U.S. patent application number 13/382087 was filed with the patent office on 2012-05-24 for prophylactic, ameliorating or therapeutic agent for oral diseases.
This patent application is currently assigned to HIROSHIMA UNIVERSITY. Invention is credited to Hiroki Nikawa.
Application Number | 20120128645 13/382087 |
Document ID | / |
Family ID | 43449195 |
Filed Date | 2012-05-24 |
United States Patent
Application |
20120128645 |
Kind Code |
A1 |
Nikawa; Hiroki |
May 24, 2012 |
PROPHYLACTIC, AMELIORATING OR THERAPEUTIC AGENT FOR ORAL
DISEASES
Abstract
To provide a novel lactic acid bacterium strain having a broad
antibacterial spectrum against bacteria that cause oral diseases,
from which a fermented product having good flavor and being
excellent in palatability can be produced, and a prophylactic,
ameliorating or therapeutic agent for oral diseases using the same.
A prophylactic, ameliorating or therapeutic agent for an oral
disease, which contains a lactic acid bacterial cell of one or more
kinds selected from the group consisting of Lactobacillus rhamnosus
strain KO3, Lactobacillus casei strain YU3 and Lactobacillus
paracasei strain YU4 or a culture of the cell, or an extract
thereof, as an active ingredient.
Inventors: |
Nikawa; Hiroki;
(Hiroshima-shi, JP) |
Assignee: |
HIROSHIMA UNIVERSITY
Higashi-Hiroshima-shi
JP
|
Family ID: |
43449195 |
Appl. No.: |
13/382087 |
Filed: |
July 16, 2010 |
PCT Filed: |
July 16, 2010 |
PCT NO: |
PCT/JP2010/004626 |
371 Date: |
January 3, 2012 |
Current U.S.
Class: |
424/93.45 ;
426/580; 426/590; 435/252.9 |
Current CPC
Class: |
A61K 35/747 20130101;
A23C 9/123 20130101; A23Y 2220/73 20130101; A61P 1/02 20180101 |
Class at
Publication: |
424/93.45 ;
435/252.9; 426/580; 426/590 |
International
Class: |
A61K 35/74 20060101
A61K035/74; A23C 9/123 20060101 A23C009/123; A23L 2/00 20060101
A23L002/00; C12N 1/20 20060101 C12N001/20; A61P 1/02 20060101
A61P001/02 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 16, 2009 |
JP |
2009-168122 |
Claims
1. A prophylactic, ameliorating or therapeutic agent for an oral
disease, comprising: a lactic acid bacterial cell of one or more
kinds selected from the group consisting of Lactobacillus rhamnosus
strain KO3 (NITE BP-771), Lactobacillus casei strain YU3 (NITE
BP-772) and Lactobacillus paracasei strain YU4 (NITE BP-775) or a
culture of the cell, or an extract thereof, as an active
ingredient.
2. A food, comprising: a lactic acid bacterial cell of one or more
kinds selected from the group consisting of Lactobacillus rhamnosus
strain KO3 (NITE BP-771), Lactobacillus casei strain YU3 (NITE
BP-772) and Lactobacillus paracasei strain YU4 (NITE BP-775) or a
culture of the cell, or an extract thereof.
3. The food according to claim 2, which is a fermented milk or a
fermented beverage.
4. An agent for suppression of proliferation of a cariogenic
bacterium, a periodontal disease bacterium and a candida bacterium,
comprising: a lactic acid bacterial cell of one or more kinds
selected from the group consisting of Lactobacillus rhamnosus
strain KO3 (NITE BP-771), Lactobacillus casei strain YU3 (NITE
BP-772) and Lactobacillus paracasei strain YU4 (NITE BP-775) or a
culture of the cell, or an extract thereof, as an active
ingredient.
5. The agent for suppression of proliferation of a cariogenic
bacterium, a periodontal disease bacterium and a candida bacterium
according to claim 4, wherein the cariogenic bacterium, periodontal
disease bacterium and candida bacterium are at least Streptococcus
mutans, Streptococcus sobrinus, Porphyromonas gingivalis and
Candida albicans.
6. Lactobacillus rhamnosus strain KO3 that has been deposited as
NITE BP-771, Lactobacillus casei strain YU3 that has been deposited
as NITE BP-772 and Lactobacillus paracasei strain YU4 that has been
deposited as NITE BP-775, at the Patent Microorganisms Depositary
of the National Institute of Technology and Evaluation.
7. A lactic acid bacterial cell of one or more kinds selected from
the group consisting of Lactobacillus rhamnosus strain KO3 (NITE
BP-771), Lactobacillus casei strain YU3 (NITE BP-772) and
Lactobacillus paracasei strain YU4 (NITE BP-775) or a culture of
the cell, or an extract thereof, which is used as a prophylactic,
ameliorating or therapeutic agent for an oral disease.
8. A method for prophylaxis, amelioration or treatment of an oral
disease, comprising: administering or ingesting a lactic acid
bacterial cell of one or more kinds selected from the group
consisting of Lactobacillus rhamnosus strain KO3 (NITE BP-771),
Lactobacillus casei strain YU3 (NITE BP-772) and Lactobacillus
paracasei strain YU4 (NITE BP-775) or a culture of the cell, or an
extract thereof.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a prophylactic,
ameliorating or therapeutic agent for oral diseases such as dental
caries or periodontal diseases.
BACKGROUND OF THE INVENTION
[0002] A bacterial flora in the oral cavity is constituted by 400
to 500 kinds of microorganisms. Therefore, various pathogenic
microorganisms such as cariogenic bacteria, periodontal disease
bacteria and candida bacteria are present in the oral cavity, which
causes various diseases such as dental caries, periodontal
diseases, glossitis, thrush and oral candidiasis. Furthermore, it
is reported in recent studies that candida bacteria also relate to
periodontal diseases.
[0003] Therefore, many suggestions have been heretofore made with
respect to oral compositions containing an antibacterial substance.
However, even an antibacterial agent was administered to the oral
cavity, the antibacterial agent was washed out by saliva or a food
or beverage within a short time, and thus the effect thereof had to
be said to be a transient effect.
[0004] Furthermore, in view of that a lactic acid bacterium
suppresses bacteria causing various diseases in the large
intestine, studies for applying the technique thereof to dental
diseases have been done. For example, it was reported that lactic
acid bacteria such as Lactobacillus salivarius (Patent Document 1
and Patent Document 2), Lactobacillus reuteri (Patent Document 3
and Patent Document 4), Lactobacillus paracasei (Patent Document
5), Lactobacillus delbrueckii (Patent Document 6) and Lactobacillus
fermentum (Non-patent Document 1) are effective for the prophylaxis
of dental caries and periodontal diseases.
[0005] However, there was also a problem that even a lactic acid
bacterium having an antibacterial effect against cariogenic
bacteria and periodontal disease bacteria has a narrow
antibacterial spectrum, has insufficient fermentability and thus
cannot provide a good fermented product, and cannot provide a
fermented product having good flavor and being excellent in
palatability.
PRIOR ART DOCUMENTS
Patent Document
[0006] Patent Document 1: WO 2002/016554 [0007] Patent Document 2:
WO 2003/082027 [0008] Patent Document 3: Japanese Patent
Application Laid-Open (JP-A) No. 2003-299480 [0009] Patent Document
4: Japanese Patent Application National Publication (Laid-Open) No.
2008-502360 [0010] Patent Document 5: JP-A-2008-37859 [0011] Patent
Document 6: JP-A-2008-237198
Non-patent Documents
[0011] [0012] Non-patent Document 1: Sumiyo MIMURA, Hiroki NIKAWA,
Seicho MAKIHIRA, Aya HIYAMA and Yuko TAKAMOTO, the Journal of
Nippon Academy of Dental Technology, Vol. 29, special issue, 298
(2008)
SUMMARY OF THE INVENTION
Problem to be Solved by the Invention
[0013] The present invention relates to provision of a novel lactic
acid bacterium strain, having a broad antibacterial spectrum
against bacteria that cause oral diseases, from which a fermented
product having good flavor and being excellent in palatability can
be produced, and to provision of a food and a prophylactic,
ameliorating or therapeutic agent for oral diseases containing the
same.
Means for Solving the Problem
[0014] The present inventors have done intensive studies on oral
microorganisms, and found that specific lactic acid bacterium
strains belonging to Lactobacillus rhamnosus, Lactobacillus casei
and Lactobacillus paracasei that are present in saliva have an
excellent antibacterial effect against any of cariogenic bacteria,
periodontal disease bacteria and candida bacteria, and that a
fermented product being excellent in palatability can be produced
by using these.
[0015] Therefore, the present invention relates to the following 1)
to 8).
[0016] 1) A prophylactic, ameliorating or therapeutic agent for an
oral disease, which contains a lactic acid bacterial cell of one or
more kinds selected from the group consisting of Lactobacillus
rhamnosus strain KO3 (NITE BP-771), Lactobacillus casei strain YU3
(NITE BP-772) and Lactobacillus paracasei strain YU4 (NITE BP-775)
or a culture of the cells, or an extract thereof, as an active
ingredient.
[0017] 2) A food containing a lactic acid bacterial cell of one or
more kinds selected from the group consisting of Lactobacillus
rhamnosus strain KO3 (NITE BP-771), Lactobacillus casei strain YU3
(NITE BP-772) and Lactobacillus paracasei strain YU4 (NITE BP-775)
or a culture of the cell, or an extract thereof.
[0018] 3) The food according to the above-mentioned 2), which is a
fermented milk or a fermented beverage.
[0019] 4) An agent for the suppression of the proliferation of a
cariogenic bacterium, a periodontal disease bacterium and a candida
bacterium, which contains a lactic acid bacterial cell of one or
more kinds selected from the group consisting of Lactobacillus
rhamnosus strain KO3 (NITE BP-771), Lactobacillus casei strain YU3
(NITE BP-772) and Lactobacillus paracasei strain YU4 (NITE BP-775)
or a culture of the cell, or an extract thereof, as an active
ingredient.
[0020] 5) The agent for the suppression of the proliferation of a
cariogenic bacterium, a periodontal disease bacterium and a candida
bacterium according to 4), wherein the cariogenic bacterium,
periodontal disease bacterium and candida bacterium are at least
Streptococcus mutans, Streptococcus sobrinus, Porphyromonas
gingivalis and Candida albicans.
[0021] 6) Lactobacillus rhamnosus strain KO3 that has been
deposited as NITE BP-771, Lactobacillus casei strain YU3 that has
been deposited as NITE BP-772 and Lactobacillus paracasei strain
YU4 that was deposited as NITE BP-775, at the Patent Microorganisms
Depositary of the National Institute of Technology and
Evaluation.
[0022] 7) A lactic acid bacterial cell of one or more kinds
selected from the group consisting of Lactobacillus rhamnosus
strain KO3 (NITE BP-771), Lactobacillus casei strain YU3 (NITE
BP-772) and Lactobacillus paracasei strain YU4 (NITE BP-775) or a
culture of the cell, or an extract thereof, which is used as a
prophylactic, ameliorating or therapeutic agent for an oral
disease.
[0023] 8) A method for the prophylaxis, amelioration or treatment
of an oral disease, which includes administering or ingesting a
lactic acid bacterial cell of one or more kinds selected from the
group consisting of Lactobacillus rhamnosus strain KO3 (NITE
BP-771), Lactobacillus casei strain YU3 (NITE BP-772) and
Lactobacillus paracasei strain YU4 (NITE BP-775) or a culture of
the cell, or an extract thereof.
Effects of the Invention
[0024] According to the present invention, a food with high
palatability, a medicament, an oral composition and the like, which
exert a prophylactic, ameliorating or therapeutic effect for
various oral diseases such as dental caries, periodontal diseases,
glossitis, thrush and oral candidiasis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1a is a graph showing the growth-inhibiting effect
against Streptococcus mutans.
[0026] FIG. 1b is a graph showing the growth-inhibiting effect
against Streptococcus mutans.
[0027] FIG. 2 is a graph showing the growth-inhibiting effect
against Streptococcus sobrinus.
[0028] FIG. 3a is a graph showing the growth-inhibiting effect
against Porphyromonas gingivalis.
[0029] FIG. 3b is a graph showing the growth-inhibiting effect
against Porphyromonas gingivalis.
[0030] FIG. 4a is a graph showing the growth-inhibiting effect
against Candida albicans.
[0031] FIG. 4b is a graph showing the growth-inhibiting effect
against Candida albicans.
[0032] FIG. 5 is a graph showing the biofilm formation-inhibiting
effect against Candida albicans.
[0033] FIG. 6 is a graph showing the result of the evaluation on
taste (Visual Analogue Scale (VAS) method).
[0034] FIG. 7 is a graph showing the antibacterial action of
yogurt. (A): S. mutans ingbritt strain, (B): S. sobirinus strain
B13
[0035] FIG. 8a is a graph showing the effect of decreasing the
numbers of cariogenic bacteria and periodontal disease bacteria in
the human oral cavity. (A): cariogenic bacteria, (B): P. intermedia
(Pi)
[0036] FIG. 8b is a graph showing the effect of decreasing the
numbers of cariogenic bacteria and periodontal disease bacteria in
the human oral cavity. (C): T. forsythensis (Tf), (D): F. nucleatum
(Fuso)
MODES FOR CARRYING OUT THE INVENTION
[0037] The lactic acid bacteria used for the fermented milk of the
present invention are lactic acid bacteria belonging to
Lactobacillus rhamnosus, Lactobacillus casei and Lactobacillus
paracasei; specifically, they are Lactobacillus rhamnosus strain
KO3 that was deposited as NITE BP-771 on Jun. 10, 2009,
Lactobacillus casei strain YU3 that has been deposited as NITE
BP-772 on Jun. 10, 2009, and Lactobacillus paracasei strain YU4
that has been deposited as NITE BP-775 on Jun. 24, 2009, at the
Patent Microorganisms Depositary of the National Institute of
Technology and Evaluation (Address: 2-5-8 Kazusakamatari,
Kisarazu-shi, Chiba 292-0818, Japan).
[0038] The KO3 strain was first separated from human saliva by the
present inventors, and was identified as Lactobacillus rhamnosus
since the base sequence of 16S rRNA shows a homology of 100% with
the base sequence of Lactobacillus rhamnosus strain IDCC3201
between 1485/1485, and bears the aspect of a cram-positive bacillus
under a microscope after gram staining. The major mycological
properties of the KO3 strain are shown below.
[0039] 1) Gram-positive lactobacillus, 2) homo-type lactic
fermentation, 3) catalase negative, 4) no endospore formability, 5)
culturable under aerobic conditions, and 6) producing
exopolysaccharide.
[0040] The YU3 strain was first separated from human saliva by the
present inventors, and was identified as Lactobacillus casei since
the base sequence of 16S rRNA shows a homology of 100% with the
base sequence of Lactobacillus casei ATCC 334 between 1485/1485,
and bears the aspect of a cram-positive bacillus under a microscope
after gram staining. The major mycological properties of the YU3
strain are shown below.
[0041] 1) Gram-positive lactococcus, 2) homo-type lactic
fermentation, 3) catalase negative, 4) no endospore formability, 5)
culturable under aerobic conditions, and 6) producing
exopolysaccharide.
[0042] The YU4 strain was first separated from human saliva by the
present inventors, and was identified as Lactobacillus paracasei
since the base sequence of 16S rRNA shows a homology of 1477/1477
(100%) with the Partial Sequence of Lactobacillus paracasei strain
DJ1 16S ribosomal RNA gene, and bears the aspect of a gram-positive
bacillus under a microscope after gram staining. The major
mycological properties of the YU4 strain are shown below.
[0043] 1) Gram-positive lactococcus, 2) homo-type lactic
fermentation, 3) catalase negative, 4) no endospore formability, 5)
culturable under aerobic conditions, and 6) producing
exopolysaccharide.
[0044] In the present invention, the above-mentioned lactic acid
bacterial cell that has been recovered by culturing the bacterium
according to a general method for culturing a lactic acid bacterium
and has been separated from the culture by cell-collecting means
such as centrifugation may be used directly. Alternatively, the
cultured and fermented liquid (culture supernatant), a concentrated
liquid thereof, or a cytoplasma or cell wall fraction obtained by
treating the bacterial cell through an enzyme or a physical means
may also be used. Furthermore, not only a viable cell but also a
killed cell may be used.
[0045] The medium for culturing the lactic acid bacterium used in
the present invention includes various media such as a fruit juice
medium, a vegetable juice medium, a milk medium, a skimmed milk
medium or a medium containing a milk component, and a
semi-synthetic medium free of a milk component. Examples of such
the medium may include a reduced skimmed milk medium produced
through heat-sterilized skimmed milk, a skimmed milk medium to
which yeast extract has been added, an MRS medium, and a GAM
medium.
[0046] No particular limitation imposed on the culturing method, so
long as the method allows favorable growth of the target cells.
Examples of the method include stationary culture, neutralization
culture (as a constant pH), batch culture and continuous
culture.
[0047] The extract of lactic acid bacterial cell or the culture of
the cell of the present invention means various solvent extract
liquids obtained by extracting the cell or the culture of the cell
with a solvent, a diluted liquid thereof, a concentrated liquid
thereof or a dried powder thereof.
[0048] As the extraction solvent used for obtaining the extract of
the present invention, either a polar solvent or nonpolar solvent
can be used, or these can be used after mixing. Examples may
include water; alcohols such as methanol, ethanol, propanol and
butanol; polyols such as propylene glycol and butylene glycol;
ketones such as acetone and methyl ethyl ketone; esters such as
methyl acetate and ethyl acetate; chain and cyclic ethers such as
tetrahydrofuran and diethyl ether; polyethers such as polyethylene
glycol; hydrocarbons such as hexane, cyclohexane and petroleum
ethers; aromatic hydrocarbons such as benzene and toluene;
pyridines, and the like, and among these, esters such as ethyl
acetate and alcohols such as ethanol are preferable.
[0049] Although the conditions for extraction differ depending on
the solvent to be used, it is preferable to conduct extraction, for
example, by using from 1 to 10 parts by mass of a solvent with
respect to 1 part by mass of a culture liquid at a temperature of
from 0 to 50.degree. C., preferably of from 25 to 37.degree. C.,
for from 0.5 hour to 3 hours.
[0050] The above-mentioned extract may be used as is, or may be
used after diluting, concentrating or lyophilizing the extract and
thereafter forming the product into a powder or paste if necessary.
Alternatively, the extract may also be used after appropriate
purification by a purification technique such as liquid-liquid
distribution.
[0051] The "oral disease" in the prophylactic, ameliorating or
therapeutic agent for an oral disease of the present invention
refers to oral diseases that are caused by cariogenic bacteria,
periodontal disease bacteria and candida bacteria, and examples may
include dental caries; periodontal diseases such as gingivitis and
periodontitis, oral candidiasis such as glossitis, thrush and
angular stomatitis, and the like.
[0052] Examples of the cariogenic bacteria may include
Streptococcus mutans and Streptococcus sobrinus; examples of the
periodontal disease bacteria may include Porphyromonas gingivalis,
Prevotella intermedia, Treponema denticola, Tannerella
forsythensis, Actinobacillus actinomycetemcomitans, Fusobacterium
nucleatum and the like; and examples of the candida bacteria may
include Candida albicans, Candida glabrata, Candida tropicalis and
the like.
[0053] As shown in the following Examples, the lactic acid
bacterium of the present invention has a growth suppressing effect
against any of Streptococcus mutans and Streptococcus sobrinus that
are cariogenic bacteria, Porphyromonas gingivalis that is a
periodontal disease bacterium, and Candida albicans that is a
candida bacterium. Furthermore, it has an effect of suppressing the
proliferation of Fusobacterium bacteria such as Fusobacterium
nucleatum that is known as a subgingival plaque former in the oral
cavity. Furthermore, a fermented milk prepared by using the lactic
acid bacterium has good flavor and texture, and even it is present
together with other lactic acid bacterium in an existing fermented
milk such as Lactobacillus bulgaricus, the taste thereof is not
impaired or can further be improved.
[0054] Therefore, the lactic acid bacterial cell or the culture of
the cell, or the extract thereof can be a prophylactic,
ameliorating or therapeutic agent for oral diseases, or an agent
for suppressing the proliferation of cariogenic bacteria,
periodontal disease bacteria and candida bacteria. Such
prophylactic, ameliorating or therapeutic agent for oral diseases
and agent for suppressing the proliferation of cariogenic bacteria,
periodontal disease bacteria and candida bacteria can be used as
they are, as foods, medicaments, oral compositions and the like,
for the prophylaxis, amelioration or treatment of oral diseases
such as dental caries, periodontal diseases and oral candidiasis
that are caused by pathogenic microorganisms in the oral cavity, or
for the suppression of the proliferation of cariogenic bacteria,
periodontal disease bacteria and candida bacteria, and can also be
used as materials for incorporating in foods, medicaments or oral
compositions. Furthermore, the foods can also be health foods,
supplements or functional foods such as foods for specified health
use, and have a product concept of the prophylaxis, amelioration or
the like of tooth decay, periodontal diseases and other oral
infectious diseases and include a label indicating such effects as
necessary.
[0055] The form in the case when used as a medicament is preferably
a form of oral administration, and the dosage form thereof may
include various forms such as liquid agents; solid agents such as
pills, granules, fine granules, powders and tablets; or capsules in
which the liquid agent or solid agent is encapsulated, oral sprays,
troches, and the like. In order to prepare pharmaceutical
formulations and supplements having such various dosage forms,
other excipients, binders, bulking agents, disintegrating agents,
surfactants, lubricants, dispersing agents, buffer agents,
preservatives, flavoring agents, fragrances, coating agents,
carriers, diluents and the like that are pharmaceutically
acceptable can be used in suitable combination to the extent that
the actions of the cell and culture of the present invention are
not obstructed.
[0056] The content of the lactic acid bacterial cell or the culture
of the cell, or the extract thereof of the present invention in the
oral pharmaceutical formulation when it is used as such formulation
is from 1% by mass to 50% by mass, preferably from 10% by mass to
20% by mass in the whole composition.
[0057] Examples of the form in the case of use as a food may
include beverages such as fruit juice or vegetable juice beverages,
carbonated beverages, tea-based beverages, milk beverages,
fermented milks, fermented fruit juices, fermented vegetable
juices, alcoholic beverages and soft drinks; various foods such as
jelly-like foods, various snacks, baked confectioneries, cakes,
chocolates, jams, breads, gums, candies, soups, pickles and foods
boiled in soy; supplements having the same forms as those for the
above-mentioned oral pharmaceutical formulation (pills, capsules,
syrups and the like); and the like.
[0058] The culture of the lactic acid bacterium of the present
invention becomes a fermented food such as yogurts, cheeses, miso,
soybean sauces and pickles, and such fermented milks or cheeses can
be used as a material to form breads, snacks, cakes and the like
for the prophylaxis or amelioration of tooth decay and periodontal
diseases.
[0059] The fermentation by utilizing the lactic acid bacterium of
the present invention is preferably a method including preparing a
starter in advance, and fermenting by inoculating the starter to a
raw material substance for fermentation. Typical examples of the
starter may include those obtained by inoculating a lactic acid
bacterium into a raw material substance for fermentation that has
been subjected to a general sterilization treatment in advance,
such as a 10% powdered skimmed milk to which a yeast extract has
been added, and conducting culturing. In addition, if necessary, a
substance for promoting fermentation such as carbon sources such as
glucose, starch, sucrose, lactose, dextrin, sorbitol and fractose;
nitrogen sources such as yeast extract and peptone; vitamins;
minerals; and the like can be added to the raw material substance
for fermentation.
[0060] It is adequate that the amount of inoculation of a lactic
acid bacterium is generally selected from an amount by which cell
is included in amount of about 1.times.10.sup.6 cells or more,
preferably around 1.times.10.sup.7 cells, in 1 mL of a liquid
containing the raw material substance for fermentation. The
culturing conditions are generally selected from a fermentation
temperature of from about 20 to 42.degree. C., preferably from
about 25 to 37.degree. C., and a fermentation time of from about 5
to 72 hours. The thus-obtained lactic acid fermented product has a
card-like form (yogurt-like form), and the product can directly
become a solid food. The lactic acid fermented product having a
card-like form can be formed into a desired beverage form by
further homogenizing it.
[0061] Examples of the specific form in the case when the
prophylactic, ameliorating, therapeutic agent for oral diseases, or
the like of the present invention is used as an oral composition
may include mouth rinses, mouthwashes, toothpastes, tooth powders,
tooth liquids, oral ointments, gel agents, pills, granules, fine
granules, gummy jellies, troches, tablets, capsules, candies,
chewing gums and the like, and preferable examples may include
toothpastes, mouth rinses, gummy jellies and troches.
[0062] The content of the lactic acid bacterium of the present
invention in the above-mentioned medicament or food is not
specifically limited and may be suitably adjusted depending on a
daily dose and the like; for example, when the dosage form is a
liquid, the concentration of the lactic acid bacterial cell is
preferably from 1.times.10.sup.6 cells/ml to 1.times.10.sup.8
cells/ml, and when the dosage form is a solid, the concentration is
preferably from 1.times.10.sup.7 cells/g to 1.times.10.sup.10
cells/g.
[0063] When the lactic acid bacterium of the present invention is
administered as a viable cell, it is preferably administered by
from 1.times.10.sup.8 to 5.times.10.sup.10 cfu/day per an adult
human.
[0064] Hereinafter the present invention will be explained in more
detail with referring to the Examples and Test Examples.
EXAMPLES
Preparation Example 1
Preparation of Lactic Acid Bacterial Cells
[0065] An MRS medium (Difco) is sterilized at 121.degree. C. for 20
minutes, and Lactobacillus rhamnosus strain KO3 (this was deposited
as NITE BP-771 with the Patent Microorganisms Depositary of the
National Institute of Technology and Evaluation (Address: 2-5-8
Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan) (Jun. 10,
2009)) is inoculated, cultured at 37.degree. C. for 48 hours in the
air and washed with distilled water, ultrapure water, a buffer
liquid or the like, thereby a cell can be obtained.
[0066] Similarly, an MRS medium (Difco) is sterilized at
121.degree. C. for 20 minutes, and Lactobacillus casei strain YU3
(this was deposited as NITE BP-772 with the Patent Microorganisms
Depositary of the National Institute of Technology and Evaluation
(Address: 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba 292-0818,
Japan) (Jun. 10, 2009)) is inoculated, cultured at 37.degree. C.
for 48 hours in the air and washed with distilled water, ultrapure
water, a buffer liquid or the like, thereby a cell can be
obtained.
[0067] Furthermore, an MRS medium (Difco) is sterilized at
121.degree. C. for 20 minutes, and Lactobacillus paracasei strain
YU4 (this was deposited as NITE BP-775 with the Patent
Microorganisms Depositary of the National Institute of Technology
and Evaluation (Address: 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba
292-0818, Japan) (Jun. 24, 2009)) is inoculated, cultured at
37.degree. C. for 48 hours in the air and washed with distilled
water, ultrapure water, a buffer liquid or the like, thereby a cell
can be obtained.
[0068] In addition, Lactobacillus Fermentum used for comparison was
cultured in a similar manner to that mentioned above by using
Lactobacillus Fermentum strain SU3 collected from the human oral
cavity described in the above-mentioned Non-patent Document 1, and
subjected to the tests.
Test Example 1
Antibacterial Actions Against Cariogenic Bacteria, Periodontal
Disease Bacteria and Candida Bacteria Test Method
[0069] Each cell that had been stored at -80.degree. C. was thawed
at an ordinary temperature, collected by centrifugation (3000 rpm
for 5 min), and washed twice by sterilized distilled water (MQ
water) to adjust to 0.3 at OD600 (about 1.0.times.10.sup.9 cfu/ml).
500 .mu.l of each suspension liquid was inoculated into 15 ml of
MRS Broth or Brain Heart Infusion Broth (hereinafter referred to as
BHI Broth: Difco), and stationary culture was conducted at
37.degree. C. for 48 hours. Thereafter centrifugation was conducted
at 3000 rpm for 5 minutes (room temperature), and the supernatant
was collected and used for an antibacterial assay.
<Test Strains>
[0070] Streptococcus mutans Ingbritt and Streptococcus sobrinus B13
were used as cariogenic bacteria (mutans streptococci). For the
preculturing of these bacteria, Tryptic Soy Broth (hereinafter
referred to as TSBY: Difco) to which 5% of Yeast Extract (Difco)
had been added was used, and the culturing was conducted at
37.degree. C. for 24 hours. Thereafter the bacteria were washed
twice with MQ water, adjusted to 0.3 at OD600 (Streptococcus mutans
Ingbritt; 1.0.times.10.sup.8 cfu/ml, Streptococcus sobrinus B13;
1.0.times.10.sup.8 cfu/ml) and the thus-prepared strain was
used.
[0071] Porphyromonas gingivalis strain 381 was used as a
periodontal disease bacterium; this was precultured by using a GAM
medium to which 1% hemin and 0.2% vitamin K had been added, at
37.degree. C. for 7 days in an anaerobic jar containing AneroPack
(MCG) (BBL, Cookeysville, USA) (10% CO.sub.2). Thereafter the
bacterium was washed twice with phosphate buffer (pH 7.4; PBS),
adjusted to 0.3 at OD600 (about 0.5.times.10.sup.7 cfu/ml) and the
thus-prepared strain was used.
[0072] Furthermore, Candida albicans MYA274 was used as candida;
this was precultured at 37.degree. C. for 24 hours by using
Sabouraud Dextrose Broth (Difco), washed twice with MQ water,
adjusted to 0.3 at OD600 (Candida albicans MYA274;
1.0.times.10.sup.7 cells/ml) and the thus-prepared strain was
used.
<Antibacterial Assay>
[0073] One ml of TSBY for the cariogenic bacterium, a GAM medium or
BHI medium to which 1% of hemin and 0.2% of vitamin K had been
added for the periodontal disease bacterium or Sabouraud Broth for
the candida, 1 ml of each supernatant, and 50 .mu.l of the
bacterial suspension of the lactic acid bacterium that had been
adjusted to 0.3 at OD600 were inoculated into a 24-well plate, and
a turbidness was measured at 37.degree. C. in 24 hours. As a
control, a control to which 50 .mu.l of a bacterial suspension had
been inoculated, wherein the bacterial suspension was obtained by
adding 1 ml of a similar medium to that used in the preculture of
the lactic acid bacterium, i.e., MRS Broth or BHI Broth, to 1 ml of
TSBY or Sabouraud Broth and adjusting the medium to 0.3 at OD600,
was used. Furthermore, four similar samples were made for each
supernatant, and for the control, and an average value.+-.SD was
calculated.
[0074] The results are shown in FIGS. 1a and 1b, FIG. 2, FIGS. 3a
and 3b, and FIGS. 4a and 4b.
[0075] The strains YU3, YU4 and KO3 showed a high
proliferation-suppressing effect against all of Streptococcus
mutans, Streptococcus sobrinus, Porphyromonas gingivalis and
Candida albicans. Furthermore, the antibacterial effects thereof
were superior to that of known Lactobacillus Fermentum strain
SU3.
Test Example 2
Biofilm-Inhibiting Action
[0076] A biofilm assay by Candida albicans was conducted according
to the method of Nikawa et al., 1996 (Nikawa, H., Nishimura, H.
Yamamoto, T., Hamada, T. & Samaranayake, L. P.: The role of
saliva and serum in Candida albicans biofilm formation on denture
acrylic surfaces. Microbial Ecol Health & Dis 9, 35-48, 1996).
Using a resin for a denture base (Bio Resin, Shofu, Kyoto),
50.times.50.times.0.2 mm of the resin sample was polymerized
according to a general procedure at a liquid mixing ratio as
instructed by the manufacturer. This was cut into
10.times.10.times.0.2 mm by using a resin cutter and used for a
biofilm assay.
[0077] The resin plate was put on the bottom surface of a 24-well
plate, and 500 .mu.l of human blood serum (Type, Sigma co.human
male AB plasma) was added thereto, and incubation was conducted at
37.degree. C. for 1 hour. Thereafter the human blood serum was
removed. 50 .mu.l of a suspension liquid of Candida albicans strain
MYA274, which had been adjusted to 0.3 at OD600, was inoculated
onto the surface of the sample, and the sample was stood still at
37.degree. C. for 2 hours to promote the fixing. Thereafter 1 ml of
the MRS culture supernatant and 1 ml of Sabouraud Broth were added,
and culturing was conducted at 37.degree. C. for 72 hours. The
resin sample was removed gently so that the biofilm formed on the
surface of the sample was not broken, and washed with 400 ml of MQ
water for 5 seconds to remove excess bacteria. The amount of the
biofilm formed on the resin sample was examined by extracting ATP
and quantifying the ATP by using a luminometer (AB2200
Luminescencer PSN, ATTO, Tokyo). In addition, a control to which
the same amount of MRS Broth had been added instead of the culture
supernatant of the lactic acid bacterium was used. Furthermore,
four similar samples were made for each supernatant, and for the
control, and an average value.+-.SD was calculated.
[0078] The results are shown in FIG. 5.
[0079] The strains YU3, YU4 and KO3 strongly suppressed the biofilm
formation of Candida albicans.
Example 1
Production of Fermented Milk (1)
[0080] Lactobacillus rhamnosus strain KO3 (NITE BP-771) was
cultured in an MRS medium at 37.degree. C. for 18 hours to give a
preculture liquid. The liquid was washed twice with MQ water and
centrifuged to collect the bacteria. The above-mentioned bacteria
of the strain KO3 that had been washed, and lactose in an amount to
be 0.1 to 10% against 100 mL of a milk were added to a commercially
available milk to which "Danone Yogurt" (Danone Japan Co., Ltd.;
bifidobacteria are used) had been added, and cultured at 37.degree.
C. for 24 hours to give a fermented milk.
[0081] Meanwhile, it is considered that, in the case when a small
amount of "Danone Yogurt" (bifidobacteria are used) is added and
general culturing is conducted, only a starter used in the yogurt
proliferates since bifidobacteria are obligatory anaerobes.
Test Example 3
Evaluation of Fermented Milk (1)
[0082] A questionnaire about taste was conducted on 127 subjects by
a Visual Analogue Scale (VAS) method. Each subject ate a yogurt and
thereafter filled in a scale bar which comprised a line of 10 cm on
which "very delicious" was described on the left end, "very
distasteful" was described on the right end, and "normal" was
described on the center, and the results were quantified by
measuring the distances from the left end and obtaining the average
value thereof. The results are shown in FIG. 6.
[0083] Furthermore, in the summary of the free opinions that were
written at the same time, the subjects who wrote "tasty" or "mild"
accounted for 72% in total, and thus it was confirmed that all of
the fermented milks of the present invention had good taste.
Example 2
Production of Fermented Milk (2)
[0084] Raw materials containing a milk are mixed homogeneously,
heated and sterilized. This was cooled, and fermented by using S.
thermophilus as a starter and using L. bulgaricus and L. rhamnosus
strain KO3 at from 37 to 40.degree. C. to produce a yogurt (L8020).
Furthermore, a placebo yogurt was produced by only L. bulgaricus
using S. thermophilus as a starter.
Test Example 4
Evaluation of Fermented Milk (2)
[0085] Using the L8020 yogurt prepared in Example 2, antibacterial
tests against cariogenic bacteria were conducted.
[0086] S. mutans ingbritt strain and S. sobirinus strain B13 as
mentioned above were used as the cariogenic bacteria. Each strain
was precultured in TSBY, and thereafter washed three times with
sterilized distilled water to adjust to 1.times.10.sup.8 cfu/ml.
1.5 mL of TSBY was dispensed into a 24-well plate, and 100 .mu.L of
the bacterial liquid was inoculated into each well.
[0087] The inside of the intercell containing each yogurt was put
into a 24-well plate, and the amounts of the bacteria after 24
hours of culturing were compared.
[0088] The measurement was conducted by using the amount of the
bacteria after 24 hours as the amount of ATP. Four same samples
were made for each, and an average value.+-.SD was obtained. The
results are shown in FIG. 7.
[0089] In FIG. 7 (A) and (B), the amount of ATP (pmol/well) is
shown in the longitudinal axis of the graph; a tendency of slight
increase was recognized in placebo against both the Ingbritt strain
and strain B-13, whereas a significant suppression effect was also
recognized in the state of the L8020 yogurt to which KO3 had been
added.
Test Example 5
Evaluation of Fermented Milk (3)
[0090] Human tests were conducted by using the L8020 yogurt
prepared in Example 2.
[0091] Forty subjects from 19 to 25 years of age were divided into
two groups according to a random number table. The subjects in
Group 1 continued to eat the placebo yogurt and the subjects in
Group 2 continued to eat the L8020 yogurt, once a day at lunchtime
for 2 weeks.
[0092] The carriage numbers of the cariogenic bacteria and
periodontal disease bacteria in the oral cavity were calculated by
collecting saliva by using a kit of BML and quantifying the
cariogenic bacteria by a culture method or the periodontal disease
bacteria by using a PCR-invader method or invader method for 4
kinds of bacteria: P. intermedia (Pi), T. forsythensis (Tf) and F.
nucleatum (Fuso).
[0093] The collection of saliva was conducted at 3 days before
initiation of tasting by collecting saliva on ice after stimulation
for 5 minutes by a gum chewing method, and the number of carriage
in the oral cavity for each of the above-mentioned bacteria was
calculated and used as the pre-value of the above-mentioned
tests.
[0094] After the tasting for 2-weeks, about 5 mL of stimulated
saliva was collected on ice in a similar manner by a gum chewing
method, and the number of carriage in the oral cavity for each of
the above-mentioned bacteria was calculated and the effect in the
above-mentioned test was evaluated. The results are shown in FIG.
8.
[0095] As is apparent from FIG. 8 (A), when the pre-value of the
number of carriage in the oral cavity of the cariogenic bacterium
was defined as 100, the value was decreased to about 60% by the
placebo yogurt. On the other hand, significant decrease to about
15% was observed in the L8020 yogurt. It was clarified from this
result that the carriage of the cariogenic bacterium in the oral
cavity is decreased significantly and effectively by ingesting the
L8020 yogurt for 2 weeks.
[0096] As is apparent from FIG. (B), when the pre-value of the
number of carriage in the oral cavity of the Pi bacterium was
defined as 100, the value was increased to about 180% by the
placebo yogurt. On the other hand, significant decrease to about
50% was observed in the L8020 yogurt. It was clarified from this
result that the carriage of the Pi bacterium in the oral cavity is
decreased significantly and effectively by ingesting the L8020
yogurt for 2 weeks.
[0097] As is apparent from FIG. (C), when the pre-value of the
number of carriage in the oral cavity of the Tf bacterium was
defined as 100, the value was increased to about 105% by the
placebo yogurt. On the other hand, significant decrease to about
60% was observed in the L8020 yogurt. It was clarified from this
result that the carriage of the Tf bacterium in the oral cavity is
decreased significantly and effectively by ingesting the L8020
yogurt for 2 weeks.
[0098] As is apparent from FIG. 8 (D), when the pre-value of the
number of carriage in the oral cavity of the Fuso bacterium was
defined as 100, the value was increased to about 160% by the
placebo yogurt. On the other hand, significant decrease to about
60% was observed in the L8020 yogurt. It was clarified from this
result that the carriage of the Tf bacterium in the oral cavity is
decreased significantly and effectively by ingesting the L8020
yogurt for 2 weeks.
[0099] For the Pg bacterium, since the number of the carrier was
small, a significant change was not observed.
[0100] It was clarified by the above-mentioned results that the
L8020 yogurt significantly decreases Pi bacterium, Tf bacterium,
and Fusobacterium bacterium that is known as a subgingival plaque
former, among cariogenic bacteria and periodontal disease bacteria,
and thus has a very high effect against bacteria relating to dental
caries and periodontal diseases as compared to a general
yogurt.
* * * * *