Note: Descriptions are shown in the official language in which they were submitted.
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NOVEL ?~ACTIC ACID BACTERIA
BACKGROUND OF THEM INVENTION
1. Field of the Invention
The present invention relates to some novel lactic acid
bacteria which inhibit the production of dental plaque in
human mouths. More specifically, the production of water-
insoluble glucan (mutan), a major component of dental
plaque, which is produced by bacteria normally inhibiting in
human mouths, can be inhibited by the novel bacteria. Oral
anaerobic bacteria causing gingivitis, periodontitis, and
accompanied halitosis (malodor) can be inhibited by the
novel bacteria, too. These lactic acid bacteria belong to
Enterococcus spp., Lactobacillus spp., Lactococcus spp., and
Stretococcus spp. which inhibit the production of water-
insoluble glucan or antagonize against the bacteria playing
a role in forming water-insoluble glucan, or inhibit the
growth of anaerobic bacteria causing gingivitis and
periodontitis.
2. Description of the Prior Art
Lactic acid bacteria generally ferment carbohydrates to
lactic acid. Lactic acid bacteria live in the oral cavities
and the alimentary tracts of men and animals and are
utilized for the manufacture of fermentative foods, such as
yogurt, cheese, etc. In addition, they are used for the
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production of biologically active materials, such as
medicines. Representatives of these lactic acid-producing
bacteria are Streptococcus thermophilus, Enterococcus
faecalis, Enterococcus durans, Lactococcus lactis,
Lactobacillus acidophilus, Lactobacillus bulgaricus,
Lactobacillus casei, and Lactobacillus plantarum. As
inhabitants in the entrails of men and animals, these Gram-
positive lactic acid bacteria are known to play an important
role in maintaining the entrails healthy by the production
of lactic acid and antibacterial materials which inhibit the
growth of pathogenic bacteria.
The most important component of dental plaque is
glucan. Glucan is either water-soluble glucan, dextran
having 1,6-a linkage as a predominant linkage, or water-
insoluble glucan (mutan) having 1,3-a linkage as a
predominant linkage. The solubility in water is inversely
proportional to the number of 1,3-a linkage. Therefore,
water-insoluble glucan (mutan) serves as a main matrix of
dental plaque. Dextranase (a-1,6 glucan hydrolase) which
digests dextran, was tested as to its ability to prevent
dental plaque. But the effectiveness value of dextranse to
prevent dental plaque was questionable (Essential Dental
Microbiology: Appleton & Lange, Norwalk, San Mateo, p.337,
1991), because dextranase can not digest mutan, the main
matrix of dental plaque. Mutanase (endo-a-1,3-glucanase)
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which decomposes mutan was found to have some effect on the
digestion of dental plaque. The decomposition effect of the
mutanase on dental plaque, however, was trivial and it took
too much time to express its effectivity. Therefore, these
enzymes were found to have an insignificant effect on dental
plaque formation in human oral cavity.
With regard to dental plaque prevention with lactic
acid bacteria, European patent publication number 0-524-732-
A2 disclosed the use of Streptococcus salivarius which was
capable of extracellular production of dextranase. But its
effect on preventing dental plaque is questionable because
dextranase can not digest mutan, the main matrix of dental
plaque. Streptococcus salivarius is not used as the starter
bacteria fermenting milk.
i5
SUMMARY 0~' THE INVF,,N~ION
As a consequence of intensive and thorough researches
on the inhibitory activity of lactic acid bacteria against
the production of water-insoluble glucan or dental plaque
and the growth of anaerobic bacteria, the present study has
been based on the presumption that some lactic acid bacteria
inhabiting in human mouths may be able to inhibit the
production of water-insoluble glucan or dental plaque or the
growth of anaerobic bacteria causing gingivitis and
periodontitis. Through many clinical experiments, these
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novel strains have been found and proved to have the ability
to inhibit the production of water-insoluble glucan or
dental plaque significantly, and to inhibit the growth of
anaerobic bacteria causing gingivitis and periodontitis.
They was named Enterococcus spp. 1357, Lactobacillus spp.
V20, and Lactococcus spp. 1370, respectively. They are now
deposited in the Korean Collection for Type Cultures, Korean
Research Institute of Bioscience and Biotechnology, on 30,
Jul. and 11, Dec. 1997 (deposition No. KCTC 0360BP for
Enterococcus spp. 1357, KCTC 0361BP for Lactobacillus spp.
V20, KCTC 0415BP for Lactococcus spp. 1370).
Glucan is either water-soluble or water-insoluble
(mutan), each being synthesized from sucrose by the
glucosyltransferase secreted from Streptococcus mutans, the
most important bacteria among dental plaque-forming
bacteria. However, only the water-insoluble glucan, mutan,
is the main matrix of dental plaque.
Enterococcus spp. 1357, Lactobacillus spp. V20, and
H20z-producing Streptococci such as Streptococcus oralis,
Streptococcus mitior, Streptococcus mitis, and Streptococcus
sanguis (Bergey's Manual of Systematic Bacteriology vol. 2:
Williams & Wilkins, Baltimore, London, Los Angeles, Sydney,
1986) have a growth-inhibitory activity on Streptococcus
mutans. When.Streptococcus mutans was cultured with
Enterococcus spp. 1357, Lactobacillus spp. V20, or
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Streptococcus oralis (ATCC 35037) as a representative of
H202-producing Streptococci in the broth, the colony-forming
number of Streptococcus mutans was decreased to about one
hundredth compared with that of Streptococcus mutans alone.
The production of water-insoluble glucan or dental plaque
was also suppressed significantly due to inhibition of
Streptococcus mutans.
When high molecular weight dextran was added into the
culture broth of Streptococcus mutans, the
glucosyltransferase binding to water-insoluble glucan was
interfered, and then the following synthesis of water-
insoluble glucan was inhibited (Shigeyuki H., et al.,
Journal of General Microbiology, 116:51, 1980). Lactococcus
spp. 1370 produced the large amounts of water-soluble
glucan~ when Streptococcus mutans was incubated with this
Lactococcus spp. 1370, the synthesis of water-insoluble
glucan was suppressed.
Generally, dental plaque, adherent to the surface of
teeth, provides a suitable habitat at which Streptococcus
mutans as well as other bacteria proliferate and causes the
dental caries formed, and it was an object of the present
study to find novel bacteria which could inhibit the
production of water-insoluble glucan or dental plaque by
Streptococcus mutans in the mouth.
Anaerobic bacteria occur in high proportions in
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periodontitis as well as gingivitis of oral cavities. The
proportions of anaerobic bacteria increase significantly
(above 905 of microflora in periodontitis lesion) with
increasing severity of the diseases. Predominant anaerobic
bacteria causing gingivitis are Prevotella intermedia and
Fusobacterium nucleatum. Predominant anaerobic bacteria
causing periodontitis include Porphyromonas gingivalis,
Actinobacillus actinomycetemcomitans, Prevotella intermedia,
and Fusobacterium nucleatum (Contemporary Oral Microbiology
and Immunology, Mosby-Year Book, St. Louis, 1992). These
anaerobic bacteria produce malodorous components such as
volatile sulfur compounds in mouth. The predominant
volatile sulfur compounds are hydrogen sulfide from L-
cysteine and methyl mercaptan from L-methionine (Persson,
S., Oral Microbiol Immunol, 7:378, 1992). Lactobacillus
spp. V20 and HZOz-producing Streptococci such as
Streptococcus oralis, Streptococcus mitior, Streptococcus
mitis, and Streptococcus sanguis produce hydrogen peroxide
inhibiting the growth of anaerobic bacteria causing
gingivitis and periodontitis, so improving and preventing
the lesions, and decreasing the accompanied halitosis. The
hydrogen peroxide is an oxidizing agent inactivating enzymes
by converting functional -SH groups to the oxidized S-S form
and is used as a disinfectant against bacteria, especially
anaerobics.
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Another object of this research was to provide foods or
beverages employing the lactic acid bacteria. When the
foods containing the lactic acid bacteria capable of
directly inhibiting the production of water-insoluble glucan
or having the growth-inhibitory activity on the
microorganisms which contribute to the formation of dental
plaque are eaten, the lactic acid bacteria naturally
suppress the formation of dental plaque and consequently
prevent dental caries formation. And when the foods contain
the lactic acid bacteria capable of inhibiting the growth of
anaerobic bacteria causing gingivitis and periodontitis, the
lactic acid bacteria naturally improve and prevent
gingivititis, periodontitis, and accompanied halitosis.
$RIEF DESCRIPTION OF THE DRAWING
Above and other objects and aspects of the invention
will become apparent from the following description of
embodiment with reference to the accompanying drawing which
shows the inhibitory effect of the novel strain on the
production of artificial dental plaque on orthodontic wires
(Fig. 1) .
p~ETATLED DESCRIPTTON OF PROCESS TO THE INVE'~TTON
Lactic acid bacteria were taken from human bodies,
streaked on Brain Heart Infusion agar, and cultured at 37°C.
*rB
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_g_
Thereafter, separated bacterial colonies were tested whether
they could suppress the production of the water-insoluble
glucan that Streptococcus mutans (Ingbritt strain) produced.
In a cuvette, 3 mL of a Brain Heart Infusion medium
supplemented with 0.5% yeast extract and 5o sucrose was
inoculated with 0.1 mL of Streptococcus mutans and 0.1 mL of
a culture broth of the separated bacteria. As a control,
Streptococcus mutans was inoculated alone in a Brain Heart
Infusion medium containing yeast extract and sucrose. The
cuvette was placed at an angle of 30° to the horizontal
plane of an incubator and incubated for 1 day at 37°C, in
order for the Streptococcus mutans to produce water-
insoluble glucan. After removing the culture broth, the
cuvette was washed with 4 mL of distilled water and then,
filled with 3 mL of distilled water. Its absorbance (OD) at
550 nm was measured by a spectrophotometer. Because the OD
value was proportional to the water-insoluble glucan
produced, the bacteria which brought about a significantly
lower OD value compared with the control, were isolated as
dental plaque-inhibitory strains.
To evaluate the capability producing hydrogen peroxide,
the isolated strains were streaked on the Brain Heart
Infusion agar containing 0.25mg/mL 3,3',5,5'-
tetramethylbenzidine dihydrochloride and 0.01mg/mL
horseradish peroxidase and incubated in anaerobic incubator
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for 48 hours. Lactobacillus spp. V20 formed the blue
colonies, indicating that Lactobacillus spp. V20 had the
capability to produce hydrogen peroxide.
The microbiological properties of the isolated strains,
such as morphological and physiological properties (Table
1), and sugar catabolytic ability (Table 1) were
investigated.
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TABLE 1
Morphological and Physiological Properties
Isolated Bacterial
Strains
Pro
erties
p
Enterococcus Lactobacillus Lactobacillus
spp. 1357 spp. V20 spp. 1370
Morphology coccus, bacillus, coccus, chain
chain chain
Gram stain positive positive positive
Spore forming - - -
Catalase - - -
Activity
Culture + - +
Temp. 10C
Culture + + -
Temp. 45C
pH 9 . 6 + - -
1~ 40 bile acid + - +
6.5% NaCl + - -
Growth on MRS - + -
medium
Acetoin + +
Production
Hippurate + -
Hydrolysis
Pyrrolidonylary + -
amidase
a- - -
Galactosidase
o
nidase
Glucu
o
sidase
Galac
Alkaline - -
phosphatase
Leucine + +
arylamidase
3J A r ; nine + _
d i by rolase
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TABLE 2
Catalytic Activities on Sugars
Carbohydrate Isolated Bacterial
Strains
Enterococcus Lactobacillus Lactococcus
spp. 1357 spp. V20 spp. 13i0
Arabinose - - -
Amygdalin + - -
Cellobiose + + +
Esculin + + -
Fructose + + +
Galactose + + +
Glucose + + +
Lactose + + +
Maltose + + +
Mannitol - - +
Mannose + + +
I~ Melezitose - - -
Raffinose - - -
Rhamnose - - -
Salicin + - -
Sorbitol - - -
Trehalose + + +
Inulin - -
Starch + +
Glycogen - -
As mentioned above, the novel lactic acid bacteria
being able t;, inhibit the production of dental plaque were
isolated and assayed in comparison with the control in
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vitro. Further, the novel properties were tested in vivo,
that is, the isolated novel bacteria were applied to the
human oral cavity.
EXAMPLE I
Inhibition of the Production of Water-Insoluble Glucan in
Disposable Cuvette
An equal amount of M17 medium was mixed with MRS medium
and supplemented with 0.5% yeast extract, 5% sucrose and
O.1M TES (pH 8.0). Three milliliters of the constituted
medium were transferred to a disposable cuvette which was,
then, inoculated with 751 of Streptococcus mutans overnight
culture. The cuvette was placed at an angle of 30° to the
horizontal plane in an incubator and cultured at 30°C for 1
day. The content was removed and then, the cuvette was
washed with 3mL of distilled water. Thereafter, the cuvette
was filled with 4mL of distilled water and the absorbency at
550nm was measured by a spectrophotometer. This measurement
was repeated three times and the average value was obtained
(Table 3) .
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TABLE 3
Inhibitory Effect of Lactic Acid Bacteria on the Production
of Water-Insoluble Glucan
Samples Test Bacterial Strains OD (550nm)
Control I Str. mutans 2.122
Control II Streptococcus thermophilus 2.325
+ Str. mutans
Group I Enterococcus spp. 1357 0.434
Group II Enterococcus spp. 1357 0.713
+ Str. mutans
Group III Lactobacillus spp. V20 0.506
Group IV Lactobacillus spp. V20 1.154
+ Str. mutans
Group V Lactococcus spp. 1370 0.576
Group VI Lactococcus spp. 1370 1.020
+ Str. mutans
Group VII Streptococcus oralis 0.511
Group VIII Streptococcus oralis 0.980
+ Str. mutans
As shown in Table 3, the optical densities of Control
group I and Control group II were 2.122 and 2.325 in
absorbency at 550nm whereas those of Test group II, Test
group IV, Test group VI, and Test group VIII were 0.713,
1.154, 1.020, and 0.980, respectively. This reduction in
the absorbency meant that these bacteria inhibited the
Streptococcus mutans' production of water-insoluble glucan.
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EXAMPLE II
Artificial Dental Plaque Formation on Orthodontic Wire
An equal amount of M17 medium was mixed with MRS medium
and supplemented with 0.5~ yeast extract, 5~S sucrose,- and
O.1M TES (pH 8.0). One hundred and fifty milliliters of the
constituted medium were poured into beaker. 0.016 inch
stainless steel orthodontic wires (45mg) were immersed in
the medium. Streptococcus mutans was inoculated at the
concentration of 2.5X106 per mL of the medium. Thereafter,
the lactic acid bacterial strains were inoculated in the
medium at the concentration of 5 times higher than that of
Streptococcus mutans and incubated in a COZ incubator at
37°C for 6.5 hours with shaking. Only water-insoluble
glucan or plaque was attached on the wires (McCabe, R.M.,
et. Al., Archs oral Boil., 12:1653, 1967). The wires were
transferred to fresh beakers and photographed (Fig. 1).
Fig. 1(A) is a photograph of the culture of Streptococcus
mutans alone while Fig. 1(B) is that of the co-culture of
Streptococcus mutans and Lactococcus spp. 1370.
The weights of the artificial plaques formed on the
wires were measured and the results are shown in Table 4.
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TABLE 4
Inhibitory Activity of Lactic Acid Bacteria
on the Formation of Artificial Plaque
Samples Test Bacterial Strains Weight of
Produced Plaque
Control I Str. mutans 75.4 mg
Control II Streptococcus thermophilus 92.3 mg
+ Str. mutans
Group I Enterococcus spp. 1357 0.0 mg
+ Str. mutans
Group II Lactobacillus spp. V20 30.9 mg
+ Str, mutans
Group III Lactococcus spp. 1370 0.0 mg
+ Str. mutans
Group IV Streptococcus oralis 0.0 mg
+ Str. mutans
In the control group I and the control group II, an
artificial plaque of 75.4 mg and 92.3 mg was formed while no
artificial plaque was formed in Test group I, Test group III
and Test group IV. In Test group II, the plaque weight was
reduced to 30.9 mg. Thus, it is clearly shown that these
bacteria have a potent inhibitory activity on the production
of dental plaque by Streptococcus mutans.
EXAMPLE III
Reduction of the Dental Plaque Index in Human Mouths
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In order to evaluate the reduction effect of the novel
lactic acid bacterial strains on plaque index in human
mouths, experiments were performed in thirty-eight persons
to achieve the plaque scores by Quigley and Hein Plaque
S Index (Harper, D.S., et. Al., J Periodontol, 61:352, 1990).
Thirty-eight young adults, 22 to 26 years of age,
volunteered to participate in this study. All volunteers
were received thorough oral prophylaxis, and suspended all
oral hygiene. Volunteers ate and drank as usual, but
stopped brush washing. Baseline plaque scores were assessed
at 24 hours after receiving oral prophylaxis. Plaque scores
were performed by Quigley and Hein Plaque Index after
disclosing all plaque except third molars. The volunteers
were randomly assigned to two groups (each nineteen
persons), group I mouthrinsing with Lactococcus spp. 1370
while group II mouthrinsing with Lactobacillus spp. V20.
Test bacterial suspensions were prepared by incubating
either Lactococcus spp. 1370 or Lactobacillus spp. V20 in
milk for 24 hours. Volunteers rinsed immediately once after
oral prophylaxis and twice after meals with 20 mL of
Lactococcus spp. 1370 or Lactobacillus spp. V20 culture in
milk (109 CFU/mL) for 2 minutes. Plaque scores were again
assessed at 24 hours after receiving oral prophylaxis. The
plaque scores of total teeth except third molars were
averaged and statistically analyzed in each group. The
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results indicated that plaque index reduction of 0.97 in the
group I and 0.55 in the group II at 24 hours after receiving
oral prophylaxis (Table 5). The reductions of plaque index
were statistically significant (p<0.05), i.e. Lactococcus
sp. 1370 and Lactobacillus sp. V20 reduced plaque formation
in the oral cavity significantly.
TABLE 5
Reduction of the Plaque Index by Bacterial Mouthrinse
Mean Mean Plaque Score
Used Bacteria Baseline afer Bacterial Difference
Plaque Score Mouthrinse
Lactococcus 2.17 1.20 -0.97*
spp. 1370
Lactobacillus 2.15 1.60 -0.55*
spp. V20
p« .ul py pairea z zest
EXAMPLE IV
Inhibition of the Growth of Anaerobic Bacteria
in Mixed Culture
Lactobacillus spp. V20 was cultured in MRS media for 24
hours. Prophyromonas gingivalis, Prevotella intermedia, and
Fusobacterium nucleatum were cultured in the anaerobic
bacteria culture broth containing Brain Heart Infusion media
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18.5 g, yeast extract 5.0 g, hemin solution 10 mL (dissolved
50 mg hemin in 1 N sodium hydroxy solution 1 mL and added
with distilled water 100 mL), and vitamin K solution 0.2 mL
(vitamin K solution 0.15 mL mixed with 95°s ethanol 30 mL)
per liter in anaerobic incubator for 36 hours.
Actinobacillus actinomycetemcomitans was cultured in the
TSBV media containing Tryptic soy broth 30 g, yeast extract
1.0 g, horse serum 100 mL, bacitracin 75 mg, and vancomycin
5 mg per liter in anaerobic incubator for 36 hours. Culture
suspension 0.1 mL of Lactobacillus spp. V20 and each
anaerobic bacterium at the concentration of 1.4X108 per mL
were inoculated singly or in combination in the media
containing anaerobic bacteria culture broth or TSBV media
3.7 mL mixed with MRS broth 0.3 mL, and cultured in
anaerobic incubator for 36 hours. The culture suspension
was diluted and inoculated on MRS agar, anaerobic bacteria
culture agar containing 3o sheep blood or TSBV agar. At 72
hours after culturing, the number of colonies was counted.
The colony-forming units of Lactobacillus spp. V20 and each
anaerobic bacterium were increased after being cultured
singly, whereas the colony of each anaerobic bacterium was
not found after being cultured in combination with
Lactobacillus spp. V20. When anaerobic bacteria was
cultured with Lactobacillus casei which did not produce
hydrogen peroxide, the colony-forming units were not
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decreased significantly (Table 5). When Streptococcus
oralis (ATCC 35037) as a representative of H202-producing
Streptococci was cultured with each anaerobic bacterium by
the above mentioned method, anaerobic bacteria did not form
a colony on the media.
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TABLE 6
Colony-forming Units after Culturing
Colony-forming Colony-forming
Inoculated units of units of
bacteria Lactobac illus anaerobe after
after culture(/mL) culture (/mL)
Lactobacillus spp. 8.2 X 108
V20
Lactobacillus casei 9.0 X 108
Porphyromonas 1.9 X lOg
gingivalis
Actinobacillus 2.0 X 108
actinomycetemcomitans
Prevotella intermedia 1.8 X 108
Fusobacterium 1.8 X 10$
nucleatum
Lactobacillus spp.
V20 + Porphyromonas 8.0 X 108 0
gingivalis
Lactobacillus spp.
V20 + Actinobacillus 8.8 X I08 0
actinomycetemcomitans
Lactobacillus spp.
V20 + Prevotella 8.9 X 108 0
intermedia
Lactobacillus spp.
V20 + Fusobacterium 7.8 X 108 0
nucleatum
Lactobacillus casei
+
Porphyromonas 9.1 X 108 1.5 X 108
gingivalis
Lactobacillus casei
+
Actinobacillus 9.3 X 108 I.8 X 108
actinomycetemcomitans
Lactobacillus casei 8.9 X 108 1.6 X 108
+
Prevotella intermedia
Lactobacillus casei
+
Fusobacterium 9.8 X 108 1.6 X 10g
nucleatum
*rB
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Hereafter were presented the examples in which the
novel lactic acid bacteria were practically applied.
USE EXAMPLE I: Yogurt
A broth culture containing the novel lactic acid
bacterial strains was added at an amount of 0.1 vol. percent
to the food just before fermentation and subjected to
fermentation along with the existing bacteria to produce
yogurt foods. The resulting yogurt foods were tasted by 10
panelists. They noted no different flavor between the test
samples and the commercially available foods (controls).
Before a sealing step in the manufacture procedure, the
lactic acid bacterial strains were added at an amount of 0.2
vol. percent. A response that these test samples thus
obtained were not different from the control foods in taste
was drawn from 10 panelists who took part in the tasting
tests.
USE EXAMPLE II: Butter
Before a packaging step, butter foods which were
manufactured by a typical procedure were added with 0.2 wt.
percent of the freeze-dried lactic acid bacterial strains.
These butter foods thus obtained were given as taste
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samples.
USE EXAMPLE III: Cheese
Before a packaging step, cheese foods which were
manufactured by a typical procedure were added with 0.2 wt.
percent of the freeze-dried lactic acid bacterial strains.
These cheese foods thus obtained were given as taste
samples.
USE EXAMPLE IV: Freeze-dried lactic acid bacteria
The novel lactic acid bacteria were cultured and
freeze-dried (lyophilized). These freeze-dried bacteria in
capsule', tablet, and small package could be taken singly or
with other bacteria or materials. These freeze-dried
products thus obtained were given taste samples.
Accordingly, the lactic acid bacterial strains were
applied for various foods, including gum, shortening, ice
cream, margarine, kimchi, etc.
From the examples above, it is apparent that the novel
lactic acid strains have a potent and lasting inhibitory
effect on the production of water-insoluble glucan or dental
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plaque in human mouth, or on the growth of anaerobic
bacteria causing gingivitis, periodontitis, and accompanied
halitosis.
In addition, the novel strains were found to be able to
be applied for various foods as well as directly to the
teeth.
Many modifications and variations of the present
invention are possible in the light of the above techniques.
