U.S. patent application number 09/173935 was filed with the patent office on 2002-01-17 for plaque-inhibiting oral compositions.
This patent application is currently assigned to Novozymes A/S. Invention is credited to TSUCHIYA, RIE.
Application Number | 20020006385 09/173935 |
Document ID | / |
Family ID | 27221156 |
Filed Date | 2002-01-17 |
United States Patent
Application |
20020006385 |
Kind Code |
A1 |
TSUCHIYA, RIE |
January 17, 2002 |
PLAQUE-INHIBITING ORAL COMPOSITIONS
Abstract
The present invention relates to oral compositions comprising
plaque-inhibiting or plaque-removing enzymes, in particular at
least one starch-hydrolysing enzyme, e.g. an .alpha.-amylase or a
debranching enzyme such as a pullulanase, and/or at least one
starch-modifying enzyme, e.g. a transglucosidase or a CGTase, and
to a method for inhibiting plaque formation or removing plaque
using such oral compositions.
Inventors: |
TSUCHIYA, RIE; (MAGNOLIAVEJ
5B, DK) |
Correspondence
Address: |
STEVE T ZELSON
NOVO NORDISK OF NORTH AMERICA
405 LEXINGTON AVENUE
SUITE 6400
NEW YORK
NY
101746401
|
Assignee: |
Novozymes A/S
Bagsvaerd
DK
|
Family ID: |
27221156 |
Appl. No.: |
09/173935 |
Filed: |
October 16, 1998 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60065944 |
Oct 27, 1997 |
|
|
|
Current U.S.
Class: |
424/49 |
Current CPC
Class: |
A61Q 11/00 20130101;
A61Q 11/02 20130101; A61K 8/66 20130101 |
Class at
Publication: |
424/49 |
International
Class: |
A61K 007/16 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 17, 1997 |
DK |
1191/97 |
Claims
1. An oral care composition comprising a plaque-inhibiting and/or
plaque-removing effective amount of at least one enzyme selected
from starch-hydrolysing enzymes and starch-modifying enzymes.
2. The oral care composition according to claim 1, wherein the
starch-modifying enzyme is a CGTase.
3. The oral care composition according to claim 2, wherein the
CGTase is derived from a strain selected from a strain of Bacillus
autolyticus, a strain of Bacillus cereus, a strain of Bacillus
circulans, a strain of Bacillus circulans 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,
and a strain of Thermoanaerobacterium thermosulfurigenes.
4. The oral care composition according to claim 1, wherein the
starch-modifying enzyme is a transglucosidase.
5. The oral care composition according to claim 4, wherein the
transglucosidase is derived from Aspergillus niger.
6. The oral care composition according to claim 1, wherein the
starch-hydrolysing enzyme is an .alpha.-amylase.
7. The oral care composition according to claim 6, wherein the
.alpha.-amylase is a bacterial .alpha.-amylase.
8. The oral care composition according to claim 6, wherein the
.alpha.-amylase is selected from .alpha.-amylases derived from
Bacillus subtilis; .alpha.-amylases derived from Bacillus
amyloliquefaciens; .alpha.-amylases derived from Bacillus
stearothermophilus; .alpha.-amylases derived from Aspergillus
oryzae: and .alpha.-amylases derived from non-pathogenic
microorganisms.
9. The oral care composition according to claim 6, wherein the
.alpha.-amylase is a fungal .alpha.-amylase.
10. The oral care composition according to claim 1, wherein the
starch-hydrolysing enzyme is a debranching enzyme.
11. The oral care composition according to claim 10, wherein the
starch-hydrolysing enzyme is a pullulanase.
12. The oral care composition according to claim 1, further
comprising an enzyme selected from dextranases and mutanases.
13. The oral care composition according to claim 12, comprising a
mutanase derived from Trichoderma harzianum and/or a dextranase
derived from a strain of Paecilomyces.
14. An oral care product comprising an oral care composition
according to claim 1.
15. The oral care product of claim 14 which is selected from a
toothpaste, a tooth gel, a tooth powder, a mouthwash, a denture
cleaning composition, a chewing gum, a lozenge, a dental floss and
a toothpick.
16. A method for inhibiting plaque formation or removing plaque,
comprising contacting the teeth and/or gums with an oral care
composition according to claim 1 for a period of time sufficient to
obtain a plaque-inhibiting or plaque-removing effect.
17. A method for inhibiting plaque formation or removing plaque,
comprising contacting the teeth and/or gums with an oral care
product according to claim 14 for a period of time sufficient to
obtain a plaque-inhibiting or plaque-removing effect.
18. The method of claim 17, comprising brushing the teeth on a
regular basis with an oral care product in the form of a toothpaste
or tooth gel comprising at least one enzyme selected from
starch-hydrolysing enzymes and starch-modifying enzymes.
19. The method of claim 17, comprising rinsing the mouth on a
regular basis with an oral care product in the form of a mouthwash
comprising at least one enzyme selected from starch-hydrolysing
enzymes and starch-modifying enzymes.
20. The method of claim 17, comprising chewing an oral care product
in the form of a chewing gum comprising at least one enzyme
selected from starch-hydrolysing enzymes and starch-modifying
enzymes.
Description
FIELD OF THE INVENTION
[0001] 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
[0002] 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.
[0003] 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
.alpha.-1,6 glucosidic linkages, and a major component of
water-insoluble extracellular polysaccharides called "mutan"
comprised of a backbone with .alpha.-1,3-glycosidic linkages and
branches with .alpha.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.
[0004] 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.
[0005] 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.
[0006] 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.
[0007] JP patent publication 8012544 (Lion) describes a plaque
preventing effect of dextranase, mutanase and triclosan and/or
biosol.
[0008] U.S. Pat. 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 .alpha.-1,3-glucosidic bonds and that such
polysaccharide material termed mutan is not attacked by
dextranase.
[0009] 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).
[0010] 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 .alpha.-1,3 glucanase from Cladosporium
resinae.
[0011] U.S. Pat. No. 4,438,093 (The Research Foundation for
Microbial diseases of Osaka) describes oral compositions comprising
a dextranase and a .alpha.-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 .alpha.-1,3-glucanase is derived from a
bacteria within the genus Pseudomonas,
[0012] 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.
[0013] U.S. Pat. No. 5,145,665 (Henkel) discloses a composition for
the care of the mouth and teeth comprising a dextranase and/or
.alpha.-1,3-glucanase for cleaving polysaccharides in the
mouth.
[0014] 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
[0015] U.S. Pat. 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.
[0016] 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
[0017] 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.
[0018] 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.
[0019] 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.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The term "starch-hydrolysing enzyme" in the context of the
present application refers to any enzyme, such as an
.alpha.-amylase (E.C. 3.2.1.1), which functions to hydrolyse
linkages in starch.
[0021] 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).
[0022] 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 least one starch-modifying
enzyme. Said effect of such enzymes has not been foreseen
before.
[0023] 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.
[0024] 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).
[0025] 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 circulans, a strain of Bacillus
circulans 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 thermoamylolyricum, a strain of
Clostridium thermosaccharolyticum, or a strain of
Thermoanaerobacterium thermosulfurigenes.
[0026] 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.
[0027] In another embodiment of the invention the oral care
composition comprises a starch-hydrolysing enzyme.
[0028] This will typically be an .alpha.-amylase, such as a
bacterial .alpha.-amylase, such as BAN.TM. or Maltogenase.TM. (both
available from Novo Nordisk), or an .alpha.-amylase derived from
Bacillus subtilis; an .alpha.-amylase derived from Bacillus
amyloliquefaciens; an .alpha.-amylase derived from Bacillus
stearothermophilus; an .alpha.-amylase derived from Aspergillus
oryzae; or an .alpha.-amylase derived from a non-pathogenic
microorganism.
[0029] The .alpha.-amylase may also be a fungal .alpha.-amylase,
such as Fungamyl.TM., which is available from Novo Nordisk.
[0030] 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 Promozyme.TM..
[0031] 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.
[0032] In another preferred embodiment the oral care composition of
the invention comprises at least one starch-hydrolysing enzyme as
defined above, in particular a bacterial .alpha.-amylase, and a
mutanase and/or a dextranass.
[0033] The mutanase may be derived from a strain of Trichodermna
sp., in particular T. harzianum, especially T. harzianum CBS 243.71
(available from Novo Nordisk).
[0034] The dextranase may be derived from a strain of Paecilomyces
sp., in particular Paecilomyces lilacinus (available from Novo
Nordisk).
[0035] In a second aspect the invention relates to an oral care
product comprising an oral care composition of the invention.
[0036] 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.
[0037] 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.
[0038] 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.
[0039] Mouthwashes typically comprise a water/alcohol solution,
flavouring agents, humectants, sweeteners, foaming agents and
colorants.
[0040] 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 enzyme(s) to the gum base, the temperature of
the gum base should not be too high, e.g. preferably not more than
about 60.degree. C., more preferably not more than about 50.degree.
C.
[0041] Abrasive polishing materials for use in oral care products
according to the invention include alumina and hydrates thereof,
such as alpha alumina trihydrate. 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, hydrogels and
aerogels and the like. Also suitable as abrasive agents are calcium
pyrophosphate, water-insoluble alkali metaphosphates, dicalcium
phosphate and/or its dihydrate, dicalcium orthophosphate,
tricalcium phosphate, particulate hydroxyapatite and the like. It
is also possible to employ mixtures of these substances.
[0042] 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 lies in the range from 10% to 70% by
weight of the final toothpaste product.
[0043] 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%,
preferably 5 to 70% by weight in a toothpaste.
[0044] 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.
[0045] 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%,
more preferably from 0.25 to 10% by weight of the final
product.
[0046] 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.
[0047] 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.
[0048] Suitable sweeteners include saccharin and/or other
sweeteners suitable for use in oral care products.
[0049] Flavouring agents, such as spearmint and peppermint, are
usually present in low amounts, such as from 0.01% to about 5% by
weight, especially from 0.1% to 5%.
[0050] Water is usually added in an amount giving e.g. a toothpaste
a flowable form, i.e. an amount of from 40% to 70% by weight of the
final product.
[0051] In addition, water-soluble anti-bacterial agents, such as
chlorhexidine digluconate, hexetidine, alexidine, 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.
[0052] 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.
[0053] A toothpaste produced from an oral composition of the
invention may e.g. comprise the following ingredients (in weight %
of the final toothpaste composition):
1 Abrasive material 10 to 70% Humectant 0 to 80% Thickener 0.1 to
20% Binder 0.01 to 10% Sweetener 0.1% to 5% Foaming agent 0 to 15%
Starch-degrading enzyme(s) and/or 0.0001% to 20% starch-modifying
enzyme(s) Other enzymes 0 to 20% Peroxide 0 to 1%
[0054] A mouthwash produced from an oral care composition of the
invention may e.g. comprise the following ingredients (in weight %
of the final mouthwash composition):
2 0-20% Humectant 0-2% Surfactant 0-5% Starch-degrading enzyme(s)
and/or starch-modifying enzyme(s) 0-5% Other enzymes 0-20% Ethanol
0-2% Other ingredients (e.g. flavour, sweetener or other active
ingredients such as fluorides). 0-70% Water
[0055] The mouthwash composition may be buffered with an
appropriate buffer, e.g. sodium citrate or phosphate in the
pH-range 6-7.5.
[0056] The mouthwash may be in non-diluted form (i.e. to be diluted
before use) or in diluted (ready-to-use) form.
[0057] The oral care compositions and products of the present
invention can be made using methods which are common in the oral
product field.
[0058] 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 inhibitioniprevention of plaque formation and/or removal of
plaque.
[0059] 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.
[0060] 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.
[0061] 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.
[0062] 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.
[0063] The invention will be further illustrated in the following
non-limiting examples.
MATERIALS AND METHODS
[0064] Materials
[0065] 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)
[0066] Maltogenase: Novamyl.TM. (a bacterial maltogenic
.alpha.-amylase) available from Novo Nordisk A/S.
[0067] Fungamyl.TM. (a fungal .alpha.-amylase) available from Novo
Nordisk A/S.
[0068] Transglucosidase produced by Aspergillus niger (available
from Amona Pharmaceuticals Co.).
[0069] BAN.TM. (a bacterial .alpha.-amylase) available from Novo
Nordisk A/S.
[0070] CGTase produced by Thermobacterium sp., available from Novo
Nordisk A/S.
[0071] Promozyme.TM. (a bacterial pullulanase) available from Novo
Nordisk A/S
[0072] Microorganisms
[0073] Streptococcus sobrinus strain CBS 350.71 identifiable as OMZ
176
[0074] Actinomyces viscosus DSM 43329
[0075] Fusobacterium nucleatum subsp. polymorphum DSM 20482
[0076] Solutions
[0077] Erythrosin B (Sigma)
[0078] Equipment
[0079] Chromameter CR-200 (Minolta)
[0080] Preparation of hydroxyapatite disks
[0081] Hydroxyapatite (HA) disks are prepared by compressing 250 mg
of hydroxyapatite in a disk die at about 5,900 kg (13,000 lbs) of
pressure for 5 minutes. The disks are then sintered at 600.degree.
C. for 4 hours and finally hydrated with sterile deionized
water.
[0082] Sterilisation of hydroxypapatite disks
[0083] HA disks are sterilised at 180.degree. C. for two hours.
[0084] Mutan preparation
[0085] Mutan is prepared by growing Streptococcus sobrinus CBS
350.71 at pH 6.5, 37.degree. C. (kept constant), and with an
aeration rate of 75 rpm in a medium comprised of the following
components;
3 NZ-Case 6.5 g/l Yeast Extract 6 g/l (NH.sub.4).sub.2SO.sub.4 20
g/l K.sub.2PO.sub.4 3 g/l Glucose 50 g/l Pluronic PE6100 0.1%
[0086] After 35 hours, sucrose is added to a final concentration of
60 g/l to induce glucosyltransferase. 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% (pH is adjusted to pH 7.0 with acetic
acid) and the solution is stirred overnight at 37.degree. 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.
[0087] Determination of dextranase activity (KDU)
[0088] 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:
4 Substrate Dextran 500 (Pharmacia) Reaction time 20 minutes
Temperature 40.degree. C. pH 5.4
[0089] A detailed description of Novo Nordisk's analytical method
(AF 120) is available on request.
[0090] Determination of mutanase activity (MU)
[0091] One Mutanase Unit (MU) is the amount of enzyme which under
standard conditions liberates 1 .mu.mol reducing sugar (calculated
as glucose) per minute.
5 Standard Conditions Substrate 1.5% mutan Reaction time 15 minutes
Temperature 40.degree. C. pH 5.5
[0092] A detailed description of Novo Nordisk's analytical method
(AF 180/1-GB) is available from Novo Nordisk A/S on request.
[0093] Determination of BAN.TM. (Bacteria Amylase Novo) activity
(KNU) (Kilo Novo-.alpha.-amylase Units)
[0094] The standard activity is determined relative to an
analytical standard under the following conditions:
6 Substrate: p-nitrophenyl-alpha-D-maltoheptaoside (pNP-G7)
Temperature: 37.degree. C. pH: 7.1
[0095] A detailed description of Novo Nordisk's analytical method
(Novo Nordisk publication AF215) is available on request.
[0096] Determination of Fungamyl.RTM. activity (FAU)
[0097] One Fungal .alpha.-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 .alpha.-amylase based upon the following standard
conditions:
7 Substrate: soluble starch Reaction time: 7-20 minutes Temperature
37.degree. C. pH: 4.7
[0098] A detailed description of Novo Nordisk's method of analysis
is available on request.
[0099] Determination of Maltogenase activity (MANU)
[0100] 1 Maltogenic Amylase Novo Unit (MANU) is defined as the
amount of enzyme which under standard conditions hydrolyzes 1
.mu.mole of maltotriose per minute. Copies of the analytical method
are available on request.
[0101] Determination of AMG.TM. activity (AGU)
[0102] One Novo Amyloglucosidase Unit (AGU) is defined as the
amount of enzyme which hydrolyzes 1 pmole maltose per minute under
the following standard conditions;
8 Substrate: maltose Temperature: 25.degree. C. pH: 4.3 (acetate
buffer) Reaction time 30 minutes
[0103] A detailed description of the analytical method (AF 22) is
available on request.
[0104] Determination of Transolucosidase activity (AGU)
[0105] Transglucosidase activity is determined using the method
described above for determining AMG.TM. activity, and is expressed
using the same units (AGU).
[0106] Determination of CGTase activity (KNU)
[0107] The CGTase KNU enzymatic activity was measured by a slightly
modified procedure of the Phadebas amylase test (Pharmacia).
Phadebas tablets (Phadebas.TM. 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.degree. C., pH
6.2, in 0.15 nM calcium for 15 minutes. The absorbance of the
resulting blue solution, determined at 620 nm, corresponds to the
enzymatic activity.
[0108] 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 Termamyl.TM. (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 .alpha.-Amylase
Unit (KNU) is defined as the amount of enzyme which, under standard
conditions (i.e. at 37.degree. C.+/-0.05; 0.0003 M Ca.sup.2+; 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.
[0109] CGTase activity was determined by incubating diluted enzyme
with substrate in 10 mM sodium citrate, pH 6.0 for 4-10 minutes at
60.degree. C.
[0110] Determination of Promozyme.TM. activity (PUN)
[0111] 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
.mu.mole glucose per minute under the following standard
conditions:
9 Substrate: 0.2% pullulan Temperature: 40.degree. C. pH: 5.0
(0.05M citrate buffer) Reaction time: 30 minutes
[0112] A detailed description of the analysis method (AF190) is
available on request.
[0113] Assessment of the plaque inhibition effect
[0114] 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 nucleatum) and
various enzymes in a Brain Heart Infusion Medium (Difco) containing
0.2% sucrose.
[0115] 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 (ie. 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
[0116] Plaque preventing effect of different starch-hydrolysing
enzymes
[0117] Three oral microorganisms, Streptococcus sobrinus,
Actinomyces viscosus and Fusobacterium nucleatum, respectively,
were cultivated anaerobically overnight at 37.degree. 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 PRS 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.
[0118] The plaque-prevention results of a number of different
enzymes are shown below in Table 1.
10TABLE 1 Plaque inhibiting/preventing activity of selected
starch-hydrolysing enzymes Enzyme Activity Plaque intensity (%)
None (control) 100 Maltogenase .TM. (bacterial 200 MANU/ml 36.8**
maltogenic .alpha.-amylase) Fungamyl .TM. 40 FAU/ml 45.3** (fungal
.alpha.-amylase) Transglucosidase 32 AGU/ml 54.2** derived from A.
niger Promozyme .TM. (bacterial 10 PUN/ml 45.0* pullulanase)
*statistically significant, p < 0.01 **statistically
significant, p < 0.001
[0119] As can be seen in the table above, statistically significant
plaque prevention/inhibition effects are obtained with all
starch-hydrolysing enzymes at the activity levels listed.
Example 2
[0120] Plaque preventing effect of a starch-hydrolysing enzyme a
starch-modifying enzyme in combination with a mutanase and a
dextranase
[0121] Three oral microorganisms, Streptococcus sobrinus,
Actinomyces viscosus and Fusobacterium nucleatum, respectively,
were cultivated anaerobically overnight at 37.degree. C. in the
presence of 1 MU/ml mutanase and 1 KDU/ml dextranase and the 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.
11TABLE 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 BAN .TM. (Bacterial 12 KNU/ml 42.7 .alpha.-amylase)
CGTase 0.32 KNU/ml 40.4 *: All culture broths contained 1 MU/ml
mutanase and 1 kDU/ml dextranase
[0122] As can be seen, the use of the two enzymes BAN.TM. and the
CGTase together with a mutanase and a dextranase resulted in an
improved plaque inhibition compared to mutanase and dextranase
alone. The additional effect of the combination with mutanase and
dextranase was particularly pronounced for CGTase. For both BAN.TM.
and CGTase, there was a synergistic effect of combining them with
mutanase and dextranase, since use of BAN.TM. or CGTase alone did
not provide a plaque reduction in this test.
Example 3
[0123] Enzyme protein dose response curves for plaque
inhibition/prevention effect
[0124] Three oral microorganisms, Streptococcus sobrinus,
Actinomyces viscosus and Fusobacterium nucleatum, respectively,
were cultivated anaerobically overnight at 37.degree. C. in the
presence of various enzymes (Fungamyl.TM., Promozyme.TM. and
Maltogenase.TM., 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 FIGS.
1, 2 and 3.
* * * * *