Note: Descriptions are shown in the official language in which they were submitted.
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TITLE: Plaque-inhibiting oral compositions
FIELD OF THE INVENTION
The present invention relates to oral compositions comprising plaque-
inhibiting
or plaque-removing enzymes and to a method for inhibiting plaque formation or
removing plaque using such oral compositions.
BACKGROUND OF THE INVENTION
Dental plaque is a mixture of bacteria, epithelial cells, leukocytes,
macrophages and
other oral exudates that forms on the surface of unclean teeth. The bacteria
produce
highly branched polysaccharides which together with microorganisms from the
oral
cavity form an adhesive matrix for the continued proliferation of plaque. Left
untreated,
the formation of dental plaque will eventually lead to dental caries, gingival
inflammation,
periodontal disease, and eventually tooth loss. As plaque continues to
accumulate, rock-
hard white or yellowish deposits arise. These deposits are called calcified
plaque,
calculus or tartar, and are formed in the saliva from plaque and minerals, in
particular
calcium.
Oral polysaccharides are produced from sucrose introduced into the mouth, e.g.
as a food or beverage constituent, by the action of cariogenic microorganisms
such as
Streptococcus mutans or Streptococcus sanguis growing in the oral cavity.
These oral
polysaccharides comprise water-soluble dextran having primarily a-1,6
glucosidic
linkages, and a major component of water-insoluble extracellular
polysaccharides called
"mutan" comprised of a backbone with a-1,3-glycosidic linkages and branches
with a-
1,6-glycosidic linkages. Mutan binds to hydroxyapatite (constituting the hard
outer
porous layer of the teeth) and to acceptor proteins on the cell surface of
said cariogenic
bacteria adhering to the teeth surface.
Numerous attempts have been made to combat this process by either
eliminating the plaque or by altering the environment by changing the
composition of
the plaque, and many toothpastes and other oral care compositions have, with
varying
success, aimed at removing or inhibiting plaque. The goal of effectively
inhibiting
plaque in particular has remained elusive, however, and the need remains for
oral care
compositions that in normal use are effective at inhibiting plaque formation
and the
accompanying dental diseases that can result from plaque formation.
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WO 97/06775 discloses oral compositions comprising an oxidoreductase, and
optionally a dextranase and/or a mutanase, for bleaching of teeth, but no
plaque-
inhibiting or plaque-removing effects of such compositions are described or
suggested.
To prevent the formation of dental caries, plaque and tartar, it has been
suggested to
add a dextranase and/or a mutanase and/or other enzymes to oral care
compositions and
products.
JP patent publication 8012544 (Lion) describes a plaque preventing effect of
dextranase, mutanase and triclosan and/or biosol.
US patent No. 4,353,891 (Guggenheim et al.) concerns plaque removal using
mutanase from Trichoderma harzianum CBS 243.71 to degrade mutan synthesized by
cultivating Streptococcus mutans strain CBS 350.71 identifiable as OMZ 176. It
is
stated that the critical ingredient in dental plaque is a water-insoluble
polysaccharide with
a-1,3-glucosidic bonds and that such polysaccharide material termed mutan is
not
attacked by dextranase.
Guggenheim et al. (1972), Caries Res. 6, p. 289-297, disclose that the extent
of
dental plaque of rats is not significantly affected by the simultaneous use of
a dextranase
and a 1,3-glucanase (mutanase).
Hare et al. (1978), Carbohydrate Research 66, p. 245-264, found that a
synergistic
effect is obtained when hydrolysing and solubilizing oral glucans with a
bacterial
dextranase in combination with bacterial a-1,3 glucanase from Cladosporium
resinae.
US patent No. 4,438,093 (The Research Foundation for Microbial diseases of
Osaka)
describes oral compositions comprising a dextranase and a a-1,3-glucanase
(mutanase),
both being present in an amount of 0.5 to 100 enzyme units per gram of said
oral
composition, in an enzyme unit ratio of 1:2 to 2:1. Said dextranase is derived
from a
bacteria within the genus Corynbacterium and said a-1,3-glucanase is derived
from a
bacteria within the genus Pseudomonas.
GB 2,206,585 (Dental Chem Co. Ltd.) describes a tooth-cleaning agent
containing
hydroxyapatite as a polishing agent, with a laevanase, dextranase and mutanase
immobilized on the hydroxyapatite.
US patent No. 5,145,665 (Henkel) discloses a composition for the care of the
mouth
and teeth comprising a dextranase and/or a 1,3-glucanase for cleaving
polysaccharides in
the mouth.
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FR 2,651,433 (DANA) concerns dentifrice products containing a dextranase to
act
on recent plaque, a mutanase to act on old and insoluble plaque, and a mixture
of other
enzymes having bactericidal action
US patent No. 5,320,830 (Proctor & Gamble) describes toothpaste compositions
for the reduction of plaque and gingivitis comprising a) a surfactant, b) an
enzyme, c) a
chelating agent d) a fluoride source, e) a silica abrasive and f) a carrier.
The enzyme is an
endoglucanase, papain, a dextranase and/or a mutanase.
It has now surprisingly been found that oral care compositions comprising one
or more starch-hydrolysing or starch-modifying enzymes are effective for
inhibiting/preventing dental plaque formation and/or for removing plaque.
SUMMARY OF THE INVENTION
The object of the present invention is thus to provide oral compositions that
are
effective for inhibiting/preventing plaque formation and for removing plaque,
as well as
a method for inhibiting plaque formation or for removing plaque.
A first aspect of the invention thus relates to an oral care composition
comprising a plaque-inhibiting and/or plaque-removing effective amount of at
least one
starch-hydrolysing enzyme and/or at least one starch-modifying enzyme.
In a second aspect, the invention relates to a method for inhibiting plaque
formation or removing plaque, comprising contacting the teeth and/or gums with
an
oral care composition comprising a plaque-inhibiting and/or plaque-removing
effective
amount of at least one starch-hydrolysing enzyme and/or at least one starch-
modifying
enzyme for a period of time to obtain a plaque-inhibiting or plaque-removing
effect.
In a third aspect, the invention relates to the use of one or more starch-
hydrolysing enzymes andlor one or more starch-modifying enzymes for the
preparation
of a composition for the inhibition/prevention of plaque formation and/or
removal of
plaque.
DETAILED DESCRIPTION OF THE INVENTION
The term "starch-hydrolysing enzyme" in the context of the present application
refers to any enzyme, such as an a-amylase (E.C. 3.2.1.1 ), which functions to
hydrolyse linkages in starch.
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The term "starch-modifying enzymes" used in the context of the invention
refers
to the group of enzymes within E.C. 2.4.1., and includes in particular
transglycosidases (E.C. 2.4.1.18) and CGTases (E.C. 2.4.1.19).
The inventors have found that dental plaque can be inhibited/prevented or
removed by the use of at least one starch-hydrolysing enzyme and/or at feast
one
starch-modifying enzyme. Said effect of such enzymes has not been foreseen
before.
In the first aspect the invention relates to an oral care composition
comprising a
plaque-inhibiting and/or plaque-removing effective amount of at least one
starch-
hydrolysing enzyme and/or at least one starch-modifying enzyme.
In an embodiment of the invention the starch-modifying enzyme is a CGTase
(E.C. 2.4.1.19) or a transglucosidase (2.4.1.18).
When the starch-modifying enzyme is a CGTase, it may be derived from a strain
of Bacillus autolyticus, a strain of Bacillus cereus, a strain of Bacillus
circulars, a strain of
Bacillus circulars var. alkalophilus, a strain of Bacillus coagulans, a strain
of Bacillus
firmus, a strain of Bacillus halophilus, a strain of Bacillus macerans, a
strain of Bacillus
megaterium, a strain of Bacillus ohbensis, a strain of Bacillus
stearothermophilus, a strain
of Bacillus subtilis, a strain of Klebsiella pneumoniae, a strain of
Thermoanaerobacter
sp., a strain of Thermoanaerobacter ethanolicus, a strain of
Thermoanaerobacter finnii, a
strain of Clostridium thermoamylolyticum, a strain of Clostridium
thermosaccharolyticum,
or a strain of Thermoanaerobacterium thermosulfurigenes.
When the starch-modifying enzyme is a transglucosidase, it may be derived from
Aspergillus niger, e.g. the product sold by Amamo Pharmaceutical Co., Japan.
In another embodiment of the invention the oral care composition comprises a
starch-hydrolysing enzyme.
This will typically be an a-amylase, such as a bacterial a-amylase, such as
BAN'"
or Maltogenase"" (both available from Novo Nordisk), or an a-amylase derived
from
Bacillus subtilis; an a-amylase derived from Bacillus amyloliquefaciens; an a-
amylase
derived from Bacillus stearothermophilus; an a-amylase derived from
Aspergillus oryzae;
or an a-amylase derived from a non-pathogenic microorganism.
The a-amylase may also be a fungal a-amylase, such as FungamylT"", which is
available from Novo Nordisk.
The starch-hydrolysing enzyme may in another embodiment of the invention be a
debranching enzyme, in particular a pullulanase (E.C. 3.2.1.41 ), such as
PromozymeTM.
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In a preferred embodiment the oral care composition comprises at least one
starch-modifying enzyme as defined above, in particular a CGTase, and a
mutanase
and/or a dextranase.
In another preferred embodiment the oral care composition of the invention
5 comprises at least one starch-hydrolysing enzyme as defined above, in
particular a
bacterial a-amylase, and a mutanase and/or a dextranase.
The mutanase may be derived from a strain of Trichoderma sp., in particular T.
harzianum, especially T, harzianum CBS 243.71 (available from Novo Nordisk).
The dextranase may be derived from a strain of Paecilomyces sp., in particular
Paecilomyces iilacinus (available from Novo Nordisk).
In a second aspect the invention relates to an oral care product comprising an
oral care composition of the invention.
An "oral care product" of the invention is defined as a product which can be
used for maintaining and/or improving oral hygiene in the mouth of humans and
animals, and/or preventing or treating dental diseases.
Examples of such oral care products include toothpastes, dental creams, gels
or
tooth powders, odontics, mouthwashes, denture cleaning agents, pre- or post-
brushing rinse formulations, chewing gum and lozenges. An oral care product
may
also be in the form of a dental floss or toothpick.
Toothpastes and tooth gels typically include abrasive polishing materials,
foaming agents, flavouring agents, humectants, binders, thickeners, sweeteners
and
water. As used herein, the term "toothpaste" is intended to refer to
formulations in
the form of both pastes and gels.
Mouthwashes typically comprise a water/alcohol solution, flavouring agents,
humectants, sweeteners, foaming agents and colorants.
A chewing gum according to the invention may be prepared in a manner known
per se by incorporating one or more enzymes into a conventional chewing gum
base,
e.g. based on chicle and/or one or more additional synthetic or natural
polymers, and
containing e.g. natural and/or artificial sweeteners, flavourings, etc. as
desired. Upon
addition of the enzymes) to the gum base, the temperature of the gum base
should
not be too high, e.g. preferably not more than about 60°C, more
preferably not more
than about 50°C.
Abrasive polishing materials for use in oral care products according to the
invention include alumina and hydrates thereof, such as alpha alumina
trihydrate,
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magnesium trisilicate, magnesium carbonate, sodium bicarbonate ("baking
soda"),
kaolin, aluminosilicates, such as calcined aluminum silicate and aluminum
silicate,
calcium carbonate, zirconium silicate, and also powdered plastics, such as
polyvinyl
chloride, polyamides, polymethyl methacrylate, polystyrene, phenol-
formaldehyde
resins, melamine-formaldehyde resins, urea-formaldehyde resins, epoxy resins,
powdered polyethylene, silica xerogels, hydrvgels and aerogels and the like.
Also
suitable as abrasive agents are calcium pyrophosphate, water-insoluble alkali
metaphosphates, dicalcium phosphate and/or its dehydrate, dicalcium
orthophosphate,
tricalcium phosphate, particulate hydroxyapatite and the like. It is also
possible to
employ mixtures of these substances.
Depending on the nature of the oral care product, the abrasive product may be
present in an amount of from 0 to 70°~ by weight, preferably from 1
°~ to 70%. For
toothpastes, the abrasive material content typically ties in the range from
10°~6 to 70%
by weight of the final toothpaste product.
Humectants are employed to prevent loss of water from e.g. toothpastes.
Suitable humectants for use in oral care products according to the invention
include
the following compounds and mixtures thereof: glycerol, polyol, sorbitol,
polyethylene
glycols (PEG), propylene glycol, 1,3-propanediol, 1,4-butane-diol,
hydrogenated
partially hydrolysed polysaccharides and the like. Humectants are in general
present in
an amount of from 0°~ to 80°r6, preferably 5 to 70°~ by
weight in a toothpaste.
Examples of suitable thickeners and binders which help stabilize the
dentifrice
product are silica, starch, tragacanth gum, xanthan gum, extracts of Irish
moss,
alginates, pectin, cellulose derivatives, such as hydroxyethyl cellulose,
sodium
carboxymethyl cellulose and hydroxy-propyl cellulose, polyacrylic acid and its
salts and
polyvinyl-pyrrolidone. Thickeners may be present in toothpastes, creams and
gels in an
amount of from 0.1 to 20% by weight, and binders in an amount of from 0.01 to
10°~ by weight of the final product.
As a foaming agent soap, anionic, cationic, non-ionic, amphoteric and/or
zwitterionic surfactants can be used. These may be present at levels of from 0
to
15°~, preferably from 0.1 to 13°r6, more preferably from 0.25 to
10°~ by weight of
the final product.
Surfactants are only suitable in the context of the present invention to the
extent
that they do not exert any adverse effect on the activity of the enzymes.
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Examples of surfactants include fatty alcohol sulphates, salts of sulphonated
monoglycerides or fatty acids having from 10 to 20 carbon atoms, fatty acid-
albumin
condensation products, salts of fatty acids amides and taurines and/or salts
of fatty
acid esters of isethionic acid.
Suitable sweeteners include saccharin and/or other sweeteners suitable for use
in oral care products.
Flavouring agents, such as spearmint and peppermint, are usually present in
low
amounts, such as from 0.01 °.6 to about 5 °rb by weight,
especially from 0.1 °~ to 5 °r6.
Water is usually added in an amount giving e.g. a toothpaste a flowable form,
i.e. an amount of from 40°~ to 70°r6 by weight of the final
product.
In addition, water-soluble anti-bacterial agents, such as chlorhexidine
digluconate, hexetidine, aleicidine, quaternary ammonium anti-bacterial
compounds and
water-soluble sources of certain metal ions such as zinc, copper, silver and
stannous
ions (e.g. zinc, copper and stannous chloride, and silver nitrate) may also be
included.
Also contemplated according to the invention is the addition of other anti-
calculus agents, anti-plaque agents other than the enzymes which are the
subject of
the present application, compounds which can serve as a fluoride source,
dyes/colorants, preservatives, vitamins, pH-adjusting agents, anti-caries
agents,
desensitizing agents, etc.
A toothpaste produced from an oral composition of the invention may e.g.
comprise the following ingredients (in weight °~6 of the final
toothpaste composition):
Abrasive material 10 to 70°~
Humectant 0 to 80°~
Thickener 0.1 to 20°~
Binder 0.01 to 10°.6
Sweetener 0.1 °~ to 5°~
Foaming agent 0 to 15°~
Starch-degrading
enzymes) andlor
starch-modifying enzymes) 0.0001 °~6 to 20°~
Other enzymes 0 to 20°~
Peroxide 0 to 1 °~6
A mouthwash produced from an oral care composition of the invention may e.g.
comprise the following ingredients tin weight °r6 of the final
mouthwash composition):
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0-20°~ Humectant
0-2°.6 Surfactant
0-5°~6 Starch-degrading enzymes) and/or starch-modifying enzymes)
0-5°~6 Other enzymes
0-20°6 Ethanol
0-2°~6 Other ingredients (e.g. flavour, sweetener or other active
ingredients
such as fluorides).
0-70°~ Water
The mouthwash composition may be buffered with an appropriate buffer, e.g.
sodium citrate or phosphate in the pH-range 6-7.5.
The mouthwash may be in non-diluted form (i.e. to be diluted before use) or in
diluted (ready-to-use) form.
The oral care compositions and products of the present invention can be made
using methods which are common in the oral product field.
The invention further relates to the use of one or more starch-hydrolysing
enzymes and/or one or more starch-modifying enzymes as described above for the
preparation of a composition for the inhibition/prevention of plaque formation
and/or
removal of plaque.
An oral care product in solid to flowable form such as a toothpaste will
typically
be contacted with the teeth and/or gums using a toothbrush or the like. In the
case of
a liquid oral care product such as a mouthwash, the contact will typically
take place
by rinsing the mouth.
The time period during which an oral care product according to the invention
is
contacted with the teeth and/or gums to obtain the desired plaque inhibiting
or plaque
removing effect can vary according to such factors as the nature of the
composition
or product and the need of the subject. However, contacting the oral care
product
with the teeth and/or gums for between about 30 seconds to 15 minutes will
normally
be sufficient for obtaining the desired result, e.g. contact by brushing the
teeth or
rinsing the mouth for a period of about 1-3 minutes at a time. This is
preferably
performed on a regular basis, e.g. 1-3 times a day.
After use, the oral care product is typically removed from the mouth, e.g. by
spitting it out, and the mouth may subsequently be rinsed with a liquid such
as tap
water.
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While not wishing to be bound by any particular theory, it is believed that
the
surprising plaque-inhibiting effect found according to the present invention
may be due
to the fact that the starch-hydrolysing and/or starch-modifying enzymes react
with
sucrose to produce a coupling sugar before enzymes produced by the
microorganisms
gain access to the sucrose. Regardless of the mechanism of action, the plaque-
inhibiting effect observed by use of the enzymes in question is surprising and
is not
believed to have been described or suggested previously.
The invention will be further illustrated in the following non-limiting
examples.
MATERIALS AND METHODS
M~tPrials
Dextranase produced by Paecilomyces lilacinum (available from Novo Nordisk
A/S).
Mutanase produced by Trichoderma harzianum CBS 243.71 (available from Novo
Nordisk
A/S)
Maltogenase: Novamyl'"" (a bacterial maltogenic a-amylase) available from Novo
Nordisk
A/S.
Fungamyl'"' (a fungal a-amylase) available from Novo Nordisk A/S.
Transglucosidase produced by Aspergillus niger (available from Among
Pharmaceuticals
Co.).
BAN" (a bacterial a-amylase) available from Novo Nordisk A/S.
CGTase produced by Thermobacterium sp., available from Novo Nordisk A/S.
Promozyme'"" (a bacterial pullulanase) available from Novo Nordisk A/S
Microorganisms
Streptococcus sobrinus strain CBS 350.71 identifiable as OMZ 176
Actinomyces viscosus DSM 43329
Fusobacterium nucleatum subsp. polymorphism DSM 20482
of i ns
Erythrosin B (Sigma)
Eauioment
Chromameter CR-200 (Minolta)
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Preparation of hvdroxyaoat~tR ~~aks
Hydroxyapatite (HA) disks are prepared by compressing 250 mg of hydroxyapatite
in
a disk die at about 5,900 kg (13,000 Ibs) of pressure for 5 minutes. The disks
are then
5 sintered at 600°C for 4 hours and finally hydrated with sterile
deionized water.
sterilisation of hvdroxvapatite disks
HA disks are sterilised at 180°C for two hours.
10 Mutan prenara i n
Mutan is prepared by growing
Streptococcus sobrinus
CBS 350.71 at pH 6.5,
37C
(kept constant), and withtion rate of 75 rpm in a medium comprised
an aera of the
following components:
NZ-Case 6.5 g/I
Yeast Extract 6 g/I
(NH4)zS04 20 g/I
K2P04 3 g/I
Glucose 50 g/I
Pluronic PE6100 0.1 r6
After 35 hours, sucrose is added to a final concentration of 60 g/l to induce
glucosyitransferase. The total fermentation time is 75 hours. The supernatant
from the
fermentation is centrifuged and filtered (sterile). Sucrose is then added to
the
supernatant to a final concentration of 5°r6 (pH is adjusted to pH 7.0
with acetic acid)
and the solution is stirred overnight at 37°C. The solution is filtered
and the insoluble
mutan is harvested on Propex and washed extensively with deionized water
containing
1 °~ sodium benzoate, pH 5 (adjusted with acetic acid). Finally, the
insoluble mutan is
lyophilized and ground.
Determination of dextranase activity (KDU)
One Kilo Novo Dextranase Unit (1 KDU) is the amount of enzyme which breaks
down dextran forming reducing sugar equivalent to 1 g maltose per hour in the
Novo
Nordisk method for determination of dextranase based on the following standard
conditions:
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Substrate Dextran 500 (Pharmacia)
Reaction time 20 minutes
Temperature 40°C
pH 5.4
A detailed description of Novo Nordisk's analytical method (AF 120) is
available on
request.
Determination of mutanase activity (MU)
One Mutanase Unit (MU) is the amount of enzyme which under standard conditions
liberates 1 ~mol reducing sugar (calculated as glucose) per minute.
Standard Conditions
Substrate 1.5°~6 mutan
Reaction time 15 minutes
Temperature 40°C
pH 5.5
A detailed description of Novo Nordisk's analytical method (AF 180/1-GB) is
available
from Novo Nordisk A/S on request.
Determination of BAN'" (Bacterial Amylase Novo) activity (KNU) (Kilo Novo-a
amylase
ni
The standard activity is determined relative to an analytical standard under
the
following conditions:
Substrate: p-nitrophenyl-alpha-D-maltoheptaoside (pNP-G7)
Temperature: 37°C
pH: 7,1
A detailed description of Novo Nordisk's analytical method (Novo Nordisk
publication
AF215) is available on request.
Determination of Funqamvl~ activity (FAU)
One Fungal a-amylase Unit (1 FAU) is the amount of enzyme which breaks down
5.26 g
starch (Merck, Amylum solubile Erg. B.6, Batch 9947275) per hour at Novo
Nordisk's
standard method for determination of a-amylase based upon the following
standard
conditions:
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Substrate: soluble starch
Reaction time: 7-20 minutes
Temperature 37°C
pH: 4.7
A detailed description of Novo Nordisk's method of analysis is available on
request.
Determination of Maltoyg2~se activity (MANU)
1 Maltogenic Amylase Novo Unit (MANU) is defined as the amount of enzyme which
under standard conditions hydrolyzes 1 Nxnole of maltotriose per minute.
Copies of the
analytical method are available on request.
Determination of AMG'"" activity (AGUI
One Novo Amyloglucosidase Unit (AGU) is defined as the amount of enzyme which
hydrolyzes 1 wmole maltose per minute under the following standard conditions:
Substrate: maltose
Temperature: 25°C
pH : 4.3 (acetate buffer)
Reaction time 30 minutes
A detailed description of the analytical method (AF 22) is available on
request.
Determination of Transglucosidase activity (AGUI
Transglucosidase activity is determined using the method described above for
determining AMGT'" activity, and is expressed using the same units (AGU).
Determination of CGTase activity IKNUI
The CGTase KNU enzymatic activity was measured by a slightly modified
procedure
of the Phadebas amylase test (Pharmacia). Phadebas tablets (Phadebas'"'
Amylase Test,
Pharmacia) are used as a substrate. This substrate is a cross-linked insoluble
blue-colored
starch polymer, which is mixed with bovine serum albumin and a buffer
substance. After
suspension in water, starch is hydrolyzed by the enzyme, thereby yielding blue
fragments. The determination is carried out after incubation at 60°C,
pH 6.2, in 0.15 nM
calcium for 15 minutes. The absorbance of the resulting blue solution,
determined at fi20
nm, corresponds to the enzymatic activity.
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The enzyme activity is compared to that of an enzyme standard, and the
activity is
expressed in the same unit as that of the enzyme standard. The enzyme standard
was
TermamylT"" (Novo Nordisk A/D, Denmark), the amylolytic activity of which has
been be
determined using potato starch as a substrate. This method is based on the
break-down
of modified potato starch by the enzyme, and the reaction is followed by
mixing samples
of the starch/enzyme solution with an iodine solution, initially, a blackish-
blue color is
formed, but during the break-down of the starch the blue color gets weaker and
gradually turns into a reddish-brown, which is compared to a colored glass
standard.
One Kilo Novo a-Amylase Unit (KNU) is defined as the amount of enzyme which,
under
standard conditions (i.e. at 37°C +/- 0.05; 0.0003 M Ca2+; and pH 5.6)
dextrinizes
5.26 g starch dry substance, Merck Amylum soluble. The activity is expressed
below in
Novo Units (NU) per ml.
CGTase activity was determined by incubating diluted enzyme with substrate in
10
mM sodium citrate, pH 6.0 for 4-10 minutes at 60°C.
Determination of Promozyme'" activity (PUN)
One Pullulanse Unit Novo (PUN) is defined as the amount of enzyme which
hydrolyzes pullulan, liberating reducing carbohydrate with a reducing
carbohydrate with a
reducing power equivalent to 1 pmole glucose per minute under the following
standard
conditions:
Substrate: 0.2°~ pullulan
Temperature: 40°C
pH: 5.0 (0.05M citrate buffer)
Reaction time: 30 minutes
A detailed description of the analysis method (AF190) is available on request.
Assessment of the alaaue inhj-bition effect
The method used for assessing the plaque removal effect is based on the method
described by Kao in JP 2250816. According to the present method,
hydroxyapatite
disks, sterilised as described above, become coated with a biofilm by being
placed
overnight in the presence of three strains of oral microorganisms
(Streptococcus
sobrinus, Actinomyces viscosus and Fusobacterium nucleatumJ and various
enzymes
in a Brain Heart Infusion Medium (Difco) containing 0.2°r6 sucrose.
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14
To test plaque inhibition effect, 0.1 % Erythrosin B in PBS (phosphate
buffered
saline) is used to stain plaque present on the hydroxyapatite disks red. The
intensity of
the red colour (referred to as a~") is measured on a Chromameter CR-200. The
maximum a~' value is 60. Values below that indicate a less intensive red
colour (i.e.
less plaque present). An a~ value of zero indicates no red colouring (i.e. no
plaque). A
plaque inhibition effect is expressed as a relative figure based on the value
of a* for a
non-treated biofilm being 100%..
EXAMPLES
Example 1
Plague preventinct effect of different starch-hvdrolvsina and starch-modifying
enzymes
Three oral microorganisms, Streptococcus sobrinus, Actinomyces viscosus and
Fusobacterium nucieatum, respectively, were cultivated anaerobically overnight
at
37°C in the presence of various enzymes. Hydroxyapatite disks coated
with sterilised
saliva were immersed in a culture broth during cultivation so that an oral
biofilm was
formed on the disks. After cultivation, the disks were briefly rinsed with a
phosphate
buffered saline and then incubated in a 1 ml 0.1 % Erythrosin B in PBS for 1
minute to
stain plaque present on the hydroxyapatite disks red. The Erythrosin B
solution was
removed and the disks were rinsed with PBS for a few minutes. The disks were
air-
dried overnight. The intensity of the red colour (a'~) was measured on a
Chromameter
CR-200 and compared to that of the non-treated disks.
The plaque-prevention results of a number of different enzymes are shown below
in Table 1.
Atf,E''~ ED ~
CA 02305804 2000-04-04
20357PC1' . , ,'
Table 1: Plaque inhibiting/preventing activity of selected starch-hydrolysing
and
starch-modifying enzymes
Enzyme Activity Plaque intensity (%)
None (control) 100
Maltogenase~" (bacterial200 MANU/ml 36.8"'
maltogenic a-amylase)
Fungamyl"" 40 -FAU/ml 45.3 ~'
(fungal a-amylase)
Transglucosidase 32 AGU/ml 54.2~'~
derived from A.
niger
Promozyme~' (bacterial10 PUN/ml 45.0'
pullulanase)
~" statistically significant, p < 0.01
* ~" statistically significant, p < 0.001
5 As can be seen in the table above, statistically significant plaque
prevention/inhibition effects are obtained with all starch-hydrolysing and
starch-
modifying enzymes at the activity levels listed.
Example 2
10 Plague preventing effect of a starch-hydrolysing enzyme, a starch-modifying
enzyme
in combination with a mutanase and a dextranase
Three oral microorganisms, Streptococcus sobrinus, Actinomyces viscosus and
Fusobacterium nucieatum, respectively, were cultivated anaerobically overnight
at
37°C in the presence of 1 MU/ml mutanase and 1 KDU/ml dextranase and
the
15 enzyme listed in the Table below. Hydroxyapatite disks coated with
sterilised saliva
were immersed in a culture broth during cultivation so that an oral biofilm
was formed
on the disks. After cultivation, the disks were treated as describe in Example
1, and
the intensity of the red colour was determined.
Ar~~~r~D Std
CA 02305804 2000-04-04
WO 99/20239 PCT/DK98/00452
16
Table 2: Plaque inhibiting/preventing activity of a starch-modifying enzyme
and a
starch-hydrolysing enzyme together with mutanase and dextranase
Enzyme Activity Plaque intensity (~)
Control" 100
BANTM (Bacteria!12 KNU/ml 42.7
a-amylase)
CGTase 0.32 KNU/ml 40.4
Hn culture aroths contained 7 MU/ml mutanase and 1 kDU/ml dextranase
As can be seen, the use of the two enzymes BANT"" and the CGTase together
with a mutanase and a dextranase resulted in an improved plaque inhibition
compared
to mutanase and dextranase atone. The additional effect of the combination
with
mutanase and dextranase was particularly pronounced for CGTase. For both
BANT""
and CGTase, there was a synergistic effect of combining them with mutanase and
dextranase, since use of BANT"" or CGTase alone did not provide a plaque
reduction in
this test.
Example 3
Enzvme arotein dose response curves for plague inhibition/orevention effect
Three oral microorganisms, Streptococcus sobrinus, Actinomyces viscosus and
Fusobacterium nucleatum, respectively, were cultivated anaerobically overnight
at
37°C in the presence of various enzymes (FungamylT"", PromozymeT"" and
MaltogenaseT"", all from Novo Nordisk A/S>. Hydroxyapatite disks coated with
sterilised
saliva were immersed in a culture broth during cultivation so that an oral
biofilm was
formed on the disks. After cultivation, the disks were briefly rinsed with a
phosphate-
buffered saline and then incubated in a 1 ml 0.1 °~ Erythrosin B
solution in PBS for
1 minute to stain plaque present on the hydroxyapatite disks red. The
Erythrosin B
solution was removed and the disks were rinsed with PBS for a few minutes. The
disks were air-dried overnight. Biofilm remaining on the disks was determined
by
measuring the intensity of the red colour (a'") using a Chromameter CR-200,
the
obtained values being compared to values for non-treated disks. The results
are shown
in the attached Figures 1, 2 and 3.
