against glucan
production and plaque formation is described (U.S. Pat. No.
6,036,952). The mechanism of action has to be elucidated.
[0020] A further approach to inhibit caries is to neutralise the
low pH in the plaque. Urea and arginine are components of saliva.
Urea is present in concentrations of 3-10 mmol/L without major
differences between caries free and caries affected persons. The
concentration of free arginine differs between 4 and 40 .mu.mol/L.
Caries free individuals have a higher average of free arginine
concentrations in saliva than caries affected persons (van
Wuyckhuyse et al., J. Dent. Res. 74 (1995), 686-690).
[0021] Some plaque bacteria like Streptococcus sanguis and
Actinomyces naeslundi are capable of cleaving urea or arginine
resulting in the formation of ammonia. The alkaline ammonium rises
the pH of the plaque and therefore reduces caries (Curran et al.,
Appl. Environm. Microbiol. 61 (1995), 4494-4496; Morou-Bermudez and
Burne, Infect. Immun. 68 (2000), 6670-6676). Accordingly, these
bacteria are suggested to be used to treat caries. Another approach
suggested for treating caries is that by proteolyses of PRP-1 and
PRP-3 arginine rich peptides are created, that can, after further
proteolysis by oral bacteria like S. sanguis, S. oralis and S.
mitis, lead to a higher pH in the plaque. By application of a
recombinant variant of these peptides, the sucrose dependent
decrease of the pH is inhibited (Li et al., Infect. Immun. 68
(2000), 5425-5429). Moreover, it is described that by using a urea
containing chewing gum after sucrose intake the drop of pH can be
inhibited and, accordingly, for example, S. mutans may not
contribute so much to caries.
[0022] However, as is evident from the above, the prior art
provides recombinant microorganisms and/or live microorganisms for
use in treating caries which may be harmful and which are not food
grade organisms. Alternatively, the prior art provides agents which
may not be stable enough for a prolonged period in the oral cavity
to exert their potential anticariogenic effect. In addition, the
agents of the prior art so far suggested to be useful for treating
caries, e.g., enzyme preparations, chemical compounds, etc. may not
be cold-stable, pH-stable and/or thermostable which renders them
rather ineffective. Furthermore, some of them bear the risk of
adverse side effects. For example, streptococcal antigens which are
suggested to be used for vaccination against caries may cause
severe problems associated with vaccination. To summarize, the
prior art does not provide an agent which is not harmful for the
subject in need of caries prophylaxis and/or treatment, which can
be effectively and easily used for treating caries and which can be
cheaply produced in large amounts. Hence, there is a need for an
agent which fulfils the aforementioned desirable criteria and which
is useful for preventing and/or treating caries.
[0023] It, thus, follows that the technical problem underlying the
present invention is to comply with the needs described above. The
solution to this technical problem is achieved by providing the
embodiments characterized in the claims.
[0024] Accordingly, in a first aspect the present invention relates
to a microorganism belonging to the group of lactic acid bacteria
characterized in that it is capable of specifically binding to
Streptococcus mutans, wherein the specific binding is
[0025] (i) resistant to heat treatment; and/or
[0026] (ii) resistant to protease treatment; and/or
[0027] (iii) calcium-dependent; and/or
[0028] (iv) formed within a pH range between 4.5 and 8.5 and/or
[0029] (v) formed in the presence of saliva.
[0030] Preferably, the specific binding can be assayed as
follows:
[0031] (a) growing said microorganism to stationary phase;
[0032] (b) mixing said microorganism with Streptococcus mutans
which has been grown to stationary phase;
[0033] (c) incubating the mixture obtained in step (b) under
conditions allowing the formation of aggregates of said
microorganism and Streptococcus mutans; and
[0034] (d) detecting aggregates by the occurrence of a pellet.
[0035] The specific binding is preferably assayed as described in
Example 3 herein below. Microorganisms belonging to the group of
lactic acid bacteria are preferably mixed with S. mutans in
volumetric ratios of 3:1 to 60:1 (S. mutans: lactobacilli). Both,
the lactic acid bacteria and S. mutans are grown to stationary
phase as described in Example 1. Preferably, the optical density is
measured photometrically at a wavelength of 600 nm. The mentioned
ratios correspond to a ratio of colony forming units from 1:50 to
1:2.5. Preferably, an OD.sub.600=1 in 1 ml correlates to
3.times.10.sup.8 colony forming units of S. mutans. Preferably, an
OD.sub.600=1 in 1 mL correlates to 7.times.10.sup.9 colony forming
units of lactobacilli of the present invention. Preferably, for
assaying the aggregation reaction, the bacteria are in a volume of
2 ml in 15 ml Falcon tubes. If necessary, the culture suspensions
are diluted with PBS-butter to obtain volumetric ratios mentioned
above, while keeping the final volume at 2 ml. Preferably, the
mixture is vortexed for about 15 seconds and then left undisturbed
for at least 5, 10, 15 minutes and more preferably for at least 20
minutes at room temperature, i.e. any temperature between
16.degree. C. and 25.degree. C. An aggregation is visible as an
immediate turbity of the suspension and, after at least 20 minutes
an aggregation is visible by aggregates that settle as a visible
pellet (exemplarily shown in FIG. 1, left Falcon tube), whereas
non-S. mutans aggregating mixtures stay in suspension (exemplarily
shown in FIG. 1, right Falcon tube). As a control, self-aggregation
of the respective lactic acid bacterium and the S. mutans strain
can be assayed by omitting either S. mutans or the lactic acid
bacterium.
[0036] Additionally, the specific binding does not require
magnesium. This characteristic can be tested as described in the
appended Examples.
[0037] All the above-mentioned characteristics render the
microorganism of the present invention belonging to the group of
lactic acid bacteria a suitable agent for preventing and/or
treating caries which is caused by S. mutans. Accordingly, the
microorganism of the present invention exerts an anticariogenic
effect and is thus a useful agent for preventing and/or treating
caries. "Caries" or "dental caries" or "cavity" are interchangeable
terms for a chronic infectious disease associated with soft decayed
area in a tooth which progressively leads to the death of a tooth.
It usually occurs in children and young adults but can affect any
person. It is the most important cause of tooth loss in younger
people. Caries can be diagnosed by methods known in the art (see,
for example, Anqmar-Mansson and ten Bosch, Adv. Dent. Res. 7
(1993), 70-79)
[0038] The term "preventing caries" includes prophylaxis of caries.
Accordingly, a subject who has never been encountered with
Streptococcus mutans, the causative agent of caries, but is at a
risk of being encounterred, i.e. infected with Streptococcus mutans
benefits, for example, from the compositions of the present
invention which comprise the microorganism or a mutant or
derivative of the present invention or an analog or fragment
thereof as described herein insofar as said subject will not suffer
from caries. Hence, the compositions of the present invention are,
for example, useful for being administered to infants or children
for prophylaxis of caries since the infant's oral cavity is
normally free of Streptococcus mutans. However, the compositions of
the present invention are not limited to administration to infants
or children.
[0039] The term "treating caries" includes administration of the
compositions of the present invention to a subject suffering from
caries for the purpose of diminishing the amount of cells of
Streptococcus mutans and/or for completely depleting Streptococcus
mutans from the mouth, in particuler from the oral cavity including
teeth. Of course, after having been cured from Streptococcus
mutans, it is envisaged that the respective subject has benefits
from the compositions of the present invention as regards their
prophylactic anti-caries effects exerted on Streptococcus
mutans.
[0040] Optionally, the microorganism of the present invention is a
probiotic microorganism which has, besides its anticariogenic
effects, beneficial effects to the host organism to which it is
administered. A "probiotic", by the generally accepted definition,
is a "live microbial feed supplement which beneficially affects the
host animal by improving its intestinal microbial balance". The
microorganism of the present invention, if it has probiotic
properties, may be included in functional or health food stuff
which is described herein below.
[0041] Streptococcus mutans occurs as part of the normal flora in
the mouth. It is involved in the cause of dental caries. Dental
plaque adheres to the fissures and pits of the teeth adjacent to
the gums. It consists initially of glycoprotein which is
precipitated and is adsorbed onto the tooth enamel. Oral bacteria
then become associated with the glycoprotein. Dietary sucrose is an
important contributor to caries production, particularly if the
sucrose is in the form of sticky sweet foods some of which can
remain in the mouth for some time. The sucrose is thus more
completely metabolised by Streptococcus mutans to form acid. Drinks
which contain sucrose are swallowed and so the sucrose spends less
time in the mouth. It is essential that dental plaque is controlled
by the use of regular tooth-brushing and the use of toothpicks and
dental floss. The addition of 1 ppm of fluoride to drinking water
has proved very effective in reducing caries. The possibility of
using a vaccine against Streptococcus mutans has been rejected.
However, by the surprising finding of the present invention that
naturally-occurring microorganisms belonging to the group of lactic
acid bacteria preferably to the genus of Lactobacillus are capable
of specifically binding to
[0042] Streptococcus mutans, it is possible to effectively prevent
and/or treat caries since the microorganisms of the present
invention aggregate and flush away Streptococcus mutans due to, for
example, salivary flow from the mouth including the tooth surface
and the oral cavity. Accordingly, the present invention provides
easily administrable bacteria which are food-grade organisms that
may, in addition to their anticariogenic properties, be useful as
probiotics.
[0043] When screening a private collection to identify
microorganisms for the capability to bind to Streptococcus mutans,
it was surprisingly found that naturally-occurring microorganisms
belonging to the group of lactic acid bacteria, preferably to the
genus of Lactobacillus are capable of specifically binding to
Streptococcus mutans which is the causative agent of caries. By
specifically binding to Streptococcus mutans, the microorganism
belonging to the group of lactic acid bacteria, preferably to the
genus of Lactobacillus disclosed herein, inter alia, bind to and
aggregate Streptococcus mutans and thus, in consequence, flush away
Streptococcus mutans by the natural flow of salivary, thereby
preventing and/or treating caries. On top of this, the
microorganisms of the present invention do preferably not bind
other microorganisms present in the oral cavity which is described
herein and in particular in Example 4 herein below. Thus, the
microenvironment of the oral cavity is not disturbed since only S.
mutans as the causative agent of caries is depleted. To the best
knowledge, S. mutans does not have any beneficial effects to the
oral cavity and, thus, its loss has no adverse effect to the
respective host.
[0044] Strikingly, the specific binding of the microorganism, in
particular of the Lactobacillus species disclosed herein to
Streptococcus mutans is resistant to heat treatment and/or
resistant to protease treatment. In addition, the specific binding
is dependent on calcium and/or independent of magnesium and stable
at an acidic point of 4.5 and it occurs in the presence of saliva
which renders it in particular suitable for oral applications or as
additive for food, feed or drinks which may contain higher
concentrations of calcium, such as milk. Remarkably, thermally
inactivated or lyophilised analogs or (a) fragment(s) of said
microorganisms disclosed herein are still capable of specifically
binding to Streptococcus mutans. This surprising effect is
advantageous for using said analog(s) or fragment(s) of said
microorganisms as well as mutants or derivatives thereof in
compositions for use in the mammals, preferably, humans or animals
to prevent and/or treat caries. In particular said analogs or
fragments can be easily added to any composition, e.g. cosmetic or
pharmaceutical composition, food or feedstuff or drinks and the
like. Additionally, the production of such analogs or fragments is
cheap and easy and they can be stored for prolonged periods of time
without loosing their capability to specifically bind to
Streptococcus mutans. A further advantage of the microorganism of
the present invention is that it retains its capability to
specifically bind to S. mutans if it is lyophilised or spray-dried
or dried. This makes it a favourable ingredient for the
compositions disclosed herein.
[0045] Other embodiments and advantages of the invention are set
forth in part in the description herein, and in part, may be
obvious from the description, or may be learned from the practice
of the invention.
[0046] Before the present invention is described in detail, it is
to be understood that this invention is not limited to the
particular methodology, protocols, bacteria, vectors, and reagents
etc. described herein as these may vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to
limit the scope of the present invention which will be limited only
by the appended claims. Unless defined otherwise, all technical and
scientific terms used herein have the same meanings as commonly
understood by one of ordinary skill in the art.
[0047] Preferably, the terms used herein are defined as described
in "A multilingual glossary of biotechnological terms: (IUPAC
Recommendations)", Leuenberqer, H. G. W, Nagel, B. and Kolbl, H.
eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).
Throughout this specification and the claims which follow, unless
the context requires otherwise, the word "comprise", and variations
such as "comprises" and "comprising", will be understood to imply
the inclusion of a stated integer or step or group of integers or
steps but not the exclusion of any other integer or step or group
of integer or step. Several documents are cited throughout the text
of this specification. Each of the documents cited herein
(including all patents, patent applications, scientific
publications, manufacturer's specifications, instructions, etc.),
whether supra or infra, are hereby incorporated by reference in
their entirety. Nothing herein is to be construed as an admission
that the invention is not entitled to antedate such disclosure by
virtue of prior invention.
[0048] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the", include plural
referents unless the context clearly indicates otherwise. Thus, for
example, reference to "a reagent" includes one or more of such
different reagents, and reference to "the method" includes
reference to equivalent steps and methods known to those of
ordinary skill in the art that could be modified or substituted for
the methods described herein.
[0049] When used in the context of the present invention, the term,
microorganism belonging to the group of lactic acid bacteria" or
"microorganism of the present invention" encompasses (a)
microorganism(s) which belong(s) to bacteria, in particular
belonging to gram-positive fermentative eubacteria, more
particularly belonging to the family of lactobacteriaceae including
lactic acid bacteria. In addition, said term also encompasses
derivatives or mutants or analogs or fragments, such as a membrane
fraction as described herein, of said microorganims(s) which retain
the capability to specifically bind to S. mutans. The terms
"derivative", "mutants", "analogs" and "fragments" are described
elsewhere herein. Lactic acid bacteria are from a taxonomical point
of view divided up into the subdivisions of Streptococcus,
Leuconostoc, Pediococcus and Lactobacillus. The microorganism of
the present invention is preferably a Lactobacillus species.
Members of the lactic acid bacteria group normally lack porphyrins
and cytochromes, do not carry out electron-transport
phosphorylation and hence obtain energy only by substrate-level
phosphorylation. I.e. in lactic acid bacteria ATP is synthesized
through fermentation of carbohydrates. All of the lactic acid
bacteria grow anaerobically, however, unlike many anaerobes, most
lactic acid bacteria are not sensitive to oxygen and can thus grow
in its presence as well as in its absence. Accordingly, the
bacteria of the present invention are preferably aerotolerant
anaerobic lactic acid bacteria, preferably belonging to the genus
of Lactobacillus.
[0050] The lactic acid bacteria of the present invention are
preferably rod-shaped or spherical, varying from long and slender
to short bent rods, are moreover preferably immotile and/or
asporogenous and produce lactic acid as a major or sole product of
fermentative metabolism. The genus Lactobacillus to which the
microorganism of the present invention belongs in a preferred
embodiment is divided up by the following characteristics into
three major subgroups, whereby it is envisaged that the
Lactobacillus species of the present invention can belong to each
of the three major subgroups:
[0051] (a) homofermentative lactobacilli [0052] (i) producing
lactic acid, preferably the L-, D- or DL-isorner(s) of lactic acid
in an amount of at least 85% from glucose via the Embden-Meyerhof
pathway; [0053] (ii) growing at a temperature of 45.degree. C., but
not at a temperature of 15.degree. C.; [0054] (iii) being long-rod
shaped; and [0055] (iv) having glycerol teichoic acid in the cell
wall;
[0056] (b) homofermantative lactobacilli [0057] (i) producing
lactic acid, preferably the L- or DL-isomer(s) of lactic acid via
the Embden-Meyerhof pathway; [0058] (ii) growing at a temperature
of 15.degree. C., showing variable growth at a temperature of
45.degree. C.; [0059] (iii) being short-rod shaped or coryneform;
and [0060] (iv) having ribitol and/or glycerol teichoic acid in
their cell wall;
[0061] (c) heterofermentative lactobacilli [0062] (i) producing
lactic acid, preferably the DL-isomer of lactic acid in an amount
of at least 50% from glucose via the pentose-phosphate pathway;
[0063] (ii) producing carbondioxide and ethanol [0064] (iii)
showing variable growth at a temperature of 15.degree. C. or
45.degree. C.; [0065] (iv) being long or short rod shaped; and
[0066] (v) having glycerol teichoic acid in their cell wall.
[0067] Based on the above-described characteristics, the
microorganisms of the present invention can be classified to belong
to the group of lactic acid bacteria, particularly to the genus of
Lactobacillus. By using classical systematics, for example, by
reference to the pertinent descriptions in "Bergey's Manual of
Systematic Bacteriology" (Williams & Wilkins Co., 1984), a
microorganim of the present invention can be determined to belong
to the genus of Lactobacillus. Alternatively, the microorganisms of
the present invention can be classified to belong to the genus of
Lactobacillus by methods known in the art, for example, by their
metabolic fingerprint, i.e. a comparable overview of the capability
of the microorganism(s) of the present invention to metabolize
sugars or by other methods described, for example, in Schleifer et
al., System. Appl. Microb., 18 (1995), 461-467 or Ludwig et al.,
System. Appl. Microb., 15 (1992), 487-501. The microorganisms of
the present invention are capable of metabolizing sugar sources
which are typical and known in the art for microorganisms belonging
to the genus of Lactobacillus. In a preferred embodiment, however,
the microorganism of the present invention has a metabolic
fingerprint selected from the group consisting of:
[0068] (i) it metabolizes D-lactose, but not L-sorbose and/or
D-saccharose and/or D-inuline,
[0069] (ii) it metabolizes inuline,
[0070] (iii) it metabolizes L-sorbose, but not D-lactose and/or
D-saccharose and/or inuline, and
[0071] (iv) it metabolizes L-sorbose, D-lactose and inuline.
[0072] Preferably, the microorganism of the present invention has a
metabolic fingerprint selected from the group consisting of:
[0073] (i) it metabolizes D-lactose, but not L-sorbose,
D-saccharose and inuline,
[0074] (ii) it metabolizes L-sorbose, D-lactose and inuline, but
not D-saccharose,
[0075] (iii) it metabolizes L-sorbose, but not D-lactose,
D-saccharose and inuline, and
[0076] (iv) it metabolizes L-sorbose, D-lactose, D-saccharose, but
not inuline.
[0077] Of course, the microorganism of the present invention is not
limited to the metabolization of the sugars mentioned in the
aforementioned metabolic fingerprint pattern, but may be capable of
metabolizing further sugars which are commonly metabolized by
Lactobacillus species.
[0078] The affiliation of the microorganisms of the present
invention to the genus of Lactobacillus can also be characterized
by using other methods known in the art, for example, using
SDS-PAGE gel electrophoresis of total protein of the species to be
determined and comparing them to known and already characterized
strains of the genus Lactobacillus. The techniques for preparing a
total protein profile as described above, as well as the numerical
analysis of such profiles, are well known to a person skilled in
the art. However, the results are only reliable insofar as each
stage of the process is sufficiently standardized. Faced with the
requirement of accuracy when determining the attachment of a
microorganism to the genus of Lactobacillus, standardized
procedures are regularly made available to the public by their
authors such as that of Pot et al., as presented during a
"workshop" organized by the European Union, at the University of
Ghent, in Belgium, on Sep. 12 to 16, 1994 (Fingerprinting
techniques for classification and identification of bacteria,
SDS-PAGE of whole cell protein). The software used in the technique
for analyzing the SDS-PAGE electrophoresis gel is of crucial
importance since the degree of correlation between the species
depends on the parameters and algorithms used by this software.
Without going into the theoretical details, quantitative comparison
of bands measured by a densitometer and normalized by a computer is
preferably made with the Pearson correlation coefficient. The
similarity matrix thus obtained may be organized with the aid of
the UPGMA (unweighted pair group method using average linkage)
algorithm that not only makes it possible to group together the
most similar profiles, but also to construct dendograms (see
Kersters, Numerical methods in the classification and
identification of bacteria by electrophoresis, in Computer-assisted
Bacterial Systematics, 337-368, M. Goodfellow, A. G. O'Donnell Ed.,
John Wiley and Sons Ltd, 1985).
[0079] Alternatively, the affiliation of said microorganisms of the
present invention to the genus of Lactobacillus can be
characterized with regard to ribosomal RNA in a so called
Riboprinter..RTM.. More preferably, the affiliation of the newly
identified species of the invention to the genus Lactobacillus is
demonstrated by comparing the nucleotide sequence of the 16S
ribosomal RNA of the bacteria of the invention, or of their genomic
DNA which codes for the 16S ribosomal RNA, with those of other
genera and species of lactic acid bacteria known to date. Another
preferred alternative for determining the attachment of the newly
identified species of the invention to the genus Lactobacillus is
the use of species-specific PCR primers that target the 16S-23S
rRNA spacer region. Another preferred alternative is RAPD-PCR
(Nigatu et al. in Antonie van Leenwenhoek (79), 1-6, 2001) by
virtue of that a strain specific DNA pattern is generated which
allows to determine the affiliation of an identified microorganisms
in accordance with the present invention to the genus of
Lactobacillus. Further techniques useful for determining the
affiliation of the microorganism of the present invention to the
genus of Lactobacillus are restriction fragment length polymorphism
(RFLP) (Giraffa et al., Int. J. Food Microbiol. 82 (2003),
163-172), fingerprinting of the repetitive elements (Levers et al.,
FEMS Microbiol. Lett. 205 (2001) 31-36) or analysis of the fatty
acid methyl ester (FAME) pattern of bacterial cells (Heyrman et
al., FEMS Microbiol. Lett. 181 (1991), 55-62). Alternatively,
lactobacilli can be determined by lectin typing (Annuk et al., J.
Med. Microbiol. 50 (2001), 1069-1074) or by analysis of their cell
wall proteins (Gatti et al., Lett. Appl. Microbiol. 25 (1997),
345-348.
[0080] In accordance with the present invention, the microorganisms
are preferably lactic acid bacteria belonging to the genus of
Lactobacillus, more preferably Lactobacillus species as described
herein. Even more preferably the Lactobacillus of the present
invention is Lactobacillus paracasei or Lactobacillus rhamnosus.
However, the Lactobacillus species are not limited thereto. In a
particular preferred embodiment the microorganisms of the present
invention are "isolated" or "purified". The term "isolated" means
that the material is removed from its original environment, e.g.
the natural environment if it is naturally occurring. For example,
a naturally-occurring microorganism, preferably a Lactobacillus
species, separated from some or all of the coexisting materials in
the natural system, is isolated. Such a microorganism could be part
of a composition, and is to be regarded as still being isolated in
that the composition is not part of its natural environment. The
term "purified" does not require absolute purity; rather, it is
intended as a relative definition. Individual microorganisms
obtained from a library have been conventionally purified to
microbiological homogeneity, i.e. they grow as single colonies when
streaked out on agar plates by methods known in the art.
Preferably, the agar plates that are used for this purpose are
selective for Lactobacillus species. Such selective agar plates are
known in the art.
[0081] In a particularly preferred embodiment of the present
invention, the microorganism of the present invention is selected
from the group consisting of Lactobacillus paracasei or
Lactobacillus rhamnosus having DSMZ accession number DSM 16667 (L.
paracasei ssp. paracasei Lb-Ob-K1), DSMZ accession number DSM 16668
(L. paracasei ssp. paracasei Lb-Ob-K2), DSMZ accession number DSM
16669 (L. paracasei ssp. paracasei Lb-Ob-K3), DSMZ accession number
DSM 16670 (L. paracasei ssp. paracasei Lb-Ob-K4), DSMZ accession
number DSM 16671 (L. paracasei ssp. paracasei Lb-Ob-K5), DSMZ
accession number DSM 16672 (L. rhamnosus Lb-Ob-K6) and DSM
accession number DSM 16673 (L. rhamnosus Lb-Ob-K7) or a mutant or
derivative thereof, wherein said mutant or derivative retains the
capability to specifically bind to Streptococcus mutans. The term
"Lactobacillus paracasei or Lactobacillus rhamnosus having DSMZ
accession number" relates to cells of a microorganism belonging to
the species Lactobacillus paracasei or Latobacillus rhamnosus
deposited with the Deutsche Sammlung fur Mikroorganismen and
Zellkulturen GmbH ("DSMZ") on Aug. 26, 2004 and having the
following deposit numbers DSM 16667, 16668, 16669, 16670, 16671,
16672 or 16673. The DSMZ is located at the Mascheroder Weg 1 b,
D-38124 Braunschweig, Germany. The aforementioned DSMZ deposits
were made pursuant to the terms of the Budapest treaty on the
international recognition of the deposit of microorganisms for
purposes of patent procedure.
[0082] "A mutant or derivative" of the microorganism of the present
invention, preferably of the deposited Lactobacillus paracasei or
Lactobacillus rhamnosus cells has preferably the same
characteristics as the respective deposited strains, i.e. it
retains the capability to specifically bind to Streptococcus
mutans, preferably with the binding characteristics as described
hereinabove. For example, said derivative can be genetically
engineered. In the context of the present invention the term
"genetically engineered" is used in its broadest sense for methods
known to the person skilled in the art to modify desired nucleic
acids in vitro and in vivo such that genetic modifications are
affected and genes are altered by recombinant DNA technology.
Accordingly, it is preferred that said methods comprise cloning,
sequencing and transformation of recombinant nucleic acids. For
this purpose appropriate vectors including expression vectors for
Lactobacillus species as, for example, described in EP-B1 506 789,
EP-B1 316 677, EP-B1 251 064, EP-B1 218 230, EP-B1 133 046 or WO
89/01970.
[0083] Primers, enzymes, further host cells for cloning of
intermediate constructs and the like can be used and are known by
the skilled artisan. Preferably, genetically engineered mutants
comprise cells of the microorganism of the present invention,
preferably of the deposited Lactobacillus species harbouring
recombinant nucleic acids either comprised in their bacterial
chromosome or on (a) plasmid(s) or comprised in their bacterial
chromosome and/or (a) plasmid(s). Said recombinant nucleic acids
are preferably foreign to the microorganism of the presnt
invention. By "foreign" it is meant that the polynucleotide or
nucleic acid molecule is either heterologous with respect to the
host cell, this means derived from a cell or organism with a
different genomic background, or is homologous with respect to the
host cell but located in a different genomic environment than the
naturally occurring counterpart of said nucleic acid molecule. This
means that, if the nucleic acid molecule is homologous with respect
to the host cell, it is not located in its natural location in the
genome of said host cell, in particular it is surrounded by
different genes. In this case the polynucleotide may be either
under the control of its own promoter or under the control of a
heterologous promoter. The vector or nucleic acid molecule
according to the invention which is present in the host cell may
either be integrated into the genome of the host cell or it may be
maintained in some form extrachromosomally. In this respect, it is
also to be understood that the nucleic acid molecule of the
invention can be used to restore or create a mutant gene via
homologous recombination.
[0084] Plasmids may be low, medium or high copy number plasmids.
Said genetically engineered mutants may harbour nucleic acids
encoding a glucanase or mutanase which is capable of degrading the
mutan specific 1,3-glycosidic bond of saccharose subunits. Fungal
glucanases are, for example, described in Fuglsang et al., J. Biol.
Chem. 275 (2000), 2009-2018. It is also envisaged that genetically
engineered mutants comprise cells harbouring recombinant nucleic
acids encoding antibodies which are preferably secreted or anchored
in the bacterial cell wall. The term "antibody" encompasses intact
antibodies as well as antibody fragments thereof, like, separated
light and heavy chains, Fab, Fab/c, Fv, Fab', F(ab')2. The term
"antibody" also comprises humanized antibodies, bifunctional
antibodies and antibody constructs, like single chain Fvs (scfv) or
antibody-fusion proteins. It is also envisaged in context of this
invention that the term "antibody" comprises antibody constructs
which may be expressed in cells of the derivative of the deposited
microorganism of the present invention, e.g. antibody constructs
which may be transformed via, inter alia, vectors by methods known
in the art. It is in particular envisaged that such antibody
constructs specifically recognize, for example, the streptococcal
antigen I/II. Such an approach is, for example, described in
Krueger el: al., Nat. Biotechnol. 20 (2002), 702-706 or Shiroza,
Biochim Biophys Acta 1626 (2003), 57-64.
[0085] Secretion of the expressed antibody is preferably achieved
by operatively linking the nucleic acid encoding an antibody to a
secretion signal sequence. Anchoring in the bacterial cell wall
could be achieved by making use of the mechanism of the enzyme
sortase. Namely, surface proteins of gram-positive bacteria are
linked to the bacterial cell wall by a mechanism that involves
cleavage of a conserved Leu-Pro-X-Thr-Gly (LPXTG) motif and that
occurs during assembly of the peptidoglycan cell wall. Accordingly,
the nucleic acid molecule encoding an antibody may be fused to a
sequence encoding the aforementioned conserved motif which is used
by sortase to anchor proteins in the bacterial cell wall.
[0086] It is also envisaged that the microorganism of the present
invention, preferably the deposited Lactobacillus species be
genetically modified to harbor a nucleic acid molecule encoding
reuterin which is an antimicrobial substance effective, inter alia,
against Streptococcus mutans. Reuterin is, for example, described
in Talarico et al., Chemother. 33 (1989), 674-679.
[0087] A mutant of the microorganism of the present invention,
preferably a mutant of the deposited Lactobacillus strains is
preferably artificially mutated. In accordance with the present
invention, the term "mutated" means (a) permanent modification(s)
of genetic material, i.e. nucleic acids, caused, for example,
naturally or by physical means or chemical
compounds/substances/agents, such as EMS or ENU. Said modifications
include point mutations, like transitions or transversions,
deletion/insertion/addition of one or more bases within a nucleic
acid/gene/chromosome thereby modifying the nucleic
acid/gene/chromosome which can cause, inter alia, aberrant gene
expression/transcription/translation or inactive gene products,
constitutive active/inactive gene products leading to e.g.
dominant-negative effects. Preferablly, a mutation leads to in
increased capability of specifically binding Streptococcus mutans.
Thus, it is also preferred that the mutant cells of the deposited
microorganism which harbour (a) mutation(s) in (a) desired gene(s)
or in which (a) mutation(s) in (a) desired gene(s) is induced by
methods known to the person skilled in the art. It is also known in
the prior art that mutated or genetically engineered bacterial
cells can be selected by any suitable method/phenotype. In the
context of the present invention, a mutant having an increased
capability to specifically bind to Streptococcus mutans can be
tested in accordance with the methods described in the appended
Examples. The term "mutant", however, also includes cells of the
microorganism of the present invention, preferably cells of the
deposited microorganism which harbour naturally-occurring,
spontaneous mutations in their genome, i.e. bacterial chromosome.
"Spontaneous mutations" are mutations that arise naturally, i.e.,
without direct genetic manipulation by man, or by exposure to a
mutagen. Selection of spontaneous mutants can be accomplished by
culturing the strain and selecting the desired variants by, for
example, the variant bacterium's capability to show an improved .
Methods for selection of spontaneous mutants are well known in the
art (see, for example, Sambrook, Russell "Molecular Cloning, A
Laboratory Manual", Cold Spring Harbor Laboratory, N.Y. (2001);
Ausubel, "Current Protocols in Molecular Biology", Green Publishing
Associates and Wiley Interscience, N.Y. (1989)). For example, such
mutations may occur during cultivation, for example, during the
normal cell division process coupled with DNA replication or during
passaging and/or preserving the mutant of the microorganism of the
present invention.
[0088] The oral cavity is home to many different species of
streptococci and it is not surprising, considering they share the
same habitat, that they have many features in common. Thus, it is
preferable that the microorganism of the present invention binds
specifically to Streptococcus mutans. Accordingly, the term
"specifically binding" in the context of the present invention
means that the microorganism of the present invention, preferably a
microorganism belonging to the genus of Lactobacillus binds to
Streptococcus mutans but does not bind to most other, preferably to
no other species belonging to the genus Streptococcus. Other
species belonging to the genus of Streptococcus are those described
in Example 4. Namely, the microorganism of the present invention
does preferably not bind to bacteria belonging to the species of
Streptococcus salivarius, preferably belonging to the subspecies
thermophilus, to the species Streptococcus oralis, to the species
Streptococcus mitis and/or to the species Streptococcus sanguinis.
More preferably, it does not bind to Streptococcus salivarius ssp.
thermophilus (identified by API 50 CH (Biomerieux, France),
Streptococcus oralis (DSMZ 20066), Streptococcus oralis (DSMZ
20395), Streptococcus oralis (DSMZ 20627), Streptococcus mitis
(DSMZ 12643) and/or Streptococcus sanguinis (DSMZ 20567). In
addition, said microorganism preferably does not bind to bacteria
belonging to genera other than Streptococcus, e.g. belonging to the
genus of Staphylococcus. More preferably, it does not bind to
bacteria belonging to the species Staphylococcus epidermidis. Most
preferably, it does not bind to Staphylococcus epidermidis (DSMZ
1798) and/or Staphylococcus epidermidis (DSMZ 20044) For the test
of specific binding, preferably each of the aforementioned oral
bacteria are preferably mixed in a volumetric ratio of 3:1 with
Lactobacillus cultures of the present invention and aggregation is
preferably assayed as described herein and for example in Example
3.
[0089] It was shown that the Lactobacillus paracasei, preferably L.
paracasei ssp. paracasei of the present invention does not
aggregate any of the aforementioned oral bacterial belonging to the
genus of Streptococcus and it does not bind the bacteria belonging
to the genus of Staphylococcus mentioned herein above. The
Lactobacillus rhamnosus strains of the present invention were shown
to not aggregate all of the above mentioned Streptococcus and
Staphylococcus species, apart from Streptococcus salivarius ssp.
thermophilus. Preferably the term "specifically binding" also means
that a microorganism of the present invention binds to such
Streptococcus mutans strains which have the capability to be a
cariogenic dental pathogen.
[0090] The specific binding reaction comprises binding and,
preferably, aggregating Streptococcus mutans cells as described
herein by the microorganism of the present invention in the mouth.
This specific binding leads, in consequence, to flushing away the
Streptococcus mutans cells by, for example, salivary flow or by a
mouth rinse or mouth wash and the like as described herein. The
mouth defines the oral cavity of mammals, preferably 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). Preferably, the specific binding
reaction of the microorganisms of the present invention to
Streptococcus mutans prevents Streptococcus mutant cells from
attaching to the surface of a tooth or teeth (or while not being
bound by theory could lead to detachment of Streptococcus mutans
cells from the surface of a tooth or teeth) In consequence, the
specific binding reaction results in flushing away Streptococcus
mutans cells out of the mouth, thereby diminishing the causative
agent of caries and, thus, preventing and/or treating caries.
[0091] It is believed that the microorganism of the present
invention may bind specifically to the streptococcal antigen I/II
which is also known as antigen B, IF, P1, SR, MSL-1 or PAc.
However, the microorganism of the present invention may bind to any
other protein or surface structure of S. mutans , thereby
aggregating S. mutans and flushing it out of the oral cavity as
described herein. It is known that Streptococcus mutants binds via
said streptococcal antigen I/II to the pellicle. Accordingly, when
the microorganism of the present invention may bind, for example,
to said streptococcal antigen I/II, Streptococcus mutans is
hampered to bind to the surface of teeth which thus helps to
prevent and/or treat caries.
[0092] The pellicle is a clear, thin covering containing proteins
and lipids (fats) found in saliva. It is formed within seconds
after a tooth surface is cleaned. Pellicle formation is the first
step in dental plaque formation. Dental plaque is a soft deposit
that accumulates on the teeth. Plaque can be defined as a complex
microbial community, with greater than 10.sup.10 bacteria per
milligram. It has been estimated that as many as 400 distinct
bacterial species may be found in plaque. In addition to the
bacterial cells, plaque contains a small number of epithelial
cells, leukocytes, and macrophages. The cells are contained within
an extracellular matrix, which is formed from bacterial products
and saliva. The extracellular matrix contains protein,
polysaccharide and lipids. One of the proteins present in saliva is
agglutinin which is on the one hand thought to lead to a partial
removal of Streptococcus mutans from the mouth, however, is on the
other hand suspected to facilitate adhesion of Streptococcus mutans
to the surface of teeth, thereby facilitating the initial
attachment of Streptococcus mutans to teeth and, thus, onset of
caries.
[0093] Whether the microorganism of the present invention
specifically binds to Streptococcus mutans as defined herein above
can easily be tested, inter alia, by comparing the reaction of said
microorganism of the present invention with S. mutans cells with a
microorganism also belonging to the genus of Lactobacillus that
does not specifically bind to Streptococcus mutans by preferably
employing the method as described in the appended Examples herein
below.
[0094] Preferably, the microorganism of the present invention is
capable of specifically binding to Streptococcus mutans serotype c
(DSMZ 20523) and/or serotype e (NCTC 10923) and/or serotype f (NCTC
11060). This means that the microorganism of the present invention
binds to Streptococcus mutans serotype c, serotype e or serotype f.
Preferably, this means that the microorganism of the present
invention binds to Streptococcus mutans serotype c and serotype e
or serotype f. This also means that the microorganism of the
present invention binds to Streptococcus mutans serotype c and
serotype f or serotype e or that the microorganism of the present
invention binds to Streptococcus mutans serotype e and serotype f
or c. More preferably this means that the microorganism of the
present invention binds to Streptococcus mutans serotype c,
serotype e and serotype f. In accordance with the present invention
a "serotype" is an antigenic property of a bacterial cell,
preferably of a Streptococcus mutans cell identified by serological
methods known in the art.
[0095] As described above, the specific binding of the
microorganism of the present invention to Streptococcus mutans is
resistant to heat treatment. Accordingly, the microorganism of the
present invention is treated with heat, for example, at a
temperature above 15.degree. C. or 37.degree. C. More preferably,
the cells are incubated at a temperature of more than 55.degree.
C., even more preferably of more than 65.degree. C., particularly
preferred of more than 95.degree. C. and most preferred at
121.degree. C. After cooling down, the capability of the
microorganism of the present invention to specifically bind the S.
mutans is determined as described herein.
[0096] The corresponding temperature can depend on the specific
Lactobacillus species but can be easily determined by the skilled
person by routine experimentation, e.g. by incubating the
corresponding cells at different temperatures and determining the
amount of Lactobacillus cells which is still capable of
specifically binding to Streptococcus mutans by using methods as
those shown in the examples herein.
[0097] Generally, the heat treatment should last for a period of
time of at least 1 minute. Preferably, the heat treatment lasts for
a period of time of at least n minutes, wherein n is an integer in
the range of 2 to 60, with n=20 being particularly preferred.
However, there is in principle no upper limit for the time of
incubation. However, it is preferably no longer than 4, 3, 2 or 1
hour(s). The most preferred heat treatment is at least 20 minutes
at a temperature of 121.degree. C. in a saturated steam having an
atmospheric pressure of 2 bar. The most preferred heat treatment is
considered as abolishing any function of a protein and of any
vitality of cells which thus distinguishes the microorganism of the
present invention from other microorganism in that it is still
capable of the specifically binding to S. mutans. Hence, it is very
useful for any food, feed, drink or composition of the present
invention if it is desired that the microorganism should not be
alive.
[0098] The specific binding of the microorganism of the present
invention is furthermore characterized by its resistance to
protease treatment which is treatment with a protease selected from
the group consisting of pronase E, proteinase K, trypsin and
chymotrypsin. These proteinases are proteases which show no
specificity and, thus, are considered as degrading any protein
being on the cell surface of a microorganism. Other proteases,
which are known to have preferences for certain patterns of amino
acid residues are elastase, thrombin, aminopeptidase I,
carboxypeptidase, dostripain, endoproteinase, papain, pepsin or
proteases. The latter proteases could also be used to test whether
the specific binding of the microorganism of the present invention
to S. mutans is resistant to the latter more specific proteases.
Thus, after protease treatment which is described in the appended
Examples, the microorganism of the present invention is still
capable of specifically binding to Streptococcus mutans.
[0099] In addition, the specific binding of the microorganism of
the present invention is furthermore characterized by its
dependency on calcium. Preferably, the specific binding takes place
in the presence of a concentration of calcium ions between 0.05 mM
and 500 mM, preferably between 1 mM and 100 mM. Particularly
preferred the calcium concentration is between 2 mM and 30 mM. The
dependency of the specific binding on calcium can be tested as
described in the appended Examples.
[0100] Moreover, the specific binding to the microorganism of the
present invention is maintained over a pH range between 4.0 and
9.0, preferably between 4.0 and 7.0 In particular, the pH value at
which the specific binding takes still place is preferably 4.5.
Assaying of the maintenance of the specific binding over the pH
range described above is shown in the appended Examples.
[0101] Furthermore, the specific binding is independent of
magnesium. Thus, it is not necessary that magnesium ions or
magnesium salts are present which is demonstrated in the appended
Examples.
[0102] A still further characteristic of the specific binding is
its occurrence in the presence of saliva. Saliva is an exogenous
secrete which is synthesized by the salivary glands. It is a
complex liquid containing, apart from about 99% water a
multiplicity of organic and inorganic compounds. Physiological
ingredients of saliva are, inter alia, enzymes, e.g., amylases,
carboanhydrases, lysozyme, peroxidases or proteins, e.g., mucins,
lactoferrin, proline-rich proteins, cystatines, histatines or
statherines or soluble IgA. Thus, although a variety of potentially
interfering substances are present in saliva, the specific binding
of the microorganism of the present invention was not disturbed or
hampered. For testing the specific binding in the presence of
saliva, it is preferred that saliva is used which contains
preferably the Streptococcus species described in Example 4 and/or
the Staphylococcus species of Example 4. If, however, Lactobacillus
rhamnosus species of the present invention are tested for specific
binding to S. mutans in the presence of saliva, it is preferred
that Streptococcus salivarius ssp. thermophilus is omitted. The
specific binding is assayed as described herein.
[0103] The aforementioned characteristics of the microorganism of
the present invention belonging to the group of lactic acid
bacteria renders it to be a robust and effective agent for
preventing and/or treating caries since it is mainly administered
in various forms to the mouth including the oral cavity and teeth
where, inter alia, saliva including certain proteases and low pH
values after ingestion of carbohydrate containing food stuff is
present. Moreover, the resistance to heat has beneficial effects in
adding the microorganism of the present invention as additive to
food stuff during the preparation of said food stuff. Namely, food
stuff is often heat sterilized, pre-cooked, pasteurized and the
like which is detrimental for viability of microorganisms.
[0104] In another aspect the present invention relates to an analog
or fragment of the microorganism of the present invention, which is
thermally inactivated or lyophilized, wherein said analog or
fragment retains the capability of specifically binding
Streptococcus mutans.
[0105] According to the present invention the term "analog of the
microorganism of the present invention" includes a dead or
inactivated cell of the microorganism of the present invention,
preferably of the Lactobacillus species disclosed herein which is
no longer capable to form a single colony on a plate specific for
microorganisms belonging to the genus of Lactobacillus. Said dead
or inactivated cell may have either an intact or broken cell
membrane. Methods for killing or inactivating cells of the
microorganism of the present invention are known in the art.
El-Nezami et al., J. Food Prot. 61 (1998), 466-468 describes a
method for inactivating Lactobacillus species by UV-irradiation.
Preferably, the cells of the microorganism of the present invention
are thermally inactivated or lyophilised as described in the
appended Examples. Lyophilization of the cells of the present
invention has the advantage that they can be easily stored and
handled while retaining their capability to specifically bind to S.
mutans. Moreover, lyophilised cells can be grown again when applied
under conditions known in the art to appropriate liquid or solid
media. Lyophilization is done by methods known in the art.
Preferably, it is carried out for at least 2 hours at room
temperature, i.e. any temperature between 16.degree. C. and
25.degree. C. Moreover, the lyophilized cells of the microorganism
of the present invention are stable for at least 4 weeks at a
temperature of 4.degree. C. so as to still specifically bind to S.
mutans as is shown in Example 7 herein below. Thermal inactivation
can be achieved by incubating the cells of the microorganism of the
present invention for at least 2 hours at a temperature of
170.degree. C. Yet, thermal inactivation is preferably achieved by
autoclaving said cells at a temperature of 121.degree. C. for at
least 20 minutes in the presence of saturated steam at an
atmospheric pressure of 2 bar. In the alternative, thermal
inactivation of the cells of the microorganism of the present
invention is achieved by freezing said cells for at least 4 weeks,
3 weeks, 2 weeks, 1 week, 12 hours, 6 hours, 2 hours or 1 hour at
-20.degree. C. It is preferred that at least 70%, 75% or 80%, more
preferably 85%, 90% or 95% and particularly preferred at least 97%,
98%, 99% and more particularly preferred, 99.1%, 99.2%, 99.3%,
99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% and most particularly
preferred 100% of the cells of the analog of the microorganism of
the present invention are dead or inactivated, however, they have
still the capability to specifically bind to S. mutans. Whether the
analog or fragment of the microorganism of the present invention is
indeed dead or inactivated can be tested by methods known in the
art, for example, by a test for viability.
[0106] The term "analog of the microorganism of the present
invention" also encompasses lysates or fractions of the
microorganism of the present invention, preferably of the
Lactobacillus species disclosed herein. According to the present
invention the term "lysate" means a solution or suspension in an
aqueous medium of cells of the microorganism of the present
invention that are broken. However, the term should not be
construed in any limiting way. The cell lysate comprises, e.g.,
macromolecules, like DNA, RNA, proteins, peptides, carbohydrates,
lipids and the like and/or micromolecules, like amino acids,
sugars, lipid acids and the like, or fractions of it. Additionally,
said lysate comprises cell debris which may be of smooth or
granular structure. Methods for preparing cell lysates of
microorganism are known in the art, for example, by employing
French press, cells mill using glass or iron beads or enzymatic
cell lysis and the like. In addition, lysing cells relates to
various methods known in the art for opening/destroying cells. The
method for lysing a cell is not important and any method that can
achieve lysis of the cells of the microorganism of the present
invention may be employed. An appropriate one can be chosen by the
person skilled in the art, e.g. opening/destruction of cells can be
done enzymatically, chemically or physically. Non-limiting examples
for enzymes and enzyme cocktails are proteases, like proteinase K,
lipases or glycosidases; non-limiting examples for chemicals are
ionophores, detergents, like sodium dodecyl sulfate, acids or
bases; and non-limiting examples of physical means are high
pressure, like French-pressing, osmolarity, temperature, like heat
or cold. Additionally, a method employing an appropriate
combination of an enzyme other than the proteolytic enzyme, an
acid, a base and the like may also be utilized. For example, the
cells of the microorganism of the present invention are lysed by
freezing and thawing, more preferably freezing at temperatures
below -70.degree. C. and thawing at temperatures of more than
30.degree. C., particularly freezing is preferred at temperatures
below -75.degree. C. and thawing is preferred at temperatures of
more than 35.degree. C. and most preferred are temperatures for
freezing below -80.degree. C. and temperatures for thawing of more
than 37.degree. C. It is also preferred that said freezing/thawing
is repeated for at least 1 time, more preferably for at least 2
times, even more preferred for at least 3 times, particularly
preferred for at least 4 times and most preferred for at least 5
times.
[0107] Accordingly, those skilled in the art can prepare the
desired lysates by referring to the above general explanations, and
appropriately modifying or altering those methods, if necessary.
Preferably, the aqueous medium used for the lysates as described is
water, physiological saline, or a buffer solution. An advantage of
a bacterial cell lysate is that it can be easily produced and
stored cost efficiently since less technical facilities are needed.
According to the invention, lysates are also preparations of
fractions of molecules from the above-mentioned lysates. These
fractions can be obtained by methods known to those skilled in the
art, e.g., chromatography, including, e.g., affinity
chromatography, ion-exchange chromatography, size-exclusion
chromatography, reversed phase-chromatography, and chromatography
with other chromatographic material in column or batch methods,
other fractionation methods, e.g., filtration methods, e.g.,
ultrafiltration, dialysis, dialysis and concentration with
size-exclusion in centrifugation, centrifugation in
density-gradients or step matrices, precipitation, e.g., affinity
precipitations, salting-in or salting-out
(ammoniumsulfate-precipitation), alcoholic precipitations or other
proteinchemical, molecular biological, biochemical, immunological,
chemical or physical methods to separate above components of the
lysates. In a preferred embodiment those fractions which are more
immunogenic than others are preferred. Those skilled in the art are
able to choose a suitable method and determine its immunogenic
potential by referring to the above general explanations and
specific explanations in the examples herein, and appropriately
modifying or altering those methods, if necessary.
[0108] "A fragment of the microorganism of the present invention"
encompasses any part of the cells of the microorganism of the
present invention. Preferably, said fragment is a membrane fraction
obtained by a membrane-preparation. Membrane preparations of
microorganisms belonging to the genus of Lactobacillus can be
obtained by methods known in the art, for example, by employing the
method described in Rollan et al., Int. J. Food Microbiol. 70
(2001), 303-307, Matsuguchi et al., Clin. Diagn. Lab. Immunol. 10
(2003), 259-266 or Stentz et al., Appl. Environ. Microbiol. 66
(2000), 4272-4278 or Varmanen et al., J. Bacteriology 182 (2000),
146-154. Alternatively, a whole cell preparation is also envisaged.
Preferably, the herein described derivative or fragment of the
microorganism of the present invention retains the capability of
specifically binding to Streptococcus mutans which is described in
detail herein.
[0109] Another aspect of the present invention is a composition
comprising a microorganism belonging to the group of lactic acid
bacteria which is capable of specifically binding to Streptococcus
mutans or a mutant, derivative, analog or fragment of this
microorganism. Preferably, this microorganism is a microorganism of
the present invention or a mutant or derivative thereof or said
analog or fragment of said microorganism. In a preferred
embodiment, said composition comprises a microorganism as described
above in an amount between 10.sup.2 to 10.sup.12 cells, preferably
10.sup.3 to 10.sup.8 cells per mg in a solid form of the
composition. Preferably, this microorganism is a microorganism of
the present invention. In case of a liquid form of compositions,
the amount of the microorganisms is between 10.sup.2 to 10.sup.13
cells per ml. However, for specific compositions the amount of the
microorganism may be different as is described herein. A preferred
composition of the present invention does not contain lactose in a
range between 1% (w/w) and 6% (w/w). It is also preferred that the
composition contains not more than 1%(w/w) lactose, e.g. it
contains less than 1%, preferably less than 0.9% (w/w), 0.8% (w/w)
lactose, etc. or that the composition contains more than 6%, 7%, 8%
etc. (w/w) lactose. Alternatively, but also preferred is that the
composition does not contain lactose.
[0110] In a still further aspect, the present invention provides a
method for the production of an anticariogenic composition
comprising the steps of formulating a microorganism belonging to
the group of lactic acid bacteria which is capable of specifically
binding to Streptococcus mutans or a mutant, derivative, analog or
fragment of this microorganism with a cosmetically, orally or
pharmaceutical acceptable carrier or excipient. Preferably, this
microorganism is a microorganism of the present invention and the
mutant, derivative, analog or fragment is one of those of the
present invention. A preferred anticariogenic composition of the
present invention does not contain lactose in a range between 1%
(w/w) and 6% (w/w). It is also preferred that the composition
contains not more than 1%(w/w) lactose, e.g. it contains less than
1%, preferably less than 0.9% (w/w), 0.8% (w/w) lactose, etc. or
that the anticariogenic composition contains more than 6%, 7%, 8%
etc. (w/w) lactose. Alternatively, but also preferred is that the
anticariogenic composition does not contain lactose.
[0111] The term "composition", as used in accordance with the
present invention, relates to (a) composition(s) which comprise(s)
at least one microorganism or mutant or derivative as described
above, preferably of the present invention or analog or fragment of
said microorganism. It is envisaged that the compositions of the
present invention which are described herein below comprise the
aforementioned ingredients in any combination. It may, optionally,
comprise at least one further ingredient suitable for preventing
and/or treating caries. Accordingly, it may optionally comprise any
combination of the hereinafter described further ingredients. The
term "ingredients suitable for preventing and/or treating caries"
encompasses compounds or compositions and/or combinations thereof
which either inhibit the binding of Streptococcus mutans to the
surface of teeth, to pellicles and/or which inactivate
Streptococcus mutans. More preferably, said term encompasses
compounds or compositions and/or combinations thereof which may
inhibit the adhesion of Streptococcus mutans to the surface of
teeth, inhibit the activity of glycosyltransferases of
Streptococcus mutans, inhibit or inactivate Streptococcus mutans,
inhibit the agglutinin-dependent binding of Streptococcus mutans
and/or inhibit the saccharose-dependent binding of Streptococcus
mutans as will be described below.
[0112] In particular, it is envisaged that the composition
optionally further comprises compounds which inhibit the adhesion
of Streptococcus mutans to the tooth surface. Accordingly, it is
envisaged that such a compound is an inhibitor of the competence
signal peptide (CSP) of Streptococcus mutans. Said inhibitor is
described in CA 2,302,861 as being a derivative or fragment of said
CSP which competitively inhibits binding of said CSP to its natural
receptor, a histidine kinase receptor, or which is an antibody
against said CSP. Said inhibitor prevents the development of a
biofilm environment of dental plaque on the surface of teeth and,
thus, prevents binding of Streptococcus mutans. Alternatively, the
composition of the present invention may optionally further
comprise polypeptide fragments of the Streptococcus mutans I/II
antigen that are useful in treating and/or preventing dental
caries. Such polypeptide fragments are described in U.S. Pat. No.
6,500,433. Namely, said polypeptide fragments may have the ability
to adhere to the mammalian tooth surface by binding to agglutinin
in a competitive manner with naturally occurring Streptococcus
mutans antigen I/II, thus preventing or diminishing the adhesion of
S. mutans to the tooth. Some of the peptides of U.S. Pat. No.
6,500,433 have been shown to inhibit adhesion of S. mutans to a
tooth surface model (whole human saliva adsorbed to the wells of
polystyrene microtitre plates or hydroxyapatite beads).
Accordingly, U.S. Pat. No. 6,500,433 describes these peptides to
comprise one or more adhesion sites and will adhere to a mammalian
tooth in a competitive manner with naturally occurring SA I/II.
Another optional ingredient of the composition of the present
invention is the fimbrial-associated adhesion protein from
Streptococus mutans, SmaA, or a fragment thereof as described in WO
00/66616. The SmaA protein which is involved in the present
invention is an adhesion from fimbriae of S. mutans which mediates
attachment of the bacteria to the salivary pellicle, believed to be
via binding to the 52 kd salivary protein, amylase. The mature SmaA
protein has a molecular weight of about 65 kilodaltons (kd) as
measured on a reducing polyacrylamide gel, exhibits the ability to
bind amylase, and is the major immunodominant fimbrial protein of
S. mutans. Accordingy, SmaA is believed to compete with
Streptococcus mutans for adhesion sites on the surface of
teeth.
[0113] As described above, it is envisaged that compounds which
inhibit Streptococcus mutans glycosyltransferase activity are
optionally further comprised in the composition of the present
invention. For example, US 2004/0057908 describes a mixture of
terpenoids and flavonoids which inhibit the activity of said
glycosyltransferases. Duarte et al., Biol. Pharm. Bull. 26 (2003),
527-531 describe a novel type of propolis and its chemical
fractions on glycosyltransferases and on growth and adherence of
Streptococcus mutans. Accordingly, said novel type of propolis and
its chemical fractions are contemplated to be an optional further
ingredient of the composition of the present invention. Koo et al.,
J. Antimicrob. Chemother. 52 (2003), 782-789 describe that apigenin
and tt-farnesol inhibit Streptococcus mutans biofilm accumulation
and polysaccharide production. Hence, apigenein and tt-farnesol are
contemplated to be optionally comprised in the composition of the
present invention. Since carbohydrate fatty acid esters are
described in Devulapalle et al., Carbohydr. Res. 339 (2004),
1029-1034 to effect glycosyltransferase activity, said carbohydrate
fatty acid esters are contemplated to be optionally comprised in
the composition of the present invention.
[0114] Direct inhibition of Streptococcus mutans is, for example,
described in WO 2004/000222. Namely, genetically modified
bacteriophages specific for Streptococcus mutans are used for
treating bacterial caries caused by Streptococcus mutans. WO
2004/017988 describes a composition of biologically active protease
and at least one biologically active glycosidase which is used for
treating bacterial caries. Imazato et al., Biomaterials 24 (2003),
3605-3609 describes that methacryloyloxydodecylpyridinium bromide
(MDPB) is useful for inhibiting growth of Streptococcus mutans.
Accordingly, it is envisaged that the aforementioned compounds may
optionally be further comprised in the composition of the present
invention.
[0115] Bovine milk lactoferrin described by Mitoma et al., J. Biol.
Chem. 276 (2001), 18060-18065 or extracts of Helichrysum italicum
described by Nostro et al., Lett. Appl. Microbiol. 38 (2004),
423-427 which inhibit agglutinin-dependent or saccharose-dependent
binding of Streptococcus mutans are contemplated to be optionally
further comprised in the composition of the present invention.
[0116] Moreover, the composition of the present invention may
optionally further comprise a mutanase (1,3-glucanase) which is,
for example, described in DE 2152620 or Fuglsang (2000), loc. cit.
or an antibiotic against Streptococcus mutans, for example, those
described in U.S. Pat. No. 6,342,385; U.S. Pat. No. 5,932,469; U.S.
Pat. No. 5,872,001 or U.S. Pat. No. 5 833 958. In addition, it is
noted that the composition of the present invention may optionally
comprise one or more of the aforementioned optional ingredients
which are suitable for preventing and/or treating caries. Thus,
said composition may contain at least two, three, four, five, etc.,
i.e. "n" optional ingredients, wherein "n" is an integer greater
than 2 which is not limited. Said optional ingredients may be
combined in any possible combination.
[0117] The composition may be in solid, liquid or gaseous form and
may be, inter alia, in the form of (a) powder(s), (a) tablet(s),
(a) film preparation(s), (a) solution(s) (an) aerosol(s), granules,
pills, suspensions, emulsions, capsules, syrups, liquids, elixirs,
extracts, tincture or fluid extracts or in a form which is
particularly suitable for oral administration. Liquid preparations
suitable for oral administration, for example syrups can be
prepared, using water, conventional saccharides such as sucrose,
sorbitol and fructose, glycols such as polyethylene glycol and
propylene glycol, oils such as sesame seed oil, olive oil and
soybean oil, antiseptics such as p-hydroxybenzoate ester,
preservatives such as p-hydroxybenzoate derivatives, for example
p-hydroxybenzoate methyl and sodium benzoate, and other materials
such as flavors, for example strawberry flavor or peppermint.
[0118] Further, preparations suitable for oral administration, for
example tablets, powders and granules can be produced, using
conventional saccharides such as sucrose, glucose, mannitol, and
sorbitol, starch such as potato, wheat and corn, inorganic
materials such as calcium carbonate, calcium sulfate, sodium
hydrogen carbonate, and sodium chloride, plant powders such as
crystal cellulose, licorice powder and gentian powder, excipients
such as pinedex, disintegrators such as starch, agar, gelatin
powder, crystal cellulose, carmellose sodium, carmellose calcium,
calcium carbonate, sodium hydrogen carbonate and sodium alginate,
lubricants such as magnesium stearate, talc, hydrogenated vegetable
oils, macrogol, and silicone oil, binders such as polyvinyl
alcohol, hydroxypropyl cellulose, methyl cellulose, ethyl
cellulose, carmellose, gelatin, and starch glue fluid, surfactants
such as fatty acid ester, and plasticizers such as glycerin. A film
preparation(s) can be prepared by methods known in the art. An
example for the preparation of a film is given in Example 19
herein.
[0119] In case of ordinary oral administration, the dose of the
microorganism or analog or fragment of the present invention could
be (in dry weight) as described hereinabove with respect to the
cell number or with respect to the mass, for example, 1 .mu.g to 50
g, 1 .mu.g to 10 g, 1 .mu.g to 5 mg, 1 .mu.g to 1 mg or any other
weight per subject per day or in several portions daily. In case of
dosing to non-human animals, further, the dose varies depending on
the age and species of an animal and the nature or severity of the
symptom thereof. Without any specific limitation, the dose for
animals is 0.1 mg to 10 g per 1 kg body weight, preferably 1 mg to
1 g per 1 kg body weight once daily or in several portions daily.
However, these doses and the number of dosages vary depending on
the individual conditions.
[0120] Preferably, the composition of the present invention is a
cosmetic composition further comprising a cosmetically acceptable
carrier or excipient. More preferably, said cosmetic composition is
a dentifrice, chewing gum, lozenge, mouth wash, mouse rinse or
dental floss, which has an anticariogenic activity. A preferred
cosmetic composition of the present invention does not contain
lactose in a range between 1% (w/w) and 6% (w/w). It is also
preferred that the cosmetic composition contains not more than
1%(w/w) lactose, e.g. it contains less than 1%, preferably less
than 0.9% (w/w), 0.8% (w/w) lactose, etc. or that the cosmetic
composition contains more than 6%, 7%, 8% etc. (w/w) lactose.
Alternatively, but also preferred is that the cosmetic composition
does not contain lactose.
[0121] The cosmetic composition of the present invention comprises
the microorganism, mutant, derivative, analog or fragment thereof
as described above in connection with the composition of the
invention and further a cosmetically or orally acceptable carrier.
Preferably, as mentioned in connection with the composition of the
present invention the microorganism, mutant, derivative, analog or
fragment thereof is a microorganism, mutant, derivative, analog or
fragment of the present invention. Preferably the cosmetic
composition of the present invention is for use in oral
applications. Accordingly, it may be in the form of a toothpaste,
dentifrice, tooth powder, topical oral gel, mouth rinse, denture
product, mouthspray, lozenge, oral tablet, or chewing gum.
[0122] The term "orally or cosmetically acceptable carrier" as used
herein means a suitable vehicle, which can be used to apply the
present compositions to the oral cavity in a safe and effective
manner. Such vehicle may include materials such as fluoride ion
sources, additional anticalculus agents, buffers, other abrasive
materials, peroxide sources, alkali metal bicarbonate salts,
thickening materials, humectants, water, surfactants, titanium
dioxide, flavor system, sweetening agents, xylitol, coloring
agents, and mixtures thereof. The term "safe and effective amount"
as used herein, means a sufficient amount to clean teeth and reduce
stain/plaque/gingivitis/calculus without harming the tissues and
structures of the oral cavity.
[0123] The pH of the present herein described compositions ranges
preferably from about 3.0 to about 9.0, with the preferred pH being
from about 5.5 to about 9.0 and the most preferred pH being 7.0 to
about 8.5 or 9.0.
[0124] The cosmetical composition is a product, which in the
ordinary course of usage, is not intentionally swallowed for
purposes of systemic administration of particular therapeutic
agents, but is rather retained in the oral cavity for a time
sufficient to contact substantially all of the dental surfaces
and/or oral tissues for purposes of oral activity. The oral
composition may be a single phase oral composition or may be a
combination of two or more oral compositions.
[0125] The term "dentifrice", as used herein, means paste, gel, or
liquid formulations unless otherwise specified. The dentifrice
composition may be in any desired form, such as deep striped,
surface striped, multilayered, having the gel surrounding the
paste, or any combination thereof. The dentifrice composition may
be contained in a physically separated compartment of a dispenser
and dispensed side-by-side. Dentifrice compositions are, for
example, described in EP-B1 0 617 608.
[0126] Preferred dentifrice compositions are described in Examples
13 to 16. In addition to the above described components, the
embodiments of this invention can contain a variety of optional
dentifrice ingredients some of which are described below. Optional
ingredients include, for example, but are not limited to,
adhesives, sudsing agents, flavouring agents, sweetening agents,
additional antiplaque agents, additional abrasives, and colouring
agents. These and other optional components are further described,
for example, in U.S. Pat. No. 5,004,597; U.S. Pat. No. 4,885,155;
U.S. Pat. No. 3,959,458; and U.S. Pat. No. 3,937,807.
[0127] For example, the toothpaste may include surfactants,
chelating agents, fluoride sources, teeth whitening actives and
teeth color modifying substances, thickening agents, humectants,
flavouring and sweetening agents, alkali metal bicarbonate salt,
miscellaneous carriers and/or other active agents.
[0128] One of the preferred optional agents of the present
invention is a surfactant, preferably one selected from the group
consisting of sarcosinate surfactants, iselhionate surfactants and
taurate surfactants. Preferred for use herein are alkali metal or
ammonium salts of these surfactants. Most preferred herein are the
sodium and potassium salts of the following: lauroyl sarcosinate,
myristoyl sarcosinate, palmitoyl sarcosinate, stearoyl sarcosinate
and oleoyl sarcosinate.
[0129] Another preferred optional agent is a chelating agent such
as tartaric acid and pharmaceutically-acceptable salts thereof,
citric acid and alkali metal citrates and mixtures thereof.
Chelating agents are able to complex calcium found in the cell
walls of the bacteria. Chelating agents can also disrupt plaque by
removing calcium from the calcium bridges, which help hold this
biomass intact.
[0130] It is common to have an additional water-soluble fluoride
compound present in dentifrices and other oral compositions in an
amount sufficient to give a fluoride ion concentration in the
composition at 25.degree. C., and/or when it is used of from about
0.0025% to about 5.0% by weight, preferably from about 0.005% to
about 2.0% by weight, to provide additional anticaries
effectiveness. A wide variety of fluoride ion-yielding materials
can be employed as sources of soluble fluoride in the present
compositions. Examples of suitable fluoride ion-yielding materials
are found in U.S. Pat. No. 3,535,421 and U.S. Pat. No. 3,678,154.
Representative fluoride ion sources include stannous fluoride,
sodium fluoride, potassium fluoride, sodium monofluorophosphate and
many others. Stannous fluoride and sodium fluoride are particularly
preferred, as well as mixtures thereof.
[0131] Teeth whitening actives that may be used in the oral care
compositions of the present invention include bleaching or
oxidizing agents such as peroxides, perborates, percarbonates,
peroxyacids, persulfates, metal chlorites, and combinations
thereof. Suitable peroxide compounds include hydrogen peroxide,
urea peroxide, calcium peroxide, and mixtures thereof. A preferred
percarbonate is sodium percarbonate. Other suitable whitening
agents include potassium, ammonium, sodium and lithium persulfates
and perborate mono- and tetrahydrates, and sodium pyrophosphate
peroxyhydrate. Suitable metal chlorites include calcium chlorite,
barium chlorite, magnesium chlorite, lithium chlorite, sodium
chlorite, and potassium chlorite. The preferred chlorite is sodium
chlorite. Additional whitening actives may be hypochlorite and
chlorine dioxide. In addition to bleaching agents as teeth
whitening agents, teeth color modifying substances may be
considered among the oral care actives useful in the present
invention. These substances are suitable for modifying the color of
the teeth to satisfy the consumer. These substances comprise
particles that when applied on the l:ooth surface modify that
surface in terms of absorption and, or reflection of light. Such
particles provide an appearance benefit when a film containing such
particles is applied over the surfaces of a tooth or teeth.
[0132] In preparing toothpaste or gels, it is necessary to add some
thickening material to provide a desirable consistency of the
composition, to provide desirable active release characteristics
upon use, to provide shelf stability, and to provide stability of
the composition, etc. Preferred thickening agents are carboxyvinyl
polymers, carrageenan, hydroxyethyl cellulose, laponite and water
soluble salts of cellulose ethers such as sodium
carboxymethylcellulose and sodium carboxymethyl hydroxyethyl
cellulose. Natural gums such as gum karaya, xanthan gum, gum
arabic, and gum tragacanth can also be used. Colloidal magnesium
aluminum silicate or finely divided silica can be used as part of
the thickening agent to further improve texture.
[0133] Another optional component of the topical, oral carriers of
the compositions of the subject invention is a humectant. The
humectant serves to keep toothpaste compositions from hardening
upon exposure to air, to give compositions a moist feel to the
mouth, and, for particular humectants, to impart desirable
sweetness of flavor to toothpaste compositions. The humectant, on a
pure humectant basis, generally comprises from about 0% to about
70%, preferably from about 5% to about 25%, by weight of the
compositions herein. Suitable humectants for use in compositions of
the subject invention include edible polyhydric alcohols such as
glycerin, sorbitol, xylitol, butylene glycol, polyethylene glycol,
and propylene glycol, especially sorbitol and glycerin.
[0134] Flavoring and sweetening agents can also be added to the
compositions. Suitable flavoring agents include oil of wintergreen,
oil of peppermint, oil of spearmint, clove bud oil, menthol,
anethole, methyl salicylate, eucalyptol, cassia, 1-menthyl acetate,
sage, eugenol, parsley oil, oxanone, alpha-irisone, marjoram,
lemon, orange, propenyl guaethol, cinnamon, vanillin, thymol,
linalool, cinnamaldehyde glycerol acelal known as CGA, and mixtures
thereof. Flavouring agents are generally used in the compositions
at levels of from about 0.001% to about 5%, by weight of the
composition.
[0135] Sweetening agents which can be used include sucrose,
glucose, saccharin, dextrose, levulose, lactose as described herein
above, mannitol, sorbitol, fructose, maltose, xylitol, saccharin
salts, thaumatin, aspartame, D-tryptophane, dihydrochalcones,
acesulfame and cyclamate salts, especially sodium cyclamate and
sodium saccharin, and mixtures thereof. A composition preferably
contains from about 0.1% to about 10% of these agents, preferably
from about 0.1% to about 1%, by weight of the composition.
[0136] The present invention may also include an alkali metal
bicarbonate salt. Alkali metal bicarbonate salts are soluble in
water and unless stabilized, tend to release carbon dioxide in an
aqueous system. Sodium bicarbonate, also known as baking soda, is
the preferred alkali metal bicarbonate salt. The present
composition may contain from about 0.5% to about 30%, preferably
from about 0.5% to about 15%, and most preferably from about 0.5%
to about 5% of an alkali metal bicarbonate salt.
[0137] Water employed in the preparation of commercially suitable
oral compositions should preferably be of low ion content and free
of organic impurities. Water generally comprises from about 10% to
about 50%, and preferably from about 20% to about 40%, by weight of
the aqueous toothpaste compositions herein. These amounts of water
include the free water which is added plus that which is introduced
with other materials, such as with sorbitol. Titanium dioxide may
also be added to the present composition. Titanium dioxide is a
white powder, which adds opacity to the compositions. Titanium
dioxide generally comprises from about 0.25% to about 5% by weight
of the dentifrice compositions. The pH of the present compositions
is preferably adjusted through the use of buffering agents.
Buffering agents, as used herein, refer to agents that can be used
to adjust the pH of the compositions to a range of about 4.5 to
about 9.5. Buffering agents include monosodium phosphate, trisodium
phosphate, sodium hydroxide, sodium carbonate, sodium acid
pyrophosphate, citric acid, and sodium citrate. Buffering agents
can be administered at a level of from about 0.5% to about 10%, by
weight of the present compositions. The pH of dentifrice
compositions is measured from a 3:1 aqueous slurry of dentifrice,
e.g., 3 parts water to 1 part toothpaste.
[0138] Other optional agents that may be used in the present
compositions include dimethicone copolyols selected from alkyl- and
alkoxy-dimethicone copolyols, such as C12 to C20 alkyl dimethicone
copolyols and mixtures thereof. Highly preferred is cetyl
dimethicone copolyol marketed under the Trade Name Abil EM90. The
dimethicone copolyol is generally present in a level of from about
0.01% to about 25%, preferably from about 0.1% to about 5%, more
preferably from about 0.5% to about 1.5% by weight. The dimethicone
copolyols aid in providing positive tooth feel benefits. Other
useful carriers include biphasic dentifrice formulations such as
those disclosed in U.S. Pat. No. 5,213,790; U.S. Pat. No.
5,145,666; U.S. Pat. No. 5,281,410; U.S. Pat. No. 4,849,213 and
U.S. Pat. No. 4,528,180.
[0139] The present cosmetic compositions may also include other
active agents, such as antimicrobial agents. Included among such
agents are water insoluble non-cationic antimicrobial agents such
as halogenated diphenyl ethers, phenolic compounds including phenol
and its homologs, mono and poly-alkyl and aromatic halophenols,
resorcinol and its derivatives, bisphenolic compounds and
halogenated salicylanilides, benzoic esters, and halogenated
carbanilides. The water soluble antimicrobials include quaternary
ammonium salts and bis-biquanide salts, among others. Triclosan
monophosphate is an additional water soluble antimicrobial agent.
The quaternary ammonium agents include those in which one or two of
the substitutes on the quaternary nitrogen has a carbon chain
length (typically alkyl group) from about 8 to about 20, typically
from about 10 to about 18 carbon atoms while the remaining
substitutes (typically alkyl or benzyl group) have a lower number
of carbon atoms, such as from about 1 to about 7 carbon atoms,
typically methyl or ethyl groups. Dodecyl trimethyl ammonium
bromide, tetradecylpyridinium chloride, domiphen bromide,
N-tetradecyl-4-ethyl pyridinium chloride, dodecyl dimethyl
(2-phenoxyethyl) ammonium bromide, benzyl dimethylstearyl ammonium
chloride, cetyl pyridinium chloride, quaternized
5-amino-1,3-bis(2-ethyl-hexyl)-5-methyl hexa hydropyrimidine,
benzalkonium chloride, benzethonium chloride and methyl
benzethonium chloride are exemplary of typical quaternary ammonium
antibacterial agents. Other compounds are
bis[4-(R-amino)-1-pyridinium] alkanes as disclosed in U.S. Pat. No.
4,206,215. Other antimicrobials such as copper bisglycinate, copper
glysinate, zinc citrate, and zinc lactate may also be included.
Enzymes are another type of active that may be used in the present
compositions. Useful enzymes include those that belong to the
category of proteases, lytic enzymes, plaque matrix inhibitors and
oxidases: Proteases include papain, pepsin, trypsin, ficin,
bromelin; cell wall lytic enzymes include lysozyme; plaque matrix
inhibitors include dextranses, mutanases; and oxidases include
glucose oxidase, lactate oxidase, galactose oxidase, uric acid
oxidase, peroxidases including horse radish peroxidase,
myeloperoxidase, lactoperoxidase, chloroperoxidase. The oxidases
also have whitening/cleaning activity, in addition to
anti-microbial properties. Such agents are disclosed in U.S. Pat.
No. 2,946,725 and in U.S. Pat. No. 4,051,234. Other antimicrobial
agents include chlorhexidine, triclosan, triclosan monophosphate,
and flavor oils such as thymol. Triclosan and other agents of this
type are disclosed in U.S. Pat. No. 5,015,466 and U.S. Pat. No.
4,894,220. These agents, which provide anti-plaque benefits, may be
present at levels of from about 0.01% to about 5.0%, by weight of
the dentifrice composition.
[0140] The term "chewing gum" as defined herein means a
confectionery composition which is suitable for chewing and which
comprises 2% or greater, by weight of the composition, of
elastomer. Suitable lozenge and chewing gum components are, for
example, disclosed in U.S. Pat. No. 4,083,955; U.S. Pat. No.
6,770,264 or U.S. Pat. No. 6,270,781. Preferred lozenges are those
described in Examples 11 and 12. A preferred chewing gum
composition is described in Example 17. Compositions of the present
invention preferably comprise an elastomer, or mixture of several
different elastomers. Elastomeric materials are generally known in
the art but illustrative examples include styrene-butadiene rubber
(SBR); synthetic gums; polyisobutylene and isobutylene-isoprene
copolymers; natural gums; chicle; natural rubber; jelutong; balata;
guttapercha; lechi caspi; sorva; and mixtures thereof. Compositions
of the present invention preferably comprise from about 2% to about
30%, more preferably from about 5% to about 25%, by weight, of
elastomer. These levels are determined by the desired final texture
of the chewing gum since when the total level of elastomer is below
about 2% the base composition lacks elasticity, chewing texture,
and cohesiveness whereas at levels above about 30% the formulation
is hard, rubbery and maintains a tight chew. Elastomer solvents are
also preferably present in compositions of the present invention
since they aid softening of the elastomer component. Preferred
examples of elastomer solvents for use herein include the
pentaerythritol ester of partially hydrogenated wood rosin,
pentaerythritol ester of wood rosin, glycerol ester of partially
dimerized rosin, glycerol ester of polymerised rosin, glycerol
ester of tall oil, wood or gum rosin, glycerol ester of partially
hydrogenated rosin, methyl ester of partially hydrogenated rosin,
and mixtures thereof. Compositions of the present invention
preferably comprise from about 2% to about 50%, more preferably
from about 10% to about 35%, by weight, of elastomer solvent.
[0141] Lozenges for use in accordance with this invention can be
prepared, for example, by art-recognized techniques for forming
compressed tablets where the disaccharide is dispersed on a
compressible solid carrier, optionally combined with any
appropriate tableting aids such as a lubricant (e.g.,
magnesium-stearate) and is compressed into tablets. The solid
carrier component for such tableting formulations can be a
saliva-soluble solid, such as a cold water-soluble starch or a
monosaccharide, so that the lozenge will readily dissolve in the
mouth to release the contained disaccharide acid in saliva solution
for contact with and absorption by the oral/pharyngeal mucosa when
the lozenge is held in the mouth. The pH of the above-described
formulations can range from about 4 to about 8.5.
[0142] Lozenges for use in accordance with the present invention
can also be prepared utilizing other art-recognized solid unitary
dosage formulation techniques.
[0143] A mouth wash or mouth rinse of the present invention could
preferably be as follows:
[0144] A Olium menthae 1.2 parts [0145] Tinctura Arnicae 3.0 parts
[0146] Tinctura Myrrhae 3.0 parts [0147] Tween 5.0 parts
[0148] B Spiritus 90% 50.0 parts
[0149] C Sodium Benzoate 0.2 parts [0150] Sweetening agent (e.g.
aspartane) 0.02 parts [0151] Agua destilata ad 100,
[0152] A is to be well mixed, B is added under stirring and C is
added subsequently. The resulting clear liquid is to be filtered
within 48 hours after preparation. Another preferred mouth wash is
described in Example 18.
[0153] Regardless of the dosage form, liquid or solid, in one
preferred embodiment of the present invention the dosage form is
held in the patient's mouth for a period of time to promote contact
of the microorganism or analog or fragment of amicroorganism of the
present invention with the patient's oral cavity.
[0154] Another preferred composition of the present invention is a
pharmaceutical composition comprising the microorganism or a
derivative or mutant or an analog or fragment thereof as described
above in connection with the pharmaceutical composition further
comprising a pharmaceutical acceptable carrier or excipient.
Preferably, the microorganism, mutant, analog, derivative or
fragment thereof is a microorganism, mutant, derivative or fragment
of the present invention.
[0155] In addition, the present invention relates to the use of a
microorganism or a derivative or mutant or an analog or fragment
thereof as described above in connection with the composition of
the present invention for the preparation of a composition,
preferably a pharmaceutical or cosmetic compound for the
prophylaxis against caries. Preferably, the microorganism, mutant,
derivative, analog or fragment thereof is a microorganism, mutant,
derivative or fragment of the present invention.
[0156] Pharmaceutical compositions comprise a therapeutically
effective amount of a microorganism or derivative or mutant of the
present invention or an analog or fragment of said microorganism of
the present invention described in connection with the composition
of the present invention and can be formulated in various forms,
e.g. in solid, liquid, powder, aqueous, lyophilized form.
[0157] The pharmaceutical composition may be administered with a
pharmaceutically acceptable carrier to a patient, as described
herein. In a specific embodiment, the term "pharmaceutically
acceptable" means approved by a regulatory agency or other
generally recognized pharmacopoeia for use in animals, and more
particularly in humans. A preferred pharmaceutical composition of
the present invention does not contain lactose in a range between
1% (w/w) and 6% (w/w). It is also preferred that the pharmaceutical
composition contains not more than 1% (w/w) lactose, e.g. it
contains less than 1%, preferably less than 0.9% (w/w), 0.8% (w/w)
lactose, etc. or that the pharmaceutical composition contains more
than 6%, 7%, 8% etc. (w/w) lactose. Alternatively, but also
preferred is that the pharmaceutical composition does not contain
lactose.
[0158] The term "carrier" refers to a diluent, adjuvant, excipient,
or vehicle with which the therapeutic is administered. Such a
carrier is pharmaceutically acceptable, i.e. is non-toxic to a
recipient at the dosage and concentration employed. It is
preferably isotonic, hypotonic or weakly hypertonic and has a
relatively low ionic strength, such as provided by a sucrose
solution. Such pharmaceutical carriers can be sterile liquids, such
as water and oils, including those of petroleum, animal, vegetable
or synthetic origin, such as peanut oil, soybean oil, mineral oil,
sesame oil and the like. Saline solutions and aqueous dextrose and
glycerol solutions can also be employed as liquid carriers,
particularly for injectable solutions. Suitable pharmaceutical
excipients include starch, glucose, sucrose, gelatin, malt, rice,
flour, chalk, silica gel, sodium stearate, glycerol monostearate,
talc, sodium ion, dried skim milk, glycerol, propylene, glycol,
water, ethanol and the like. The excipient may contain lactose as
described herein above, most preferably it is lactose-free. The
composition, if desired, can also contain minor amounts of wetting
or emulsifying agents, or pH buffering agents. These compositions
can take the form of solutions, suspensions, emulsion, tablets,
pills, capsules, powders, sustained-release formulations and the
like. Oral formulation can include standard carriers such as
pharmaceutical grades of mannitol, starch, magnesium stearate,
sodium saccharine, cellulose, magnesium carbonate, etc. Examples of
suitable pharmaceutical carriers are described in "Remington's
Pharmaceutical Sciences" by E.W. Martin. Skim milk, skim milk
powder, non-milk or non-lactose containing products may also be
employed. The skim milk powder is conventionally suspended in
phosphate buffered saline (PBS), autoclaved or filtered to
eradicate proteinaceous and living contaminants, then freeze dried
heat dried, vacuum dried, or lyophilized. Some other examples of
substances which can serve as pharmaceutical carriers are sugars,
such as glucose and sucrose; starches such as corn starch and
potato starch; cellulose and its derivatives such as sodium
carboxymethycellulose, ethylcellulose and cellulose acetates;
powdered tragancanth; malt; gelatin; talc; stearic acids; magnesium
stearate; calcium sulfate; calcium carbonate; vegetable oils, such
as peanut oils, cotton seed oil, sesame oil, olive oil, corn oil
and oil of theobroma; polyols such as propylene glycol, glycerine,
sorbitol, manitol, and polyethylene glycol; agar; alginic acids;
pyrogen-free water; isotonic saline; cranberry. extracts and
phosphate buffer solution; skim milk powder; as well as other
non-toxic compatible substances used in pharmaceutical formulations
such as Vitamin C, estrogen and echinacea, for example. Wetting
agents and lubricants such as sodium lauryl sulfate, as well as
colouring agents, flavouring agents, lubricants, excipients,
tabletting agents, stabilizers, anti-oxidants and preservatives,
can also be present.
[0159] Preferably, the oral formulation contains lactose as
described herein and is most preferably lactose-free. Various
carriers and/or excipients suitable for oral administration which
are well known in the art may be used for the purpose of this
invention. The non-cariogenic composition may, if desired, further
contain various known additives such as, for example,
preservatives, hardening agents, lubricants, emulsifiers,
stabilizers, essence and the like. Such compositions will contain a
therapeutically effective amount of the aforementioned compounds,
preferably in purified form, together with a suitable amount of
carrier so as to provide the form for proper administration to the
patient. The formulation should suit the mode of
administration.
[0160] Generally, the ingredients are supplied either separately or
mixed together in unit dosage form, for example, as a dry
lyophilised powder or water free concentrate in a hermetically
sealed container such as an ampoule or sachette indicating the
quantity of active agent. Where the composition is to be
administered by infusion, it can be dispensed with an infusion
bottle containing sterile pharmaceutical grade water or saline.
[0161] The pharmaceutical composition of the invention can be
formulated as neutral or salt forms. Pharmaceutically acceptable
salts include those formed with anions such as those derived from
hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and
those formed with cations such as those derived from sodium,
potassium, ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. In
vitro assays may optionally be employed to help identify optimal
dosage ranges. The precise dose to be employed in the formulation
will also depend on the route of administration, and the
seriousness of the disease or disorder, and should be decided
according to the judgment of the practitioner and each patient's
circumstances. Effective doses may be extrapolated from
dose-response curves derived from in vitro or animal model test
systems. Preferably, the pharmaceutical composition is administered
directly or in combination with an adjuvant. Adjuvants may be
selected from the group consisting of a chloroquine, protic polar
compounds, such as propylene glycol, polyethylene glycol, glycerol,
EtOH, 1-methyl L-2-pyrrolidone or their derivatives, or aprotic
polar compounds such as dimethylsulfoxide (DMSO), diethylsulfoxide,
di-n-propylsulfoxide, dimethylsulfone, sulfolane,
dimethylformamide, dimethylacetamide, tetramethylurea, acetonitrile
or their derivatives. These compounds are added in conditions
respecting pH limitations. The composition of the present invention
can be administered to a vertebrate. "Vertebrate" as used herein is
intended to have the same meaning as commonly understood by one of
ordinary skill in the art. Particularly, "vertebrate" encompasses
mammals, and more particularly humans.
[0162] The term "administered" means administration of a
therapeutically effective dose of the aforementioned composition.
By "therapeutically effective amount" is meant a dose that produces
the effects for which it is administered, preferably this effect is
anticariogenic. The exact dose will depend on the purpose of the
treatment, and will be ascertainable by one skilled in the art
using known techniques. As is known in the art and described above,
adjustments for systemic versus localized delivery, age, body
weight, general health, sex, diet, time of administration, drug
interaction and the severity of the condition may be necessary, and
will be ascertainable with routine experimentation by those skilled
in the art.
[0163] The methods are applicable to both human therapy and
veterinary applications. The compounds described herein having the
desired therapeutic activity may be administered in a
physiologically acceptable carrier to a patient, as described
herein. Depending upon the manner of introduction, the compounds
may be formulated in a variety of ways as discussed below. The
concentration of therapeutically active compound in the formulation
may vary from about 0.1-100 wt %. The agents maybe administered
alone or in combination with other treatments.
[0164] The administration of the pharmaceutical composition can be
done in a variety of ways as discussed above, including, but not
limited to, orally, subcutaneously, intravenously, intra-arterial,
intranodal, intramedullary, intrathecal, intraventricular,
intranasally, intrabronchial, transdermally, intranodally,
intrarectally, intraperitoneally, intramuscularly, intrapulmonary,
vaginally, rectally, or intraocularly.
[0165] Preferably the administration is orally or buccal. The
attending physician and clinical factors will determine the dosage
regimen. As is well known in the medical arts, dosages for any one
patient depends upon many factors, including the patient's size,
body surface area, age, the particular compound to be administered,
sex, time and route of administration, general health, and other
drugs being administered concurrently. A typical dose can be, for
example, in the range of 0.001 to 1000 .mu.g; however, doses below
or above this exemplary range are envisioned, especially
considering the aforementioned factors.
[0166] The dosages are preferably given once a week, however,
during progression of the treatment the dosages can be given in
much longer time intervals and in need can be given in much shorter
time intervals, e.g., daily. In a preferred case the immune
response is monitored using herein described methods and further
methods known to those skilled in the art and dosages are
optimized, e.g., in time, amount and/or composition. Progress can
be monitored by periodic assessment. The pharmaceutical composition
of the invention may be administered locally or systemically. It is
also envisaged that the pharmaceutical compositions are employed in
co-therapy approaches, i.e. in co-administration with other
medicaments or drugs, for example other drugs for preventing,
treating or ameliorating caries which are described herein.
[0167] Another preferred composition of the present invention is a
food or feed composition comprising a microorganims, mutant,
derivative, analog or fragment thereof as described in connection
with the composition of the present invention , further comprising
an orally acceptable carrier or excipient. Preferably, the
microorganism, mutant, derivative, analog or fragment thereof is a
microorganism, mutant, derivative or fragment of the present
invention.
[0168] "Food" or "feed" comprises any latable, palatable and/or
drinkable stuff for mammals, for example, humans or animals, e.g.,
pets as described herein. Food and feedstuff is described herein
elsewhere. An "orally acceptable carrier" is described herein above
and is preferably not toxic and of food and/or feed grade. Yet,
this term also encompasses the carriers mentioned in connection
with the pharmaceutical composition of the present invention. A
preferred food or feed composition of the present invention does
not contain lactose in a range between 1% (w/w) and 6% (w/w). It is
also preferred that the food or feed composition contains not more
than 1%(w/w) lactose, e.g. it contains less than 1%, preferably
less than 0.9% (w/w), 0.8% (w/w) lactose, etc. or that the food or
feed composition contains more than 6%, 7%, 8% etc. (w/w) lactose.
Alternatively, but also preferred is that the food or feed
composition does not contain lactose.
[0169] The present invention provides furthermore the use of a
microorganism or a derivative or mutant thereof or an analog or
fragment disclosed in connection with the composition of the
present invention herein for the preparation of an anticariogenic
composition which is preferably a dentifrice, chewing gum, lozenge,
mouth wash, mouse rinse or dental floss as described herein above.
Preferably, the microorganism, mutant, derivative, analog or
fragment thereof is a microorganism, mutant, derivative or fragment
of the present invention.
[0170] Another aspect of the present invention is a method for the
production of an anticariogenic composition comprising the steps of
formulating a microorganism or derivative or mutant thereof or an
analog or fragment of a microorganism described in connection with
the composition of the present invention with a cosmetically,
pharmaceutically or orally acceptable carrier or excipient.
Preferably, the microorganism, mutant, derivative, analog or
fragment thereof is a microorganism, mutant, derivative or fragment
of the present invention.
[0171] A method for the production of an anticariogenic foodstuff
or feedstuff wherein the method comprises the step of adding a
microorganism or derivative or mutant or an analog or fragment
thereof which are disclosed herein in connection with the
composition of the present invention is also provided by the
present application. Preferably, the microorganism, mutant,
derivative, analog or fragment thereof is a microorganism, mutant,
derivative or fragment of the present invention.
[0172] In accordance with the present invention, the term
"foodstuff" encompasses all eatable and drinkable food and drinks.
Accordingly, the microorganism or analog or fragment may be
included in a food or drink. These are, for example, gum, spray,
beverage, candies, infant formula, ice cream, frozen dessert, sweet
salad dressing, milk preparations, cheese, quark, lactose-free
yogurt, acidified milk, coffee cream or whipped cream and the
like.
[0173] Milk-based products are envisaged within the framework of
the invention. Milk is however understood to mean that of animal
origin, such as cow, goat, sheep, buffalo, zebra, horse, donkey, or
camel, and the like. The milk may be in the native state, a
reconstituted milk, a skimmed milk or a milk supplemented with
compounds necessary for the growth of the bacteria or for the
subsequent processing of fermented milk, such as fat, proteins of a
yeast extract, peptone and/or a surfactant, for example. The term
milk also applies to what is commonly called vegetable milk, that
is to say extracts of plant material which have been treated or
otherwise, such as leguminous plants (soya bean, chick pea, lentil
and the like) or oilseeds (colza, soya bean, sesame, cotton and the
like), which extract contains proteins in solution or in colloidal
suspension, which are coagulable by chemical action, by acid
fermentation and/or by heat. Finally, the word milk also denotes
mixtures of animal milks and of vegetable milks.
[0174] Where the microorganism or analog or fragment of this
invention are added to yogurt and the like having similar contents,
it is sufficient to add the microorganism of this invention at a
concentration of about 10.sup.5-10.sup.7 cells/ml. In such a case,
it is possible to completely prevent or inhibit dental caries
induced by cariogenic strains of S. mutans without significant side
effect upon the quality of the drink per se.
[0175] Such food drink or feed can be produced by a general method
for producing foods and drinks or feeds, including adding the
active ingredient to a raw or cooked material of the food, drink or
feed. The food, drink or feed in accordance with the present
invention can be molded and granulated in the same manner as
generally used for foods, drinks or feeds. The molding and
granulating method includes granulation methods such as fluid layer
granulation, agitation granulation, extrusion granulation, rolling
granulation, gas stream granulation, compaction molding
granulation, cracking granulation, spray granulation, and injection
granulation, coating methods such as pan coating, fluid layer
coating, and dry coating, puff dry, excess steam method, foam mat
method, expansion methods such as microwave incubation method, and
extrusion methods with extrusion granulation machines and
extruders.
[0176] The food, drink or feed according to the present invention
includes foods, drinks or feeds comprising the active ingredient.
The food, drink or feed to be used in the present invention
includes any food, drink or feed. The active ingredient in the
food, drink or feed is not specifically limited to any
concentration as long as the resulting food, drink or feed can
exert its activity of specifically binding to Streptococcus mutans.
The concentration of the active ingredient is preferably 0.001 to
100% by weight, more preferably 0.01 to 100% by weight and most
preferably 0.1 to 100% by weight of the food, drink or feed
comprising such active ingredient or with respect to the cell
number those described herein.
[0177] Specific foods or drinks, to which the active ingredient is
added, include, for example, juices, refreshing drinks, soups,
teas, sour milk beverages, dairy products such as fermented milks,
ices, butter, cheese, processed milk and skim milk, meat products
such as ham, sausage, and hamburger, fish meat cake products, egg
products such as seasoned egg rolls and egg curd, confectioneries
such as cookie, jelly, snacks, and chewing gum, breads, noodles,
pickles, smoked products, dried fishes and seasonings. The form of
the food or drink includes, for example, powder foods, sheet-like
foods, bottled foods, canned foods, retort foods, capsule foods,
tablet foods and fluid foods. The food or drink with an activity to
specifically bind to Streptococcus mutans to be ingested by
infants, are preferably nutritious compositions for infants. Such
nutritious composition for infants includes modified milk prepared
for infants, protein-decomposed milk, specific nutritionally
modified milk or baby foods and foods prepared for toddlers. The
form of the nutritious composition for infants includes but is not
specifically limited to powder milks dried and pulverized and baby
foods and also include general foods such as ice cream, fermented
milk, and jelly for infantile ingestion.
[0178] The nutritious composition for infants in accordance with
the present invention is principally composed of protein, lipid,
saccharide, vitamins and/or minerals. In the nutritious
composition, the active ingredient is blended with these
components.
[0179] The protein includes milk proteins such as skim milk,
casein, cheese whey, whey protein concentrate and whey protein
isolates and their fractions such as alpha s-casein, beta-casein,
alpha-lactoalbumin and beta-lactoglobulin . Further, egg protein
such as egg yolk protein, egg white protein, and ovalbumin, or
soybean protein such as defatted soybean protein, separated soybean
protein, and concentrated soybean protein can be used. Other than
these, proteins such as wheat gluten, fish meat protein. cattle
meat protein and collagen may also be used satisfactorily. Further,
fractions of these proteins, peptides from the acid or enzyme
treatment thereof, or free no acids maybe used satisfactorily as
well.
[0180] The free amino acids can serve as nitrogen sources and can
additionally be used to give specific physiological actions. Such
free amino acids include, for example, taurine, arginine, cysteine,
cystine and glutamine. The lipid includes animal fats and oils such
as milk. fat , lard, beef fat and fish oil, vegetable oils such as
soybean oil. rapeseed oil, corn oil, coconut oil, palm oll, palm
kernel oil, safflower oil, perilla oil, linseed oil, evening
primrose oil, medium chain fatty acid triglyceride, and cotton seed
oil, bacterially generated fats and oils, and fractionated oils
thereof, hydrogenated oils thereof, and ester exchange oils
thereof. The amount of lipid to be blended varies depending on the
use. The saccharide includes, for example, one or more of starch,
soluble polysaccharides, dextrin, monosaccharides such as sucrose,
lactose as described herein, maltose, glucose, and fructose and
other oligosaccharides. The total amount of such saccharide is
preferably 40 to 80% by weight to the total solid in the nutritious
composition. Further, artificial sweeteners such as aspartame may
be used satisfactorily. The amount of an artificial sweetener is
appropriately 0.05 to 1.0% by weight per the total solid in the
nutritious composition.
[0181] The vitamins include, but are not limited to, lycopene as an
essential component and additionally include, for example, vitamins
such as vitamin A, vitamin B group, vitamins C, D, and E and
vitamin K group, folic acid, pantothenic acid, niootinamide,
carnitine, choline, inositol and biotin as long as such vitamins
can be administered to infants. Such vitamins are preferably from
10 mg to 5 g by weight per the total solid in the nutritious
composition for infants.
[0182] Further, the minerals include calcium, magnesium, potassiw,
sodium, iron, copper, zinc, phosphorus, chlorine, manganese,
selenium and iodine. Such minerals are preferably from 1 mg to 5 g
by weight per the total solid in the nutritious composition for
infants . Other than those components described above, the
nutritious composition for infants of the present invention may be
blended with any component desirably blended in nutritious
compositions, for example, dietary fiber, nucleotides, nucleic
acids, flavors, and colorants. The food or drink of the present
invention can be used as a health food or drink or a functional
food or drink to prevent and/or treat caries.
[0183] When the food or drink according to the present invention is
ingested, the amount to be ingested is not specifically limited.
The amount to be ingested is generally 0.1 to 50 g, preferably 0.5
g to 20 g daily, based on the total amount of active ingredient .
The food or drink is continuously ingested at this amount for a
period from a single day up to 5 years, preferably from 2 weeks to
one year. Herein, the amount ingested can be adjusted to an
appropriate range depending on the severity of the symptom of the
individual ingesting the food or drink, the age and body weight
thereof, and the like.
[0184] The feed of the present invention maybe any feed comprising
the active ingredient. The feed includes, for example, pet feeds
for dogs, cats and rats, cattle feeds for cows and pigs, chicken
feeds for chicken and turkeys, and fish cultivation feeds for porgy
and yellowtail.
[0185] The feed can be produced by appropriately blending the
active ingredient of the present invention in a raw feed material
including, for example, cereals, brans, oil-seed meals,
animal-derived raw feed materials, other raw feed materials and
purified products. The cereals include, for example, mile, wheat,
barley, oats, rye, brown rice, buckwheat, fox-tail millet, Chinese
millet, Deccan grass, corn, and soybean.
[0186] The brans include, far example, rice bran, defatted rice
bran, bran, lowest-grade flour, wheat germ, barley bran. screening
pellet, corn bran, and corn germ.
[0187] The oil-seed meals include, for example, soybean meal,
soybean powder, linseed meal, cottonseed meal, peanut meal,
safflower meal, coconut meal, palm meal, sesame meal, sunflower
meal, rapeseed meal, kapok seed meal and mustard meal.
[0188] The animal-derived raw feed materials include , for example
, fish powders, import meal, whole meal, and coast meal, fish
soluble, meat powder, meat and bone powder, blood powder,
decomposed hair, bone powder, byproducts from butchery, feather
meal, silkworm pupa, skim milk, casein, dry whey and krill.
[0189] Other raw feed materials include, for example, plant stems
and leaves such as alfalfa, hey cube, alfalfa leaf meal, and locust
leaf powder, byproducts from corn processing industries, such as
corn gluten meal, corn gluten feed and corn steep liquor, starch,
sugar, yeast, byproducts from fermentation industry such as beer
residue, malt root, liquor residue and soy sauce residue, and
agricultural byproducts such as citrus processed residue, soybean
curd residue, coffee residue, and cocoa residue, cassava, horse
bean, guar meal, seaweed, spirulina and chlorella.
[0190] The purified products include, for example, proteins such as
casein and albumin, amino acids, starch, cellulose, saccharides
such as sucrose and glucose, minerals and vitamins, In case of
providing to animals the feed according to the present Invention,
the amount of the feed to be ingested is not specifically limited
but is preferably for example, 0.1 mg to 50 g per 1 kg body weight
per day, preferably 0.5 mg to 20 g per 1 kg body weight per day,
based on the amount of the active ingredient. The feed is
continuously ingested at this amount for a period from a single day
up to 5 years, preferably from 2 weeks to one year. Again, the
amount ingested can be adjusted to an appropriate range depending
on the species, age and body weight of the animal ingesting the
feed, and the like.
[0191] Furthermore, the present invention relates to an additive
for foods, drinks and feeds, which, due to the presence of a
microorganism or derivative or mutant or analog or fragment thereof
as described in connection with the composition of the present
invention is, inter alia, capable of specifically binding to
Streptococcus mutans so as to prevent and/or treat caries.
Preferably, the microorganism, mutant, derivative, analog or
fragment thereof is a microorganism, mutant, derivative or fragment
of the present invention. The additive for foods or drinks includes
the additive for nutritious compositions for infants. The additive
for foods can be produced by a general method for producing
additives for foods, drinks or feeds. If necessary, additives for
general use in foods, drinks or feeds, for example, additives
described in Food Additive Handbook (The Japan Food Additives
Association; issued on Jan. 6, 1997) may be added satisfactorily,
including sweeteners, colorants, preservatives, thickeners and
stabilizers, anti-oxidants, color fixing agents, bleaches,
antiseptics, gum base, bitters, enzymes, brightening agents,
acidifier, seasonings, emulsifiers, enhancers, agents for
manufacture, flavors, and spice extracts. Further, conventional
saccharides, starch, inorganic materials, plant powders,
excipients, disintegrators, lubricants, binders, surfactants, and
plasticizers mentioned previously for pharmaceutical tablets may be
added satisfactorily.
[0192] The additives include the following additives.
[0193] The sweeteners include aspartame, licorice, stevia, xylose
and rakanka (Momordica grosvenori fruit). The colorants include
carotenoid and turmeric oleoresin, flavonold, caramel color,
spirulina color, chlorophyll, purple sweet potato color, purple yam
color, perilla color, and blueberry color.
[0194] The preservatives include, for example, sodium sulfite,
benzoates, benzoin extract, sorbates, and propionates. The
thickeners and stabilizers include, for example, gums such as gum
arable and xanthan gum, alginates, chitin, chitosan, aloe extract,
guar gum, hydroxypropyl cellulose, sodium casein, corn starch.
carboxymethyl cellulose, gelatin, agar, dextrin, methyl cellulose,
polyvinyl alcohol, microfiber cellulose, microcrystalline
cellulose, seaweed cellulose, sodium polyacrylate, sodium
polyphosphate, carrageenan or yeast cell wall.
[0195] The anti-oxidants include, for example, vitamin C group,
sodium ethylenediaminetetraacetate, calcium
ethylenediaminetetraacelate, erythorbic acid, oryzanol, catechin,
quercetin, clove extract, enzyme-treated rutin, apple extract,
sesame seed extract, dibutylhydroxytoluene, fennel extract,
horseradish extract, water celery extract, tea extract,
tocopherols, rapeseed extract, coffee bean extract, sunflower seed
extract, ferulio acid, butylhydroxyanisole, blueberry leaf extract.
propolis extract, pepper extract, garden balsam extract, gallic
acid, eucalyptus extract, and rosemary extract. The color fixing
agents include, for example, sodium nitrite. The bleaches include,
for example, sodium sulfite.
[0196] The antiseptics include, for example, o-phenyl phenol. The
gum base includes, for example, acetylricinoleate methyl, urushi
wax, ester gum, elerni resin, urucury wax, kaurigum, carnaubawax,
glycerin fatty acid ester, spermaceti wax, copaibabalsam, copal
resin, rubber, rice bran wax, cane wax, shellac, jelutong, sucrose
fatty acid ester, depolymerized natural rubber, paraffin wax, fir
balsam, propylene glycol fatty acid ester, powdered pulp, powdered
rice hulls, jojoba oil, polyisobutylene, polybutene,
microcrystalline wax, mastic gum, bees wax and calcium
phosphate.
[0197] The bitters include, for example, iso-alpha-bitter acid,
caffeine, kawaratake (Coriolus versieolor) extract, redbark
cinchona extract, Phellodendron bark extract, gentian root extract,
spice extracts, enzymatically modified naringin, Jamaica cassia
extract, theabromine, naringin, cassia extract, absinth extract,
isodonis extract, olive tea, bitter orange (Citrus aurantium)
extract, hop extract and wormwood extract.
[0198] The enzymes include, for example, amylase, trypsin or
rennet.
[0199] The brightening agents include, for example, urushi wax and
japan wax. The acidifier include, for example, adipic acid,
itacania acid, citric acids, succinic acids, sodium acetate,
tartaric acids, carbon dioxide, lactic acid, phytic acid, fumario
acid, malic acid and phosphoric acid. The seasonings include, for
example, amino acids such as asparagine, aspartic acid, glutamic
acid, glutamine, alanine, isoleucine, glycine, serine, cystine,
tyrosine, leucine, and praline, nucleic acids such as sodium
inosinate, sodium uridinate, sodium guanylate, sodium cytidylate,
calcium ribonucleotide and sodium ribonucleotide, organic acids
such as citric acid and succinic acid, potassium chloride, sodium
chloride-decreased brine, crude potassium chloride, whey salt,
tripotassium phosphate, dipotassium hydrogen phosphate, potassium
dihydrogen phosphate, disodium hydrogen phosphate, sodium
dihydrogen phosphate, trisodium phosphate and chlorella
extract.
[0200] The enhancers include, for example, zinc salts, vitamin C
group, various amino acids, 5-adenylic acid, iron chloride,
hesperidin, various calcined calcium, various non-calcined calcium,
dibenzoylthiamine, calcium hydroxide, calcium carbonate, thiamine'
hydrochloride salt, Dunallella. Oarotene, tocopherol, nicotinic
acid, carrot carotene, palm oil carotene, calcium pantothenate,
vitamin A, hydroxyproline, calcium dihydrogen pyrophosphate,
ferrous pyrophosphate, ferric pyrophosphate, ferritin, heme iron,
rnenaquinone, folic acid and riboflavine.
[0201] The agents for manufacture include, for example, processing
auxiliaries such as acetone and ion exchange resin. The flavors
include, for example, vanilla essence and the spice extracts
include, for example, capsicum extract.
[0202] These various additives can be added to the active
ingredient, taking into consideration the mode of administration,
in accordance with the present invention.
[0203] The anticariogenic composition of the present invention
encompasses an amount of a microorganism or derivative or mutant
thereof of the present invention or analog or fragment thereof as
described in connection with the composition of the present
invention. Preferably, the microorganism, mutant, derivative,
analog or fragment thereof is a microorganism, mutant, derivative
or fragment of the present invention. It is envisaged that the
compositions and in particular the anticariogenic composition
comprise a microorganism of the present invention as described in
connection with the composition of the present invention in the
form of a probiotic microorganism. Namely, in addition to the
probiotic effect, the probiotic microorganism of the present
invention is useful for treating and/or preventing caries. The
amount of said probiotic microorganism is high enough to
significantly positively modify the condition to be treated,
preferably caries, but low enough to avoid serious side effects (at
a reasonable benefit/risk ratio), within the scope of sound medical
judgment. An effective amount of said probiotic microorganism will
vary with the particular goal to be achieved, the age and physical
condition of the patient being treated, the severity of the
underlying disease, the duration of treatment, the nature of
concurrent therapy and the specific microorganism employed. The
effective amount of said probiotic microorganism will thus be the
minimum amount which will provide the desired specific binding to
Streptococcus mutans. The presence of, for example,
1.times.10.sup.9 bacteria, as viable or non-viable whole cells, in
0.05 ml solution of phosphate buffered saline solution, or in 0.05
ml of suspension of agar, or the dry weight equivalent of cell wall
fragments, is effective when administered in quantities of from
about 0.05 ml to about 20 ml.
[0204] A decided practical advantage is that the probiotic organism
may be administered in a convenient manner such as by the oral
route. Depending on the route of administration, the active
ingredients which comprise said probiotic organisms may be required
to be coated in a material to protect said organisms from the
action of enzymes, acids and other natural conditions which may
inactivate said organisms. In order to administer probiotic
organisms by other than parenteral administration, they should be
coated by, or administered with, a material to prevent
inactivation. For example, probiotic organisms may be
co-administered with enzyme inhibitors or in liposomes. Enzyme
inhibitors include pancreatic trypsin inhibitor,
diisopropylfluorophosphate (DFP) and trasylol. Liposomes include
water-in-oil-in-water P40 emulsions as well as conventional and
specifically designed liposomes which transport lactobacilli or
their by-products to the urogenital surface. Dispersions can also
be prepared, for example, in glycerol, liquid polyethylene glycols,
and mixtures thereof, and in oils. Generally, dispersions are
prepared by incorporating the various sterilized probiotic
organisms into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and the freeze-drying technique
which yield a powder of the active ingredient plus any additional
desired ingredient from previously sterile-filtered solution
thereof. Additional preferred methods of preparation include but
are not limited to lyophilization and heat-drying.
[0205] The anticariogenic composition also encompasses products
intended to be administered orally, or buccal, which comprise an
acceptable pharmaceutical carrier as described herein to which, or
onto which, cells of a microorganism of the present invention as
described in connection with the composition of the present
invention which is preferably a microorganism of the present
invention is added in fresh, concentrated or dried form, for
example. Of course, also a derivative or fragment of said
microorganism can be added or any combination of said
microorganism, derivative and/or fragment thereof which are
disclosed herein. These products may be provided in the form of an
ingestible suspension, a gel, a diffuser, a capsule, a hard gelatin
capsule, a syrup, or in any other galenic form known to persons
skilled in the art.
[0206] When the probiotic organisms are suitably protected as
described above, the active compound may be orally administered,
for example, with an inert diluent or with an assimilable edible
carrier, or it may be enclosed in hard or soft shell gelatin
capsule, or it may be compressed into tablets designed to pass
through the stomach (i.e., enteric coated), or it may be
incorporated directly with the food of the diet. For oral
therapeutic administration, the probiotic organisms may be
incorporated with excipients and used in the form of ingestible
tablets, buccal tablets, troches, capsules, elixirs, suspensions,
syrups, wafers, and the like. Compositions or preparations
according to the present invention are prepared so that an oral
dosage unit form contains, for example, about 1.times.10.sup.9
viable or non-viable e.g., lactobacilli per ml. The probiotic
organism is compounded for convenient and effective administration
in effective amounts with a suitable pharmaceutically or food
acceptable carrier in dosage unit form as hereinbefore disclosed. A
unit dosage form can, for example, contain the principal active
compound in an amount approximating 10.sup.9 viable or non-viable,
e.g., lactobacilli, per ml. In the case of compositions containing
supplementary ingredients such as prebiotics, the dosages are
determined by reference to the usual dose and manner of
administration of the said ingredients.
[0207] The Figures show:
[0208] FIG. 1: Aggregation of Streptococcus mutans by Lactobacillus
species
[0209] The figure shows a mixture of an aggregating Lactobacillus
with S. mutans (left tube) in comparison with a mixture of a
non-aggregating Lactobacillus with S. mutans (right tube). The
experiment has been performed as described in Example 3 and the
tubes were left undisturbed for 20 minutes to allow the aggregates
to settle.
[0210] FIG. 2: Microscopic picture of an aggregating Lactobacillus
species with Streptococcus mutans
[0211] The figure shows a microscopic picture of the aggregate
between Lactobacillus and S. mutans shown in FIG. 1 (left tube).
The picture was taken at a 1000-fold magnification using a
phase-contrast microscope.
[0212] A better understanding of the present invention and of its
many advantages will be had from the following examples, offered
for illustrative purposes only, and are not intended to limit the
scope of the present invention in any way.
EXAMPLE 1
Storage and Growth
[0213] Storage and growth of strains can occur according to
ordinary procedures. For example, strains can be stored as frozen
stocks at -80.degree. C. 1 ml of a culture can be grown to
stationary phase (OD600/mL 4-8) in MRS-Medium and mixed with 500
.mu.l of a sterile 50% glycerine solution and frozen. Cultures of
S. mutans can be grown in TSY-media to stationary phase (OD600/mL
1-2) and treated as mentioned above Cultivation of S. mutans (DSMZ
20523, serotype c; NCTC 10923, serotype e; NCTC 11060, serotype f
as well as non serotyped isolates) as well as cultivation of
lactobacilli can be done in 5 ml in closed Falcon tubes at
37.degree. C. without shacking over night.
[0214] In particular, the strains used in the present application
were stored as frozen stocks at -80.degree. C. 1 ml of a culture
grown to stationary phase (OD600/mL 4-8) in MRS-broth was mixed
with 500 .mu.l of a sterile 50% glycerol solution and frozen.
[0215] In particular, cultures of S. mutans were grown in TSY-broth
to stationary phase (OD600/mL 1-2) and treated as mentioned
above.
[0216] Cultivation of S. mutans (DSMZ 20523, serotype c; NCTC
10923, serotype e; NCTC 11060, serotype f and other non serotyped
isolates--isolated by OrganoBalance) and cultivation of
lactobacilli was done in 5 ml in closed Falcon tubes at 37.degree.
C. without shacking over night.
EXAMPLE 2
Taxonomic Classification of Strains
[0217] The taxonomic classification of the strains was done
according to their carbohydrate fermentation pattern. This was
determined using the API 50 CH (bioMerieux, France) system and
analyzed using APILAB PLUS software version 3.3.3 (bioMerieux,
France).
EXAMPLE 3
Test on Aggregation of Streptococcus Mutans
[0218] Mixing of the lactobacilli with S. mutans was done in
volumetric ratios of 3:1 to 60:1 (S. mutans: lactobacilli), this
corresponds to a ratio of colony forming units from 1:50 to 1:2,5.
An optical density measured at a wavelength of 600 nm in 1 ml means
preferably for S. mutans 3.times.10.sup.8 colony forming units and
for lactobacilli preferably 7.times.10.sup.9 colony forming units.
Mixing was done in 2 mL volume in 15 mL Falcon tubes. The culture
suspensions were diluted with PBS-buffer to obtain the volumetric
ratios mentioned above while keeping the final volume at 2 ml. The
mixture was vortexed for 15 seconds. An aggregation is visible as
an immediate turbidity of the suspension. The tubes were left
undisturbed for 20 min, after that period of time the aggregates
settle as a visible pellet whereas non-aggregating mixtures stay in
suspension.
[0219] As a control, self-aggregation of the respective
Lactobacillus strain and the S. mutans strains was always
investigated by performing the test with only the Lactobacillus or
the S. mutans strain added to the tube. An aggregation of S. mutans
by Lactobacillus is shown in FIGS. 1 (left tube) and 2.
[0220] The lactobacilli strains of the present invention, in
particular those deposited with the DSMZ exhibited aggregation of
all S. mutans serotypes without showing a self-aggregation
behaviour.
[0221] Media:
TABLE-US-00001 MRS-broth: MRS-mixture (Difco, USA) 55 g/L pH: 6.5
TSY-broth: TSY-mixture (Difco, USA) 30 g/L Yeast extract (Deutsche
3 g/L Hefewerke, Germany)
[0222] Buffer:
TABLE-US-00002 PBS-buffer: Na.sub.2HPO.sub.4*2H.sub.20 1.5 g/L
KH.sub.2PO.sub.4 0.2 g/L NaCl 8.8 g/L pH adjusted with HCl
EXAMPLE 4
Specificity of the Aggregation Towards Typical Members of the Oral
Flora
[0223] The Lactobacillus cultures were grown as in Example 1.
[0224] The oral bacteria--namely: Streptococcus salivarius subsp.
thermophilus (isolated by OrganoBalance, identified by API 50 CH
(Biomerieux, France) according to manufacturers instructions);
Streptococcus oralis (DSMZ 20066); Streptococcus oralis (DSMZ
20395); Streptococcus oralis (DSMZ 20627); Staphylococcus
epidermidis (DSMZ 1798); Staphylococcus epidermidis (DSMZ
20044);Streptococcus mitis (DSMZ 12643); Streptococcus sanguinis
(DSMZ 20567)--were grown in 5 rnL BHI-medium in closed 15 mL Falcon
tubes at 37.degree. C. over night. Each of the oral bacteria were
preferably mixed in a volumetric ratio of 3:1 with Lactobacillus
cultures and aggregation was assayed as in Example 3. For each
testing of aggregation/non-aggregation only one of the
aforementioned bacteria is preferably used to immediately determine
the outcome of the testing.
[0225] As a control, a self-aggregation of the respective oral
bacteria as well as the tested Lactobacillus strains was always
investigated by performing the test with only the lactobacilli or
the oral flora strains added to the tube.
[0226] The mentioned L. paracasei subsp. paracasei strains did not
aggregate the oral bacteria mentioned above. The L. rhamnosus
strains aggregated Streptococcus salivarius subsp thermophilus.
TABLE-US-00003 BHI-broth: BHI-mixture (Difco, USA) 37 g/L pH:
7.2
EXAMPLE 5
Temperature Resistance of the Aggregating Capacity of the
Lactobacilli
[0227] The bacteria were grown as in Example 1.
[0228] The grown lactobacilli cultures were incubated at
121.degree. C. at 2 bar in satured steam for 20 min (autoclaved).
After cooling of the autoclaved cultures to room temperature, the
lactobacilli were mixed in a volumetric ratio of 1:3 with grown S.
mutans cultures and aggregation was assayed as in example 3
including the control experiments.
[0229] Aggregation was also assayed using the oral bacteria as
outlined in Example 4.
[0230] It was found that the aggregation behaviour of the
lactobacilli was not changed by the autoclaving procedure towards
the tested S. mutans serotypes or towards the oral bacteria.
EXAMPLE 6
Dependency of the Aggregation on pH-Value
[0231] The bacteria were grown as in Example 1.
[0232] 0.5 ml of the lactobacilli and 1.5 ml of S. mutans were
harvested by centrifugation at 3200*g for 10 min and the
supernatant was discarded. The cells were resuspended in their
original volume (0.5 ml and 1.5 ml, respectively) in different
PBS-buffers adjusted to different pH-values. The pH-values of the
buffers were adjusted to values from 7.0 to 3.0 in steps of 0.5
pH-units. Cultures were resuspended in buffers of the respective
pH-value that was to be used for the aggregation behaviour
assay.
[0233] Afterwards the lactobacilli were preferably mixed in a
volumetric ratio of 1:3 with S. mutans cultures and aggregation was
assayed as in example 3 including the control experiments. No
visible aggregation of S. mutans by the lactobacilli occurred at pH
values lower than 4.5.
EXAMPLE 7
Sensitivity of the Aggregation Behaviour to Lyophilisation.
[0234] The bacteria were grown as in Example 1.
[0235] Aliquots of 1 ml of the lactobacilli cultures were harvested
by centrifugation at 3200*g for 10 minutes. The supernatant was
discarded and the pellets were lyophilised at room temperature
under vacuum for two hours. Resulting dry pellets of each tested
Lactobacillus strain were stored at room temperature and at
4.degree. C., respectively, for 1 day, 1 week, 2 weeks, 3 weeks and
4 weeks. After the storage time, lyophilised pellets were
resuspended in 1 ml PBS-buffer, pH 7.0. The resuspended
lactobacilli were mixed in a volumetric ratio of 1 : 3 with freshly
grown S. mutans cultures and aggregation was assayed as in example
3 including the control experiments.
[0236] The aggregation behaviour of the mentioned lactobacilli
towards S. mutans was not changed by the lyophilization or the
storage procedures.
EXAMPLE 8
Test on Protease Resistance
[0237] The bacteria were grown as in Example 1.
[0238] Proteases used were Pronase E, Proteinase K, Trypsin,
Chymotrypsin (all obtained from Sigma, Germany). Aliquots of 1 ml
of the lactobacilli were washed in PBS-buffer by harvesting the
cells by centrifugation at 3200*g for 10 minutes and resuspending
the pellet in 1 ml PBS-buffer (pH 7.0). Afterwards the cells were
harvested again as described above and resuspended in PBS-buffer
(pH 7.0) containing the respective protease at a final
concentration of 2.5 mg/mL. The suspension was incubated for 1 hour
at 37.degree. C. Afterwards the cells were washed and resuspended
in PBS-buffer (pH 7.0) as described above.
[0239] The aggregation was assayed as in example 3 including the
control experiments.
[0240] The aggregation behaviour of the mentioned lactobacilli
towards S. mutans was not changed by treatment with any of the
mentioned proteases.
EXAMPLE 9
Ion Dependency of the Aggregation Behaviour
[0241] The bacteria were grown as in Example 1.
[0242] Aliquots of 1 ml of the lactobacilli were washed in 1 ml 200
mM EDTA solution twice as described above. Afterwards the cells
were harvested and resuspended in 1 ml PBS-buffer (pH 7.0).
[0243] The aggregation was assayed as in Example 3 and a complete
loss of the aggregation ability was observed. Resuspension of the
lactobacilli in 1 ml of a 2 mM calcium solution after the two times
washing in 200 mM EDTA-solution restored the ability to aggregate
S. mutans. Resuspension of the EDTA washed cells in up to 100 mM
magnesium solution did not restore the ability to aggregate S.
mutans.
EXAMPLE 10
Test of Aggregation in the Presence of Saliva
[0244] The bacteria were grown as in Example 1.
[0245] 2 ml aliquots of S. mutans cultures were harvested as
described above and resuspended in 2 ml of saliva. The saliva was
provided by two volunteers and used immediately after winning.
[0246] The aggregation was assayed as in Example 3.
[0247] The aggregation behaviour of the mentioned lactobacilli
towards S. mutans did not change in the presence of saliva.
EXAMPLE 11
Lozenge Composition (I)
[0248] The lozenge composition is preferably prepared as is
described in Example 4 on page 8 of DE-C2 36 45 147, wherein, in
addition to the ingredients mentioned in said Example 4, the
microorganism of the present invention is added in an amount of
10.sup.2 to 10.sup.12, preferably 10.sup.3 to 10.sup.8 cells per mg
of the lozenge.
EXAMPLE 12
Lozenge Composition (II)
[0249] The lozenge composition is preferably prepared as is
described in Example 5 on page 8 of DE-C2 36 45 147, wherein, in
addition to the ingredients mentioned in said Example 4, the
microorganism of the present invention is added in an amount of
10.sup.2 to 10.sup.12, preferably 10.sup.3 to 10.sup.8 cells per mg
of the lozenge.
EXAMPLE 13
Dentifrice Composition
[0250] The dentifrice composition is preferably prepared as is
described in Example 3 on page 8 of DE-C2 36 45 147, wherein, in
addition to the ingredients mentioned in said Example 4, the
microorganism of the present invention is added in an amount of
10.sup.2 to 10.sup.12, preferably 10.sup.3 to 10.sup.8 cells per mg
of the dentifrice.
EXAMPLE 14
Chalk-Based Dentifrice Composition
[0251] The chalk-based dentifrice composition is preferably
prepared as is described in chapter 7.1.4.4 "Rezepturbeispiel" on
page 205 of the textbook "Kosmetik", W. Umbach (editor), 2.sup.nd
edition, Thieme Verlag, 1995, wherein, in addition to the
ingredients mentioned in said chapter on page 205, the
microorganism of the present invention is added in an amount of
10.sup.2 to 10.sup.12, preferably 10.sup.3 to 10.sup.8 cells per mg
of the chalk-based dentifrice.
EXAMPLE 15
Gel-Dentifrice on Basis of Silicic Acid/Sodium Fluoride
[0252] The gel-dentifrice on basis of silicic acid/sodium fluoride
dentifrice composition is preferably prepared as is described in
chapter 7.1.4.4 "Rezepturbeispiel" on page 205 of the textbook
"Kosmetik", W. Umbach (editor), 2.sup.nd edition, Thieme Verlag,
1995, wherein, in addition to the ingredients mentioned in said
chapter on page 205, the microorganism of the present invention is
added in an amount of 10.sup.2 to 10.sup.12, preferably 10.sup.3 to
10.sup.8 cells per mg of the gel-dentifrice on basis of silicic
acid/sodium fluoride.
EXAMPLE 16
Dentifrice Composition Against Tartar
[0253] The dentifrice composition against tartar is preferably
prepared as is described in chapter 7.1.4.4 "Rezepturbeispiel" on
page 206 of the textbook "Kosmetik", W. Umbach (editor), 2.sup.nd
edition, Thieme Verlag, 1995, wherein, in addition to the
ingredients mentioned in said chapter on page 206, the
microorganism of the present invention is added in an amount of
10.sup.2 to 10.sup.12, preferably 10.sup.3 to 10.sup.8 cells per mg
of the dentifrice against tartar.
EXAMPLE 17
Chewing Gum Composition
[0254] The chewing gum composition is preferably prepared as is
described in Example 6 on page 9 of DE-C2 36 45 147, wherein, in
addition to the ingredients mentioned in said Example 4, the
microorganism of the present invention is added in an amount of
10.sup.2 to 10.sup.12, preferably 10.sup.3 to 10.sup.8 cells per mg
of the chewing gum.
EXAMPLE 18
Concentrated Mouthwash Composition
[0255] The concentrated mouth wash composition is preferably
prepared as is described in chapter 7.1.4.4 "Rezepturbeispiel" on
page 206 of the textbook "Kosmetik", W. Umbach (editor), 2.sup.nd
edition, Thieme Verlag, 1995, wherein, in addition to the
ingredients mentioned in said chapter on page 206, the
microorganism of the present invention is added in an amount of
10.sup.2 to 10.sup.13, cells per ml of the concentrated mouthwash
composition.
EXAMPLE 19
Film Preparation
[0256] Preparation of Films:
[0257] 1. Water Phase [0258] heat water to 60.degree. C. [0259]
aspartame (sweetener) is added under stirring [0260] aspartame is
dissolved completely [0261] a polymeric water-soluble film former,
like, for example, Kollicoat IR (polyethylenglycol on
polyvinylalcohol) or PVP (polyvinylpyrrolidon) or natural polymers
such as alginates are added under stirring until they are dissolved
[0262] after 10 min. the rest of the foam is removed [0263] the
microorganism of the present invention in an amount of 10.sup.2 to
10.sup.12, preferably 10.sup.3 to 10.sup.8 cells per final aroma
film is added after cooling down of the mixture; alternatively, the
mutant or derivative of the microorganism of the present invention
or an analog or fragment of the microorganism of the present
invention can be added
[0264] 2. Oily Phase [0265] menthol is dissolved in peppermint-oil
[0266] polysorbat 80 is added to the peppermint-oil-menthol-mix
under stirring [0267] this mixture is then added to
propylene-glykole under stirring [0268] optional colorants (such as
pigments, lakes) can be added
[0269] 3. [0270] under stirring the oily phase is slowly mixed with
the water phase
[0271] 4. [0272] the thin films are mechanically generated using a
cutting device
[0273] Sample formulations:
TABLE-US-00004 formulation I formulation II weight composition
weight composition [g] in film [%] [g] in film [%] Phase I
aspartame 0.7 1.4 0.7 1.8 Kollicoat IR 35.0 68.5 25.0 65.8 ascorbic
acid -- -- 1.0 2.6 cherry flavour 6.0 15.8 water demin. 85.0 --
80.0 Phase II menthol 1.4 2.7 -- peppermint oil 5.6 11.0 --
polysorbat 80 0.7 1.4 -- propylene glykol 7.0 13.7 5.0 13.2 green
lake 0.7 1.4 -- azorubin lake -- -- 0.3 0.8 sum 136.1 100.0 118.0
100.0 solid content 51.1 38.0
[0274] Other embodiments and uses of the invention will be apparent
to those skilled in the art from consideration of the specification
and practice of the invention disclosed herein. All references
cited herein, for any reason, including all publications, all U.S:
and foreign patents and all U.S. and foreign patent applications,
are specifically and entirely incorporated by reference for all
purposes. It is intended that the specification and examples be
considered exemplary only with the true scope and spirit of the
invention indicated by the following claims.
* * * * *