Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
GLUCOSYLTRANSFERASE INHIBITORS, AS WELL AS DENTAL
CARIES PREVENTION METHODS AND ANTICARIOUS FOODS
USING THE SAME
BACKGROUND OF THE INVENTION
1) Field of the Invention
The present invention relates to glucosyl-
transferase inhibitors and methods for preventing den-
tal caries by using the same, and more specifically to
glucosyltransferase inhibitors comprising as an active
ingredient an extract from fermented tea leaves having
glucosyltransferase inhibitory action, methods for pre-
venting dental caries by using the same and anticarious .
beverages and foods containing the same.
2) Description of the Related Art
Although a variety of theories were proposed in
the past as to a possible cause for dental caries, it
is now recognized that dental caries is a type of bac-
2o terial infectious disease based on the chemicoparasitic
theory proposed by Miller.
The mechanism of occurrence of dental caries on
the basis of the above theory is as follows. Namely,
an enzyme called "glucosyltransferase", which is pro-
duced by oral streptococci, notably by Streptococcus
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- 2 -
mutans produces a sticky, insoluble polysaccharide
(glucan) by using as a substrate sugar in the mouth.
Cells of streptococcus mutans (hereinafter abbreviated as
"s. mutans") adhere to the surfaces of teeth by the
glucan so produced, so that they form granules (dental
plaques). Various microorganisms led by s. mutans are
symbiotically growing in the dental plaques. Organic
acids are produced by the metabolic activities of these
microorganisms. The pH on the surfaces of the teeth
drops as a result of the action of these organic acids,
resulting in decalcification of the enamel surfaces.
Consequently, dental caries takes place and proceeds.
Further, the formation of dental plaques is also
believed to cause paradental diseases and bad breath in
addition to dental caries.
Based on the findings described above, various
investigations have been conducted with a view toward
developing, as preventive measures for dental caries,
antibacterial agents for oral microorganisms, in-
hibitors for glucosyltransferase, and enzymes capable
of decomposing polysaccharides which glucosyltrans-
ferase forms using sugar as a substrate. However, no
anticarious method having satisfactory effects has yet
been found.
Prevention of dental plaque formation by sup-
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pressing the activities of glucosyltransferase is con-
sidered likely to become an effective means for the
prevention of occurrence of dental caries especially in
view of the fact that dental plaque formed by oral
streptococci led by S. mutans acts as a cause far dental
caries. To date, however, no practically usable
glucosyltransferase activity inhibition substance has
yet been found.
SUMMARY OF THE INVENTION
The present inventors have carried out an ex-
tensive investigation with a view toward finding a sub-
stance which can effectively inhibit glucosyl-
transferase but has no adverse effects on the human
body. As a result, it has been revealed that a sub-
stance capable of extremely effectively inhibiting the
activities of glucosyltransferase produced by S. mutans
is contained in an extract of fermented tea leaves and
this substance is not adsorbed even by synthetic adsor-
bents, leading to the completion of the present inven-
tion.
An object of the present invention is therefore
to provide a glucosyltransferase inhibitor which com-
prises as an active ingredient an extract from fer-
mented tea leaves.
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Another object of the present invention is to
provide a glucosyltransferase inhibitor which comprises
as an active ingredient a non-adsorbed fraction of an
extract of fermented tea leaves. The non-adsorbed
fraction has been obtained by subjecting the extract to
adsorption treatment while using a synthetic adsorbent
and is other than extract fractions adsorbed on the
synthetic adsorbent.
A further object of the present invention is to
provide a food or beverage for the prevention of dental
caries. The food or beverage comprises an effective
amount of the above inhibitor.
A still further object of the present invention
is to provide a method for the prevention of dental
caries, which comprises taking a food or beverage con-
taming an effective amount of the glucosyltransferase
inhibitor.
In one aspect of the present invention, there is
thus provided a glucosyltransferase inhibitor compris-
ing as an active ingredient an extract of fermented tea
leaves.
In another aspect of the present invention, there
is also provided a glucosyltransferase inhibitor, which
comprises as an active ingredient a non-adsorbed frac-
tion of an extract of fermented tea leaves, said non-
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adsorbed fraction having been obtained by subjecting
the extract to adsorption treatment while using a
synthetic adsorbent and being other than extract frac-
tions adsorbed on the synthetic adsorbent.
In a further aspect of the present invention,
there is also provided a food or beverage for the pre-
vention of dental caries, which comprises an effective
amount of one of the above inhibitors.
In a still further aspect of the present inven-
tion, there is also provided a method for the pre-
vention of dental caries, which comprises taking a food
or beverage containing an effective amount of one of
the above inhibitors.
In a still further aspect of the present inven-
tion, there is also provided use of an extract of fer-
mented tea leaves for the production of a glucosyl-
transferase inhibitor.
Catechins have already been known to have
proliferation inhibitory action against S. mutans.
Their activity is however extremely weak so that a
practical level of anticarious activity cannot be ex-
pected at concentrations actually usable in respect of
taste and flavor.
In view of the fact that, as will be demonstrated
in the subsequent example, excellent glucosyltrans-
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ferase inhibition activity is also found on the non-
adsorbed fraction which does not contain catechins and
caffeine at all or contain them in extremely small
amounts, it is evident that the effects of the present
invention are not related to catechins.
The glucosyltransferase inhibitor of the present
invention can therefore be used as a new method for the
prevention of dental caries, which makes use of the
glucosyltransferase inhibitory activity.
In particular, use of the non-adsorbed fraction
as an active ingredient makes it possible to add the
same at desired concentrations to various beverages,
drinks and foods because this fraction has strong
glucosyltransferase inhibitory activity and moreover
does not have a characteristic taste, smell or the
like. It is therefore possible to produce beverages,
drinks and foods having extremely good anticarious ac-
tivity.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The active ingredient of the glucosyltransferase
inhibitor according to the present invention is present
in an extract of fermented tea leaves. Described spe-
cifically, it is contained in a fraction not adsorbed
on a synthetic adsorbent when the extract is treated
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with the synthetic adsorbent. The above fraction may
hereinafter be called a "non-adsorbed fraction".
The non-adsorbed fraction contains no catechins
and caffeine, representative components of tea, or con-
s tains them only in extremely small amounts.
In the production of the glucosyltransferase in-
hibitor of the present invention, Oolong tea leaves,
black tea leaves, Pu-erh tea leaves and the like are
all usable as fermented tea leaves to be employed as a
raw material for extraction.
Examples of the solvent usable for the extraction
include water alone and desired mixtures of water and
one or more polar solvents such as lower alcohols,
e.g., methanol and ethanol, and acetone. Since effi-
cient extraction of the active ingredient of the pres-
ent invention is not feasible if a polar solvent is
used alone, the polar solvent must be used in the form
of a mixture with water, with their preferred mixing
ratio being such that the solvent amounts to 90 wt.% or
less. Among these solvents, it is preferable to use
water, ethanol or a mixture thereof from the standpoint
of safety in view of the fact that the extract and the
like are eventually incorporated in oral preparations
or foods.
Although no particular limitation is imposed on
CA 02039538 2001-O1-31
_ g _
the ratio of fermented tea leaves to the solvent upon
extraction, it is preferable to use the solvent in an
amount 2-1,000 times by weight, especially 20-100 times
by weight the amount of the fermented tea leaves in
view of the extracting operation and efficiency.
The extraction temperature can conveniently be
set within the range of from room temperature to the
boiling point of the solvent under normal pressure.
The extraction time can preferably range from 10
minutes to 24 hours although it varies depending on the
temperature of the extraction.
To obtain a non-adsorbed fraction from the above-
obtained extract of the fermented tea leaves, it is
necessary to treat the extract with a synthetic adsor-
bent.
Examples of the synthetic adsorbent employed for
the fractionation of the extract of the fermented tea
leaves include aromatic synthetic adsorbents produced
by polymerization of styrene and divinylbenzene and
methacrylic synthetic adsorbents produced by
polymerization of methacrylic acid. A typical commer-
cial product of methacrylic synthetic adsorbent is
"DIAION'M HP21"(trade name, products of Mitsubishi Kasei
Corp., Tokyo, Japan).
The synthetic adsorbent treatment can be effected
Trademark
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preferably by packing a column with the adsorbent,
charging the extract of the fermented tea leaves into
the column and then washing the resin with water.
Upon treatment of the extract of the fermented
tea leaves with such a synthetic adsorbent, it is
preferable to subject the extract to pretreatment in
order to achieve complete fractionation. The pretreat-
ment may comprise removal of any organic solvent from
the extract, for example, by concentration of the ex-
tract under reduced pressure, followed by full dilution
of the resultant concentrate with water.
The extract of the fermented tea leaves and the
non-adsorbed fraction, both obtained in the above-
described manner, can be used in any form - fox exam-
ple, as they are, in other words, directly after the
extraction and the synthetic adsorbent treatment; as
concentratest or as dried products obtained by removing
the solvent from the extract and the non-adsorbed
eluate fraction. It is however preferable to use them
in a dried form from the standpoints of storability and
safety, i.e., the complete elimination of the organic
solvent.
The glucosyltransferase inhibitor of the present
invention is formulated into a preparation by mixing
the above extract or non-adsorbed fraction with various
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components which have conventionally been used.
111ustrative preparations of the glucosyltrans-
ferase of the present invention include oral prepara-
tions for the prevention of dental caries, such as
tooth pastes, mouth washes and troches; and prepara-
tions suitable for addition to foods, beverages and
drinks, e.g., sweetenings such as sugar, Castilla
(sponge-cake-like product containing additional
sweetening and a different texture), soft bean jelly,
sponge cake, butter cake, Bavarian cream, custard
cream, butter cream, custard pudding, cookies, jams,
lactic acid bacteria beverages, carbonated beverages,
coffee drinks, coffee jelly, caramels, ice cream, chew-
ing gum, juices, candies and chocolate. Upon produc-
tion of these oral preparations, foods, beverages and
drinks, commonly-used, suitable ingredients can also be
used in combination as needed. Examples of additives
for oral preparations include calcium carbonate, cal-
cium hydrogenphosphate, silica, magnesium carbonate,
glycerin, sorbitol, propylene glycol, polyethylene
glycol, carboxymethylcellulose, methylcellulose, sodium
alginate, carageenan, carboxylvinyl polymer, sodium
dioctylsulfosuccinate, sodium laurylsulfate, sodium
dodecylbenzenesulfonate, butyl paraoxybenzoate,
hinokitiol, allantoin, glytylricin, alcohol, gum
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- 11
arabic, starch, corn starch, saccharin sodium,
stevioside, glucose, lactose, magnesium stearate,
monopotassium phosphate, dipotassium phosphate,
menthol, eucalyptus oil, peppermint, spearmint, and
pigments. In addition, fluorides such as sodium fluo-
ride and sodium monofluorophosphate; antiinflammatory
agents such as lysozyme chloride and azulene; sodium
chloride; and the like can also be added as needed.
On the other hand, beverages and drinks can be
produced by adding those routinely employed as raw
materials for beverages and drinks as needed, includ-
ing, far example, glucose, fructose, sucrose, maltose,
sorbitol, stevioside, corn syrup, lactose, citric acid,
tartric acid, malic acid, succinic acid, lactic acid,
L-ascorbic acid, dl-a-tocopherol, sodium erysorbate,
glycerin, propylene glycol, glycerin fatty acid esters,
polyglycerin fatty acid esters, sucrose fatty acid
esters, sorbitan fatty acid esters, propylene glycol
fatty acid esters, gum arabic, carageenan, casein, gel-
atin, pectin, agar, vitamin Bs, nicotinamide, calcium
panthothenate, amino acids, calcium salts, pigments,
essences, preservatives, etc.
For the production of anticarious foods,
beverages and drinks - such as those described in sub-
sequent examples - by adding the glucosyltransferase
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inhibitor of the present invention to foods, beverages
and drinks, the glucosyltransferase inhibitor can be
added in combination with sugar. In such cases, it is
possible to add, in place of the glucosyltransferase
and sugar, such anticarious sugars as illustrated in
Examples 9-11 to be described subsequently.
Tea has been widely taken for many years through-
out the world. Its extract and the non-adsorbed frac-
tion which is obtained from the extract do not there-
fore present any question of safety. It is however
preferred to control the content of the active in-
gredient in the glucosyltransferase inhibitor of the
present invention within the range of 0.0001-10% in
terms of dry weight from the standpoints of the effects
of the glucosyltransferase inhibition activity and the
taste, flavor, color tone and the like when the
glucosyltransferase inhibitor is added.
The present invention will hereinafter be de-
scribed in further detail by the following examples of
the preparation methods of the extract of fermented tea
leaves and the non-adsorbed fraction, a glucosyltrans-
ferase inhibition activity test and the preparation of
oral sanitizers. It is however to be noted that the
present invention is by no means limited to the follow-
ing examples.
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Example 1 (Preparation of extract)
Oolong tea leaves (100 g) was placed in a
2,000-me Erlenmeyer flask, followed by the addition of
1,000 mE of hot water. The flask was heated at 9~°C
for 15 minutes over a water bath to conduct extraction.
The extract was filtered through "Celite" (trade mark),
and the filtrate thus obtained was lyophilized to ob-
tain 16.5 g of an extract.
The above procedures were repeated in respect of
black tea leaves and Pu-erh tea leaves, whereby ex-
tracts were obtained in amounts of 16.4 g and 17.4 g,
respectively.
Example 2 (Preparation of extract)
Oolong tea leaves (100 g) was placed in a
2,000-mE Erlenmeyer flask, followed by the addition of
1,000 me of 50 vol.% ethanol. At room temperature,
the tea leaves were extracted for 3 hours with gentle
stirring every hour. The extract was filtered through
"Celite°' (trade mark), and the filtrate thus obtained
was concentrated under reduced pressure to remove
ethanol. Water was added to the residue, followed by
lyophilization to obtain 29.2 g of an extract.
The above procedures were repeated in respect of
black tea leaves and Pu-erh tea leaves, whereby ex-
tracts were obtained in amounts of 30.4 g and 31.3 g,
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respectively.
Example 3 (Preparation of non-adsorbed fraction)
Oolong tea extract (15 g) which had been obtained
following the procedures of Example 1 was dissolved in
600 ml of water. The solution was charged into a
column (4.4 x 20 cm) which was packed with "DIAION HP
21", whereby adsorption took place. A fraction not ad-
sorbed on the adsorbent was combined with a washing ob-
tamed by washing the column with 2 a of water after
the adsorbing operation, whereby a non-adsorbed frac-
tion was obtained. The non-adsorbed fraction was con-
centrated under reduced pressure and then lyophilized,
whereby 6.4 g of a sample was obtained.
The above procedures were repeated in respect of
black tea leaves and Pu-erh tea leaves, whereby non-
adsorbed fractions were obtained in amounts of 7.7 g
and 6.3 g, respectively.
Example 4 (Assay of glucosyltransferase inhibition
activity)
(Enzyme solution)
As an enzyme solution, was used an extract which
was obtained by culturing S. mucans MT8148 strain on
Todd-Hewitt medium and then extracting hyphae with 8M
urea in accordance with the method proposed by Hamada
et al. [S. Hamada et al., ~7. Gen. Microbiol., 135, 335-
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344 (1989)].
(Measuring method)
A reaction system was prepared by adding 0.15 ml
of an aqueous solution of a sample, said solution hav-
5 ing been prepared to contain the sample at the con-
centration of 2,000 ppm, the enzyme solution prepared
from the s. mutans strain and water to 0.6 mE of 500 mM
sodium phosphate buffer which had pH 6.0 and contained
5% of sucrose, 0.5% of dextran T10 and 0.5% of sodium
azide. The water was added in an amount to give a to-
tal volume of 3 mE. They were reacted in a glass test
tube. In the above measurement, the amount of the en-
zyme was set such that the absorbance at 550 nm became
about 1.0 when reacted at 37°C for 3 hours.
Resultant insoluble glucan was subjected to
ultrasonic disintegration and the absorbance (A) at
550 nm was measured. Using as control (B) the absor-
bance obtained from the use of water in place of the
sample solution, the inhibition rate (%) was determined
in accordance with the following calculation formula:
100 x (B - A)
Inhibition rate (%) = B
(Measurement results)
The glucosyltransferase inhibition activities of
the extracts of the various fermented tea leaves, said
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extracts having been obtained in Examples 1, 2 and 3,
and of caffeine and catechins, which are representative
components of tea leaves, were measured using the above
reaction system. The results are summarized in Table
1.
Table 1
Inhibition
Sample tested rate, %
Oolong tea extract of Example 1 51
Black tea extract of Example 1 87
Pu-erh tea extract of Example 1 72
Oolong tea extract of Example 2 59
Black tea extract of Example 2 89
Pu-erh tea extract of Example 2 75
Non-adsorbed fraction of Oolong 81
tea extract of Example 3
Non-adsorbed fraction of black 94
tea extract of Example 3
Non-adsorbed fraction of Pu-erh 76
tea extract of Example 3
Gallocatechin 15
Epigallocatechin 11
Catechin 13
Epicatechin 14
Epicatechin gallate 19
Epigallocatechin gallate 17
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Example 5 (Tooth paste)
(Composition) (Parts by weight)
Calcium hydrogenphosphate 42
Glycerin 18
Carageenan 0.9
Sodium laurylsulfate 1.2
Saccharin sodium 0.09
Butyl paraoxybenzoate 0.005
Extract of fermented tea leaves*
0.05
Perfume 1
Water Balance
TOTAL 100
* Oolong tea extract obtained in Example 1.
Example 6 (Mouth wash)
(Composition) (Parts by weight)
Sodium laurylsulfate 0.8
Glycerin
Sorbitol 5
Ethyl alcohol 15
Extract of fermented tea leav es* 0.05
1-Menthol 0.05
Perfume 0.04
Saccharin sodium 0.1
Water Balance
TOTAL 100
* Black tea extract obtained in Example 1.
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- 18 -
Example 7 (Troches)
(Composition) (Parts by weight)
Gum arabic
Glucose
Extract of fermented tea leaves* 0.02
Potassium dihydrogenphosphate 0.2
Dipotassium hydrogenphosphate 0.1
Lactose 1~
Essence 0.1
Magnesium stearate Balance
TOTAL 100
* Pu-erh tea extract obtained in Example 2.
Example 8 (Preparation of anticarioussugar (powder))
(Procedures)
A liquid mixture of the below-described
composi-
tion was heated at 80-90C to dissolvethe solid in-
gredients. The resultant solution transferred
was to a
rectangular stainless vat and then sed to dry up
cau in
a dryer controlled at 105C. During
the drying, the
solution was mixed every hour. Afterthe drying,
the
resultant solid was ground to convertit into the
form
of a powder sugar.
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(Composition) (Parts by weight)
sugar 200
Extract of fermented tea leaves* 1
Water 30
*As the extract of fermented tea leaves, any one
of Oolong tea extract, black tea extract and
Pu-erh tear extract, which were obtained in
Examples 1-3, is usable.
Example 9 (Preparation of anticarious sugar (powder))
(Procedures)
Using the below-described composition, sugar ac-
cording to the present invention was spray granulated
in a spray granulation apparatus ("Flow Coater Multi
TLO-5M", trade name; manufactured by Okawa Mfg. Co.,
Ltd., Tokyo, Japan). Namely, sugar was placed in a
batch container and then pre-dried at the hot air
temperature of 90°C for about 2 hours. Fermented tea
extract which had been dissolved in water was sprayed
against the sugar at the rate of 100 mE/min for 30
seconds by a spray gun. The spraying was then stopped
and intermediate drying was conducted for 20 minutes.
Spraying and intermediate drying were repeated 4 times,
followed by finish drying for 20 minutes. The
resultant sugar was cooled for 20 minutes, whereby an
anticarious sugar was obtained.
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(Composition) (Parts by weight)
Sugar 200
Extract of fermented tea leaves* 1
Water 10
*As the extract of fermented tea leave, any one
of Oolong tea extract, black tea extract and
Pu-erh tear extract, which were obtained in
Examples 1-3, is usable.
Example 10 (Preparation of anticarious sugar (syrup))
(Procedures)
An extract (0.75 part) of fermented tea leaves
was added to 50 parts of hot water, so that the former
was dissolved. Sugar (150 parts) was added to the
solution, whereby an anticarious sugar (syrup) accord-
ing to the present invention was obtained.
As the extract of fermented tea leaves, any one
of Oolong tea extract, black tea extract and Pu-erh
tear extract, which were obtained in Examples 1 and 2,
is usable.
Example 11 (Comparison of the sweetness of anticarious
sugar)
An anticarious sugar according to the present in-
vention was compared in sweetness with sugar and palat-
inose by organoleptic evaluation as will be described
next.
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- 21 -
(Test samples)
Anticarious sugar of the present invention:
The anticarious sugar obtained in Example 8
by using the Oolong tea extract of Example 1.
Sugar: Refined sugar (prime grade)
Palatinose: Crystalline palatinose
(Evaluation methods)
A 5% sugar solution was prepared as a control.
Using the anticarious sugar, 3%, 4%, 5%, 6% and
7% solutions were prepared. In addition, 6%, 8%, 10%,
12% and 14% solutions were also prepared from the pal-
atinose. These solutions were subjected at room
temperature to organoleptic evaluation by 10 panelists
in accordance with the pair testing method. The
anticarious sugar solution and palatinose solution,
which were comparable in sweetness with the 5% sugar
solution, were determined.
The results are summarized in Table 2.
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fable 2
Sample Sweetness
Sugar 1.0
Anticarious sugar 1.0
Palatinose 0.4
It is envisaged from the above results that the
anticarious sugar of the present invention exhibits
substantially the same degree of sweetness as sugar but
palatinose, an anticarious sugar substitute, shows
sweetness as low as less than half of sugar.
Example 12 (Solubility of anticarious sweetening)
The readiness of dissolution of sugar, an
anticarious sugar and palatinose in water were compared
in terms of solubility in the following manner.
(Test samples)
Anticarious sugar of the present invention:
The anticarious sugar obtained in Example 8
by using the Oolong tea extract of Example 1.
Sugar: Refined sugar (prime grade)
Palatinose: Crystalline palatinose
(Evaluation methods)
At 10°C, 30°C, 50°C and 70°C, the sugar,
anticarious sugar and palatinose were independently
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- 23 -
added to distilled water as much as they were dissolved
completely. Their temperature-dependent solubilities
are shown in Table 3.
Table 3
Temperature
Sample
10C 30C 50C 70C
Sugar 63 68 72 74
Anticarious sugar63 68 72 74
Palatinose 21 I 32 44 58
Solubility (g-solid/100 g-solution)
As is apparent from the foregoing results, the
anticarious sugar according to the present invention
shows the same solubility as sugar. In contrast, pal-
atinose which is a typical anticarious sugar substitute
has low solubility especially at low temperatures. It
is therefore evident that the anticarious sugar accord-
ing to the present invention is easier to use compared
with palatinose.
Example 13 (Anti-glucan formation test)
With respect to three samples consisting of sugar
(refined sugar, prime grade), an anticarious sugar (Ex-
ample 8) and palatinose (crystalline palatinose), the
amounts of glucan independently produced by them were
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- 24 -
compared in the following manner. To a mixture con-
silting of 0.3 mE of a 10% sample solution, 0.3 mE of
1 M sodium phosphate buffer - which had pH 6.0 and con-
tained 1.0% of dextran T10 and 1.0% of sodium azide -
and a glucosyltransferase enzyme solution, water was
added in an amount to give a total volume of 3 mE so
that a reaction system was prepared. They were reacted
in a glass test tube. In the above test, the amount of
the enzyme was set such that the absorbance at 550 nm
became about 1.0 when the sugar (refined sugar, prime
grade) was reacted at 37°C for 3 hours.
Insoluble glucan thus formed was subjected to
ultrasonic disintegration, and the absorbance (A) at
550 nm was measured. Using as control (B) the absor-
bance achieved when the sugar was employed as a test
sample, the relative amount (%) of the so-formed glucan
to the sugar was determined in accordance with the
below-described calculation formula. The results are
given in Table 4.
As the glucosyltransferase enzyme solution, was
used an extract which was obtained by culturing s.
mutans MT8148 strain on Todd-Hewitt medium and then ex-
tracting hyphae with 8M urea in accordance with the
method proposed by Hamada et al. [S. Hamada et al., J.
Gen. Microbiol., 135, 335-344 (1989)].
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(Results)
fable 4'c
Relative amount
sample of formed glucan,
%
Sugar 100
Anticarious sugar 6
Palatinose 2
As is clearly understood from the above results,
the anticarious sugar of the present invention tends to
form much less glucan compared with sugar and has
glucan-formation inhibitory activity close to palat-
inose, although it contains sugar as its principal in-
gredient.
Example 14 (Anti-plaque test)
With respect to sugar (refined sugar, prime
grade), an anticarious sugar (which had been obtained
in Example 10 using the Oolong tea extract) and palat-
inose (crystalline palatinose), an in vitro plaque
formation test was conducted. The test was carried out
in the following manner. Culture media were prepared
by adding the test samples at the concentration of 5%
to Dulbecco's modified Eagle's medium, respectively.
6-mE Portions of the media were added to glass test
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- 26 -
tubes, respectively, to which S, nmtans MT8148 strain
was planted. After the cells were cultured at 37°C for
1 day under anaerobic conditions, the amounts of plaque
adhered on the walls of the respective test tubes were
weighed. The results are summari2ed in Table 5.
Table 5
Amount of plaque
Sample formed, mg
Sugar 25.62 1.78*
Anticarious sugar 3.21 0.54*
Palatinose I 2.01 0.22*
* Average ~ S.D.
Example 15 (Chewing gum)
(Composition) (Parts by weight)
Gum base 20
Calcium carbonate 2
Steviosite 0.1
Extract of fermented tea leaves* 0.01
Lactose 76.89
Essence 1
TOTAL 100
* Oolong tea extract obtained in Example 3.
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Example 16 (Juice)
(Composition) (Parts by weight)
Condensed frozen tangerine juice 5
Liquid sugar (fructose, glucose) 11
Citric acid 0.2
L-Ascorbic acid 0.02
Non-adsorbed fraction* 0.01
Essence 0.2
Color 0.1
Water Balance
TOTAL 100
* Non-adsorbed fraction of the black
tea extract
obtained in Example 3.
Example 17 (Candies)
(Composition) (Parts by weight)
Sorbitol powder 99.745
Essence 0.2
Non-adsorbed fraction* 0.005
Sorbitol seeds 0.05
TOTAL 100
* Non-adsorbed fraction of the Pu-erhtea extract
obtained in Example 3.
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