U.S. patent application number 13/981194 was filed with the patent office on 2013-12-26 for oral health improving compositions.
This patent application is currently assigned to BASF SE. The applicant listed for this patent is Markus Pompejus. Invention is credited to Markus Pompejus.
Application Number | 20130344010 13/981194 |
Document ID | / |
Family ID | 45509475 |
Filed Date | 2013-12-26 |
United States Patent
Application |
20130344010 |
Kind Code |
A1 |
Pompejus; Markus |
December 26, 2013 |
Oral Health Improving Compositions
Abstract
The present invention is concerned with microorganisms or
fragments thereof as sensorically neutral oral care agents,
particularly for prevention of dental calculus, as anti-caries
agents and/or anti-oral malodor agents. The invention is
furthermore concerned with compositions comprising microorganisms
or fragments thereof for reducing mutans Streptococci. Such
compositions can be used in oral care compositions, e.g. for caries
prophylaxis, or for prophylaxis of dental calculus or oral malodor.
They may also or instead be used for prevention or treatment of
oral malodor. As the microorganisms and fragments thereof according
to the present invention have a very low, unobtrusive smell and
taste, they are particularly suited as sensorically neutral agents
for preventment of dental calculus, caries, oral biofilm formation
and/or for prevention or treatment of oral malodor. The
microorganisms and fragments thereof, and also compositions
comprising such microorganisms and fragments, can thus
advantageously be used in food and feed compositions, particularly
in pet foods. Furthermore, the invention is concerned with methods
of preparing such microorganisms, fragments, compositions, foods
and feeds.
Inventors: |
Pompejus; Markus; (White
Plains, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pompejus; Markus |
White Plains |
NY |
US |
|
|
Assignee: |
BASF SE
Ludwigshafen
DE
|
Family ID: |
45509475 |
Appl. No.: |
13/981194 |
Filed: |
January 12, 2012 |
PCT Filed: |
January 12, 2012 |
PCT NO: |
PCT/EP2012/050463 |
371 Date: |
July 23, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61435472 |
Jan 24, 2011 |
|
|
|
Current U.S.
Class: |
424/50 |
Current CPC
Class: |
A23L 33/135 20160801;
A23K 10/18 20160501; A61Q 11/00 20130101; A61P 1/02 20180101; A23K
50/42 20160501; A23K 10/16 20160501; A61K 35/74 20130101; A61K 8/99
20130101 |
Class at
Publication: |
424/50 |
International
Class: |
A61K 8/99 20060101
A61K008/99; A61Q 11/00 20060101 A61Q011/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 24, 2011 |
EP |
11151924.5 |
Claims
1. A method for providing oral care to a human or animal,
comprising administering a composition comprising a binder
microorganism or fragment thereof as a sensorically neutral oral
care agent to a human or animal in need thereof, wherein the binder
microorganism is a lactic acid bacterium, wherein the binder
microorganism or fragment thereof is capable of binding to a
microorganism of the group of mutans Streptococci, and wherein the
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; and/or (vi) independent of magnesium.
2. A method for preparing a cosmetic, pharmaceutical or veterinary
composition comprising formulating a binder microorganism or
fragment thereof into a composition, wherein the composition is an
oral care composition, wherein the binder microorganism is used in
a sensorically neutral amount, wherein the binder microorganism is
a lactic acid bacterium, wherein the binder microorganism or
fragment thereof is capable of binding to a microorganism of the
group of mutans Streptococci, and wherein the 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; and/or (vi) independent of magnesium.
3. The method according to claim 2, wherein the binder
microorganism or fragment thereof is capable of binding to at least
one strain, at least two strains, or at least three strains of
mutans Streptococci selected from the group consisting of
Streptococcus mutans serotype c (DSMZ 20523), Streptococcus mutans
serotype e (NCTC 10923), Streptococcus mutans serotype f (NCTC
11060), Streptococcus sobrinus DSMZ 20742, Streptococcus ratti DSMZ
20564, Streptococcus cricetus DSMZ 20562, Streptococcus ferus DSMZ
20646 and Streptococcus macacae DSMZ 20714.
4. The method according to claim 2, wherein the binder
microorganism or fragment thereof is not capable of binding to at
least one, at least two, at least three, or any microorganism
selected from the group consisting of Streptococcus salivarius ssp.
thermophilus, Streptococcus oralis DSMZ 20066, Streptococcus oralis
DSMZ 20395, Streptococcus oralis DSMZ 20627, Streptococcus mitis
DSMZ 12643 and Streptococcus sanguinis DSMZ 20567.
5. The method according to claim 2, wherein the binder
microorganism is in a thermally inactivated or lyophilized form,
and/or wherein the binder microorganism is of the family
Lactobacillaceae, of the genus Lactobacillus, Paralactobacillus,
Pediococcus or Sharpea, of species Lactobacillus paracasei,
Lactobacillus rhamnosus, Lactobacillus casei or Lactobacillus zeae,
or is any of strains DSMZ 16667, DSMZ 16668, DSMZ 16669, DSMZ
16670, DSMZ 16671, DSMZ 16672 or DSMZ 16673, or a mutant or
derivative thereof.
6. The method according to claim 2, wherein the binder
microorganism or fragment thereof is used: as a preferably
sensorically neutral anti-dental calculus agent, or for the
preparation of a composition for prevention or treatment of dental
calculus, and/or as a sensorically neutral anti-caries agent, or
for the preparation of a sensorically neutral composition for
prevention or treatment of caries, and/or as a preferably
sensorically neutral anti-oral malodor agent, or for the
preparation of a composition for prevention or treatment of oral
malodor.
7. The method according to claim 2, wherein the binder
microorganism is selected from the group consisting of:
microorganisms which metabolize D-lactose, but not L-sorbose and/or
D-saccharose and/or D-inuline, (ii) microorganisms which metabolize
inuline, (iii) microorganisms which metabolize L-sorbose, but not
D-lactose and/or D-saccharose and/or inuline, and (iv)
microorganisms which metabolize L-sorbose, D-lactose and inuline,
and preferably, wherein the microorganism is selected from the
group consisting of: (i) microorganisms which metabolize D-lactose,
but not L-sorbose, D-saccharose and inuline, (ii) microorganisms
which metabolize L-sorbose, D-lactose and inuline, but not
D-saccharose, (iii) microorganisms which metabolize L-sorbose, but
not D-lactose, D-saccharose and inuline, and (iv) microorganisms
which metabolizes L-sorbose, D-lactose, D-saccharose, but not
inuline.
8. The method according to claim 2, wherein the binding of said
binder microorganism or fragment thereof to Streptococcus strains
can be assayed as follows: (a) growing said microorganism to
stationary phase; (b) mixing said microorganism with said
Streptococcus 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 said
Streptococcus; and (d) detecting aggregates by the occurrence of a
pellet.
9. A method of preparing a composition for prevention or treatment
of dental calculus and/or for the preparation of a sensorically
neutral composition for prevention or treatment of caries and/or
for the preparation of a composition for prevention or treatment of
oral malodor, comprising adding a binder microorganism or fragment
thereof to a base composition, wherein the binder microorganism is
added in a sensorically neutral amount, wherein the binder
microorganism is a lactic acid bacterium, wherein the binder
microorganism is capable of binding to a microorganism of the group
of mutans Streptococci, wherein the 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;
and/or (vi) independent of magnesium, and wherein the base
composition comprises a cosmetically, pharmaceutically or
veterinary compatible carrier.
10. The method according to claim 9, wherein the binder
microorganism is in a thermally inactivated or lyophilized form,
and/or wherein the binder microorganism or fragment thereof is
capable of binding to at least one strain, at least two strains, or
at least three strains of mutans Streptococci selected from the
group consisting of Streptococcus mutans serotype c (DSMZ 20523),
Streptococcus mutans serotype e (NCTC 10923) and Streptococcus
mutans serotype f (NCTC 11060), Streptococcus sobrinus DSMZ 20742,
Streptococcus ratti DSMZ 20564, Streptococcus cricetus DSMZ 20562,
Streptococcus ferus DSMZ 20646 and Streptococcus macacae DSMZ
20714, and preferably is not capable of binding to at least one, at
least two, at least three, or any microorganism selected from the
group consisting of Streptococcus salivarius ssp. thermophilus,
Streptococcus oralis DSMZ 20066, Streptococcus oralis DSMZ 20395,
Streptococcus oralis DSMZ 20627, Streptococcus mitis DSMZ 12643 and
Streptococcus sanguinis DSMZ 20567, and/or wherein the binder
microorganism is of family Lactobacillaceae, of the genus
Lactobacillus, Paralactobacillus, Pediococcus or Sharpea, or of
species Lactobacillus paracasei, Lactobacillus rhamnosus,
Lactobacillus casei or Lactobacillus zeae, or is any of strains
DSMZ 16667, DSMZ 16668, DSMZ 16669, DSMZ 16670, DSMZ 16671, DSMZ
16672 or DSMZ 16673, or a mutant or derivative thereof.
11. A composition comprising an isolated or purified binder
microorganism or isolated or purified fragment thereof in an
amount: sufficient for preventing dental calculus formation or
slowing down dental calculus formation, and/or sufficient for
preventing or reducing intensity of oral malodor, and/or sufficient
for preventing caries or slowing down caries generation, and
preferably in a sensorically neutral amount, wherein the binder
microorganism is a lactic acid bacterium, preferably wherein the
binder microorganism is of family Lactobacillaceae, of genus
Lactobacillus, Paralactobacillus, Pediococcus or Sharpea, of
species Lactobacillus paracasei, Lactobacillus rhamnosus,
Lactobacillus casei or Lactobacillus zeae, or is any of strains
DSMZ 16667, DSMZ 16668, DSMZ 16669, DSMZ 16670, DSMZ 16671, DSMZ
16672 or DSMZ 16673, or a mutant or derivative thereof, and wherein
the binder microorganism is capable of binding to a microorganism
of the group of mutans Streptococci, wherein the 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; and/or (vi) independent of magnesium, and
wherein preferably the total content of mono- and diglycerides is
at most 20 wt.-%, at most 5 wt.-%, or at most 2 wt.-% of the whole
composition.
12. The composition according to claim 11, wherein the composition
is for use with a human or animal, and wherein the composition is a
toothpaste, dentifrice, tooth powder, topical oral gel, mouth
rinse, denture product, mouthspray, lozenge, oral tablet, chewing
gum, mouth wash, dental floss, chew product or an additive for
food, feed or drinks, or wherein the composition is in the form of
a powder, tablet, film preparation, solution, aerosol, granule,
pill, suspension, emulsion, capsule, syrup, liquid, elixir,
extract, tincture or fluid extract, sheet-like food, bottled food,
canned food, retort food or fluid food, or wherein the composition
is a food or drink selected from the group consisting of gum,
spray, beverage, candy, infant formula, ice cream, frozen dessert,
sweet salad dressing, milk preparation, cheese, quark, yogurt,
acidified milk, coffee cream, whipped cream, butter, cheese,
processed milk and skimmed milk, meat product--preferably ham,
sausage, and hamburger--, fish meat, cake product, egg
product--preferably seasoned egg rolls and egg curd--,
confectionery--preferably cookie, jelly, snacks, and chewing gum--,
bread, noodles, pickle, smoked product, dried fish, seasoning.
13. The composition according to claim 11, wherein the binder
microorganism or fragment thereof: is not a genetically modified
microorganism as defined in Article 2(2) of Directive 2001/18/EC
other than obtained through the techniques of genetic modification
listed in Annex 1B to said Directive 2001/18/EC, and preferably
even excluding microorganisms obtained through the techniques of
genetic modification listed in Annex 1B to said Directive
2001/18/EC, does not comprise a product resulting from the
expression of genetic material heterologous to the order,
preferably to the family, even more preferably to the genus and
most preferably to the species of the binder microorganism.
14-15. (canceled)
16. The method of claim 1, wherein: (a) the binder microorganism or
fragment thereof is used as a sensorically neutral anti-dental
calculus agent, and wherein the composition is administered in an
amount effective to delay or slow down formation of dental calculus
in said human or animal; (b) the binder microorganism or fragment
thereof is used as a sensorically neutral anti-caries agent, and
wherein the composition is administered in an amount effective to
delay or slow down formation of caries in said human or animal;
and/or (c) the binder microorganism or fragment thereof is used as
a sensorically neutral anti-oral malodor agent, and wherein the
composition is administered in an amount effective to reduceoral
malodor in said human or animal.
17. The method of claim 1, wherein the binder microorganism is of
family Lactobacillaceae, of genus Lactobacillus, Paralactobacillus,
Pediococcus or Sharpea, of species Lactobacillus paracasei,
Lactobacillus rhamnosus, Lactobacillus casei or Lactobacillus zeae,
or is any of strains DSMZ 16667, DSMZ 16668, DSMZ 16669, DSMZ
16670, DSMZ 16671, DSMZ 16672 or DSMZ 16673.
18. The method of claim 1, wherein the binder microorganism is in a
viable, thermally inactivated or lyphilized form.
19. The method of claim 1, wherein the binder microorganism is in a
thermally inactivated form obtained by: treating in saturated steam
at a pressure of 1-5 bar, 1-3 bar, or 2 bar; treating in saturated
steam at a pressure of 2 bar at a temperature of 121.degree. C. for
20 minutes; or freezing at -20.degree. C. for at least 1 hour.
20. The method of claim 1, wherein the binder microorganism or
fragment thereof is capable of binding to at least one strain, at
least two strains, or at least three strains of mutans Streptococci
selected from the group consisting of Streptococcus mutans serotype
c (DSMZ 20523), Streptococcus mutans serotype e (NCTC 10923),
Streptococcus mutans serotype f (NCTC 11060), Streptococcus
sobrinus DSMZ 20742, Streptococcus ratti DSMZ 20564, Streptococcus
cricetus DSMZ 20562, Streptococcus ferus DSMZ 20646 and
Streptococcus macacae DSMZ 20714.
21. The method of claim 1, wherein the binder microorganism or
fragment thereof is not capable of binding to at least one, at
least two, at least three, or any microorganism selected from the
group consisting of Streptococcus salivarius ssp. thermophilus,
Streptococcus oralis DSMZ 20066, Streptococcus oralis DSMZ 20395,
Streptococcus oralis DSMZ 20627, Streptococcus mitis DSMZ 12643 and
Streptococcus sanguinis DSMZ 20567.
22. The method of claim 1, wherein the binder microorganism is
selected from the group consisting of: (i) microorganisms which
metabolize D-lactose, but not L-sorbose and/or D-saccharose and/or
D-inuline, (ii) microorganisms which metabolize inuline, (iii)
microorganisms which metabolize L-sorbose, but not D-lactose and/or
D-saccharose and/or inuline, and (iv) microorganisms which
metabolize L-sorbose, D-lactose and inuline, and preferably,
wherein the microorganism is selected from the group consisting of:
(i) microorganisms which metabolize D-lactose, but not L-sorbose,
D-saccharose and inuline, (ii) microorganisms which metabolize
L-sorbose, D-lactose and inuline, but not D-saccharose, (iii)
microorganisms which metabolize L-sorbose, but not D-lactose,
D-saccharose and inuline, and (iv) microorganisms which metabolizes
L-sorbose, D-lactose, D-saccharose, but not inuline.
Description
[0001] The present invention is concerned with microorganisms or
fragments thereof as sensorically neutral oral care agents,
particularly for prevention of dental calculus, as anti-caries
agents and/or anti-oral malodor agents. The invention is
furthermore concerned with compositions comprising microorganisms
or fragments thereof for reducing mutans Streptococci. Such
compositions can be used in oral care compositions, e.g. for caries
prophylaxis, or for prophylaxis of dental calculus or oral malodor.
They may also or instead be used for prevention or treatment of
oral malodor. As the microorganisms and fragments thereof according
to the present invention have a very low, unobtrusive smell and
taste, they are particularly suited as sensorically neutral agents
for preventment of dental calculus, caries, oral biofilm formation
and/or for prevention or treatment of oral malodor. The
microorganisms and fragments thereof, and also compositions
comprising such microorganisms and fragments, can thus
advantageously be used in food and feed compositions, particularly
in pet foods. Furthermore, the invention is concerned with methods
of preparing such microorganisms, fragments, compositions, foods
and feeds.
[0002] One of the notorious problems of tooth-bearing animals is
the decay of such teeth. This problem is of particular importance
to such animals that cannot, like sharks, shed their teeth to
continuously replace them by a new set. Among the most important
causes for tooth decay is caries. By the action of microorganisms
colonizing the oral cavity dental enamel is continuously weakened
and ultimately dissolved, leading to the formation of cariotic
lesions. Such lesions in turn are suitable for further colonization
of microorganisms, aggravating the problem of caries.
[0003] A problem unrelated to the formation of caries is the
development of oral malodor. Oral malodor is considered the result
of unwanted microorganisms colonizing the oral cavity. However, no
single microorganism has so far been indicated as the primary cause
for oral malodor. It seems to be clear, however, that the formation
of caries and of oral malodor are independent processes, as oral
malodor can also occur in the absence of teeth, and caries can
occur in the absence of oral malodor.
[0004] Another problem of oral health particularly in animals is
the formation of dental calculus. Calcified deposits on teeth are
formed by microorganisms colonizing the surfaces of teeth.
Typically, within 7-10 days calcified deposits on teeth have grown
to such extent that they are visible to the naked eye. Dental
calculus forms a focus to allow colonization of teeth by further
microorganisms. This, in turn, can lead to the development of oral
malodor, caries and inflammations of gingival tissue. There is thus
a need for the prevention of the development of dental calculus,
e.g. by slowing down dental calculus formation.
[0005] It has therefore frequently be tried to ameliorate these
problems independently. Among the most notable solutions so far
common in the art are tooth pastes and mouth washes. Such tooth
pastes and mouth washes generally comprise antimicrobially
effective agents to reduce the number of or inhibit the activity of
tooth colonizing microorganisms, particularly caries causative
microorganisms and/or oral malodor generating microorganisms.
However, due to the uncorrelatedness of caries and oral malodor
formation, many agents effective against caries are ineffective for
prevention or treatment of oral malodor, and vice versa. Also,
efficiency against dental calculus formation needs to be
improved.
[0006] Another problem is that antimicrobially effective substances
frequently exhibit a strong intrinsic smell or taste, which is
considered repulsive by consumers and particularly by pet animals.
Thus, compositions for oral care frequently comprise further
olfactorily active agents to mask or alter the taste or smell
inherent in the antimicrobially effective substances. However, such
further agents are also not readily accepted by consumers due to
their even stronger intrinsic taste or smell. This results in an
undesirably low compliance of consumers to the application
recommendations of manufacturers of such oral care
compositions.
[0007] The problem of compliance is particularly important with
children and animals. Both are prone to devising cunning ways of
avoiding oral care compositions or of misusing them, e.g. by
swallowing, such that an antimicrobially effective agent or agents
cannot fully exert their intended action. Resistance particularly
of pet animals like cats and dogs to any form of medication is
frequently lamented. Also, such animals are known to avoid
medications including antimicrobially effective oral care
compositions even when hidden in a feed they would otherwise
accept. In addition certain modes of applications for oral care
compositions are not available for animals, as they for example are
unable to gargle. Also, certain treatments of the oral cavity are
particularly stressful to animals and would require anesthesis. For
example, animals like cats and dogs will furiously resist removal
of dental calculus.
[0008] In summary, oral hygiene is considered tedious for human
beings including children and for animals including pet animals
alike. The present invention therefore intends to ameliorate the
above problems of prior art. In particular the present invention
aims at increasing compliance with oral care instructions for human
beings and for animals and particularly pet animals.
[0009] According to the invention, it is important for furthering
compliance that the agent or agents used for prevention of dental
calculus, caries and/or for prevention or treatment of oral malodor
has a low intrinsic taste and scent. Thus, instead of masking or
altering the taste and smell such agents by addition of further
strongly tasting or smelling agents the present invention is
concerned with a different approach. The inherent taste and smell
of oral care agents had so far being overlooked or not been
considered to be of major importance. Furthermore, it has now
surprisingly turned out that agents are available which are both
effective for prevention of dental calculus, caries and/or
prevention or therapy of oral malodor without having a strong
inherent taste or smell. It has also surprisingly been found that
such agents are useful to increase compliance with oral care
instructions and particularly to reduce oral care avoidance
behavior in humans, including children, and animals, including pet
animals. Also, the present invention allows to prepare oral care
compositions having other flavors or fragrances than previously
required for masking or altering the taste or smell of
anti-calculus agents, anti-caries agents and/or anti-oral-malodor
agents.
[0010] The present invention is concerned with microorganisms or
fragments thereof as sensorically neutral oral care agents. The
invention is furthermore concerned with compositions comprising
microorganisms or fragments thereof for reducing mutans
Streptococci. Such microorganisms and fragments thereof, and also
such compositions, can be used for treatment or prevention of
dental calculus. Such microorganisms, fragments or compositions can
also be used in caries treatment or prophylaxis. They may also or
instead be used for prevention or treatment of oral malodor. As the
microorganisms and fragments thereof according to the present
invention have a very low, unobtrusive smell and taste, they are
particularly suited as sensorically neutral agents for preventment
of caries, oral biofilm formation including formation of dental
calculus and/or for prevention or treatment of oral malodor. The
microorganisms and fragments thereof, and also compositions
comprising then, can thus advantageously be used in food and feed
compositions, particularly in pet foods. Furthermore, the invention
is concerned with methods of preparing such microorganisms,
fragments, compositions, foods and feeds.
[0011] According to the invention, the microorganism or fragment
thereof useful as a sensorically neutral oral care agent,
preferably is a lactic acid bacterium.
[0012] As indicated above, it has now surprisingly been found that
lactic acid bacteria can be used as sensorically neutral oral care
agents, particularly for prophylaxis and treatment of dental
calculus, as anti-caries and/or anti-oral malodor agents. This
could not be expected, as lactic acid bacteria are commonly known
to exhibit a strongly sour or acidic flavor and/or smell, e.g. in
yoghurt and other milk products.
[0013] According to the present invention, a lactic acid bacterium
is any microorganism taxonomically of order Lactobacillales, and
preferably is of family Lactobacillaceae. A microorganism according
to the invention is considered to belong to a specified taxonomic
group if it is more similar to a type strain within this taxonomic
group or within a lesser taxonomic rank than to any type strain
belonging to a taxonomic group other than the group in question or
its lesser ranks, wherein said analysis is based on a comparison of
their respective genetic material excluding plasmids and
non-integrated viruses. Thus, a microorganism is considered to
belong to the family of Lactobacillaceae if it is, as defined in
the previous sentence, genetically more similar to a type strain of
a species or genus within the family of Lactobacillaceae compared
to type strains of a species or genus belonging to another family
within the order of Lactobacillales.
[0014] Similarity according to the present invention is assessed
using the Needleman-Wunsch global alignment algorithm. This
algorithm is the standard algorithm to find the optimum alignment
including gaps of two nucleic acid sequences along their entire
length. The algorithm of Needleman and Wunsch has been published in
1970 J. Mol. Biol. 48, 443-453, wherein a penalty for a gap of n
positions is computed according to the formula
gap opening penalty+(n-1).times.gap extension penalty.
[0015] There is no penalty for hanging ends.
[0016] Gap open penalty in the context of the present invention is
10.0. The gap extension penalty in the context of the present
invention is 0.5. The scoring matrix for comparing nucleic acid
sequences in the context of the present invention is the unitary
DNA identity matrix, which assigns a score of 1 for each identical
base "substitution", and -10000 for all other base substitutions.
Sequence alignments can be performed with these parameters e.g. via
publicly available tools offered by the EBI.
[0017] A particular advantage inherent in microorganisms of the
family of Lactobacillaceae is that they can generally be considered
save for consumption by human beings and animals. For example,
lactic acid bacteria have been used for a long time in the
manufacture of foods for example by processing milk. They are also
easy to handle due to their low danger to human or animal health
and they are easy to cultivate in large quantities, e.g. in batches
of 500 l of culture medium. Particularly preferred methods of
cultivation will be described later.
[0018] Among the members of family of Lactobacillaceae, such lactic
acid bacteria are particularly preferred according to the present
invention which belong to genus Lactobacillus, Paralactobacillus,
Pediococcus or Sharpea, wherein microorganisms of genus
Lactobacillus are most preferred. Such microorganisms have been
extensively used in the preparation of foods and feeds; they are
easy to handle and can be produced in large quantities.
Furthermore, within the family of Lactobacillaceae it is
particularly among genus Lactobacillus that microorganisms can be
obtained which are both sensorically neutral and effective as oral
care agents, particularly as anti-dental calculus agents,
anti-caries agents and/or anti-oral-malodor agents.
[0019] 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: [0020] (a) homofermentative
lactobacilli [0021] (i) producing lactic acid, preferably the L-,
D- or DL-isomer(s) of lactic acid in an amount of at least 85% from
glucose via the Embden-Meyerhof pathway; [0022] (ii) growing at a
temperature of 45.degree. C., but not at a temperature of
15.degree. C.; [0023] (iii) being long-rod shaped; and [0024] (iv)
having glycerol teichoic acid in the cell wall; [0025] (b)
homofermentative lactobacilli [0026] (i) producing lactic acid,
preferably the L- or DL-isomer(s) of lactic acid via the
Embden-Meyerhof pathway; [0027] (ii) growing at a temperature of
15.degree. C., showing variable growth at a temperature of
45.degree. C.; [0028] (iii) being short-rod shaped or coryneform;
and [0029] (iv) having ribitol and/or glycerol teichoic acid in the
cell wall; [0030] (c) heterofermentative lactobacilli [0031] (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; [0032] (ii) producing carbondioxide and ethanol [0033]
(iii) showing variable growth at a temperature of 15.degree. C. or
45.degree. C.; [0034] (iv) being long or short rod shaped; and
[0035] (v) having glycerol teichoic acid in the cell wall.
[0036] Based on the above-described characteristics, the
microorganisms preferred according to the present invention can be
classified to belong to the group of lactic acid bacteria,
particularly to the genus of Lactobacillus.
[0037] In a preferred embodiment, the microorganism of the present
invention has a metabolic fingerprint selected from the group
consisting of: [0038] (i) it metabolizes D-lactose, but not
L-sorbose and/or D-saccharose and/or D-inuline, [0039] (ii) it
metabolizes inuline, [0040] (iii) it metabolizes L-sorbose, but not
D-lactose and/or D-saccharose and/or inuline, and [0041] (iv) it
metabolizes L-sorbose, D-lactose and inuline.
[0042] Preferably, the microorganism of the present invention has a
metabolic fingerprint selected from the group consisting of: [0043]
(i) it metabolizes D-lactose, but not L-sorbose, D-saccharose and
inuline, [0044] (ii) it metabolizes L-sorbose, D-lactose and
inuline, but not D-saccharose, [0045] (iii) it metabolizes
L-sorbose, but not D-lactose, D-saccharose and inuline, and [0046]
(iv) it metabolizes L-sorbose, D-lactose, D-saccharose, but not
inuline.
[0047] Of course, the microorganism of the present invention is not
limited to the metabolization of the aforementioned sugars of the
metabolic fingerprint patterns, but may be capable of metabolizing
further sugars.
[0048] Within the present invention, the term "microorganism" not
only refers to such microorganisms which, when placed in the
appropriate culturing conditions, can multiply (viable
microorganisms). It is a particular advantage of the present
invention that instead of such viable microorganisms also the
corresponding thermally inactivated or lyophilized microorganisms
can be used. This is particularly useful as some consumers may
object to the idea of consuming viable microorganisms, or giving
such viable microorganisms to children or pet animals even though
they are assured that such viable microorganisms are beneficial to
their health. As the present invention is concerned with increasing
compliance with oral care instructions, it is a particular
advantage that even such unfounded objections can be taken care of.
Thus, unless explicitly mentioned this description and the
accompanying examples, figures and claims do not differentiate
between viable, thermally inactivated or lyophilized
microorganisms.
[0049] According to the present invention, thermally inactivated
cells preferably are obtained by autoclaving viable microorganism
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. Such preparation of thermally inactivated microorganisms can
be achieved using standard laboratory equipment, as the process of
autoclaving is a standard technique known in the art of
microbiology and corresponding biotechnological engineering. It is
a very fast technique and is proven to inactivate microorganisms of
family Lactobacillaceae and particularly of genus
Lactobacillus.
[0050] Alternatively, thermal inactivation of microorganisms
according to the present invention can be achieved by freezing such
cells to a temperature of -20.degree. C. Such freezing is also easy
to perform using standard laboratory equipment.
[0051] Regardless of the mode of thermal inactivation, i.e. by
autoclaving or by freezing, it is according to the present
invention preferred that the concentration of viable microorganisms
is reduced by the treatment by at least 85%, 90% or 95%, and
particularly preferred by 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%. Particularly preferred is such autoclavation
or freezing that reduces the concentration of viable microorganisms
by at least 3 orders of magnitude (i.e. by a factor of 1000), even
more preferably by at least 4 orders of magnitude and even more
preferably by at least 5 orders of magnitude.
[0052] A particular advantage of the microorganisms of the present
invention is that they retain their usefulness as sensorically
neutral oral care agents, preferably anti-dental calculus agent,
anti-caries and/or anti-oral-malodor agents even after such
inactivation by autoclaving or freezing. In such thermally
inactivated form, the microorganisms of the present invention are
particularly easy to store even at 20.degree. C. in a standard
atmosphere having 80% relative humidity at this temperature. This
storability is of particular importance, as the inactivated
microorganisms according to the present invention allow to produce
foods or feeds comprising such inactivated forms without corrupting
the "best before" date.
[0053] The same advantages can be obtained according to the present
invention by using lyophilized microorganisms of the present
invention, i.e. lyophilized microorganisms of family
Lactobacillaceae and particularly lyophilized lactic acid
microorganisms of genus Lactobacillus. Lyophilization according to
the present invention is preferably for at least 2 hours at room
temperature, i.e. at a temperature between 16.degree. C. and
25.degree. C.
[0054] In addition to or alternatively to microorganisms of the
present invention, for example in viable, thermally inactivated or
lyophilized form, fragments thereof can be used. A fragmentation of
microorganisms can be easily performed using standard methods known
in the art, particularly by cell lysis and/or pasteurization. The
method used for lysing or fragmenting a microorganism according to
the present invention including thermally inactivated and/or
lyophilized forms thereof is of no particular concern. Appropriate
methods can be chosen for example among chemical methods including
enzymatic treatment, preferably by one or more proteases, for
example by proteinase K, lipases or glycosidases; non-limiting
examples for other chemicals are ionophores, detergents, for
example sodium dodecyl sulfate, acids or bases; non-limiting
examples of physical means are high pressure, like French-pressing,
osmolarity and milling for example using glass or iron beats.
Particularly preferred methods for producing thermally inactivated
and/or lyophilized microorganisms according to the present
invention and fragments thereof are described in WO 2006/027265 A1,
pages 25-28 and WO 2009/149816 A1, pages 28-34. "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 binder
microorganisms belonging to the family of Lactobacillaceae,
preferably 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, Matsuquchi 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 fragment of the binder
microorganism of the present invention retains the capability of
specifically binding to a mutans Streptococcus and more preferably
Streptococcus mutans, which is described in detail herein.
[0055] The microorganisms used in the present invention, i.e.
lactic acid bacteria preferably of family Lactobacillaceae or of
genus Lactobacillus including respective thermally inactivated or
lyophilized forms thereof and fragments thereof, are preferably
used as anti-caries agents. According to the present invention, the
term "anti-caries agent" is defined as any agent which, by its mere
presence in the oral cavity of a human or animal including pet
animals like dogs, cats, rats, mice, hamsters, guinea pigs or
monkeys, reduces the risk of developing new cariotic lesions. It is
a particular advantage of the present invention that the
microorganism used as anti-caries agent does not have to be
antimicrobially effective on its own. Thus, it is not necessary
according to the present invention that microorganisms or fragments
thereof used as anti-caries agents reduce in a co-cultivation the
number of caries-generating microorganisms, particularly
microorganisms belonging to the group of mutans Streptococci.
[0056] It is therefore another advantage according to the present
invention that the microorganisms used as anti-caries agents have a
very limited impact on the normal microflora of the oral cavity of
a human being or of an animal, particularly a pet animal. Suitable
microorganisms will be described below.
[0057] The microorganism used in the present invention, i.e. lactic
acid bacteria preferably of family Lactobacillaceae or of Genus
lactobacillus, including respective thermally inactivated or
lyophilized forms thereof and fragments thereof, are preferably
used as anti-dental calculus agents. According to the present
invention, the term "anti-dental calculus agent" is defined as any
agent which, by its mere presence in the oral cavity of a human or
animal including pet animals, reduces the risk of developing dental
calculus, or which slows down further development of dental
calculus. It is not required that the anti-dental calculus agent of
the present invention actually removes dental calculus already
formed on a tooth.
[0058] Also, the microorganisms used in the present invention, i.e.
lactic acid bacteria preferably of family Lactobacillaceae or of
Genus lactobacillus including respective thermally inactivated or
lyophilized forms thereof and fragments thereof, are preferably
used as anti-oral malodor agents. According to the present
invention, the term "oral malodor" indicates any unpleasant smell
originating in the oral cavity. The term thus is used according to
the present invention both for weak forms of malodor, also termed
"bad breath", which are not considered an illness, and also for
such forms of malodor which, for example due to their intensity or
scent, are considered to be the symptom of an illness; such latter
forms of oral malodor are also called halitosis. Clinically
relevant forms of oral malodor, i.e. halitosis, can have various
causes. Most notable for all forms of oral malodor are infections
by microorganisms colonizing the oral cavity, wherein such
microorganisms digest organic material, for example dead epithelial
cells, other microorganisms or residual food or feed. Preferably,
the present invention is set to delay onset of oral malodor, and/or
to reduce the intensity or alter the scent of oral malodor, be it
clinically not relevant bad breath or clinically relevant
halitosis. However, unless explicitly mentioned herein, the term
"oral malodor" does not cover malodors originating beyond the oral
cavity, e.g. in the esophagus or stomach. Such malodors may for
example become noticeable during eructation.
[0059] Within the present invention, the term "oral cavity"
indicates that cavity which extends from lips and teeth up to but
not including the uvula.
[0060] 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).
[0061] Also according to the present invention, the term
"sensorically neutral" is defined as such agent which can replace
up to 2 wt.-% of wholemeal wheat flour such that at most 5 out of
10 trained panelists will consider the taste and scent of such
mixture of wholemeal wheat flour and microorganisms and/or
fragments according to the invention to be noticeably altered,
judging on the basis of a scale ranging from no difference,
slightly altered, noticeably altered to clearly altered.
Preferably, up to 5 wt.-% of wholemeal wheat flour can be replaced
by the microorganisms and/or fragments thereof such that at most 5
out of 10 panelists consider the taste and/or scent of such mixture
to be noticeably altered.
[0062] According to the invention, there is thus also provided a
composition comprising [0063] a binder microorganism or fragment
thereof in an amount both (a) sensorically neutral and (b)
effective for reducing mutans Streptococci in the oral cavity of a
human or animal and/or effective for reducing oral malodor, wherein
the microorganism is a lactic acid bacterium.
[0064] It has now surprisingly been found that to exert an
anti-caries effect by a sensorically neutral agent, it is
sufficient to use a binder lactic acid bacterium for reducing
mutans Streptococci in the oral cavity. As described above, such
binder lactic acid bacteria, thermally inactivated or lyophilized
forms thereof and also fragments of such binder lactic acid
bacteria can be used as anti-caries agent without having an
anti-microbial effect, i.e. without killing cariogenic
microorganisms themselves or repressing their growth. Instead, it
is sufficient that a binder microorganism or fragment thereof binds
to one or more species of mutans Streptococci. Such binding can
lead to an agglutination of bound mutans Streptococci, which in
turn are removed from the oral cavity by normal swallowing of
saliva and during eating and drinking. As described in WO
2008/074473 A2 removal of mutans Streptococci from the oral cavity
significantly reduces the risk of biofilm formation in the oral
cavity and on teeth, thereby indirectly reducing the risk of
developing caries. This particularly holds true also for such
binder microorganisms which bind to Streptococcus mutans, as
described in WO 2006/027265 A1. However, it had not been known so
far that such binder microorganisms could be used as sensorically
neutral agents, i.e. in a sensorically neutral concentration as
described above and detailed hereinafter. Also, it had not been
known so far that such binding to mutans Streptococci and
particularly to Streptococcus mutans could reduce the risk of
development of oral malodor.
[0065] According to the invention, a composition is thus preferred
comprising a microorganism or fragment thereof that is capable of
specifically binding to a bacterium belonging to the group of
mutans Streptococci, wherein the specific binding is [0066] (i)
resistant to heat treatment; and/or [0067] (ii) resistant to
protease treatment; and/or [0068] (iii) calcium-dependent; and/or
[0069] (iv) formed within a pH range between 4.5 and 8.5; and/or
[0070] (v) formed in the presence of saliva.
[0071] The term "specifically binding" in the context of the
present invention means that the binder microorganism or fragment
thereof, preferably a microorganism (or corresponding fragment)
belonging to the family of Lactobacillaceae and more preferably of
genus Lactobacillus, binds to one or more strains belonging to
mutans Streptococci, preferably to Streptococcus mutans, but does
not bind to most other, preferably to no other species belonging to
the genus Streptococcus. Namely, the binder microorganism or
fragment thereof does preferably not bind to bacteria belonging to
the species of Streptococcus oralis and/or Streptococcus mitis
and/or Streptococcus sanguinis. Even more preferably, the binder
microorganism also does not bind to bacteria belonging to the
species of Streptococcus salivarius, more preferably belonging to
the subspecies thermophilus. More preferably, the binder
microorganism or fragment thereof does not bind to Streptococcus
oralis DSMZ 20066, Streptococcus oralis DSMZ 20395, Streptococcus
oralis DSMZ 20627, Streptococcus mitis DSMZ 12643 and/or
Streptococcus sanguinis DSMZ 20567. And even more preferably, the
binder microorganism or fragment thereof also does not bind to
Streptococcus salivarius ssp. thermophilus,
[0072] The specific binding reaction comprises binding and,
preferably, aggregating Streptococcus mutans or other mutans
Streptococci cells as described herein by the binder microorganism
of the present invention including a fragment thereof in the mouth.
This specific binding leads, in consequence, to flushing away the
boud cells by, for example, salivary flow or by a mouth rinse or
mouth wash and the like as described herein. Preferably, the
specific binding reaction of the binder microorganisms of the
present invention and their fragments to Streptococcus mutans
and/or other mutans Streptococci prevents such Streptococcus cells
from attaching to the surface of a tooth or teeth, or, while not
being bound by such theory, could lead to detachment of
Streptococcus cells from the surface of a tooth or teeth. In
consequence, the specific binding reaction results in flushing away
bound Streptococcus cells out of the mouth, thereby diminishing a
causative agent of biofilm formation and, thus, preventing and/or
treating caries.
[0073] It is believed that the binder microorganism or fragment
thereof 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 binder microorganism or fragment thereof 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 binder microorganism of the present invention may bind, for
example, to said streptococcal antigen I/II, Streptococcus mutans
or another respective mutans Streptococcus is hampered to bind to
the surface of teeth which thus helps to prevent and/or treat
caries.
[0074] The pellicle is a clear, thin covering containing proteins
and lipids 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 mutans Streptococci from the mouth, however, is on the
other hand suspected to facilitate adhesion of mutans Streptococci
to the surface of teeth, thereby facilitating the initial
attachment of Streptococcus cells to teeth and, thus, onset of
caries.
[0075] Preferably, the above mentioned binder microorganism
belonging to the group of lactic acid bacteria--or fragment
thereof--is capable of specifically binding to Streptococcus mutans
serotype c (DSMZ 20523) and/or serotype e (NCTC 10923) and/or
serotype f (NCTC 11060) and/or Streptococcus sobrinus DSMZ 20742
and/or Streptococcus ratti DSMZ 20564 and/or Streptococcus cricetus
DSMZ 20562 and/or Streptococcus ferus DSMZ 20646 and/or
Streptococcus macacae DSMZ 20714.
[0076] This means that the above mentioned binder microorganism
belonging to the group of lactic acid bacteria, or its fragment,
preferably binds to at least one microorganism selected from the
group consisting of Streptococcus mutans serotype c (DSMZ 20523),
serotype e (NCTC 10923), serotype f (NCTC 11060), Streptococcus
sobrinus DSMZ 20742, Streptococcus ratti DSMZ 20564, Streptococcus
cricetus DSMZ 20562, Streptococcus ferus DSMZ 20646 and
Streptococcus macacae DSMZ 20714. More preferably, the above
mentioned binder microorganism belonging to the group of lactic
acid bacteria or fragment thereof binds to any combination,
grouping or subgrouping of the above mentioned bacteria. Even more
preferably, the above mentioned binder microorganism or fragment
thereof belonging to the group of lactic acid bacteria binds to all
of the above mentioned bacteria. In accordance with the present
invention a "serotype" is an antigenic property of a bacterial
cell, preferably of a Streptococcus mutans or Streptococcus
sobrinus cell, identified by serological methods known in the
art.
[0077] As described above, the specific binding of the binder
microorganism or fragment thereof to mutans Streptococci and
preferably to Streptococcus mutans is preferably resistant to heat
treatment. Accordingly, binding is not abolished when the binder
microorganism of the present invention is treated with heat, for
example, at a temperature above 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 binder microorganism of the present
invention or its fragment to specifically bind mutans Streptococci
is determined as described herein.
[0078] The corresponding temperature can depend on the specific
binder microorganism 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 binder cells or fragments thereof which is still capable
of specifically binding to mutans Streptococci and/or Streptococcus
mutans by using methods as those shown herein. 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. Thus, the thermally inactivated form of the binder
microorganism of the present invention obtainable by autoclaving is
particularly preferred. 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.
[0079] The specific binding of the binder microorganism or its
fragment is furthermore preferably characterized by its resistance
to protease treatment. Preferably, the binding is resistant to
treatment with one or more proteases selected from the group
consisting of pronase E, proteinase K, trypsin and chymotrypsin.
These proteases 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 elastase, thrombin,
aminopeptidase I, carboxypeptidase, dostripain, endoproteinase,
papain, cathepsin B, pepsin, gastricsin, chymosin, cathepsin D. The
latter proteases could also be used to test whether the specific
binding of the binder microorganism or fragment thereof to S.
mutans or another mutans Streptococcus is resistant to the latter
more specific proteases. Thus, after protease treatment which is
described in the examples of WO 2008/074473 A2 or WO 2006/027265
A1, which are incorporated herein, the binder microorganism or
fragment is still capable of specifically binding to Streptococcus
mutans/mutans Streptococci.
[0080] In addition, the specific binding of the binder
microorganism or fragment is furthermore preferably 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 examples of WO 2008/074473 A2 or WO
2006/027265 A1, which are incorporated herein. Moreover, the
specific binding to the binder microorganism or fragment is
preferably 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 above mentioned examples.
[0081] The specific binding is preferably independent of magnesium.
Thus, it is not necessary that magnesium ions or magnesium salts
are present.
[0082] Another preferred 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 above. If, however,
Lactobacillus rhamnosus species 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. The aforementioned
characteristics of the binder microorganism or fragment thereof
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 or feed
during the preparation of said food or feed. Namely, food or feed
is often heat sterilized, pre-cooked, pasteurized and the like
which is detrimental for viability of microorganisms.
[0083] The binder microorganism of the present invention, and/or
the corresponding fragment thereof, is capable of specifically
binding to one or more mutans Streptococci and preferably to
Streptococcus mutans, wherein the specific binding is [0084] (i)
resistant to heat treatment, and [0085] (ii) resistant to protease
treatment, and [0086] (v) formed in the presence of saliva.
[0087] Such binder microorganisms and fragments thereof combine the
aforementioned advantages. In particular, since their specific
binding is resistant to heat treatment, they can be incorporated in
a product that is heat treated for sterilization, thereby
prolonging shelf life of the product without corrupting the oral
health properties conferred to the product by the microorganisms of
fragment thereof, preferably without corrupting the ant-dental
calculus effectiveness, the anti-caries effectiveness or the
ant-oral malodor effectiveness of the product. Also, due to the
protease resistance the binder microorganisms and fragments can be
incorporated in various products of different composition even
before such products are heat treated for protein denaturation. And
importantly the formation of a specific binding in the presence of
saliva allows to use the binder microorganisms and fragments
thereof also in such products which do not drain saliva from the
oral cavity. This ability in turn increases consumer compliance,
particularly in children and animals including pet animals, as such
consumers tend to be reluctant to use a product which would lead to
a dry oral cavity.
[0088] For these reasons, the binder microorganism of the present
invention, and/or the corresponding fragment thereof, is even more
preferably capable of specifically binding to one or more mutans
Streptococci and preferably to Streptococcus mutans, wherein the
specific binding is [0089] (i) resistant to heat treatment for 20
minutes at a temperature of 121.degree. C. in a saturated steam
having an atmospheric pressure of 2 bar, and [0090] (ii) resistant
to treatment by one or more enzymes selected from pronase E,
proteinase K, trypsin and chymotrypsin, and [0091] (v) formed in
the presence of saliva.
[0092] Preferably, the specific binding of the binder microorganism
of the present invention, and/or the corresponding fragment thereof
is also [0093] (iii) calcium-dependent, and [0094] (iv) formed
within a pH range between 4.5 and 8.5.
[0095] The method to determine the binding of binder microorganisms
and fragments thereof to mutans Streptococci is described in Lang
et al (2010) Journal of Dental Research 89(2) 175-179, which is
incorporated herein.
[0096] Preferably, the specific binding of the binder microorganism
or fragment thereof can be assayed as follows: [0097] (a) growing
said binder microorganism to stationary phase, or, in case a
fragment is to be tested, obtaining such fragment, [0098] (b)
mixing said binder microorganism or fragment with a mutans
Streptococcus which has been grown to stationary phase, [0099] (c)
incubating the mixture obtained in step (b) under conditions
allowing the formation of aggregates of said microorganism and said
Streptococcus, and [0100] (d) detecting aggregates by the
occurrence of a pellet.
[0101] In a preferred embodiment the bacterium belonging to the
group of mutans Streptococci used in such an assay is Streptococcus
mutans. For a specific binding to mutans Streptococci, preferably
no binding can be detected to at least one, preferably at least two
and more preferably at least three and even more preferably all
microorganisms selected from the group consisting of Streptococcus
salivarius ssp. thermophilus, Streptococcus oralis DSMZ 20066,
Streptococcus oralis DSMZ 20395, Streptococcus oralis DSMZ 20627,
Streptococcus mitis DSMZ 12643 and Streptococcus sanguinis DSMZ
20567.
[0102] In particular, binder microorganisms belonging to the group
of lactic acid bacteria, preferably of family Lactobacillaceae and
even more preferably of genus Lactobacillus, or corresponding
fragments, are preferably mixed with mutans Streptococci in
cell-to-cell ratios of 3:1 to 60:1 (mutans Streptococci:binder
microorganism). Both the lactic acid binder bacteria and mutans
Streptococci are grown in liquid culture to stationary phase.
Preferably, the optical density is measured photometrically at a
wavelength of 600 nm.
[0103] The mentioned ratios correspond to a ratio of colony forming
units from 1:50 to 1:2.5. Preferably, an OD600=1 in 1 ml
corresponds to 3.times.10.sup.8 colony forming units of a mutans
Streptococcus. Preferably, an OD600=1 in 1 ml corresponds to
7.times.10.sup.9 colony forming units of lactic acid bacteria.
Preferably, for assaying the aggregation reaction by pelleting, 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.
[0104] Preferably, the mixture of Streptococcus and binder lactic
acid bacteria or fragments thereof is vortexed for about 15 seconds
and then left undisturbed for at least 5, 10, 15 minutes and more
preferably for 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, whereas non-mutans Streptococcus aggregating
mixtures stay in suspension. As a control, self-aggregation of the
respective binder bacterium or fragment and the mutans
Streptococcus strain can be assayed by omitting either the mutans
Streptococcus or the binder microorganisms/fragment.
[0105] The aggregation of a binder microorganism and a mutans
Streptococcus according to the above described assay can be
quantified by separating the formed aggregates as described by Lang
et al (2010) Journal of Dental Research 89(2) 175-179, or by
centrifugation, e.g. at 500.times.g for 30 seconds. Subsequently,
the amount of aggregation can be determined by measuring the amount
of non-aggregated cells that are left in the supernatant. The
determination can be carried out by any suitable means known to the
person skilled in the art. Preferably, the determination is carried
out by removing a certain volume of the supernatant, e.g. 1 ml.
Subsequently, the optical density of the removed supernatant may be
measured at any suitable wavelength, known to the skilled person,
e.g. at 600 nm. The measured value after subtraction of a value a
corresponding control test without lactobacilli represents the
amount of cells that have not been aggregated.
[0106] A method for determining specific binding according to the
present invention is described in example 4 of WO 2008/74473 A2,
with corresponding instructions for cultivation of respective
microorganisms given in example 1 of that publication. Both
examples are incorporated herein for the purpose of disclosing a
method for determining specific binding according to the present
invention.
[0107] Alternatively, in order to address the possible problem of
self-aggregation a stain, preferably a fluorescent stain, can be
employed. Thus, in a more preferred embodiment, the specific
binding can be assayed as follows: [0108] (a) growing said
microorganism to stationary phase; [0109] (b) mixing said
microorganism with a bacterium belonging to the group of mutans
Streptococci which has been grown to stationary phase and which has
been stained using a suitable stain, preferably a fluorescent
stain; [0110] (c) incubating the mixture obtained in step (b) under
conditions allowing the formation of aggregates of said
microorganism and a bacterium of the group of mutans Streptococci;
and [0111] (d) detecting aggregates by the detection of the stain,
preferably a fluorescencent stain.
[0112] Again, in a preferred embodiment the bacterium belonging to
the group of mutans Streptococci used in such an assay is
Streptococcus mutans.
[0113] Preferably the aggregation assay may be carried out in that
first the binder microorganism and mutans Streptococci are grown to
stationary phase as described above. Preferably, the optical
density is measured photometrically at a wavelength of 600 nm.
Preferably, an OD600=1 in 1 ml corresponds to 3.times.10.sup.8
colony forming units of a respective mutans Streptococcus.
Preferably, an OD600=1 in 1 ml corresponds to 7.times.10.sup.9
colony forming units of binder microorganism, particularly of
family lactobacillaceae and even more preferably of genus
lactobacillus, as described herein.
[0114] Subsequently, the mutans Streptococci are stained. In a
further preferred embodiment, the binder microorganisms are
stained, whereas the Streptococci are not stained. As stain any
suitable stain can be used, preferably a fluorescence stain known
to the person skilled in the art may be used. Preferably, a
specific or unspecific fluorescence stain may be used, for example,
CFDA-SE to stain intact cells, other useful stains include
carboxyfluorescein diacetate acetoxymethyl ester, BCECF AM and
Calcein AM. Specifically, the cells are harvested, e.g. by
centrifugation, preferably at 3200.times.g for 5 min. Subsequently,
the obtained pellet may be resuspended in any suitable buffer known
the person skilled in the art, preferably in a PBS-buffer. The
amount of buffer may be calculated so that the resulting suspension
has an OD600 of, e.g., 4.2/ml. Subsequently, the suspension may be
mixed with a suitable stain, e.g. a fluorescence stain, preferably
with 5,6-carboxyfluorescein diacetate, succhinimidyl ester
(CFDA-SE), more preferably with 2 .mu.l of a CFDA-SE solution
(Invitrogen). Subsequently, the cells may be incubated for a
suitable time period, as known to the skilled person, e.g. for 2
hours, at a suitable temperature as known to the skilled person,
for instance, at 37.degree. C. In a further step, the stained cells
may be harvested, e.g. by centrifugation. Preferably, the
centrifugation is carried out at 3200.times.g for 5 min. The cells
may then be resuspended in a suitable buffer, as known to the
person skilled in the art, e.g. in 2 ml of a PBS-buffer.
[0115] For the aggregation, binder microorganisms preferably mixed
with mutans Streptococci in volumetric ratios of 3:1 to 1:3 (mutans
Streptococci:binder microorganism). More preferably, the volumetric
ratio of the mixture is 1:1. The mentioned ratios correspond to a
ratio of colony forming units from 1:50 to 1:150. For assaying the
aggregation reaction via measuring of staining, preferably of
fluorescence, the binder microorganism and the mutans Streptococci
are used in any suitable volume known to the skilled person,
preferably, in a volume of 50 .mu.l. Preferably, the mixture is
carried out in a microtiter plate, e.g. in a 96 well microtiter
plate. Subsequently, the mixture may be vortexed, preferably for 12
min at full speed. Afterwards, the mixture may be centrifuged, e.g.
for 10 seconds at 500.times.g. The supernatant may then be removed
and the pellet may be resuspended in any suitable buffer known the
person skilled in the art, preferably in PBS-buffer in any suitable
volume, e.g. in 100 .mu.l. The staining of the suspension may be
measured in the mixture by any suitable means known to the skilled
person. Preferably, in case of fluorescence, the fluorescence may
be detected in a fluorescence reader, e.g. at a wavelength of 495
nm for excitation and 525 nm for emission. As controls, binder
microorganism alone and stained mutans Streptococci alone may be
assayed. Any background staining, e.g. fluorescence, may be
measured for the tested mutans Streptococci alone and may
preferably be subtracted from the value for the aggregation with
the respective binder microorganism. An aggregation effect is
present if the background staining, e.g. fluorescence, measured as
indicated herein above, is subtracted from the measured staining,
e.g. fluorescence, in a sample containing a binder microorganism as
described herein above and a tested mutans Streptococcus, as
described herein above, and the resulting value is at least above
zero. More preferably, an aggregation effect is present if the
resulting value is reproducibly above zero in a series of tests,
carried out as described herein above. A "series of experiments"
means at least 2, preferably 3, more preferably 4 and most
preferably 5 tests.
[0116] The alternative method of testing binding can be performed
as described in example 5 of WO 2008/074473 A2, which is
incorporated herein by reference.
[0117] For fragments of binder microorganisms, binding is
essentially determined as for viable or inactivated binder
microorganisms as described above. The amount of fragments to be
used for determining binding or for preparing a food or feed
composition according to the invention instead of binder
microorganism cells is preferably the same as the amount of cell
wall material of viable or thermally inactivated binder
microorganism. For example, peptidoglycan content of binder
microorganism content can be measured by using appropriate dyes,
and the same amount of fragments based on such dye measurement can
be used.
[0118] The above mentioned binder microorganisms are preferably
lactic acid bacteria belonging to the genus of Lactobacillus, more
preferably Lactobacillus species as described herein. Even more
preferably said Lactobacillus belongs to the species of
Lactobacillus paracasei or Lactobacillus rhamnosus. However, the
Lactobacillus species are not limited thereto. The above mentioned
binder microorganisms may preferably be "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 because in that
composition is not part of its natural environment. Thus, a
microorganism grown in a pure culture is still considered an
isolated microorganism. Likewise, a fragment of a microorganism is
considered "isolated" according to the invention if it is separated
from at least some material of the respective microorganism.
[0119] 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 lactic acid bacteria, and particularly preferably to
Lactobacillus species. Such selective agar plates are known in the
art.
[0120] To obtain fragments, it is preferred to rupture viable or
thermally inactivated binder microorganism cells by methods known
in the art, for example sonication, French press or ball milling,
and to separate the fragments from other cell remains by
centrifugation. The fragment pellet can then be washed and
centrifuged again to obtain a fragments pellet.
[0121] More preferably, the above mentioned binder microorganism
belonging to the group of lactic acid bacteria is selected from the
group consisting of Lactobacillus paracasei or Lactobacillus
rhamnosus, respectively, having DSMZ accession number DSMZ 16667
(L. paracasei ssp. paracasei Lb-Ob-KI), DSMZ accession number DSMZ
16668 (L. paracasei ssp. paracasei Lb-Ob-K2), DSMZ accession number
DSMZ 16669 (L. paracasei ssp. paracasei Lb-Ob-K3), DSMZ accession
number DSMZ 16670 (L. paracasei ssp. paracasei Lb-Ob-K4), DSMZ
accession number DSMZ 16671 (L. paracasei ssp. paracasei LbOb-K5),
DSMZ accession number DSMZ 16672 (L. rhamnosus Lb-Ob-K6) and DSMZ
accession number DSMZ 16673 (L. rhamnosus Lb-Ob-K7) or a mutant or
derivative thereof, wherein said mutant or derivative retains the
capability to specifically bind to mutans Streptococci. 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 DSMZ 16667, 16668, 16669, 16670, 16671,
16672 or 16673. The DSMZ is located at the Mascheroder Weg 1b,
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.
[0122] "A mutant or derivative" of the above mentioned binder
microorganism belonging to the group of lactic acid bacteria,
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 mutans Streptococci,
preferably with the binding characteristics as described herein.
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 0 506 789 B1, EP 0 316 677 B1, EP 0 251
064 B1, EP 0 218 230 B1, EP 0 133 046 B1 or WO 89/01970.
[0123] 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 a binder microorganism belonging to the group of
lactic acid bacteria, preferably of the family of
lactobacteriaceae, even more preferably of genus lactobacillus and
most preferably one of the deposited Lactobacillus species,
harbouring a recombinant nucleic acid either comprised in their
bacterial chromosome or on one or more plasmids or comprised in
their bacterial chromosome and/or one or more plasmids. Said
recombinant nucleic acids are preferably foreign to the above
mentioned binder microorganism belonging to the group of lactic
acid bacteria. 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. In
this case the heterologous polynucleotide may be either under the
control of its own promoter or under the control of a heterologous
promoter. The above described vector or nucleic acid molecule,
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 above described nucleic acid molecule can be used to
restore or create a mutant gene via homologous recombination.
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 above
mentioned deposited microorganism, 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 et
al., Nat. Biotechnol. 20 (2002), 702-706 or Shiroza, Biochim
Biophys Acta 1626 (2003), 57-64.
[0124] 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.
[0125] It is also envisaged that the above mentioned binder
microorganism belonging to the group of lactic acid bacteria 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.
[0126] A mutant of the binder microorganism belonging to the group
of lactic acid bacteria, preferably a mutant of the deposited
Lactobacillus strains, is preferably artificially mutated. In
accordance with the present invention, the term "mutated" means one
or more permanent modifications 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. Preferably, a mutation leads to in
increased capability of specifically binding mutans Streptococci.
Thus, it is also preferred that the mutant cells of the deposited
microorganism which harbour one or more mutations in one or more
desired genes or in which one or more mutations in one or more
desired genes 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
mutans Streptococci can be tested in accordance with the methods
described above. The term "mutant", however, also includes cells of
above mentioned binder microorganism belonging to the group of
lactic acid bacteria, 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 binding to mutans
Streptococci. 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
above mentioned binder microorganism belonging to the group of
lactic acid bacteria.
[0127] However, even though genetic manipulation of microorganisms
may impart beneficial or even highly beneficial properties to the
microorganisms, it is alternatively preferred that the binder
microorganism and preferably all microorganisms used according to
the present invention is not a genetically modified microorganism
as defined in Article 2(2) of Directive 2001/18/EC in the
respective version applicable at the filing day of this
application. However, microorganisms obtained through the
techniques of genetic modification listed in Annex 1B to said
Directive 2001/18/EC according to the invention are preferably not
considered a genetically modified microorganism as defined in
Article 2 (2) of Directive 2001/18/EC. By excluding genetically
modified microorganisms, rational and irrational fears of consumers
can be avoided, thereby increasing consumer compliance with oral
healthcare instructions.
[0128] According to the present invention the binder microorganism,
preferably of the Lactobacillaceae family and even more preferably
of genus lactobacillus, most preferred one of the above mentioned
particularly preferred strains, does not comprise genetic material
which has been altered in a way that does not occur naturally by
mating and/or natural recombination. Such techniques include [0129]
(1) recombinant nucleic acid techniques involving the formation of
new combinations of genetic material by the insertion of nucleic
acid molecules produced by whatever means outside an organism, into
any virus, bacterial, plasmid or other vector system and their
incorporation into a host organism in which they do not naturally
occur but in which they are capable of continued propagation;
[0130] (2) techniques involving the direct introduction into an
organism of heritable material prepared outside the organism
including micro-injection, macro-injection and micro-encapsulation;
[0131] (3) cell fusion including protoplast fusion or hybridization
techniques where life cells with new combinations of heritable
material are formed through the fusion of two or more cells by
means of methods that do not occur naturally.
[0132] Natural processes such as conjugation, transduction and
transformation, however, are preferably not excluded according to
the present invention. Further preferably not excluded techniques
according to the present invention are, on the condition that they
do not involve the use of recombinant nucleic acid molecules or
genetically modified organisms other than those produced by one or
more of the techniques/methods listed hereinafter are [0133] (1)
mutagenesis by spontaneous or induced spontaneous mutation, and
[0134] (2) cell fusion including protoplast fusion of
microorganisms which can exchange genetic material through
traditional breading methods.
[0135] According to the invention, it is thus preferred that the
binder microorganism and preferably also any other microorganism
used according to the present invention does not comprise genetic
material of an organism of super kingdom archaea or eukaryota, of
course with the exception of such genetic material which can
naturally be found in strains of the same species or, less
preferably, at least in the same genus as the microorganism used
according to the present invention. Further preferably, the
microorganism used according to the present invention does not
comprise genetic material of a microorganism found only in a phylum
other than firmicutes and even more preferably does not comprise
genetic material found only in or taken from microorganisms of a
class other than bacilli, even more preferably does not comprise
genetic material only found in or taken from microorganisms of an
order other than Lactobacillales.
[0136] Where the microorganism used according to the present
invention and particularly the binder microorganism is of family
Lactobacillaceae, then it is preferred that the respective
microorganism does not comprise genetic material found only in or
taken from microorganisms of a family other than Lactobacillaceae.
Even more preferably, if the microorganism of the present invention
and particularly the binder microorganism is of genus
lactobacillus, then the respective microorganism does not comprise
genetic material found only in or taken from microorganisms of a
genus other than lactobacillus.
[0137] The composition according to the invention preferably is
preferably an anticariogenic food or feed composition, or an
anticariogenic pharmaceutical composition. The present invention
thus relates to the use of the above mentioned binder microorganism
or fragment thereof for the preparation of an anticariogenic
composition, preferably a pharmaceutical or cosmetic composition,
for the treatment or prevention of caries caused by mutans
Streptococci and/or Streptococcus mutans.
[0138] The term "composition", as used in accordance with the
present invention, indicates to compositions which comprise at
least one binder microorganism--possibly in thermally inactivated
or lyophilized form--or fragment thereof, preferably a deposited
microorganism as described above--possibly in thermally inactivated
or lyophilized form--or a fragment of said microorganism. It is
envisaged that the compositions as used in accordance with the
present invention 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 mutans Streptococci to the surface of teeth,
to pellicles and/or which inactivate mutans Streptococci. More
preferably, said term encompasses compounds or compositions and/or
combinations thereof which may inhibit the adhesion of mutans
Streptococci to the surface of teeth, inhibit the activity of
glycosyltransferases of mutans Streptococci, inhibit or inactivate
mutans Streptococci, inhibit the agglutinin-dependent binding of
mutans Streptococci and/or inhibit the saccharose-dependent binding
of mutans Streptococci as will be described below.
[0139] The composition of the present invention preferably is a
composition for oral health of a pet animal, and even more
preferably is a composition for prevention or reduction of dental
calculus. Such compositions are known for example from EP 01 41 645
A2, WO 0150882 A2, WO 2001/070043 A2, WO 02/078462 A1, WO
2004/082518 A2, WO 2005/092087 A2 and WO 2010/052467 A2. The foods
and feeds described in these documents and particularly in the
examples mentioned in these documents are incorporated herein as
examples of preferred base compositions. These base compositions
according to the invention are further amended by incorporating a
binder microorganism of the present invention or fragment
thereof.
[0140] It is a particular advantage of the present invention that
by the action of a binder microorganism or fragment thereof, i.e.
by specifically binding to a mutans Streptococcus and most
preferably by specifically binding Streptococcus mutans, the
build-up of dental calculus can be delayed or slowed down without
having to rely on the action of bactericidal agents or other
chemical agents which would kill microorganisms in the oral cavity.
It is suspected that the normal oral microflora is beneficial for
humans and animals, for example as such microorganisms of the
normal microflora will compete with pathogens for nutrition,
thereby limiting the growth of pathogens and avoiding infections.
The present invention allows to let the normal oral microflora
remain largely undisturbed and still confers oral care properties,
preferably the prevention or slow down of dental calculus
formation.
[0141] Another advantage of the present invention in the
prophylaxis and treatment of dental calculus is that the present
invention does not require the presence of decalcifying agents like
zinc sulfate, soluble pyrophosphates, sodium tripolyphosphate and
soluble diphosphonates. Such agents sequester calcium from the oral
cavity, thereby removing a key component of dental calculus
formation. However, tooth enamel is largely made up of hydroxyl
apatite, which is a mineral with high calcium content. Thus,
removal of calcium from the oral cavity is implicated in further
weakening of teeth, which is undesired. The present invention, on
the other hand, allows to treat or prevent formation of dental
calculus without sequestering calcium which would be required for
maintenance of healthy tooth enamel.
[0142] For best results in the prevention of dental calculus
formation, a composition of the present invention therefore
comprises a binder microorganism or fragment thereof, wherein the
binder microorganism preferably is of family Lactobacillaceae, more
preferably of genus Lactobacillus and most preferably is one of the
above mentioned deposited strains of binder microorganisms, and
further comprises decalcifying agents in a concentration such that
binding of binder microorganisms or fragments thereof to mutans
Streptococci is reduced by at most 10% as determined by
nephelometry, and preferably does not comprise such agents. In such
preferred compositions, decalcifying agents preferably are selected
from the group of zinc sulfate, zinc chloride, soluble
pyrophosphates, sodium tripolyphosphate and soluble
diphosphonates.
[0143] The present invention also provides the use of a binder
microorganism or fragment thereof as described above for the
preparation of a medicament for prevention or treatment of dental
calculus, preferably in a child or in a pet animal. As indicated
above, the binder microorganism can also be in a thermally
inactivated form, particularly in an autoclaved form, or in a
lyophilized form. The binder microorganism preferably is of the
family of Lactobacillaceae, even more preferably of genus
Lactobacillus, even more preferably of species Lactobacillus
paracasei or Lactobacillus rhamnosus. Particularly, the binder
microorganism of the present invention can be selected from the
above indicated strains of L. paracasei or L. rhamnosus,
respectively, having any DSMZ accession number of 16667 to 16673,
or a mutant or derivative thereof.
[0144] Most preferably, the composition according to the present
invention is a food or feed composition, particularly for children
or pet animals. Such oral health food or feed compositions are
sometimes termed functional food or functional feed. The terms
"food" and "feed" are used according to the invention regardless of
the nutritional value of corresponding compositions and are thus
not limited to particular nutritional purposes, even though food
and feed compositions according to the present invention can be
tailored to such particular purposes. The terms "food" and "feed"
thus indicate that the respective composition is suitable for being
placed in the oral cavity and for ingestion.
[0145] Of particular importance according to the present invention
are food or feed compositions for animals, preferably for pet
animals, and most preferably for cats, dogs, rats, hamsters and
guinea pigs. In such animals, the formation of caries is
particularly notorious and also difficult to treat, as any dental
treatment like removal of dental calculus requires anaesthesis of
the pet. The present invention is thus particularly suitable for
preventing the need for such stressful treatment.
[0146] The feed composition of the present invention preferably is
a pet feed, i.e. a composition for an animal fed, bred or kept, but
not normally used for human consumption in the European Community.
Preferably, "pet" according to the present invention is a mammal of
order carnivora, even more preferably of suborder caniformia or
suborder feliformia, and most preferably of the canidae or felidae
family. Further preferred pets are of the order rodentia, wherein
particularly preferred animals are mice, rabbits, hamsters and
guinea pigs.
[0147] For pet feed it is preferred that the compound of the
present invention is a compound feed, a complete feed, a
complementary feed or a mineral feed. According to the present
invention, the term "compound feed" means a mixture of at least two
feed materials, whether or not containing feed additives, for oral
animal feeding in the form of complete or complementary feed. The
term "complete feed" means compound feed which, by reason of its
composition, is sufficient for a daily ration. The term
"complementary feed" according to the invention means compound feed
which has a high content of certain substances but which, by reason
of its composition, is sufficient for a daily ration only if used
in combination with other feed. The term "mineral feed" means
complementary feed containing at least 40% crude ash. Finally, the
term "feed material" according to the invention means products of a
vegetable or animal origin, whose principal purpose is to meet
animal nutricial meads, in their natural state, fresh state or
preserved, and products, derived from the industrial processing
thereof, and organic or inorganic substances, whether or not
containing feed additives, which are intended for use in oral
animal feeding either directly as such or after processing, or in
the preparation of compound feed, or as carrier of pre-mixtures.
The term "oral animal feeding" means the introduction of feed into
an animal gastrointestinal tract through the mouth with the aim of
meeting the animal's nutritional needs and/or maintaining the
productivity of normally healthy animals.
[0148] According to the present invention, the composition
comprising the binder microorganism can also be a oral health
composition. Preferred examples of oral care compositions according
to the present invention are tooth paste, dentrifices, tooth
powders, topical oral jelly, mouth rinses, denture products, mouth
sprays, lozenges, oral tablets, chewing gum, dental floss or dental
tape, and particularly for animals, chew products.
[0149] A preferred oral care composition according to the present
invention comprises not only the binder microorganism or fragment
thereof, but also an orally acceptable carrier, such carrier can be
any suitable vehicle which can be applied to the oral cavity in a
safe and effective manner, such that the binder microorganism of
the present invention and/or the fragment thereof can bind to one
or more strains of mutans Streptococci and preferably to one or
more strains of Streptococcus mutans, thus exerting the anti-dental
calculus and/or anti-cariogenic and/or anti-oral-malodor effect.
The oral care composition may be a single or multiple phase
composition.
[0150] In particular, the dentrifice of the present invention can
be a paste, gel, or liquid formulation 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 0 617 608 B1.
[0151] Preferred dentifrice compositions are described in Examples
21 to 24 of WO 2008/074473 A2. In addition to the above described
components, the dentifrice compositions 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.
[0152] For example, the toothpaste may include one or more
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.
[0153] One of the preferred optional agents as used in accordance
with the present invention is a surfactant, preferably one selected
from the group consisting of sarcosinate surfactants, isethionate
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.
[0154] 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. However, as indicated above there is a preferred
maximum concentration of such agents or such compositions which are
intended for a particularly gentle treatment of teeth.
[0155] 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.
[0156] The oral care compositions according to the present
invention may also comprise teeth whitening actives, including
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.
[0157] 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 tooth 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.
[0158] 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.
[0159] 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.
[0160] 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.
[0161] 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.
[0162] 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 acetal 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.
[0163] 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. An infant food 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. Preferably, a pet food according to the present
invention only has a low content of such sugars which can be
metabolized by mutans Streptococci and preferably Streptococcus
mutans, to avoid cariogenic activity of mutans Streptococci. Thus,
the content of sucrose, cane sugar, caramel, corn syrup, corn
molasses, glucose, fructose and sorbitol is preferably kept low,
with a maximum content of 20 wt.-% of the total infant or pet food
being preferred; a maximum content of 5 wt.-% is even more
preferred, and a maximum content of 2 wt.-% being most preferred.
Also preferably, the content of each of saccharin, dextrose,
levulose, lactose, mannitol, maltose and xylitol is less than 20
wt.-%, more preferably less than 5 wt.-% and most preferably less
than 2 wt.-%, with xylitol preferably being not contained in the
pet food at all.
[0164] The oral care composition of 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. 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.
[0165] 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, preferably 4.5
to 8.5. 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.
[0166] 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.
[0167] The present 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.
[0168] The term "chewing gum" as defined herein means a
confectionery composition which is suitable for chewing and which
comprises any suitable amount of elastomer, known to the person
skilled in the art, preferably an amount of 2% or greater, by
weight of the composition. 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 19 and 20 of WO
2008/074473 A2. A preferred chewing gum composition is described in
Example 25 of WO 2008/074473 A2.
[0169] 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 as used in accordance
with 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 as used in
accordance with 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 as
used in accordance with the present invention preferably comprise
from about 2% to about 50%, more preferably from about 10% to about
35%, by weight, of elastomer solvent.
[0170] Lozenges of 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. Lozenges in accordance with the present invention can
also be prepared utilizing other art-recognized solid unitary
dosage formulation techniques.
[0171] A mouth wash or mouth rinse of the present invention could
contain EtOH or could be EtOH-free, and could contain other active
ingredients, as e.g. antimicrobials such as Chlorhexidin. A
preferred mouth wash or mouth rinse of the present invention could
be as follows:
[0172] A Olium menthae 1.2 parts [0173] Tinctura Arnicae 3.0 parts
[0174] Tinctura Myrrhae 3.0 parts [0175] Tween 5.0 parts
[0176] B Spiritus 90% 50.0 parts
[0177] C Sodium Benzoate 0.2 parts
[0178] Sweetening agent (e.g. aspartame) 0.02 parts
[0179] Aqua destilata ad 100.0
[0180] 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 26 of WO 2008074473 A2.
[0181] Regardless of the dosage form, liquid or solid, in one
preferred embodiment of the present invention the dosage form is
held in the consumers mouth, preferably the pet animal's mouth, for
a period of time to promote contact of the microorganism or analog
or fragment of a above mentioned microorganism belonging to the
group of lactic acid bacteria with the patient's oral cavity.
[0182] The terms "dental floss" and "dental tape" as used herein
refer to a material to dislodge and remove decomposing food
material that accumulated at interproximal and subgingival surfaces
and to dislodge and remove bacteria, plaque and/or calculus that
accumulated in the oral cavity. The dental floss or dental tape may
further contain, in addition to the microorganisms according to the
present invention as described herein, cleaners, abrasives, tartar
control ingredients, whiteners, surfactants and/or active
ingredients like fluorides, antimicrobials, chemotherapeutic agents
or antibiotics. Further additional agents are antiplaque agents,
flavouring agents and colouring agents. The dental floss or dental
tape may be in any suitable form, known to the person skilled in
the art, for example, in the form of PTFE (Teflon) dental flosses
as described, for instance, in U.S. Pat. No. 3,664,915, U.S. Pat.
No. 3,953,566, U.S. Pat. No. 3,962,153, U.S. Pat. No. 4,096,227,
U.S. Pat. No. 4,187,390, U.S. Pat. No. 4,256,806, U.S. Pat. No.
4,385,093, U.S. Pat. No. 4,478,665, U.S. Pat. No. 4,776,358, U.S.
Pat. No. 5,033,488, U.S. Pat. No. 5,209,251, U.S. Pat. No.
5,220,932, U.S. Pat. No. 5,518,012, U.S. Pat. No. 5,718,251, U.S.
Pat. No. 5,765,576 or U.S. Pat. No. 5,911,228, in the form of
monofilament interproximal devices as described, for instance, in
U.S. Pat. No. 3,800,812, U.S. Pat. No. 4,974,615, U.S. Pat. No.
5,760,117, U.S. Pat. No. 5,433,226, U.S. Pat. No. 5,479,952, U.S.
Pat. No. 5,503,842, U.S. Pat. No. 5,755,243, U.S. Pat. No.
5,884,639, U.S. Pat. No. 6,003,525 or U.S. Pat. No. 6,027,592, or
in the form of biocomponent tapes. Preferably, the dental floss or
dental tape may be in the form of an elastomeric coated
monofilament as described, for instance, in US 20050226820 or in
the form of an oriented thermoplastic based dental tape as
described, for instance, in US 20020144704.
[0183] The oral care cosmetic compositions as described herein,
particularly anti-dental calculus compositions, anti-caries
compositions and anti-oral malodor compositions, may be used in the
ambit of human oral administration as well as in the ambit of
veterinary oral administration, preferably for non-human mammals,
more preferably for pets. If the composition is used in the ambit
of veterinary oral administration, the composition may contain
further ingredients suitable for such an administration, as known
by a person skilled in the art.
[0184] A pharmaceutical composition according to the present
invention preferably further comprises a pharmaceutical acceptable
carrier or excipient.
[0185] Pharmaceutical compositions comprise a therapeutically
effective amount the above mentioned microorganism or fragment
thereof and can be formulated in various forms, e.g. in solid,
liquid, powder, aqueous, lyophilized form.
[0186] The pharmaceutical composition may be administered with a
pharmaceutically acceptable carrier to a patient, preferably a
human being or an animal, and most preferably a child or pet
animal. 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 as used in accordance with 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.
[0187] 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 skimmed 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 maltodextrin, 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. Skimmed milk,
skimmed milk powder, non-milk or non-lactose containing products
may also be employed. The skimmed 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.
[0188] 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.
[0189] 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.
[0190] In a further aspect, a composition of the present invention
may be produced by comprising the steps of formulating a binder
microorganism fragment thereof with a cosmetically, orally or
pharmaceutical acceptable carrier or excipient. Preferably, this
microorganism is a deposited microorganism as described herein
above or a mutant, derivative, analog or fragment thereof. A
preferred composition in accordance with 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.
[0191] The composition of the present invention, preferably a food
or feed composition including pharmaceutical compositions,
comprises a binder microorganism as described above, potentially in
a thermally inactivated or lyphilized form, in an amount of
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. In case of a liquid form
of the composition, the amount of the microorganisms is 10.sup.2 to
10.sup.13 cells per ml.
[0192] However, for specific compositions the amount of the
microorganism may be different as is described herein.
[0193] Preferably, the concentration of binder microorganisms in
the composition of the present invention is 0.01 wt.-% to 10 wt.-%,
relative to the total mass of the composition. Even more
preferably, the concentration of binder microorganisms in the
composition of the present invention is 0.025 wt.-% to 2 wt.-%. As
described above, when fragments are used instead of binder cells,
then the concentration of fragments is chosen to be the same as the
peptidoglycan content of binder cells.
[0194] In accordance with the present invention, the term food
encompasses all eatable and drinkable foods and drinks.
Accordingly, the microorganism or fragment thereof 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.
[0195] 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.
[0196] Where the binder microorganism of this invention or fragment
thereof 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, or the
equivalent amount of fragment thereof. In such a case, it is
possible to completely prevent or inhibit formation of a biofilm by
mutans Streptococci, preferably by Streptococcus mutans, and thus
to prevent or slow the development of dental calculus, oral
malodour or dental caries induced by cariogenic strains, without
significant side effect upon the quality of the drink per se.
[0197] 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.
[0198] The food, drink or feed of the present invention includes
any food, drink or feed which comprises the binder microorganism of
the invention or fragment thereof as active ingredient. 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 mutans
Streptococci. 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.
[0199] 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.
[0200] The food or drink according to the invention to be ingested
by infants, i.e. comprising the binder microorganism of fragment
thereof with an activity to specifically bind to mutans
Streptococci, is preferably a nutritious composition 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.
[0201] The nutritious composition for infants, and also the
nutritious food or feed composition for animals and particularly
pet animals, 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. 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 amino acids maybe
used satisfactorily as well. 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. For dogs, these
include arginine, methionine, histidine, phenylalanine, isoleucine,
threonine, leucine, tryptophan, lysine and valine. For cats,
taurine is also essential. 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 oil, 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.
[0202] Further preferred ingredients of food and feed, particularly
pet feed, are omega-6 fatty acids and omega-3 fatty acids.
Particularly preferred are linoleic acid, preferably in the form of
corn, soy, canola, safflower and sunflower oil, whole grains and/or
body fat of poultry; arachidonic acid, preferably in the form of
body fat of poultry, lean meat, egg yolks and/or fish oil; gamma
linolenic acid, preferably in the form of black currant seed oil,
borage oil and/or evening primrose oil; dihomogamma linolenic acid,
preferably in the form of spleen, kidney, adrenals and/or
metabolized from gla; alpha linolenic acid, preferably in the form
of flaxseed oil, canola, soy, and/or walnut oils; eicosapentaenoic
acid, preferably in the form of cold water fish and their oil;
docosahexaenoic acid, preferably in the form of cold water fish and
their oil.
[0203] 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. Preferred saccharides include glucose,
fructose, honey, galactose, lactose, sucrose, maltose, dextrins,
glycogen and starch.
[0204] However, as described above the maximum content of glucose,
fructose, honey, galactose, lactose, sucrose and maltose preferably
is 20 wt.-% of the total composition, more preferably 5 wt.-% of
the total composition, and most preferably 2 wt.-% of the total
composition. Even more preferably, the aforementioned maximum
content of 20 wt.-%, more preferably 5 wt.-% and most preferably 2
wt.-% applies to the total of all substances of the group of cane
sugar, caramel, corn molasses, corn syrup, dextrose, fructose,
galactose, glucose, honey, lactose, levulose, maltose, mannitol,
saccharin, sorbitol, sucrose and xylitol, and most preferably to
the total of mono- and disaccharides.
[0205] Also preferred are dietary fiber preferably selected form
cellulose, hemicellulose, pectin, plant gums and mucilages, beet
pulp, guar gum, gum arabic, xanthan gum and locust bean gum. 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.
[0206] 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, nicotinamide,
carnitine, choline, inositol and biotin as long as such vitamins
can be administered to infants or pet animals. Such vitamins are
preferably from 10 mg to 5 g by weight per the total solid in the
nutritious composition for infants. Preferred vitamins for
nutritious compositions for infants and/or animals, preferably pet
animals, include:
[0207] Vitamin A (Retinol), and/or beta carotene as precursor. The
vitamin is preferably present in the form of liver, fish liver oil,
carrots, green leafy vegetables, egg yolks and/or yellow fruits or
in form of synthetic forms of Vitamin A and/or beta-carotene.
[0208] Vitamin D (Calciferol). The vitamin is preferably present in
the form of halibut and/or cod liver oil, saltwater fish, cheese,
yogurt and/or eggs or in form of synthetic forms of Vitamin D.
[0209] Vitamin E (Tocopherol). The vitamin is preferably present in
the form of germ, corn, nuts, seeds, spinach and/or other green
leafy vegetables, asparagus, vegetable oils or in form of synthetic
forms of Vitamin E.
[0210] Vitamin K (Naphthoquinone). The vitamin is preferably
present in the form of cabbage, cauliflower, spinach and/or other
green leafy vegetables, cereals, soybeans, and/or other vegetables
or in form of synthetic forms of Vitamin K. Preferably, the food or
feed does not comprise menadione.
[0211] Vitamin B1 (Thiamine). The vitamin is preferably present in
the form of wheat germ, rice and/or other whole grains, lean meats
(especially pork), liver, fish, yeast, dried beans, peas and/or
soybeans or in form of synthetic forms of Vitamin B1.
[0212] Vitamin B2 (Riboflavin). The vitamin is preferably present
in the form of lean meats, liver, fish, eggs, yeast, cheese,
legumes, nuts and/or green leafy vegetables or in form of synthetic
forms of Vitamin B2.
[0213] Vitamin B3 (Niacin). The vitamin is preferably present in
the form of liver, lean meat, poultry, fish, nuts, yeast, legumes,
asparagus, seeds and/or green leafy vegetables or in form of
synthetic forms of Vitamin B3.
[0214] Vitamin B5 (Pantothenic Acid). The vitamin is preferably
present in the form of eggs, fish, lean beef, legumes, yeast,
broccoli and/or other vegetables in the cabbage family, white
and/or sweet potatoes or in form of synthetic forms of Vitamin
B5.
[0215] Vitamin B6 (Pyridoxine). The vitamin is preferably present
in the form of meat, fish, eggs, bananas and/or whole grains or in
form of synthetic forms of Vitamin B6.
[0216] Vitamin B8 (Biotin). The vitamin is preferably present in
the form of raw egg yolk, liver and/or vegetables or in form of
synthetic forms of Vitamin B8.
[0217] Vitamin B9 (Folic Acid, Folate). The vitamin is preferably
present in the form of carrots, yeast, liver, egg yolks, melon,
apricots, pumpkin, beans, rye, whole wheat and/or green leafy
vegetables or in form of synthetic forms of Vitamin B9.
[0218] Vitamin B12 (Cobalamin). The vitamin is preferably present
in the form of fish, liver, meat, poultry, eggs and/or cheese or in
form of synthetic forms of Vitamin B12.
[0219] Vitamin C (Ascorbic Acid). The vitamin is preferably present
in the form of citrus fruit juice or pulp, berries, tomatoes,
cauliflower, potatoes, green leafy vegetables and/or green peppers
or in form of synthetic forms of Vitamin C. Supplementation in an
appropriate form, preferably as calcium ascorbate, is preferred due
to its beneficial effects on dogs suffering from chronic joint and
musculoskeletal disorders. In puppies it helps to prevent the
development of such disorders.
[0220] Further, the minerals include calcium, magnesium, potassium,
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.
[0221] Other than those components described above, the nutritious
composition for infants, and also the food or feed composition for
animals, preferably pet animals, as used in accordance with the
present invention may be blended with any component desirably
blended in nutritious compositions, for example, dietary fiber,
nucleotides, nucleic acids, flavors, and colorants.
[0222] The food or drink as used in accordance with the present
invention can be used as a health food or drink or a functional
food or drink to prevent and/or treat caries and/or to prevent or
treat oral malodour and/or to prevent or treat dental calculus.
[0223] 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.
[0224] 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.
[0225] The composition of the present invention may comprise,
further to the binder microorganism or fragment thereof, cereals,
brans, oil-seed meals, animal-derived raw feed materials, other raw
feed materials and purified products. The cereals can include,
mile, wheat, barley, oats, rye, brown rice, buckwheat, fox-tail
millet, Chinese millet, Deccan grass, corn, and soybean. The brans
can include, rice bran, defatted rice bran, bran, lowest-grade
flour, wheat germ, barley bran, screening pellet, corn bran, and
corn germ. 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. 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. 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. 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.
[0226] The composition of the present invention may further
comprise one or more additives. Such 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, antioxidants, 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.
[0227] The sweeteners include Brazzein; Curculin; Erythritol;
Glycyrrhizin; Glycerol, E422; Hydrogenated starch hydrolysates;
Inulin; Isomalt, E953; Lactitol, E966; Luo han guo; Mabinlin;
Maltitol, E965; Malto-oligosaccharide; Mannitol, E421; Miraculin;
Monatin; Monellin; Pentadin; Sorbitol, E420; Stevia extracts or
steviol glycosides, particularly Stevioside, Rebaudioside A,
Rebaudioside C, Dulcoside A, Rubusoside, Steviolbioside H and
Rebaudioside B; Tagatose; Thaumatin, E957; Xylitol, E967;
Acesulfame potassium, E950; Alitame; Aspartame, E951; Salt of
aspartame-acesulfame, E962; Cyclamate, E952; Dulcin; Glucin;
Neohesperidin dihydrochalcone, E959; Neotame; P-4000; Saccharin,
E954; Sucralose, E955; licorice; xylose and rakanka (Momordica
grosvenori fruit).
[0228] 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.
[0229] The preservatives include, for example, sodium sulfite,
benzoates, benzoin extract, sorbates, and propionates.
[0230] 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.
[0231] The anti-oxidants include, for example, vitamin C group,
sodium ethylenediaminetetraacetate, calcium
ethylenediaminetetraacetate, 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.
[0232] The color fixing agents include, for example, sodium
nitrite. The bleaches include, for example, sodium sulfite.
[0233] The antiseptics include, for example, o-phenyl phenol. The
gum base includes, for example, acetylricinoleate methyl, urushi
wax, ester gum, elemi 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.
The bitters include, for example, isoalpha-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. The enzymes include, for example,
amylase, trypsin or rennet. 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 chloridedecreased brine, crude potassium chloride,
whey salt, thpotassium phosphate, dipotassium hydrogen phosphate,
potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium
dihydrogen phosphate, trisodium phosphate and chlorella
extract.
[0234] 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,
menaquinone, folic acid and riboflavine.
[0235] The agents for manufacture include, for example, processing
auxiliaries such as acetone and ion exchange resin.
[0236] The flavors include, for example, vanilla essence and the
spice extracts include, for example, capsicum extract.
[0237] These various additives can be added to the active
ingredient, taking into consideration the mode of administration,
in accordance with the present invention.
[0238] It is envisaged that the composition of the present
invention, preferably the food or feed composition or
pharmaceutical composition, comprises the above mentioned binder
microorganism belonging to the group of lactic acid bacteria in the
form of a probiotic microorganism. Namely, in addition to the
probiotic effect, the above mentioned probiotic microorganism
belonging to the group of lactic acid bacteria is useful for
treating and/or preventing biofilm formation caused by mutans
Streptococci. The amount of said probiotic microorganism is high
enough to significantly positively modify the condition to be
treated, preferably caries, dental calculus and/or oral malodour,
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 mutans Streptococci. 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. 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.
[0239] The composition of the present invention 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 the above mentioned microorganism
belonging to the group of lactic acid bacteria is added in fresh,
concentrated or dried form, for example. 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.
[0240] 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 cells per ml, 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.
[0241] Preferred ingredients in the food and feed composition
according to the present invention are alfalfa, alfalfa concentrate
powder, alfalfa dehydrated meal, alfalfa nutrient concentrate,
alpha-lipoic acid, animal digest, animal fat, argenine, artificial
flavor, ascorbic acid, asparagus, bacillus subtilis, bacon, bacon
flavors, barley, barley grass, barley malt flour, basil, beans,
beef, beef & bone meal, beef broth, beef by-products, beef
flavor, beef liver, beef meal, beef tallow, beet pulp, beet pulp
(sugar removed), beets, beta carotene, BHA, bifidobacterium longum,
bifidobacterium pseudolongum, bifidobacterium thermophilum, biotin,
blue 2, blue 2 and other color, blueberries, bone meal, borage oil,
brewers dried yeast, brewers yeast, brewers yeast extract
(saccharomyces cerevisiae fermentation solubles), brewer's rice,
broccoli, brown rice, brown rice flour, calcium, calcium ascorbate,
calcium carbonate, calcium chloride, calcium iodate, calcium
pantothenate, calcium phosphate, calcium propionate, calcium
sulfate, cane molasses, canola meal, canola oil, canthaxanthin,
caramel, caramel color, carmine, carrageenan, carrageenan gum,
carrot powder, carrots, casein, catfish, catfish meal, celery,
cellulose, cellulose powder, cheese powder, chelated cobalt,
chelated copper, chelated iron, chelated potassium, chicken,
chicken broth, chicken by products organs only, Chicken by-product,
chicken by-product meal, chicken by-products organ meat only,
chicken cartilage natural, chicken fat, chicken fat naturally,
chicken flavors, chicken giblets, chicken liver, chicken liver
digest, chicken liver flavor, chicken meal, chicken natural,
chicken stock, chicory extract, choline chloride, chondroitine
sulfate, cinnamon, citric acid, citric acid and rosemary, citric
acid and rosemary extract, citrus pectin, clove bud oil, cobalt
amino acid chelate, cobalt carbonate, cobalt proteinate, cod,
copper, copper amino acid chelate, copper amino acid complex,
copper oxide, copper proteinate, copper sulfate, corn, corn bran,
corn flour, corn germ meal, corn gluten, corn gluten meal, corn
grits, corn meal, corn oil, corn starch, corn starch-modified, corn
syrup, cracked barley, cracked pearled barley, cranberries,
cranberry powder, deboned chicken, deboned lamb, deboned turkey,
dehydrated alfalfa, dehydrated alfalfa meal, dehydrated carrots,
dehydrated potatoes, dextrose, DHA, dicalcium phosphate, DL-alpha
tocopherol acetate, DL-methionine, dried animal digest, dried
apples, dried bacillus licheniformis fermentation extract, dried
bacillus subtilis fermentation extract, dried beet pulp, dried beet
pulp (sugar removed), dried blueberries, dried brewers yeast, dried
buttermilk, dried capsicum, dried carrots, dried cellulose, dried
cheddar cheese, dried cheese, dried cheese powder, dried chicken
cartilage, dried chicken liver, dried chicken stock, dried chickory
root, dried citrus pulp, dried cooked turkey, dried cranberries,
dried egg, dried egg powder, dried egg product, dried eggs, dried
garlic, dried ginger, dried grape pomace, dried green beans, dried
kelp, dried kelp meal, dried liver digest, dried meat by-product,
dried paprika, dried parsley flakes, dried peas, dried plain beet
pulp, dried potatoes, dried spinach, dried sweet potato, dried
tomato pomace, dried vegetable fiber carrots, dried whey, dried
yam, duck, durum flour, durum semolina enriched with thiamine
mononitrate, egg noodles, egg pieces, egg product, eggs,
enterococcus faecium, ethoxyquin, eucalyptus oil, ferrous sulfate,
fiber, fish, fish broth, fish meal, fish meal natural, fish oil,
flax meal, flax seed, flaxseed meal, folic acid, folic acid
pyridoxine hydrochloride, food starch, fresh, fresh chicken, fresh
chicken by-products, fructooligosaccharides, fumaric acid, garlic,
garlic extract, garlic flavor, garlic oil, garlic powder, gelatin,
ginger, ginger extract, glucosamine, glucosamine hydrochloride,
glycerin, glycerine, glyceryl monostearate, glycine, green beans,
green tea, ground corn, ground flax seed, ground psyllium seed,
ground rice, ground wheat, ground wheat flour, ground whole grain
barley, ground whole grain corn, ground whole grain sorghum, ground
whole grain wheat, ground whole peas, ground whole wheat, ground
yellow corn, guar gum, gum arabic, halibut, herring meal, herring
oil, hydrochloric acid, inositol, iodine, iodized salt, iron amino
acid chelate, iron amino acid complex, iron oxide, iron proteinate,
iron sulfate, L-alanine, L-arginine, L-ascorbyl-2-polyphosphate,
L-ascorbyl-2-polyphosphate a, L-carnitine, L-lysine, L-lysine
monohydrochloride, L-tryptophan, lactobacillus acidophilus, lamb,
lamb broth, lamb by-product, lamb digest, lamb fat, lamb liver,
lamb meal, lamb stock, lamb tripe, lecithin, lentils, lettuce,
liver, locust bean gum, lutein, lycopene, lysine, mackerel,
magnesium oxide, malt extract, malted barley flour, managanous
sulfate, manganese amino acid chelate, manganese oxide, manganese
proteinate, manganese sulfate, manganous oxide, manganous oxide
calcium iodate, manganous proteinate, manganous sulfate, maple
syrup, marigold extract, marigold meal, meat and bone meal natural,
meat and liver meal, meat by-products, menadione
dimethylpyrimidinol bisulfite, menadione sodium bisulfite complex,
menadione vitamin K3, menhaden fish meal, menhaden fish oil, milk,
mixed tocopherols, mixed vegetable fiber carrots, modified food
starch, modified starch, molasses, monocalcium phosphate,
monosodium phosphate, natural and artificial chicken flavor,
natural and artificial flavors, natural chicken flavor, natural
color, natural flavor, natural poultry flavor, natural smoke
flavor, niacin, niacin & ferrous sulfate, non-fat yogurt, oat
bran, oat fiber, oat groats, oat meal, oats, ocean fish, ocean fish
meal, ocean whitefish, omega fatty acids, onion extract, onion
powder, pantothenate, paprika oleoresin, parsley, parsley flakes,
parsley oil, parsley powder, partially hydrogenated soybean oil,
pasta, pea fiber, pea protein, peanut hulls 10.8%, pearled barley,
peas, peppers, petrolatum, philloquinone vitamin K1, phosphoric
acid, pork broth, pork by-products, pork liver, postassium sorbate,
potassium amino acid complex, potassium chloride, potassium
citrate, potassium iodide, potassium iodine, potassium sorbate,
potato, potato fiber, potato starch, poultry, poultry by-product
meal, poultry by-products, poultry fat, poultry giblets, poultry
liver, powdered cellulose, powdered cellulose 11.1%, propionic
acid, propyl gallate and citric acid, propylene glycol, pyridoxine
hydrochloride, pysllium, rabbit, rabbit by-products, rabbit stock,
red 3, red 40 and other color, red peppers, riboflavin, rice, rice
bran, rice flour, rice gluten, rice hulls, rice protein
concentrate, rosemary, rosemary extract, rosemary extract and
citric acid, rye, sage, salmon, salmon broth, salmon meal, salmon
oil, salt, sea salt, selenium, shrimp, smoke flavor, sodium
alginate, sodium ascorbate, sodium bisulfate, sodium carbonate,
sodium caseinate, sodium chloride, sodium hexametaphosphate, sodium
metabisulfate, sodium nitrite for color retention, sodium
phosphate, sodium propionate, sodium selenite, sodium silico
aluminate, sodium tripolyphoshate, sodium tripolyphosphate, sorbic
acid, sorbitol, soy flour, soy hulls, soy lecithin, soy protein
concentrate, soy protein isolate, soya oil, soybean hulls, soybean
meal, soybean mill run, soybean oil, spearmint, spinach, spirulina,
starch, steamed bone meal, sucrose, sufficient water for
processing, sugar, sun-cured alfafa meal, sunflower meal, sunflower
oil, sweet potato powder, sweet potatoes, tallow, tapioca starch,
taurine, tetra sodium pyrophosphate, textured vegetable protein,
thiamine, thiamine hydrochloride, thiamine mononitrate, thyme,
titanium dioxide, titanium dioxide color, tomato flakes, tomato
paste, tomato pomace, tomatos, trace minerals (calcium sulfate),
trace minerals (copper sulfate), trace minerals (potassium
chloride), trace minerals (sodium tripolyphoshate), trace minerals
(zinc oxide), trace minerals (zinc proteinate), trace minerals
(zinc sulfate), tricalcium phosphate, tuna, tuna meal, turkey,
turkey broth, turkey by-product meal, turkey natural, turkey stock,
turmeric, veal, veal broth, vegetable oil, venison, venison
by-products, venison liver, venison meal, venison meat, venison
stock, vitamin A, vitamin A & D3, vitamin A acetate, vitamin
B1, vitamin B12, vitamin B12 and D3, vitamin B2, vitamin B6,
vitamin C, vitamin D3, vitamin D3 and E, vitamin E, vitamin K,
water, water cress, water sufficient for processing, watercress and
spinach, wheat, wheat bran, wheat flour, wheat germ meal, wheat
gluten, wheat middlings, wheat mill run, wheat starch, whey, white
fish, whitefish, whitefish meal, whole brown rice, whole carrots,
whole cranberries, whole eggs, whole garlic cloves, whole grain
corn, whole grain wheat, whole ground barley, whole ground brown
rice, whole ground oats, whole ground wheat, whole rice, whole
sweet potatoes, whole wheat, whole wheat flour, wild rice, xanthan
gum, yeast culture, yellow 5, yellow 6, yellow squash, yellow
zucchini, yucca schidigera, yucca schidigera extract, zinc amino
acid chelate, zinc amino acid complex, zinc oxide, zinc proteinate,
zinc proteinate and zinc sulfate.
[0242] Most preferred ingredients are: alpha-lipoic acid,
preferably in an amount sufficient to promote formation of healthy
skin and/or coat; for antibiotic purposes preferred ingredients are
alpha-lipoic acid, dried garlic, garlic extract, garlic oil and
garlic powder; lecithin, preferably as an antioxidans; cracked
barley, cracked pearled barley, ground whole grain corn, ground
whole grain sorghum, herring oil, mackerel, menhaden fish oil,
ocean fish, whole brown rice and whole sweet potatoes are preferred
as sources of energy; preferred sources of fiber are dehydrated
carrots, dried apples, ground whole grain barley, oat bran, oat
groats, oat meal, whole ground barley, whole ground oats; cobalt
proteinate (source of chelated cobalt), copper proteinate, iron
proteinate, manganese proteinate, manganous proteinate, trace
minerals and, zinc proteinate are preferred sources of minerals;
preferred sources of omega-3 fatty acids are borage oil, canola
oil, canola oil (preserved with mixed tocopherols) and flax seed;
rosemary extract and citric acid are preferred preservatives;
preferred sources of protein are beef meal, chicken meal, dried
peas, halibut, lamb meal, menhaden fish meal, ocean whitefish,
peas, venison meal, whole ground brown rice and whole ground wheat;
preferred sources of vitamins are calcium ascorbate, carrots, dried
carrots, mixed tocopherols, whole carrots; preferred replacements
for plain water, and also preferred flavours, are beef broth,
chicken broth, lamb broth, lamb stock and turkey broth.
[0243] Ingredients not most but also highly preferred are: alfalfa
concentrate powder, alfalfa dehydrated meal, chicken fat, chicken
liver, fresh, ocean fish meal and pearled barley as sources of
energy; green beans, ground whole peas, oats and pea fiber as
sources of fiber; cobalt amino acid chelate, copper amino acid
chelate, copper amino acid complex, dried kelp meal, iron amino
acid chelate, manganese amino acid chelate, manganese sulfate,
potassium amino acid complex, zinc amino acid chelate as sources of
minerals; chicory extract, ginger extract and yucca schidigera
extract as prebiotics; good preservatives are garlic, rosemary and
sage; alfalfa nutrient concentrate, catfish, catfish meal, cod,
duck, eggs, ground whole wheat, herring meal, pea protein, rabbit,
shrimp, tuna, venison, venison meat, white fish, whitefish,
whitefish meal, whole wheat, are preferred as sources of protein;
beta carotene and folic acid are preferred sources of vitamins;
chicken stock, rabbit stock, turkey stock, veal broth and venison
stock are preferred replacements for plain water, and also
preferred flavours.
[0244] A standard food of feed composition of the present
invention, preferably a pet food, preferably comprises one or more
ingredients of the group consisting of vitamin B-12, biotin,
calcium carbonate, calcium pantothenate, choline chloride, cobalt
carbonate, copper sulfate, vitamin D3 and E supplements,
DL-methionine, dried kelp, ferrous sulfate, folic acid, inositol,
manganese oxide, manganous oxide, manganous sulfate, menadione
sodium bisulfite complex, mineral supplements including zinc
sulfate, natural flavor, niacin and ferrous sulfate, niacin
supplement, potassium chloride, potassium iodide, pyridoxine
hydrochloride, riboflavin, sodium selenite, taurine, thiamine,
thiamine mononitrate, thiamine mononitrate, vitamin A, vitamin A
& D3, vitamin A acetate, vitamin B12, vitamin D3, water, zinc
oxide and zinc sulfate.
[0245] The feed as used in accordance with the present invention
maybe any feed comprising the binder microorganism or fragment
thereof as an active ingredient. The feed includes, for example,
pet feeds for dogs, cats and rats, and cattle feeds for cows and
pigs.
[0246] The feed can be produced by appropriately blending the
active ingredient as described herein above 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 as indicated above. Preferred feeds of the
present invention are chew products. Preferred products are pig
ears, bull sinews, cattle tails, oesophagus, dried muscle meat, pig
feet and dried pressed cowhide, buffalo hide or composite chew
products, containing plant materials as e.g. fibers, brans etc.
Other chew products are known in the art and are also preferred,
for example the chew products of U.S. Pat. No. 2,988,045, WO
01/50882 A2, EP 1 151 674 A1, EP 1 006 789 A1 and CH 676200 A5. The
chew product preferably is in the shape of a bone, a roll, a donut,
a bow, a pretzel, a figure eight, or a chip.
[0247] 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.
[0248] It is likewise preferred that the composition of the present
invention comprises one or more probiotic microorganisms or
products obtained thereof, independently of the binder
microorganism and fragment thereof. Preferred probiotic
microorganisms and respective products thereof are bacillus
subtilis, bifidobacterium longum, bifidobacterium pseudolongum,
bifidobacterium thermophilum, dried bacillus licheniformis
fermentation extract, dried bacillus subtilis fermentation extract,
enterococcus faecium and lactobacillus acidophilus.
[0249] Particularly preferred are food and feed compositions
further comprising, in addition to the binder microorganism or
fragment thereof, a further microorganism for preventing and/or
treating oral malodor. Most preferred are food and feed
compositions further comprising such microorganism as disclosed in
WO 2009/149816 A1, which is incorporated herein in its entirety, Of
these, the further microorganism is preferably selected from the
group consisting of Lactobacillus acidophilus having DSMZ accession
number DSMZ 19825, DSMZ 19826, DSMZ 19827.
[0250] The invention is now further described by selected examples
and embodiments. These embodiments and examples are intended to
represent certain preferred features of the present invention,
without limiting the scope of this description or the scope of the
claims. It is to be understood that the skilled artisan can devise
further working examples and embodiments by his common general
knowledge and the instructions and explanations given in this
description and the documents incorporated herein by reference.
EXAMPLE 1
Storage and Growth of Binder Microorganisms
[0251] Storage and growth of strains can be performed according to
ordinary procedures. According to this present example, strains are
stored as frozen stocks at -80.degree. C. 1 ml of a culture is
grown to stationary phase (OD600: 4-8) in MRS-Medium and mixed with
500 .mu.l of a sterile 50% glycerine solution and frozen. Cultures
of mutans Streptococci are grown in TSY-media to stationary phase
(OD600 1-2) and are treated as mentioned above for frozen
storage.
[0252] Cultivation of mutans Streptococci (S. mutans, S. sobrinus,
S. ratti, S. cricetus, S. ferus or S. macacae) as well as
cultivation of lactobacilli can be done in 5 ml in closed Falcon
tubes at 37.degree. C. without shacking over night.
[0253] 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.sub.--4-8) in MRS-broth was
mixed with 500 .mu.l of a sterile 50% glycerol solution and
frozen.
[0254] In particular, cultures of mutans Streptococci were grown in
TSY-broth to stationary phase (OD600/ml.sub.--1-2) and treated as
mentioned above.
[0255] Cultivation of mutans Streptococci (S. mutans (DSMZ 20523,
serotype c; NCTC 10923, serotype e; NCTC 11060, serotype f), S.
sobrinus DSMZ 20742, S. ratti DSMZ 20564, S. cricetus DSMZ 20562,
S. ferus DSMZ 20646 or S. macacae DSMZ 20714) and cultivation of
lactobacilli was done in 5 ml in closed Falcon tubes at 37.degree.
C. without shacking over night. For the fluorescence assays as
described in Example 5 S. mutans DSMZ 20523 was used.
[0256] For an aggregation assay the lactobacilli were grown in
MRS-medium. 5 ml MRS--medium were inoculated with 10 .mu.l of the
stock and incubated for 3 days at 37.degree. C. under aerobic
conditions. The optical density of the culture at 600 nm (OD600)
was measured. The culture was then diluted to an OD600 of 2 using
PBS-buffer. The mutans Streptococci were grown in 7 ml TSY-medium.
7 ml of TSY-medium were inoculated with 10 .mu.l of the stock and
incubated at 37.degree. C. under anaerobic conditions.
MRS-Broth:
[0257] MRS-mixture (Difco, USA) 55 g/l, pH: 6.5
TSY-Broth:
TSY-mixture (Difco, USA) 30 g/l
[0258] Yeast extract (Deutsche Hefewerke, Germany) 3 g/l
Buffer:
PBS-Buffer:
Na.sub.2HPO.sub.4 2H.sub.2O 1.5 g/l
KH.sub.2PO.sub.4 0.2 g/l
NaCl 8.8 g/l
[0259] pH adjusted with HCl
EXAMPLE 2
Taxonomic Classification of Binder Strains and Streptococcus
Strains
[0260] 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
Staining of Cells
[0261] After the lactobacilli and the mutans Streptococci were
grown as described in Example 1, the mutans Streptococci were
stained using a fluorescence strain. For this, the OD600 of the
culture was measured. The culture was harvested by centrifugation
at 3200.times.g for min. the pellet was resuspended in PBS-buffer.
The amount of buffer was calculated so that the resulting
suspension had an OD600 of 4.2 ml of that suspension were mixed
with 2 .mu.l of a CFDA-SE solution (Invitrogen, USA) that was
prepared according to the manufacturer's instructions. Staining of
the cells was carried out by incubating the mixture for 2 h at
37.degree. C. The stained cells were harvested by centrifugation at
3200.times.g for 5 min. The cells were subsequently resuspended in
2 ml PBS-buffer.
EXAMPLE 4
Pelleting Aggregation Assay of Mutans Streptococci
[0262] For the assay, mixing of lactobacilli and mutans
Streptococci was done in volumetric ratios of 3:1 to 60:1 (mutans
Streptococci:lactobacilli), this corresponds to a ratio of colony
forming units from 1:50 to 1:2.5.
[0263] An optical density measured at a wavelength of 600 nm in 1
ml means preferably for mutans Streptococci 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. The formed aggregates
were separated by centrifugation at 500.times.g for 30 seconds.
Afterwards, the amount of aggregation was quantified by measuring
the amount of non-aggregated cells that were left in the
supernatant. Correspondingly, 1 ml of the supernatant was carefully
removed to measure the optical density. The optical density was
measured at 600 nm. The value after subtraction of the respective
control experiment without lactobacilli represents the amount of
cells that have not been aggregated.
[0264] As a control, self-aggregation of the respective
Lactobacillus strain and the mutans Streptococcus strains was
always investigated by performing the test with only the
Lactobacillus or the mutans Streptococcus strain added to the tube.
An aggregation of S. mutans by Lactobacillus is shown in FIGS. 1
(left tube) and 2.
[0265] The lactobacilli strains as described herein above, in
particular those deposited with the DSMZ, exhibited aggregation of
all S. mutans serotypes without showing a self-aggregation
behaviour.
EXAMPLE 5
Fluorescence Aggregation Assay of Mutans Streptococci
[0266] For the assay, suspension of the respective lactobacillus
and the respective stained mutans Streptococcus (S. mutans DSMZ
20523 with Lb-OB-K1 (DSMZ 16667), S. mutans DSMZ 20523 with
Lb-OB-K2 (DSMZ 16668), S. mutans DSMZ 20523 with Lb-OBK3 (DSMZ
16669), S. mutans DSMZ 20523 with Lb-OB-K4 (DSMZ 16670), S. mutans
DSMZ 20523 with Lb-OB-K5 (DSMZ 16671), S. mutans DSMZ 20523 with
Lb-OB-K6 (DSMZ 16672), S. mutans DSMZ 20523 with Lb-OB-K7 (DSMZ
16673); S. sobrinus DSMZ 20742 with Lb-OB-KI (DSMZ 16667), S.
sobrinus DSMZ 20742 with Lb-OB-K2 (DSMZ 16668), S. sobrinus DSMZ
20742 with Lb-OB-K3 (DSMZ 16669), S. sobrinus DSMZ 20742 with
Lb-OB-K4 (DSMZ 16670), S. sobrinus DSMZ 20742 with Lb-OB-K5 (DSMZ
16671), S. sobrinus DSMZ 20742 with Lb-OB-K6 (DSMZ 16672), S.
sobrinus DSMZ 20742 with Lb-OB-K7 (DSMZ 16673); S. cricetus DSMZ
20562 with Lb-OB-KI (DSMZ 16667), S. cricetus DSMZ 20562 with
Lb-OB-K2 (DSMZ 16668), S. cricetus DSMZ 20562 with Lb-OB-K3 (DSMZ
16669), S. cricetus DSMZ 20562 with Lb-OB-K4 (DSMZ 16670), S.
cricetus DSMZ 20562 with Lb-OB-K5 (DSMZ 16671), S. cricetus DSMZ
20562 with Lb-OB-K6 (DSMZ 16672), S. cricetus DSMZ 20562 with
Lb-OB-K7 (DSMZ 16673); S. ratti DSMZ 20564 with Lb-OB-K1 (DSMZ
16667), S. ratti DSMZ 20564 with Lb-OB-K2 (DSMZ 16668), S. ratti
DSMZ 20564 with Lb-OB-K3 (DSMZ 16669), S. ratti DSMZ 20564 with
Lb-OB-K4 (DSMZ 16670), S. ratti DSMZ 20564 with Lb-OB-K5 (DSMZ
16671), S. ratti DSMZ 20564 with Lb-OB-K6 (DSMZ 16672), S. ratti
DSMZ 20564 with Lb-OB-K7 (DSMZ 16673);
[0267] S. ferus DSMZ 20646 with Lb-OB-K1 (DSMZ 16667), S. ferus
DSMZ 20646 with Lb-OBK2 (DSMZ 16668), S. ferus DSMZ 20646 with
Lb-OB-K3 (DSMZ 16669), S. ferus DSMZ 20646 with Lb-OB-K4 (DSMZ
16670), S. ferus DSMZ 20646 with Lb-OB-K5 (DSMZ 16671), S. ferus
DSMZ 20646 with Lb-OB-K6 (DSMZ 16672), S. ferus DSMZ 20646 with
Lb-OB-K7 (DSMZ 16673); S. macacae DSMZ 20724 with Lb-OB-K1 (DSMZ
16667), S. macacae DSMZ 20724 with Lb-OB-K2 (DSMZ 16668), S.
macacae DSMZ 20724 with Lb-OB-K3 (DSMZ 16669), S. macacae DSMZ
20724 with Lb-OB-K4 (DSMZ 16670), S. macacae DSMZ 20724 with
LbOB-K5 (DSMZ 16671), S. macacae DSMZ 20724 with Lb-OB-K6 (DSMZ
16672) and S. macacae DSMZ 20724 with Lb-OB-K7 (DSMZ 16673)) were
mixed. 50 .mu.l of the lactobacillus suspension were added to 50
.mu.l of stained mutans Streptococci in a 96 well microtiter plate.
The plate was vortexed at full speed for 12 minutes. Afterwards the
plate was centrifuged at 500.times.g for 10 seconds. The
supernatant was carefully removed and discarded. The pellet was
resuspended in 100 .mu.l of PBS-buffer The fluorescence of the
suspension was measured in a microtiterplate fluorescence reader at
a wavelength of 495 nm for excitation and 525 nm for emission. As
controls lactobacilli alone as well as stained mutans Streptococci
were treated and measured as described. The background fluorescence
measured for the respective mutans Streptococci alone was
subtracted from the value measured for the aggregation with the
respective lactobacillus. All measurements were done in triplicate.
The mutans Streptococci were aggregated by all tested lactobacilli
(see FIG. 3).
EXAMPLE 6
Specificity of the Aggregation Towards Typical Members of the Oral
Flora
[0268] The Lactobacillus cultures were grown as described in
Example 1. The oral bacteria namely: Streptococcus salivarius
subsp. thermophilus (isolated by OrganoBalance, identified by API
50 CH (Biomerieux, France) according to manufacturer's
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 ml BHI-medium in closed 15
ml Falcon tubes at 37.degree. C. over night. Each of the above
mentioned oral bacteria was preferably mixed in a volumetric ratio
of 3:1 with Lactobacillus cultures and aggregation was assayed as
in Example 4. For each testing of aggregation/non-aggregation only
one of the aforementioned bacteria is preferably used to
immediately determine the outcome of the testing.
[0269] 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.
[0270] The L. paracasei ssp. paracasei strains Lb-OB-KI (DSMZ
16667), Lb-OB-K2 (DSMZ 16668), Lb-OB-K3 (DSMZ 16669), Lb-OB-K4
(DSMZ 16670), Lb-OB-K5 (DSMZ 16671), did not aggregate the oral
bacteria mentioned above. They are thus "specifically binding to
mutans Streptococci" according to the above definition. The L.
rhamnosus strains LbOB-K6 (DSMZ 16672) and Lb-OB-K7 (DSMZ 16673)
aggregated Streptococcus salivarius subsp thermophilus. They are
nevertheless considered "specifically binding to mutans
Streptococci" according to the above less preferred definition.
BHI-Broth:
BHI-mixture (Difco, USA) 37 g/L pH: 7.2
EXAMPLE 7
Temperature Resistance of the Aggregating Capacity of the
Lactobacilli
[0271] The bacteria were grown as in Example 1. The grown
lactobacilli cultures were autoclaved at 121.degree. C. at 2 bar in
satured steam for 20 min. 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 4 including the control experiments.
Aggregation was also assayed using the oral bacteria as outlined in
Example 6.
[0272] 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 8
Aggregation by Heat-Inactivated Lactobacilli
[0273] The lactobacilli were grown as described in Example 1.
Mutans Streptococci were grown and stained as described in Examples
1 and 3. The grown lactobacilli cultures were adjusted to an OD600
of 2 as described in Example 1. 1 ml of that suspension was
autoclaved at 121.degree. C. at 2 bar for 20 min as described
above. After cooling of the autoclaved cultures to room
temperature, aggregation was measured as described in Example 5
including control experiments. The heat-inactivated lactobacilli
still aggregated all mutans Streptococci.
EXAMPLE 9
Dependency of the Aggregation on pH-Value
[0274] The bacteria were grown as in Example 1. 0.5 ml of the
lactobacilli and 1.5 ml of S. mutans were harvested by
centrifugation at 3200.times.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.
[0275] Afterwards the lactobacilli were mixed in a volumetric ratio
of 1:3 with S. mutans cultures and aggregation was assayed as in
Example 4 including the control experiments. No visible aggregation
of S. mutans by the lactobacilli occurred at pH values lower than
4.5.
EXAMPLE 10
Dependency of the Aggregation on pH-Value
[0276] The lactobacilli were grown as described in Example 1.
Mutans Streptococci were grown and stained as described in Examples
1 and 3. Afterwards the aggregation was assayed in different
pH-values. For this purpose lactobacilli as well as streptococci
were resuspended in acetate buffer adjusted to the respective pH.
pH values tested were 4.0, 4.5 and 5.0. The aggregation was assayed
as described in Example 5. No aggregation of mutans Streptococci
occurred at pH values lower than 4.5.
EXAMPLE 11
Sensitivity of the Aggregation Behaviour to Lyophilisation
[0277] The bacteria were grown as in Example 1. Aliquots of 1 ml of
the lactobacilli cultures were harvested by centrifugation at
3200.times.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 4 including the control experiments.
[0278] The aggregation behaviour of the mentioned lactobacilli
towards S. mutans was not changed by the lyophilization or the
storage procedures
EXAMPLE 12
Sensitivity of the Aggregation Behaviour to Lyophilisation
[0279] The lactobacilli were grown as described in Example 1.
Mutans Streptococci were grown and stained as described in Examples
1 and 3. The grown lactobacilli cultures were adjusted to an OD600
of 2 as described in Example 1. 1 ml of that suspension was
lyophilized at room temperature under vacuum for two hours.
Afterwards, the lyophilised pellets were resuspended in 1 ml
PBS-buffer. Aggregation was measured as described in Example 5,
including control experiments.
[0280] The aggregation behaviour of the mentioned lactobacilli
towards mutans Streptococci was not changed by the
lyophilization.
EXAMPLE 13
Test on Protease Resistance
[0281] The bacteria were grown as in Example 1. 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.times.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.
[0282] The aggregation was assayed as in Example 3 including the
control experiments. The aggregation behaviour of the mentioned
lactobacilli towards S. mutans was not changed by treatment with
any of the mentioned proteases.
EXAMPLE 14
Protease Susceptibility of Aggregation Behaviour of the
Lactobacilli
[0283] The lactobacilli were grown as described in Example 1.
Mutans Streptococci were grown and stained as described in Examples
1 and 3. Used proteases 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.times.g for 10 min 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 mgl/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. The
aggregation was assayed as described in Example 5 including control
experiments. The aggregation behaviour of the lactobacilli towards
mutans Streptococci was not changed by the treatment with any of
the mentioned proteases.
EXAMPLE 15
Ion Dependency of the Aggregation Behaviour
[0284] The bacteria were grown as in Example 1. 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).
[0285] The aggregation was assayed as in Example 4 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 16
Ion Dependency of the Aggregation Behaviour
[0286] The lactobacilli were grown as described in Example 1.
Mutans Streptococci were grown and stained as described in Examples
1 and 3. 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). The
aggregation was assayed as described in Example 5 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 mutans
Streptococci.
EXAMPLE 17
Test of Aggregation in the Presence of Saliva
[0287] The bacteria were grown as in Example 1. 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. The aggregation was assayed as in
Example 4.
[0288] The aggregation behaviour of the mentioned lactobacilli
towards S. mutans did not change in the presence of saliva.
EXAMPLE 18
Aggregation of Mutans Streptococci in the Presence of Saliva
[0289] Fresh saliva was sampled from volunteers. Saliva-flow was
induced by chewing of sugar-free chewing gum. Volunteers collected
15 ml saliva with each sampling. The freshly collected saliva was
diluted 1:2 with PBS-buffer for the assay procedure. Lactobacilli
and mutans Streptococci were cultivated as described in Example 1.
Mutans Streptococci were stained as described in Example 3, except
that after the staining procedure the stained cells were
resuspended in saliva, instead of PBS-buffer. The aggregation was
measured as described in Example 5 including control experiments.
The presence of saliva did not inhibit the aggregation.
Isolation Example 1
Obtaining of Further Lactobacilli
[0290] Lactobacilli can be obtained from any source, e.g. by oral
cavity swabs of volunteers with low amount of caries and/or dental
calculus. Also, Lactobacilli can be obtained from cell culture
collections like DSMZ and ATCC, or from other known sources of
lactic acid bacteria, e.g. plants, foods and feeds. Strains
obtained are then purified by standard microbiological techniques.
Selective media for Lactobacilli are for example described by
Rogosa et al. 1951. A selective medium for the isolation of oral
and fecal lactobacilli, J. Bacteriol. 62: 132-133.
[0291] Isolated strains are grown as described in Example 1.
Selection for binding is performed as described in Isolation
Example 2.
Isolation Example 2
[0292] Stained Streptococcus mutans (DSMZ 20523) was prepared and
distributed to 96 well microtiter plates as described above in
Example 5. Also as described in Example 5, 50 .mu.l of the strains
to be screened for binding grown according to example 1 were added
to each microtiter plate well and vortexed at full speed for 12
minutes. Afterwards, the plate was centrifuged at 500.times.g for
10 seconds and supernatant was carefully removed. The pellet was
resuspended in 100 .mu.l PBS and fluorescence of the suspension was
measured as described in Example 5.
[0293] The strains to be screened were then tested as described
above in Example 5 for binding to S. mutans DSMZ 20523, S. sobrinus
DSMZ 20742, S. cricetus DSMZ 20562, S. ratti DSMZ 20564, S. ferus
DSMZ 20646 and S. macacae DSMZ 20724. Positive binders were found
to produce aggregates.
Food and Feed Example 1
Dog Puppy Feed
[0294] Main Ingredients: beef, brown rice, canola seed, flax seed
meal, sunflower seed, buckwheat seed (soba), barley, millet.
[0295] Lesser Ingredients: carrots, red beets, broccoli, high-oleic
sunflower oil, canola oil, sea salt, oregano, garlic, a scorbic
acid, propolis, vitamins (vitamins A, B12, D3, C, E, thiamine,
riboflavin, pyridoxine, biotin, folic acid, pantothenate, niacin),
minerals (chrome, sodium selenite, iron, cooper citrate, zinc
sulfate, cobalt).
[0296] To 1 kg of a base composition having the above main and
lesser ingredients, 3 g of dried autoclaved binder microorganisms
and fragments thereof were added. The microorganism material was
prepared by growing the binder microorganism as described in
Example 1. That is, lactobacilli were grown in MRS-medium. 5 ml
MRS-medium were inoculated with 10 .mu.l of the stock and incubated
for 3 days at 37.degree. C. under aerobic conditions. The
microorganisms were pelleted by centrifugation and washed with PBS
once. The resuspended microorganisms were autoclaved as described
in Example 8. The autoclaved microorganisms were dried in an oven
at 75.degree. C. over night to obtain the dried heat-inactivated
binder microorganisms and fragments thereof. The dog puppy food was
prepared with each of the deposited Lactobacillus strains DSMZ
16667, 16668, 16669, 16670, 16671, 16672 and 16673 separately to
obtain 7 separate puppy foods.
[0297] The total content of the following parts is (in wt.-% of the
total feed):
Protein (min) 26.0 Fat (min) 14.0 Fiber (max) 5.0 Moisture (max)
10.5
Adult Dog Feed
[0298] Main Ingredient: beef, brown rice, canola seed, flax seed
meal, sunflower seed, buckwheat seed (soba), barley, millet.
[0299] Lesser Ingredients: carrots, red beets, broccoli, high-oleic
sunflower oil, canola oil, sea salt, oregano, garlic, ascorbic
acid, propolis, vitamins (i.e. vitamins A, B12, D3, C, E, thiamine,
riboflavin, pyridoxine, biotin, folic acid, pantothenate, niacin),
minerals (chrome, sodium selenite, iron, cooper citrate, zinc
sulfate, cobalt).
[0300] As described for the dog puppy food, 7 adult dog food
preparations were prepared using the deposited Lactobacillus
strains DSMZ 16667, 16668, 16669, 16670, 16671, 16672 and 16673
separately.
[0301] The total content of the following parts is (in wt.-% of the
total feed):
Protein (min) 20.0 Fat (min) 12.0 Fiber (max) 5.0 Moisture (max)
10.5
Vegetarian Dog Feed.
[0302] Main Ingredients: soymeal, brown rice, canola seed, flax
seed meal, sunflower seed, buckwheat seed, barley, millet,
[0303] Lesser Ingredients: carrots, red beets, broccoli, high-oleic
sunflower oil, canola oil, sea salt, oregano, garlic, ascorbic
acid, propolis, vitamins (i.e. vitamins A, B12, D3, C, E, thiamine,
riboflavin, pyridoxine, biotin, folic acid, pantothenate, niacin),
minerals (i.e. chrome, sodium selenite, iron, cooper citrate, zinc
sulfate, cobalt).
[0304] As described for the dog puppy food, 7 vegetarian dog food
preparations were prepared using the deposited Lactobacillus
strains DSMZ 16667, 16668, 16669, 16670, 16671, 16672 and 16673
separately.
[0305] The total content of the following parts is (in wt.-% of the
total feed):
Protein (min) 20.0 Fat (min) 12.0 Fiber (max) 9.0 Moisture (max)
10.5.
[0306] All food preparations were fed to a separate group of 5 dogs
of the appropriate age (1-8 months, 2-4 years and 2-4 years,
respectively) once per day. As required by the dogs, other standard
feed was provided at other times of the day. However, except for
the above food preparations according to the invention no nother
feed was provided that was advertised by the respective
manufacturer as particularly promoting oral health. After 6 months,
none of the dogs had developed dental calculus. Also, oral malodor
of the dogs measured by 10 volunteers 3 h after last feeding was
considered tolerable.
Food and Feed Example 2
Dog Chew Product
[0307] A strip of cow rawhide was dunked in a culture of binder
microorganism strains DSMZ 16667, 16668, 16669, 16670, 16671, 16672
and 16673, respectively. The binder microorganisms were grown as
described in Food and Feed Example 1. The rawhide strips were then
formed into a bone-like shape and autoclaved as described in Food
and Feed Example 1.
[0308] The autoclaved strips were air dried. The dried bone-like
strips were given to 5 dogs of 5-8 years which just had their
dental calculus removed. New dried bone-like strips were then given
twice per week. Except for the dried bone-like strips, the dogs
were fed as before removal of dental calculus. Within 8 months,
none of the dogs developed dental calculus again. Also, oral
malodor of the dogs measured by 10 volunteers 3 h after last
feeding was considered tolerable.
Food and Feed Example 3
Further Dog Feeds
Puppy Feed:
[0309] Main ingredients: chicken, chicken meal, barley, peas, brown
rice, ground extruded whole soybeans, chicken fat (preserved with
mixed tocopherols).
[0310] Lesser ingredients: salmon meal, natural chicken liver
flavor, brewers dried yeast, flaxseed meal, dried eggs, apples,
carrots, potassium chloride, dicalcium phosphate, minerals (i.e.
zinc proteinate, iron proteinate, copper proteinate, manganese
proteinate, sodium selenite, cobalt proteinate, calcium iodate),
salt, choline chloride, vitamins (i.e. vitamin E supplement,
1-ascorbyl-2-polyphosphate, vitamin B12 supplement, d-calcium
pantothenate, vitamin A supplement, niacin, riboflavin, folic acid,
biotin, pyridoxine hydrochloride, thiamine mononitrate, vitamin D3
supplement, calcium carbonate), yeast culture (saccharomyces
cerevisiae), dried enterococcus faecium fermentation product, dried
lactobacillus acidophilus fermentation product, dried aspergillus
niger fermentation extract, trichoderma longibrachiatum
fermentation extract, dried bacillus subtilis fermentation extract
and fermentation solubles
[0311] The total content of the following parts is (in wt.-% of the
total feed):
Protein wt.-% (min): 28.0 Fat wt.-% (min): 15.0 Fiber wt.-% (max):
3.5 Moisture wt.-% (max): 10.0.
Adult Dog Feed:
[0312] Main ingredients: chicken, chicken meal, barley, peas, oats,
brown rice, chicken fat (preserved with mixed tocopherols).
[0313] Lesser ingredients: natural chicken liver flavor, brewers
dried yeast, salmon meal, dried eggs, apple, whole flaxseed,
carrots, dicalcium phosphate, potassium chloride, salt, minerals
(i.e. zinc proteinate, iron proteinate, copper proteinate,
manganese proteinate, sodium selenite, cobalt proteinate, calcium
iodate), choline chloride, vitamins (i.e. vitamin E supplement,
1-ascorbyl-2-polyphosphate, vitamin B12 supplement, d-calcium
pantothenate, vitamin A supplement, niacin, riboflavin, folic acid,
biotin, pyridoxine hydrochloride, thiamine mononitrate, vitamin D3
supplement), glucosamine hydrochloride, chondroitin sulfate, yeast
culture (saccharomyces cerevisiae), dried enterococcus faecium
fermentation product, dried lactobacillus acidophilus fermentation
product, dried aspergillus niger fermentation extract, dried
trichoderma longibrachiatum fermentation extract, dried bacillus
subtilis fermentation extract and fermentation solubles.
[0314] The total content of the following parts is (in wt.-% of the
total feed):
Protein wt.-% (min): 26.0 Fat wt.-% (min): 13.0 Fiber wt.-% (max):
3.5 Moisture wt.-% (max): 10.0.
Weight Control Dog Feed:
[0315] Main ingredients: brown rice, chicken meal, barley, oats,
peas, chicken, chicken fat (preserved with mixed tochopherols).
[0316] Lesser ingredients: natural chicken liver flavor, brewers
dried yeast, salmon meal, dried eggs, apple, whole flaxseed,
carrots, dicalcium phosphate, potassium chloride, salt, minerals
(i.e. zinc proteinate, iron proteinate, copper proteinate,
manganese proteinate, sodium selenite, cobalt proteinate, calcium
iodate), choline chloride, vitamins (i.e. vitamin E supplement,
1-ascorbyl-2-polyphosphate, vitamin B12 supplement, d-calcium
pantothenate, vitamin A supplement, niacin, riboflavin, folic acid,
biotin, pyridoxine hydrochloride, thiamine mononitrate, vitamin D3
supplement), glucosamine hydrochloride, chondroitin sulfate, yeast
culture (saccharomyces cerevisiae), dried enterococcus faecium
fermentation product, dried lactobacillus acidophilus fermentation
product, dried aspergillus niger fermentation extract, dried
trichoderma longibrachiatum fermentation extract, dried bacillus
subtilis fermentation extract and fermentation solubles.
[0317] The total content of the following parts is (in wt.-% of the
total feed):
Protein wt.-% (min): 24.0 Fat wt.-% (min): 10.0 Fiber wt.-% (max):
3.5 Moisture wt.-% (max): 10.0.
[0318] As described for the dog puppy food of Food and Feed Example
1, 7 corresponding puppy, adult and weight control dog food
preparations were prepared using the deposited Lactobacillus
strains DSMZ 16667, 16668, 16669, 16670, 16671, 16672 and 16673
separately.
High Protein Adult Dog Feed:
[0319] Main Ingredients: De-boned chicken, chicken meal, turkey
meal, russet potato, lake whitefish, chicken fat (preserved with
mixed tocopherols).
[0320] Lesser Ingredients: Sweet potato, whole eggs, turkey, salmon
meal, salmon and anchovy oils, salmon, natural chicken flavor,
sunflower oil, sun-cured alfalfa, dried brown kelp, carrots,
spinach, peas, tomatoes, apples, psyllium, dulse, chicory root,
licorice root, tumeric root, fenugreek, glucosamine HCl,
cranberries, black currants, marigold flowers, L-carnitine, sweet
fennel, zea mays, peppermint leaf, chamomile flowers, dandelion,
summer savory, rosemary extract, chondroitin sulfate, rosehips,
vitamins (i.e. vitamin E, choline chloride, vitamin A, vitamin D3,
thiamine mononitrate, vitamin B12, folic acid, biotin), sea salt,
minerals (i.e. iron proteinate, zinc proteinate, manganese
proteinate, copper proteinate), dried Lactobacillus acidophilus
fermentation product, dried Enterococcus faecium fermentation
product.
[0321] The total content of the following parts is (in wt.-% of the
total feed):
Protein min: 42 wt.-%
Fat min: 16 wt.-%
Fiber max 3 wt.-%
Moisture max: 10 wt.-%
Ash max: 7 wt.-%.
[0322] As described for the dog puppy food of Food and Feed Example
1, 7 corresponding dog feed preparations were prepared using the
deposited Lactobacillus strains DSMZ 16667, 16668, 16669, 16670,
16671, 16672 and 16673 separately.
Vegetarian Dog Feed:
[0323] Main Ingredients: brown rice, canola seed, flax seed meal,
sunflower seed, buckwheat seed (soba), barley, millet, carrots, red
beets, broccoli, high-oleic sunflower oil.
[0324] Lesser Ingredients: canola oil, sea salt, oregano, garlic,
ascorbic acid, propolis, vitamins (i.e. vitamins A, B12, D3, C, E,
K3, thiamine, riboflavin, pyridoxine, biotin, folic acid,
pantothenate, niacin), minerals (i.e. chrome, sodium selenite,
iron, cooper citrate, zinc sulfate, cobalt).
[0325] The total content of the following parts is (in wt.-% of the
total feed):
Protein min: 20 wt.-%
Fat min: 12 wt.-%
Fiber max: 9 wt.-%
Moisture max: 10.5 wt.-%.
[0326] As described for the dog puppy food of Food and Feed Example
1, 7 corresponding dog feed preparations were prepared using the
deposited Lactobacillus strains DSMZ 16667, 16668, 16669, 16670,
16671, 16672 and 16673 separately.
FURTHER EMBODIMENTS
[0327] 1. Binder microorganism or fragment thereof, wherein the
microorganism or fragment thereof is capable of binding to at least
one, preferably at least two and more preferably at least three
Streptococcus strain of the mutans Streptococcus group, and wherein
the binding is resistant to heat treatment and/or resistant to
protease treatment and/or is calcium dependent and/or is formed
within a pH above 4.0 and/or is independent of magnesium and/or
formed in the presence of saliva as an oral care agent. [0328] 2.
Binder microorganism or fragment thereof according to embodiment 1,
wherein the binding is resistant to heat treatment at 55.degree.
C., preferably at 65.degree. C., more preferably at 95-121.degree.
C. and most preferably at 121.degree. C. [0329] 3. Binder
microorganism or fragment thereof according to embodiment 1,
wherein the binding is resistant to heat treatment for 15 min,
preferably 15-120 min, preferably 15-30 min and most preferably 20
min. [0330] 4. Binder microorganism or fragment thereof according
to embodiment 3, wherein the binding is resistant to heat treatment
in saturated steam at a pressure of 1-5 bar, preferably 1-3 bar,
more preferably 2 bar. [0331] 5. Binder microorganism or fragment
thereof according to embodiment 1, wherein the binding is resistant
to heat treatment in saturated steam at a pressure of 1-5 bar,
preferably 1-3 bar, more preferably 2 bar, at a temperature of
95-121.degree. C., more preferably at 121.degree. C., for 15 min,
preferably 15-120 min, preferably 15-30 min and most preferably 20
min. [0332] 6. Binder microorganism or fragment thereof according
to embodiment 1, wherein the binding is resistant to heat
treatment, wherein the heat treatment is in saturated steam at a
pressure of 2 bar at a temperature of 121.degree. C. for 20 min.
[0333] 7. Binder microorganism or fragment thereof according to
embodiment 1, wherein the binding is resistant to protease
treatment by pronase E, proteinase K, trypsin and/or chymotrypsin,
and preferably is also resistant to protease treatment by elastase,
thrombin, aminopeptidase I, carboxypeptidase, dostripain,
endoproteinase, papain, cathepsin B, pepsin, gastricsin, chymosin
and/or cathepsin D. [0334] 8. Binder microorganism or fragment
thereof according to embodiment 7, wherein the binding is resistant
to protease treatment by pronase E, proteinase K, trypsin and
chymotrypsin. [0335] 9. Binder microorganism or fragment thereof
according to embodiment 7, wherein the binding is resistant to
protease treatment by elastase, thrombin, aminopeptidase I,
carboxypeptidase, dostripain, endoproteinase, papain, cathepsin B,
pepsin, gastricsin, chymosin and cathepsin D. [0336] 10. Binder
microorganism or fragment thereof according to embodiment 1,
wherein the binding is resistant to heat treatment in saturated
steam at a pressure of 1-5 bar, preferably 1-3 bar, more preferably
2 bar, at a temperature of 95-121.degree. C., more preferably at
121.degree. C., for 15 min, preferably 15-120 min, preferably 15-30
min and most preferably 20 min; and also is resistant to protease
treatment by pronase E, proteinase K, trypsin and/or chymotrypsin,
and preferably is also resistant to protease treatment by elastase,
thrombin, aminopeptidase I, carboxypeptidase, dostripain,
endoproteinase, papain, cathepsin B, pepsin, gastricsin, chymosin
and/or cathepsin D. [0337] 11. Binder microorganism or fragment
thereof according to embodiment 6, wherein the binding is resistant
to protease treatment by pronase E, proteinase K, trypsin and/or
chymotrypsin, and preferably is also resistant to protease
treatment by elastase, thrombin, aminopeptidase I,
carboxypeptidase, dostripain, endoproteinase, papain, cathepsin B,
pepsin, gastricsin, chymosin and/or cathepsin D. [0338] 12. Binder
microorganism or fragment thereof according to embodiment 8,
wherein the binding is resistant to heat treatment, wherein the
heat treatment is in saturated steam at a pressure of 2 bar at a
temperature of 121.degree. C. for 20 min. [0339] 13. Binder
microorganism or fragment thereof according to embodiment 1,
wherein the binding is dependent on the presence of Ca2+ ions in a
concentration of at least 2 mM, more preferably at least 1 mM, most
preferably at least 0.05 mM. [0340] 14. Binder microorganism or
fragment thereof according to embodiment 1, wherein the binding is
dependent on the presence of Ca2+ ions in a concentration of
0.05-500 mM, more preferably 1-100 mM, most preferably 2-30 mM.
[0341] 15. Binder microorganism or fragment thereof according to
embodiment 12 or 13, wherein the binding is resistant to heat
treatment, wherein the heat treatment is in saturated steam at a
pressure of 2 bar at a temperature of 121.degree. C. for 20 min.
[0342] 16. Binder microorganism or fragment thereof according to
embodiment 12 or 13, wherein the binding is resistant to protease
treatment by pronase E, proteinase K, trypsin and/or chymotrypsin,
and preferably is also resistant to protease treatment by elastase,
thrombin, aminopeptidase I, carboxypeptidase, dostripain,
endoproteinase, papain, cathepsin B, pepsin, gastricsin, chymosin
and/or cathepsin D. [0343] 17. Binder microorganism or fragment
thereof according to embodiment 1, wherein the binding is formed at
a pH of 4.0-9.0, preferably 4.0-7.0, more preferably 4.2-5.0 and
most preferably 4.5. [0344] 18. Binder microorganism or fragment
thereof according to embodiment 16, wherein the binding is
resistant to heat treatment, wherein the heat treatment is in
saturated steam at a pressure of 2 bar at a temperature of
121.degree. C. for 20 min. [0345] 19. Binder microorganism or
fragment thereof according to embodiment 16, wherein the binding is
resistant to protease treatment by pronase E, proteinase K, trypsin
and/or chymotrypsin, and preferably is also resistant to protease
treatment by elastase, thrombin, aminopeptidase I,
carboxypeptidase, dostripain, endoproteinase, papain, cathepsin B,
pepsin, gastricsin, chymosin and/or cathepsin D. [0346] 20. Binder
microorganism or fragment thereof according to embodiment 16,
wherein the binding is dependent on the presence of Ca2+ ions in a
concentration of at least 2 mM, more preferably at least 1 mM, most
preferably at least 0.05 mM. [0347] 21. Binder microorganism or
fragment thereof according to embodiment 1, wherein the binding is
independent of the presence of magnesium ions, and preferably is
resistant to heat treatment, wherein the heat treatment is in
saturated steam at a pressure of 2 bar at a temperature of
121.degree. C. for 20 min. [0348] 22. Binder microorganism or
fragment thereof according to embodiment 1, wherein the binding is
formed in the presence of saliva, and preferably is resistant to
heat treatment, wherein the heat treatment is in saturated steam at
a pressure of 2 bar at a temperature of 121.degree. C. for 20 min.
[0349] 23. Binder microorganism or fragment thereof according to
embodiment 1, wherein the binding is resistant to heat treatment,
resistant to protease treatment, and is formed in the presence of
saliva. [0350] 24. Binder microorganism or fragment thereof
according to any of the previous embodiments, wherein the strain of
mutans Streptococcus is selected from the group consisting of
Streptococcus mutans serotype c (DSMZ 20523), Streptococcus mutans
serotype e (NCTC 10923), Streptococcus mutans serotype f (NCTC
11060), Streptococcus sobrinus DSMZ 20742, Streptococcus ratti DSMZ
20564, Streptococcus cricetus DSMZ 20562, Streptococcus ferus DSMZ
20646 and Streptococcus macacae DSMZ 20714, and preferably wherein
the specific binding of the binder microorganism or fragment
thereof can be assayed as follows: [0351] (a) growing said binder
microorganism to stationary phase, or, in case a fragment is to be
tested, obtaining such fragment, [0352] (b) mixing said binder
microorganism or fragment with a mutans Streptococcus which has
been grown to stationary phase, [0353] (c) incubating the mixture
obtained in step (b) under conditions allowing the formation of
aggregates of said microorganism and said Streptococcus, and [0354]
(d) detecting aggregates by the occurrence of a pellet. [0355] 25.
Binder microorganism or fragment thereof according to any of
embodiments 1-22,
[0356] wherein the microorganism or fragment thereof is capable of
binding to each of the strains selected from the group consisting
of Streptococcus mutans serotype c (DSMZ 20523), Streptococcus
mutans serotype e (NCTC 10923) and Streptococcus mutans serotype f
(NCTC 11060). [0357] 26. Binder microorganism or fragment thereof
according to embodiment 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24, wherein the
microorganism or fragment thereof is not capable of binding to at
least one, preferably at least two and more preferably at least
three and even more preferably all microorganisms selected from the
group consisting of Streptococcus salivarius ssp. thermophilus,
Streptococcus oralis DSMZ 20066, Streptococcus oralis DSMZ 20395,
Streptococcus oralis DSMZ 20627, Streptococcus mitis DSMZ 12643 and
Streptococcus sanguinis DSMZ 20567. [0358] 27. Binder microorganism
or fragment thereof according to embodiment 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or
25, wherein the binder microorganism is of family Lactobacillaceae,
preferably of genus Lactobacillus, genus Paralactobacillus, genus
Pediococcus or genus Sharpea, more preferably of species
Lactobacillus paracasei, species Lactobacillus rhamnosus, species
Lactobacillus casei or species Lactobacillus zeae, and most
preferably is any of strains DSMZ 16667, DSMZ 16668, DSMZ 16669,
DSMZ 16670, DSMZ 16671, DSMZ 16672 or DSMZ 16673, or a mutant or
derivative thereof. [0359] 28. Binder microorganism or fragment
thereof according to embodiment 26, wherein the microorganism or
fragment thereof is capable of binding to each of the strains
selected from the group consisting of Streptococcus mutans serotype
c (DSMZ 20523), Streptococcus mutans serotype e (NCTC 10923) and
Streptococcus mutans serotype f (NCTC 11060) and is not capable of
binding to a microorganism selected from the group consisting of
Streptococcus salivarius ssp. thermophilus, Streptococcus oralis
DSMZ 20066, Streptococcus oralis DSMZ 20395, Streptococcus oralis
DSMZ 20627, Streptococcus mitis DSMZ 12643 and Streptococcus
sanguinis DSMZ 20567. [0360] 29. Binder microorganism or fragment
thereof according to embodiment 26 or 27, wherein the microorganism
or fragment thereof, after heat treatment in saturated steam at a
pressure of 2 bar at a temperature of 121.degree. C. for 20 min,
retains the capability to bind to a mutans Streptococcus selected
from the group consisting of Streptococcus mutans serotype c (DSMZ
20523), Streptococcus mutans serotype e (NCTC 10923), Streptococcus
mutans serotype f (NCTC 11060), Streptococcus sobrinus DSMZ 20742,
Streptococcus ratti DSMZ 20564, Streptococcus cricetus DSMZ 20562,
Streptococcus ferus DSMZ 20646 and Streptococcus macacae DSMZ 20714
in the presence of at least 0.05 mM calcium ions and in the
presence of saliva and independent of a treatment by pronase E,
proteinase K, trypsin and/or chymotrypsin, wherein under these
conditions the heat treated microorganism or fragment thereof is
not capable of binding to a microorganism selected from the group
consisting of Streptococcus salivarius ssp. thermophilus,
Streptococcus oralis DSMZ 20066, Streptococcus oralis DSMZ 20395,
Streptococcus oralis DSMZ 20627, Streptococcus mitis DSMZ 12643 and
Streptococcus sanguinis DSMZ 20567. [0361] 30. Binder microorganism
according to any of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29 or 30, wherein the binder microorganism is in an inactivated
form obtainable or obtained by thermal inactivation, lyphilisation
or spray drying, wherein thermal inactivation is preferably
achieved by [0362] autoclaving cells of said binder microorganism
at a temperature of 121.degree. C. for at least 20 min in the
presence of saturated steam at an atmospheric pressure of 2 bar, or
[0363] freezing said cells for at least 1 h at -20.degree. C.
[0364] 31. Inactivated binder microorganism according to embodiment
29, obtainable or obtained by thermal inactivation at a temperature
of .gtoreq.55.degree. C., preferably at a temperature of
.gtoreq.65.degree. C., even more preferably at a temperature of
95-121.degree. C. and most preferably at a temperature of
121.degree. C. [0365] 32. Inactivated binder microorganism
according to embodiment 30, obtainable or obtained by thermal
inactivation for .gtoreq.15 min, preferably 15-120 min, preferably
15-30 min and most preferably 20 min. [0366] 33. Inactivated binder
microorganism according to embodiment 29, 30 or 31, obtainable or
obtained by thermal inactivation in saturated steam at a pressure
of 1-5 bar, preferably 1-3 bar, more preferably 2 bar, and most
preferably by thermal inactivation in saturated steam at a pressure
of 2 bar at a temperature of 121.degree. C. for 20 min. [0367] 34.
Fragment of a binder microorganism according to any of the previous
embodiments, wherein the fragment is a lysate or membrane fragment
of a binder microorganism. [0368] 35. Binder microorganism or
fragment thereof according to any of embodiments 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32 or 33, as a sensorically neutral
oral care agent. [0369] 36. Binder microorganism or fragment
thereof according to any of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, 30, 31, 32 or 33, as an anti-dental calculus agent,
sensorically neutral anti-caries agent and/or anti-oral malodor
agent. [0370] 37. Binder microorganism or fragment thereof
according to embodiment 35, as a sensorically neutral anti-dental
calculus agent, sensorically neutral anti-caries agent and/or
sensorically neutral anti-oral malodor agent. [0371] 38. Binder
microorganism or fragment thereof according to any of embodiments
34, 35 or 36, wherein the binder microorganism or fragment thereof
is an isolated or purified binder microorganism or fragment
thereof. [0372] 39. Composition comprising a binder microorganism
or fragment thereof according to any of embodiments 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37 as an oral
care agent, preferably as an anti-dental calculus agent, an
anti-caries agent and/or an anti-oral malodor agent. [0373] 40.
Composition comprising a binder microorganism or fragment thereof
according to any of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36 or 37 as a sensorically neutral oral
care agent, preferably as a sensorically neutral anti-dental
calculus agent, a sensorically neutral anti-caries agent and/or a
sensorically neutral anti-oral malodor agent. [0374] 41.
Composition comprising a binder microorganism or fragment thereof
according to any of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36 or 37 in an amount [0375] sufficient
for preventing or reducing intensity of oral malodor, and/or [0376]
sufficient for preventing caries or slowing down caries generation
and/or [0377] sufficient for preventing dental calculus formation
or slowing down dental calculus formation. [0378] 42. Composition
comprising a binder microorganism according to any of embodiments
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36 or
37, in a concentration of [0379] 10.sup.2-10.sup.13 cells/mg,
preferably 10.sup.2-10.sup.12 cells/mg, even more preferably
10.sup.3-10.sup.8 cells/mg, or [0380] 10.sup.2-10.sup.13 cells/ml,
or [0381] .gtoreq.0.01 wt.-% relative to the total composition,
preferably 0.01-10 wt.-% and more preferably 0.025-2 wt.-%, or
[0382] .gtoreq.0.01 mg dry weight/g total composition, preferably
0.01-100 mg dry weight/g total composition and more preferably
0.025-2 mg dry weight/g total composition. [0383] 43. Composition
comprising a fragment of a binder microorganism according to any of
embodiments 32, 33, 34, 35, 36 or 37, in a concentration of [0384]
.gtoreq.0.01 wt.-% relative to the total composition, preferably
0.01-10 wt.-% and more preferably 0.025-2 wt.-%, or [0385]
.gtoreq.0.01 mg dry weight/g total composition, preferably 0.01-100
mg dry weight/g total composition and more preferably 0.025-2 mg
dry weight/g total composition. [0386] 44. Composition according to
any of embodiments 38, 39, 40, 41 or 42, comprising a further
microorganism in a viable, a thermally inactivated or lyphilized
form, wherein the thermally inactivated form is obtainable or
obtained by treatment in saturated steam at a pressure of 1-5 bar,
preferably 1-3 bar, more preferably 2 bar, and most preferably by
thermal inactivation in saturated steam at a pressure of 2 bar at a
temperature of 121.degree. C. for 20 min. [0387] 45. Composition
according to embodiment 43, wherein the further microorganism is an
anti-oral malodor microorganism in a concentration sufficient for
preventing, modifying or reducing oral malodor, preferably capable
of stimulating the growth of Streptococcus salivarius but does not
stimulate the growth of Streptococcus mutans and/or Porphyromonas
gingivalis. [0388] 46. Composition according to embodiment 44,
wherein the anti-oral malodor microorganism is of species
Lactobacillus acidophilus, and preferably is any of the
Lactobacillus strains DSMZ 19825, DSMZ 19826 or DSMZ 19827. [0389]
47. Composition according to any of embodiments 38, 39, 40, 41, 42,
43, 44 or 45, [0390] wherein the composition is for use with a
human or animal, and is a toothpaste, dentifrice, tooth powder,
topical oral gel, mouth rinse, denture product, mouthspray,
lozenge, oral tablet, chewing gum, mouth wash, dental floss, chew
product or an additive for food, feed or drinks, or [0391] wherein
the composition is in the form of a powder, tablet, film
preparation, solution, aerosol, granule, pill, suspension,
emulsion, capsule, syrup, liquid, elixir, extract, tincture or
fluid extract, sheet-like food, bottled food, canned food, retort
food or fluid food or [0392] wherein the composition is a food or
drink selected from the group consisting of gum, spray, beverage,
candy, infant formula, ice cream, frozen dessert, sweet salad
dressing, milk preparation, cheese, quark, yogurt, acidified milk,
coffee cream, whipped cream, butter, cheese, processed milk and
skimmed milk, meat product--preferably ham, sausage, and hamburger,
fish meat, cake product, egg product--preferably seasoned egg rolls
and egg curd, confectionery--preferably cookie, jelly, snacks, and
chewing gum--, bread, noodles, pickle, smoked product, dried fish,
seasoning. [0393] 48. Composition according to any of embodiments
38, 39, 40, 41, 42, 43, 44 or 45, wherein the composition is a food
or feed composition. [0394] 49. Food or feed composition according
to embodiment 47, wherein the composition is for an infant or for a
pet animal, preferably a dog, cat, rat, mouse, hamster, guinea pig
or monkey. [0395] 50. Pet food or feed composition according to
embodiment 48, wherein the composition is a pet chew product, and
preferably is in the shape of a bone, a roll, a donut, a bow, a
pretzel, a figure eight, or a chip. [0396] 51. Composition
according to any of embodiments 38, 39, 40, 41, 42, 43, 44, 45, 46,
47 or 48, wherein the composition is a cosmetic, pharmaceutical or
veterinary composition. [0397] 52. Pet food or feed composition,
[0398] comprising an inactivated binder microorganism in a
thermally inactivated or lyphilized form, wherein the inactivated
microorganism is capable of binding to a Streptococcus strain of
the mutans Streptococcus group, and wherein the binding is formed
in the presence of saliva and is resistant to heat treatment and
calcium dependent, wherein the heat treatment is in saturated steam
at a pressure of 2 bar at a temperature of 121.degree. C. for 20
min, [0399] and wherein the mutans Streptococcus strain is selected
from the group consisting of Streptococcus mutans serotype c (DSMZ
20523), Streptococcus mutans sero-type e (NCTC 10923) and
Streptococcus mutans serotype f (NCTC 11060), [0400] and wherein
the inactivated binder microorganism thereof is not capable of
binding to a microorganism selected from the group consisting of
Streptococcus salivarius ssp. thermophilus, Streptococcus oralis
DSMZ 20066, Streptococcus oralis DSMZ 20395, Streptococcus oralis
DSMZ 20627, Streptococcus mitis DSMZ 12643 and Streptococcus
sanguinis DSMZ 20567, [0401] and wherein the binder microorganism
is present in a sensorically neutral amount [0402] sufficient for
preventing or reducing intensity of oral malodor, and/or [0403]
sufficient for preventing caries or slowing down caries generation
and/or [0404] sufficient for preventing dental calculus formation
or slowing down dental calculus formation. [0405] 53. Pet food
composition according to embodiment 51, wherein concentration of
inactivated binder microorganism is [0406] 10.sup.2-10.sup.13
cells/mg, preferably 10.sup.2-10.sup.12 cells/mg, even more
preferably 10.sup.3-10.sup.8 cells/mg, or [0407] 10.sup.2-10.sup.13
cells/ml, or [0408] .gtoreq.0.01 wt.-% relative to the total
composition, preferably 0.01-10 wt.-% and more preferably 0.025-2
wt.-%, or [0409] .gtoreq.0.01 mg dry weight/g total composition,
preferably 0.01-100 mg dry weight/g total composition and more
preferably 0.025-2 mg dry weight/g total composition. [0410] 54.
Pet food or feed composition, comprising a fragment according to
embodiment 33 in a concentration of [0411] .gtoreq.0.01 wt.-%
relative to the total composition, preferably 0.01-10 wt.-% and
more preferably 0.025-2 wt.-%, or [0412] .gtoreq.0.01 mg dry
weight/g total composition, preferably 0.01-100 mg dry weight/g
total composition and more preferably 0.025-2 mg dry weight/g total
[0413] 55. Use of a binder microorganism or fragment thereof
according to any of embodiments 1-37 as a sensorically neutral oral
care agent, preferably as a sensorically neutral anti-dental
calculus agent and/or sensorically neutral anti-caries agent and/or
sensorically neutral anti-oral malodor agent. [0414] 56. Use of a
binder microorganism or fragment thereof according to any of
embodiments 1-37 in the manufacture of any of [0415] a medicament
for prevention or treatment of dental calculus formation, [0416] a
medicament for prevention or treatment of caries, [0417] a
medicament for prevention or treatment of oral malodor, wherein the
binder microorganism and/or fragment thereof is in a sensorically
neutral amount.
* * * * *