U.S. patent application number 16/970018 was filed with the patent office on 2021-04-08 for bacillus bacterium, interleukin-22 production inducing agent, skin barrier function enhancing agent.
The applicant listed for this patent is ICHIBIKI CO., LTD., NATIONAL UNIVERSITY CORPORATION TOKYO MEDICAL AND DENTAL UNIVERSITY. Invention is credited to Takahiro Adachi, Noriyuki Asai, Toshihiko Kumazawa, Atsuhisa Nishimura.
Application Number | 20210100851 16/970018 |
Document ID | / |
Family ID | 1000005314094 |
Filed Date | 2021-04-08 |
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United States Patent
Application |
20210100851 |
Kind Code |
A1 |
Kumazawa; Toshihiko ; et
al. |
April 8, 2021 |
BACILLUS BACTERIUM, INTERLEUKIN-22 PRODUCTION INDUCING AGENT, SKIN
BARRIER FUNCTION ENHANCING AGENT
Abstract
A Bacillus bacterium that induces the production of
interleukin-22 is provided. The Bacillus bacterium that induces the
production of interleukin-22.
Inventors: |
Kumazawa; Toshihiko;
(Toyohashi-shi, JP) ; Nishimura; Atsuhisa;
(Toyohashi-shi, JP) ; Asai; Noriyuki;
(Toyohashi-shi, JP) ; Adachi; Takahiro; (Tokyo,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ICHIBIKI CO., LTD.
NATIONAL UNIVERSITY CORPORATION TOKYO MEDICAL AND DENTAL
UNIVERSITY |
Nagoya-shi
Tokyo |
|
JP
JP |
|
|
Family ID: |
1000005314094 |
Appl. No.: |
16/970018 |
Filed: |
February 15, 2019 |
PCT Filed: |
February 15, 2019 |
PCT NO: |
PCT/JP2019/005693 |
371 Date: |
August 14, 2020 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 17/00 20180101;
A61K 35/742 20130101; C12N 1/20 20130101; C12R 1/07 20130101 |
International
Class: |
A61K 35/742 20060101
A61K035/742; A61P 17/00 20060101 A61P017/00; C12R 1/07 20060101
C12R001/07; C12N 1/20 20060101 C12N001/20 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 16, 2018 |
JP |
2018-026444 |
Claims
1. A Bacillus bacterium that induces the production of
interleukin-22.
2. The Bacillus bacterium according to claim 1, wherein the
Bacillus bacterium has the potency to improve the viability of B
cells and the activation potency of B cells.
3. The Bacillus bacterium according to claim 1, wherein the
Bacillus bacterium is derived from foods.
4. The Bacillus bacterium according to claim 1, wherein the
Bacillus bacterium is derived from miso or natto.
5. The Bacillus bacterium according to claim 1, wherein the
Bacillus bacterium belongs to at least one selected from the group
consisting of Bacillus subtilis and Bacillus coagulans.
6. The Bacillus bacterium according to claim 1, wherein the
Bacillus bacterium is a Bacillus bacterium of Accession number NITE
BP-02583, a Bacillus bacterium of Accession number NITE BP-02584,
or a Bacillus bacterium of Accession number NITE BP-02590.
7. An interleukin-22 production inducing agent containing the
Bacillus bacterium according to claim 1.
8. A skin barrier function enhancing agent containing the Bacillus
bacterium according to claim 1.
9. The Bacillus bacterium according to claim 2, wherein the
Bacillus bacterium is derived from foods.
10. The Bacillus bacterium according to claim 2, wherein the
Bacillus bacterium is derived from miso or natto.
11. The Bacillus bacterium according to claim 3, wherein the
Bacillus bacterium is derived from miso or natto.
12. The Bacillus bacterium according to claim 2, wherein the
Bacillus bacterium belongs to at least one selected from the group
consisting of Bacillus subtilis and Bacillus coagulans.
13. The Bacillus bacterium according to claim 3, wherein the
Bacillus bacterium belongs to at least one selected from the group
consisting of Bacillus subtilis and Bacillus coagulans.
14. The Bacillus bacterium according to claim 4, wherein the
Bacillus bacterium belongs to at least one selected from the group
consisting of Bacillus subtilis and Bacillus coagulans.
15. The Bacillus bacterium according to claim 2, wherein the
Bacillus bacterium is a Bacillus bacterium of Accession number NITE
BP-02583, a Bacillus bacterium of Accession number NITE BP-02584,
or a Bacillus bacterium of Accession number NITE BP-02590.
16. The Bacillus bacterium according to claim 3, wherein the
Bacillus bacterium is a Bacillus bacterium of Accession number NITE
BP-02583, a Bacillus bacterium of Accession number NITE BP-02584,
or a Bacillus bacterium of Accession number NITE BP-02590.
17. An interleukin-22 production inducing agent containing the
Bacillus bacterium according to claim 2.
18. An interleukin-22 production inducing agent containing the
Bacillus bacterium according to claim 3.
19. A skin barrier function enhancing agent containing the Bacillus
bacterium according to claim 2.
20. A skin barrier function enhancing agent containing the Bacillus
bacterium according to claim 3.
Description
TECHNICAL FIELD
[0001] The present invention relates to a Bacillus bacterium, an
interleukin-22 production inducing agent, and a skin barrier
function enhancing agent. More particularly, the present invention
relates to Bacillus bacterium which induces the production of
interleukin-22, an interleukin-22 production inducing agent, and a
skin barrier function enhancing agent.
BACKGROUND ART
[0002] Currently, several types of interleukins, such as
interleukin-10 and interleukin-12, are known as a group of
cytokines, and interleukin-22 (IL-22) has also been reported as one
of them.
[0003] This interleukin-22 is a cytokine belonging to the
interleukin-10 family, and has been reported to have a function of
enhancing a barrier function of skin, intestine, and the like, and
is an important cytokine in improving the immune system.
[0004] Here, for example, in order to improve the immune function
by increasing the production amount of interleukin-12, a functional
food containing Bacillus natto (Bacillus subtilis), which is a kind
of Bacillus bacterium, as an active ingredient has been reported
(see Patent Document 1).
CITATION LIST
Patent Documents
[0005] [Patent Document 1] JP-B2-5090754
SUMMARY OF THE INVENTION
Problem to be Solved by the Invention
[0006] The functional food described in Patent Document 1 uses
Bacillus natto (Bacillus subtilis) to increase the production
amount of interleukin-12, and the effect of increasing (inducing)
the production of interleukin-22 has not been reported.
[0007] Thus, there is a need to find a bacterium capable of
inducing the production of interleukin-22, and in particular, there
is a need for a bacterium that is easy to ingest.
[0008] The present invention provides a Bacillus bacterium that
induces the production of interleukin-22.
Means for Solving the Problems
[0009] According to the present invention, there is provided a
Bacillus sp. bacterium shown below.
[0010] [1] A Bacillus bacterium that induces the production of
interleukin-22.
[0011] [2] The Bacillus bacterium described in [1], wherein the
Bacillus bacterium has the potency to improve the viability of B
cells and the activation potency of B cells.
[0012] [3] The Bacillus bacterium described in [1] or [2], wherein
the Bacillus bacterium is derived from foods.
[0013] [4] The Bacillus bacterium described in any one of [1] to
[3], wherein the Bacillus bacterium is derived from miso or
natto.
[0014] [5] The Bacillus bacterium described in any one of [1] to
[4], wherein the Bacillus bacterium belongs to at least one
selected from the group consisting of Bacillus subtilis and
Bacillus coagulans.
[0015] [6] The Bacillus bacterium described in any one of [1] to
[5], wherein the Bacillus bacterium is a Bacillus bacterium of
Accession number NITE BP-02583, a Bacillus bacterium of Accession
number NITE BP-02584, or a Bacillus bacterium of Accession number
NITE BP-02590.
[0016] [7] An interleukin-22 production inducing agent containing
the Bacillus bacterium described in any one of [1] to [6].
[0017] [8] A skin barrier function enhancing agent containing the
Bacillus bacterium described in any one of [1] to [6].
Effect of the Invention
[0018] The Bacillus bacterium of the present invention induces the
production of interleukin-22, and can be an active ingredient of an
interleukin-22 production inducing agent used for prevention of
diseases caused by insufficient production of interleukin-22.
[0019] The interleukin-22 production inducing agent of the present
invention induces the production of interleukin-22, and can be an
active ingredient of an interleukin-22 production inducing agent
used for prevention of a disease caused by insufficient production
of interleukin-22.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 is a graph showing the screening results of strains
with the production inducing potency of interleukin-22 in B
cells.
[0021] FIG. 2 is a graph showing the screening results of strains
with the production inducing potency of interleukin-22 in B
cells.
[0022] FIG. 3 is a graph showing the screening results of strains
with the production inducing potency of interleukin-22 in total
spleen cells.
[0023] FIG. 4 is a graph showing the screening results of strains
with the production inducing potency of interleukin-22 in total
spleen cells.
[0024] FIG. 5 is a graph showing the activation potency of B
cells.
[0025] FIG. 6 is a graph showing the activation potency of B
cells.
[0026] FIG. 7 is a diagram showing a result when measured by flow
cytometry on the production inducing potency of IL-22.
[0027] FIG. 8 is a graph showing a measurement result of the
transepidermal water loss (TEWL) in Example 5.
[0028] FIG. 9 is a graph showing a measurement result of the
transepidermal water loss (TEWL) in Example 6.
MODE FOR CARRYING OUT THE INVENTION
[0029] Hereinafter, modes for carrying out the present invention
will be described, but the present invention is not limited to the
following embodiments. That is, it should be understood that those
in which changes, improvements, and the like, have been
appropriately added to the following embodiments based on the
ordinary knowledge of a person skilled in the art without departing
from the gist of the present invention fall within the scope of the
present invention.
[0030] [1] Bacillus Bacterium:
[0031] The Bacillus bacterium of the present invention induces the
production of interleukin-22 (IL-22). Here, for example, Bacillus
subtilis, which is a kind of Bacillus bacterium, is a microorganism
that has long been involved in the Japanese diet, and for example,
in the production (fermentation) of natto, which is a traditional
fermented food of Japan, Bacillus natto, which is a kind of
Bacillus subtilis, is involved. In addition, moromi (main
fermenting mash) of miso and soy sauce often contain Bacillus
subtilis, and Bacillus subtilis has been consumed by eating these
fermented foods. Thus, for example, Bacillus subtilis has the
advantages that it is safe to ingest (i.e., highly suitable for
food) and it is easily produced since its culturing is easy.
Bacillus is an aerobic spore-bearing bacterium, and the typical
species is Bacillus subtilis. Bacillus natto is classified as a
type of Bacillus subtilis.
[0032] Since "interleukin-22" can proliferate keratinized cells to
promote skin turnover, it can be expected to be suitably used for
applications such as skin care materials and antibacterial
materials. In addition, interleukin-22 is involved in tissue
repair, cell survival and proliferation, and mucosal barrier
protection, and can be expected to have usages such as prevention
and treatment of skin diseases such as atopic dermatitis, and the
like, fatty liver disease, and infectious diseases caused by such
as Clostridium difficile.
[0033] Furthermore, it is preferable that the Bacillus bacterium of
the present invention has the potency to improve the viability of B
cells and the activation potency of B cells.
[0034] Here, B cells play a central role in humoral immunity and
are the only cells capable of producing antibodies against foreign
bodies (antigens) such as pathogens, but are not known for their
action by Bacillus bacterium such as Bacillus subtilis. In
addition, B cells, which are cells that present antigen to T cells,
are known to be indispensable cells for maintenance of activated T
cells. Therefore, strengthening the action of B cells reinforces an
action of T cells, and also strengthens the immunostimulatory
effect in the entire cells of the immune system. In the present
invention, the "activation potency of B cells" means that both the
potency of antibody production and the potency of antigen
presentation are activated.
[0035] Then, if it is possible to achieve a direct control such as
artificial reinforcement of action of B cells capable of producing
antibodies to attack foreign bodies, it can be expected to lead to
the prevention, alleviation, or treatment of immune system diseases
such as allergic diseases, infectious diseases, and autoimmune
diseases, having an effect on the action by the antibody.
[0036] As used herein, "having the potency to improve the viability
of B cells" means having the property of enhancing the "viability"
of B cells. More specifically, it means that when a ratio of the
number of viable B cells to the total number of cells in a sample
to which Bacillus bacterium is not added using experimental mouse
spleen cells is defined as a reference (reference value 100), a
value (measured value) of the ratio of the number of viable B cells
to the total number of cells in a sample to which Bacillus
bacterium is added is more than 100. The "total number of cells"
can be quantitatively determined by flow cytometry. Cells that
react with an anti-B220 antibody are referred to as B cells, the
"number of viable B cells (living cells)" is determined by
quantifying cells not stained with Propidium Iodide (PI) nucleus
staining liquid, but react with the anti-B220 antibody.
[0037] Note that "having the viability of B cells" is as described
above, but specifically, it means that a value (measured value)
obtained by a method shown in Example 2 is more than 100.
[0038] As used herein, "having an activation potency of B cells"
means having the potency (property) to activate B cells. More
specifically, it means that when a ratio of the number of activated
B cells to the number of unactivated B cells in a sample to which
Bacillus bacterium is not added using experimental mouse spleen
cells is defined as a reference (reference value 100), a value
(measured value) of the ratio of the number of activated B cells to
the number of unactivated B cells in a sample to which Bacillus
bacterium is added is more than 100. The "number of activated B
cells" is determined by measuring the number of cells reacting with
both the anti-B220 antibody and an anti-CD86 antibody by flow
cytometry. The "number of unactivated B cells" is determined by
measuring the number of cells reacting with the anti-B220 antibody
without reacting with the anti-CD86 antibody by flow cytometry.
[0039] Note that "having an activation potency of B cells" is as
described above, but specifically, it means that a measured value
obtained by a method shown in Example 1 is more than 100.
[0040] The Bacillus bacterium of the present invention may be
derived from foods, and specifically, may be derived from miso or
derived from natto. Such microorganisms derived from foods
(especially fermented foods) that have been eaten since ancient
times provide even greater safety when ingested.
[0041] Here, the Bacillus bacterium derived from miso may be
Bacillus bacterium such as Bacillus subtilis isolated in the
brewing process of miso. The Bacillus bacterium "isolated in the
brewing process of miso" refers to Bacillus bacterium fixed in
"Kura (storage)" "Muro (chamber)," and "Oke (tub)" in the brewing
process of miso. Furthermore, it refers to Bacillus bacterium which
can grow (survive) from the preparation to the ripening process of
miso. Note that, in the present invention, Bacillus bacterium
derived from miso are not limited to those directly isolated in the
brewing process of miso, but also include those isolated from miso
and subsequently cultured (subcultured). Bacillus bacterium
(Bacillus subtilis) derived from natto can be called Bacillus
bacterium isolated in the manufacturing process of natto, and also
includes Bacillus subtilis (Bacillus bacterium) isolated from
commercial (product) natto.
[0042] The Bacillus bacterium of the present invention may belong
to at least one selected from the group consisting of Bacillus
subtilis and Bacillus coagulans. Bacillus subtilis and Bacillus
coagulans (one of the spore-bearing lactobacillus) are
microorganisms that have long been involved in Japanese diet and
are safe to ingest (i.e., highly suitable for food). In addition,
these have an advantage in that they are easily produced since
their culturing is easy.
[0043] [1-1] Preferred Bacillus Bacterium:
[0044] The Bacillus bacterium of the present invention is
preferably a Bacillus bacterium (strain) (strain name "sc-09") of
Accession number NITE BP-02583 (hereinafter sometimes referred to
as "Bacillus coagulans sc-09"), a Bacillus bacterium (strain name
"bs-30") of Accession number NITE BP-02584 (hereinafter sometimes
referred to as "Bacillus subtilis bs-30") or a Bacillus bacterium
(strain name "bs-34") of Accession number NITE BP-02590
(hereinafter sometimes referred to as "Bacillus subtilis
bs-34").
[0045] These Bacillus bacteria are derived from miso, so they are
highly safe when ingested, and can directly act on B cells to exert
viability and activation potency in B cells, thereby activating the
immune system (i.e., they have a good immunostimulatory effect).
Furthermore, these Bacillus bacterium have excellent production
inducing potency of IL-22. These Bacillus bacteria may act on T
cells, and may also act on dendritic cells and the like.
[0046] Here, the Bacillus bacterium of Accession number NITE
BP-02583, the Bacillus bacterium of Accession number NITE BP-02584,
and the Bacillus bacterium of Accession number NITE BP-02590 are
all deposited with the National Institute of Technology and
Evaluation Patents and Microorganisms Depositary (NPMD).
[0047] As described above, the Bacillus bacterium of the present
invention is preferably the Bacillus bacterium of Accession number
NITE BP-02583, the Bacillus bacterium of Accession number NITE
BP-02584, or the Bacillus bacterium of Accession number NITE
BP-02590, and the Bacillus bacterium of Accession number NITE
BP-02583 is more excellent production inducing potency of
IL-22.
[0048] Among the "preferable Bacillus bacterium", the Bacillus
bacterium (strain name "se-09") of Accession number NITE BP-02583
is Bacillus coagulans derived from miso, and is excellent in IL-22
production inducing potency. In other words, it is most preferable
to employ it when it is desired to enhance the production of IL-22.
In addition, the Bacillus coagulans sc-09 can be cultivated under
anaerobic conditions and can grow in a high temperature range
(45.degree. C. to 60.degree. C.). Therefore, it can be selectively
cultivated in a high temperature range (45.degree. C. to 60.degree.
C.) where it is difficult for common bacteria to grow. Moreover, it
can be produced in a simple cultivation facility. In addition,
because of its sporulation potency, it is easy to handle spawn
(starter), such as storing them, by sporulating them.
[0049] Among the "preferred Bacillus bacterium", the Bacillus
bacterium (strain name "bs-30") of Accession number NITE BP-02584
and the Bacillus bacterium (strain name "bs-34") of Accession
number NITE BP-02590 are both Bacillus subtilis derived from miso,
and can vigorously grow in environments with good air permeability
at room temperature of about 25.degree. C. to 45.degree. C.
Further, it can be grown even at a salinity of about 7 w/v %, and
by culturing in a medium containing salinity, it is possible to
suppress the growth of common bacteria, so that it is easily
produced. In other words, it can be said that the production by a
simple culture facility is possible. In addition, because of its
sporulation potency, it is easy to handle spawn (starter), such as
storing them, by sporulating them.
[0050] Therefore, it is also preferable that the Bacillus bacterium
of the present invention is a bacterium capable of growing at a
high temperature (45.degree. C. or higher, more preferably
50.degree. C. or higher). Being able to cultivate at high
temperatures makes the culturing easier because other germs are
less likely to be mixed in (less likely to be contaminated by other
germs) as described above.
[0051] [2] Method of Preparing of Bacillus Bacterium:
[0052] The Bacillus bacterium of the present invention can be
prepared by culturing, followed by a treatment such as
sterilization, or the like. Specifically, after completion of the
culturing, the medium component is removed by means such as
centrifugation, or the like, followed by washing and purification.
Then, heat sterilization is performed, and then drying and
concentration are performed by means such as lyophilizing, reduced
pressure drying, and hot air drying, and the like. In this way,
Bacillus bacterium of the present invention can be prepared.
[0053] Note that, although there is no particular limitation on the
heat sterilization, specifically, autoclave sterilization
(121.degree. C., 20 minutes) or the same degree of sterilization is
preferable.
[0054] [3] Interleukin-22 (IL-22) Production Inducers:
[0055] IL-22 production inducing agent of the present invention
contains the Bacillus bacterium of the present invention. The IL-22
production inducing agent is capable of inducing the production of
IL-22. Note that some Bacillus bacteria are conventionally eaten
with fermented food, such as Bacillus subtilis, and some of them
are highly safe, and therefore, when Bacillus subtilis or the like
is used, IL-22 production inducing agent of the present invention
can be highly safe.
[0056] In the IL-22 production inducing agent of the present
invention, there is no particular limitation on the content ratio
as long as it contains the Bacillus bacterium of the present
invention as an active ingredient. Note that IL-22 production
inducing agent of the present invention may contain indigestible
dextrins, oligosaccharides, dextrins, silicon dioxide, and the like
as other components in addition to the Bacillus bacterium of the
present invention.
[0057] Note that the IL-22 production inducing agent of the present
invention may include a culture, a bacterial cell (which may be
either a vegetative cell or a spore), or a bacterial component,
obtained when the Bacillus bacteria of the present invention is
cultured.
[0058] Note that IL-22 production inducing agent of the present
invention may itself be used as a food and drink, a supplement, a
medicine, or the like, or may be added to a food and drink, a
supplement, a medicine, or the like and used. There are no
particular limitation for food and drinks, and the food and drinks
may include, for example, miso, instant miso soup, cooked miso
(processed miso), name-miso such as Kinzanji miso, soy sauce, soup,
season sauce, seasoning sauce, seasoning for rice, side dishes, and
sweet rice drinks (yeast drinks).
[0059] [4] Skin Barrier Function Enhancing Agent:
[0060] The skin barrier function enhancing agent of the present
invention contains Bacillus bacterium of the present invention. The
skin barrier function enhancing agent can induce the production of
IL-22 and suppress transepidermal water loss. That is, the tight
junction of the skin epithelium become stronger and the barrier
function of the skin can be enhanced (i.e., improved). When the
barrier function of the skin is improved in this way, it is
possible to keep the skin moist and to make it difficult to cause
dry skin and sensitive skin. In addition, the skin can be protected
from external stimulus such as invasion of pathogens (other
external stimulus are, for example, stimulation by exposure to
ultraviolet rays, stimulation by contact with allergens, chemicals,
dust, and the like, and stimulation by exposure to a dry
environment).
[0061] Here, the epidermal layer of the skin has a barrier function
of protecting the living body from the above-mentioned external
stimulus such as ultraviolet rays, allergens, chemicals, pathogens,
and the like, but if the tight junction of the skin epithelium is
loosened and the barrier function of the epithelial layer does not
function sufficiently, it becomes impossible to protect the skin
from the external stimulus. As a result, it causes problems such as
rough skin, stains, wrinkles, deterioration of tenseness, and
flabbiness of the skin. For this reason, it is important to keep
the barrier function of the skin normal and to improve the barrier
function when the function is deteriorated. By containing the
Bacillus bacterium of the present invention, the skin barrier
function enhancing agent of the present invention induces the
production of IL-22 by the Bacillus bacterium so that the barrier
function of the skin is exhibited (an effect of preventing
transepidermal water loss is exhibited).
[0062] Incidentally, when the amount of water loss from the
transepidermis is large (that is, when the amount of TransEpidermal
Water Loss (TEWL) is large), in addition to the skin (particularly,
the epidermal layer) becomes dry, a sufficient protective function
by the skin is not exhibited, and external stimulus such as
ultraviolet rays, allergens, chemicals, and pathogens adversely
affects the inside of the skin (occurrence of stains, itching, and
the like).
EXAMPLES
[0063] Hereinafter, the present invention will be specifically
described based on Examples, but the present invention is not
limited to these Examples.
Example 1
[0064] (Measurement Test for IL-22)
[0065] Bacillus bacteria were collected from miso and natto,
respectively, and the collected bacteria of the Bacillus bacteria
were cultured and sterilized, and then added to an experimental
mouse (C57BL/6) spleen cells and cultured, and the production
amount of IL-22 was measured after culturing. The strains used in
the tests are shown in Tables 1 and 2. Hereinafter, measurement
test for IL-22 will be described in detail.
[0066] (1) Isolation and Identification of Bacillus Bacterium:
[0067] Bacillus bacteria was collected from miso and miso brewing
process, and Bacillus bacteria were also isolated from commercial
natto. For the isolation medium, standard agar medium (manufactured
by Nissui Pharmaceutical Co., Ltd.) was used, and it was cultured
statically at 30.degree. C. and 45.degree. C. on for 1 to 2 days,
and the bacteria were purely isolated from the obtained colonies to
collect. In addition, in order to selectively obtain a Bacillus
coagulans, a Lactobacilli MRS Agar (manufactured by Wako Pure
Chemical Corporation) to which calcium carbonate was added was
used, and cultured anaerobically at 50.degree. C. for 1 to 3 days
to collect colonies in which the surrounding calcium carbonate was
dissolved.
[0068] The isolated Bacillus bacteria were subjected to Gram
staining and observed with a microscope to confirm that they were
Gram stained positive bacillus and whether they formed spores.
[0069] In addition, DNA was extracted from the isolated bacterial
cell, and 16S rDNA was amplified by PCR using primer 10F
(5'-GTTTGATCCTGGCTCA-3') and primer 1500R (5'-TACCTTGTTACGACTT-3'),
and then the bacterial species were identified by sequence analyses
of the obtained PCR products. Details of the analysis method were
based on the Japanese Pharmacopoeia 17th Edition reference
information "Rapid identification method of microorganisms by gene
analysis".
[0070] The collected Bacillus bacteria were 72 strains, of which 40
strains of Bacillus subtilis (including Bacillus natto) and 20
strains of Bacillus coagulans were included.
[0071] (2) Preparation of Bacterial Cell Suspension (Suspension of
the Bacillus Bacterium):
[0072] The isolated and identified Bacillus bacteria were cultured
by shaking at 30.degree. C. for 1 to 3 days using Nutrient Broth
medium (manufactured by Wako Pure Chemical Corporation). However,
as Bacillus coagulans, Lactobacilli MRS Broth (manufactured by Wako
Pure Chemical Corporation) was used and cultured statically at
50.degree. C. for 1 to 3 days.
[0073] After culturing, the cultures were subjected to autoclave
sterilization at 121.degree. C. for 20 minutes to obtain culture
broth for each strain.
[0074] Next, each of the obtained cultures was centrifuged at 5000
rpm for 10 minutes. Thereafter, each of them was collected, washed
3 times with distilled water, and then suspended in distilled water
and lyophilized to obtain a bacterial cell. Thereafter, each of the
obtained bacterial cells was suspended in a phosphate buffer
solution (PBS) of pH6.8 to a concentration of 1 mg/mL to prepare a
bacterial cell suspension of each strain.
[0075] (3) Preparation of Cell Suspension:
[0076] Cells collected from the spleen of experimental mouse
(C57BL/6) were collected in 50 mL conical tubes (manufactured by
FALCON), and 5 mL of erythrocyte dissolving buffer (0.155M
NH.sub.4Cl, 0.01M Tris-HCl, pH7.5) was added to suspend the cells.
Thereafter, 5 mL of phosphate buffer solution (PBS) of pH6.8 was
added to this and centrifuged at 1200 rpm for 5 minutes.
Thereafter, cells were washed twice with phosphate buffer solution
(PBS) of pH6.8 to prepare cell suspension.
[0077] (4) Cell Culture:
[0078] The cell suspension was adjusted with the basic medium so as
to be 2.times.10.sup.6 cells/mL, and 1 mL of the adjusted cell
suspension was seeded in a 24-well microplate (manufactured by
FALCON) to obtain 2.times.10.sup.6 cells/1 mL/well. Note that the
basic medium was prepared by adding fetal bovine serum
(manufactured by SAFC Biosciences) which was inactivated by heating
at 55.degree. C. for 30 minutes, to RPMI 1640 with a predetermined
0.3 g/L L-glutamic acid added (manufactured by Nacalai Tesque,
Inc.), so as to be 9 (w/v) % in a medium. The above "RPMI 1640 with
a predetermined 0.3 g/L L-glutamic acid added" is RPMI 1640 with a
L-glutamic acid (0.3 g/L) added with a penicillin-streptomycin
mixed solution (100 U/mL-100 .mu.g/mL in a medium, manufactured by
Nacalai Tesque, Inc.) and a 2-mercaptoethanol (50 .mu.M in a
medium, manufactured by Nacalai Tesque, Inc.)
[0079] After that, 10 .mu.L of each bacterial cell suspension (1
mg/mL) was added and cultured at 37.degree. C. and 5% CO.sub.2 for
2 days. A control was also prepared. This control was prepared by
culturing cells for 2 days without adding bacterial cell suspension
to the adjusted cell suspension under the same conditions
(37.degree. C., 5% CO.sub.2) as when the bacterial cells were
added.
[0080] (5) Measurement of IL-22:
[0081] After culturing for 42 hours during culturing for 2 days (48
hours), 0.67 .mu.L of BD GolgiStop (manufactured by BD) was added
to the culture liquid and mixed. Thereafter, the culture liquid was
further cultured at 37.degree. C. and 5% CO.sub.2 for 6 hours.
[0082] Thereafter, the cell culture liquid cultured in a 24-well
microplate (manufactured by FALCON) was transferred to a 1.5 mL
reaction tube (manufactured by Greiner Bio-One), centrifuged at
1200 rpm for 5 minutes, and the cells were collected. After that,
the collected cells were fixed and permeated by using BD
Cytofix/Cytoperm.TM. Fixation/Permeabilization Kit (manufactured by
BD). This procedure followed the instructions attached to
Fixation/Permeabilization Kit.
[0083] For staining of B cells, violetFluor450-labeled anti-B220
antibody (manufactured by TONBO Biosciences) was used, and further,
for staining of IL-22, PE-labeled anti-IL-22 antibody (manufactured
by affymetrix eBioscience) was used. In addition, APC-labeled
anti-CD86 antibody (manufactured by TONBO Biosciences) was used to
measure the activation status of B cells.
[0084] Staining reaction was performed by being left to stand for
60 minutes in refrigerator (5.degree. C.). Thereafter, the mixture
was centrifuged at 1200 rpm for 5 minutes, and the cells were
collected and suspended in 0.5 mL of PBS to obtain a measuring
sample.
[0085] Note that the measurement was performed using flow cytometry
(MACSQuant Analyzer manufactured by Milteny Biotec)
[0086] For the analysis, FCS data analysis software FlowJo
(manufactured by FlowJo, LLC) was used.
[0087] (Results)
[0088] (1) Measurement of IL-22-Producing Cell Amount in Spleen B
Cells:
[0089] B220 positive cells were regarded as B cells, and the ratio
of IL-22 positive cells in spleen B cells (IL-22.sup.+,
B220.sup.+/B220.sup.+) was determined for the respective measuring
samples.
[0090] The percentage of IL-22 positive cells in B cells in the
control (to which no bacterial cell suspension was added) was
defined as the standard (100), and the relative values of the
respective measuring samples were calculated to obtain the values
of IL-22-producing cell amounts of B cells (in Tables 1 and 2,
described as "IL-22 production inducing potency (spleen B
cells)".
[0091] Mean value (X.sup.-) and standard error (S.E.) were obtained
by measuring respective measuring samples once, and then repeating
the measurement 2 to 6 times for the measuring samples having the
high measurement values. The results are shown in Table 1 and Table
2.
[0092] (2) Measurement of IL-22-Producing Cell Amount in Total
Spleen Cells:
[0093] The percentage of IL-22 positive cells in total spleen cells
("IL-22 positive cells/total spleen cells") was determined for
respective measuring sample.
[0094] The percentage of IL-22 positive cells in spleen cells in
the control (to which no bacterial cell suspension was added) was
defined as the standard (100), and the relative values of the
respective measuring samples were calculated to obtain the values
of IL-22-producing cell amounts of total spleen cells (in Tables 1
and 2, described as "IL-22 production inducing potency (total
spleen cells)".
[0095] In the same manner as "(1) Measurement of IL-22-producing
cell amount in B cells" above, the mean value (X.sup.-) and the
standard error (S.E.) were obtained by measuring the respective
measuring samples once, and then further repeating the measurement
for the measuring samples having the high measurement values in the
same manner as in (1) above. The results are shown in Table 1 and
Table 2.
[0096] (3) Measurement of Activation Potency of B Cell:
[0097] The percentage of CD86 positive cells in B cells
(CD86.sup.+, B220.sup.+/CD86.sup.-, B220.sup.+) was determined.
Then, the percentage of CD86 positive cells in B cells in the
control (to which no bacterial cell suspension was added) was
defined as a reference (100), and the activation potency of B cells
was calculated (in Tables 1 and 2, described as "Activation potency
of B cells").
[0098] The measurement of the activation potency of the B cells was
similarly repeated for the measuring samples obtained by repeating
the above "(1) Measurement of IL-22-producing cell amount in B
cells" and "(2) Measurement of IL-22-producing cell amount in total
spleen cells", and the mean values (X.sup.-) and the standard error
(S.E.) were obtained. The results are shown in Table 1 and Table
2.
[0099] (4) Confirming of the Growth Temperature:
[0100] In addition, the growth temperature of each isolated strain
was confirmed. Specifically, a medium obtained by putting 10 mL
each of Nutrient Broth medium (manufactured by Wako Pure Chemical
Corporation) into a test tube (diameter 18 mm.times.180 min) and
capped with a culture stopper (SILICOSEN, manufactured by Shin-Etsu
Polymer Co., Ltd.) and sterilized with an autoclave (121.degree.
C., 20 minutes) was prepared. A loopful of colonies were
respectively inoculated into three media, and cultured statically
at 30.degree. C., 45.degree. C., and 55.degree. C., respectively.
As a control, a medium that was not ingested bacteria was prepared.
Visual confirmation (confirmation of turbidity of the medium and
presence or absence of precipitation) was performed after 2 days of
culture to determine whether or not the culture had grown
sufficiently. The determination criteria was "O" for the case of
proliferation, and "X" for the case of non-proliferation.
[0101] The results of confirmation of the growth temperature are
shown in Table 1 and Table 2 (shown in the column of "Culture
temperature" in Table 1 and Table 2). Both strains proliferated in
cultures at 30.degree. C.
TABLE-US-00001 TABLE 1 IL-22 IL-22 production production inducing
inducing Activation potency potency potency (spleen B cells) (total
spleen cells) of B cell Number Isolated Culturing temperature
Strain name X.sup.- S.E. X.sup.- S.E. X.sup.- S.E. of n from
30.degree. C. 45.degree. C. 55.degree. C. control 100 -- 100 -- 100
-- -- -- -- -- -- Bacillus subtilis (natto) bs-01 282 -- 173 -- 525
-- 1 natto .largecircle. .largecircle. X Bacillus subtilis (natto)
bs-02 355 -- 227 -- 474 -- 1 natto .largecircle. .largecircle. X
Bacillus subtilis (natto) bs-03 236 -- 217 -- 485 -- 1 natto
.largecircle. .largecircle. X Bacillus subtilis (natto) bs-04 176
-- 183 -- 561 -- 1 natto .largecircle. .largecircle. X Bacillus
subtilis (natto) bs-05 457 37 351 30 460 90 4 natto .largecircle.
.largecircle. X Bacillus subtilis (natto) bs-06 271 -- 207 -- 434
-- 1 natto .largecircle. .largecircle. X Bacillus subtilis (natto)
bs-07 427 31 313 47 321 18 4 natto .largecircle. .largecircle. X
Bacillus subtilis (natto) bs-08 353 -- 266 -- 453 -- 1 natto
.largecircle. .largecircle. X Bacillus subtilis (natto) bs-09 230
-- 211 -- 333 -- 1 natto .largecircle. .largecircle. X Bacillus
subtilis (natto) bs-10 245 -- 166 -- 215 -- 1 natto .largecircle.
.largecircle. X Bacillus subtilis (natto) bs-11 154 -- 145 -- 248
-- 1 natto .largecircle. .largecircle. X Bacillus subtilis (natto)
bs-12 332 -- 261 -- 471 -- 1 natto .largecircle. .largecircle. X
Bacillus subtilis bs-13 218 -- 160 -- 553 -- 1 miso .largecircle.
.largecircle. X Bacillus subtilis bs-14 135 -- 130 -- 206 -- 1 miso
.largecircle. .largecircle. X Bacillus subtilis bs-15 262 -- 216 --
453 -- 1 miso .largecircle. .largecircle. X Bacillus subtilis bs-16
252 -- 199 -- 622 -- 1 miso .largecircle. .largecircle. X Bacillus
subtilis bs-17 245 -- 211 -- 475 -- 1 miso .largecircle.
.largecircle. X Bacillus subtilis bs-18 262 -- 211 -- 526 -- 1 miso
.largecircle. .largecircle. X Bacillus subtilis bs-19 298 -- 224 --
311 -- 1 miso .largecircle. .largecircle. X Bacillus subtilis bs-20
193 -- 172 -- 424 -- 1 miso .largecircle. .largecircle. X Bacillus
subtilis bs-21 133 -- 110 -- 366 -- 1 miso .largecircle.
.largecircle. X Bacillus subtilis bs-22 178 -- 122 -- 535 -- 1 miso
.largecircle. .largecircle. X Bacillus subtilis bs-23 145 -- 120 --
307 -- 1 miso .largecircle. .largecircle. X Bacillus subtilis bs-24
223 -- 164 -- 378 -- 1 miso .largecircle. .largecircle. X Bacillus
subtilis bs-25 372 74 302 60 437 51 4 miso .largecircle.
.largecircle. X Bacillus subtilis bs-26 211 -- 186 -- 282 -- 1 miso
.largecircle. .largecircle. X Bacillus subtilis bs-27 161 -- 135 --
436 -- 1 miso .largecircle. .largecircle. X Bacillus subtilis bs-28
159 -- 154 -- 369 -- 1 miso .largecircle. .largecircle. X Bacillus
subtilis bs-29 166 -- 145 -- 470 -- 1 miso .largecircle.
.largecircle. X Bacillus subtilis bs-30 766 55 605 72 430 96 4 miso
.largecircle. .largecircle. X Bacillus subtilis bs-31 118 -- 126 --
280 -- 1 miso .largecircle. .largecircle. X Bacillus subtilis bs-32
267 -- 230 -- 325 -- 1 miso .largecircle. .largecircle. X Bacillus
subtilis bs-33 147 -- 149 -- 341 -- 1 miso .largecircle.
.largecircle. X Bacillus subtilis bs-34 971 53 638 60 495 38 4 miso
.largecircle. .largecircle. X Bacillus subtilis bs-35 171 -- 166 --
262 -- 1 miso .largecircle. .largecircle. X Bacillus subtilis bs-36
494 54 342 32 655 96 4 miso .largecircle. .largecircle. X Bacillus
subtilis bs-37 176 -- 183 -- 561 -- 1 miso .largecircle.
.largecircle. X Bacillus subtilis bs-38 275 -- 226 -- 449 -- 1 miso
.largecircle. .largecircle. X Bacillus subtilis bs-39 513 68 372 56
294 44 4 miso .largecircle. .largecircle. X Bacillus subtilis bs-40
249 -- 213 -- 496 -- 1 miso .largecircle. .largecircle. X
TABLE-US-00002 TABLE 2 IL-22 IL-22 production production inducing
inducing Activation potency potency potency (spleen B cells) (total
spleen cells) of B cell Number Isolated Culturing temperature
Strain name X.sup.- S.E. X.sup.- S.E. X.sup.- S.E. of n from
30.degree. C. 45.degree. C. 55.degree. C. control 100 -- 100 -- 100
-- -- -- -- -- -- Bacillus coagulans sc-01 376 70 272 52 555 46 3
miso .largecircle. .largecircle. .largecircle. Bacillus coagulans
sc-02 168 20 155 20 262 58 3 miso .largecircle. .largecircle.
.largecircle. Bacillus coagulans sc-03 179 43 173 30 441 29 3 miso
.largecircle. .largecircle. .largecircle. Bacillus coagulans sc-04
243 22 152 4 688 48 3 miso .largecircle. .largecircle.
.largecircle. Bacillus coagulans sc-05 334 75 232 47 404 19 5 miso
.largecircle. .largecircle. .largecircle. Bacillus coagulans sc-06
423 54 276 48 414 17 5 miso .largecircle. .largecircle.
.largecircle. Bacillus coagulans sc-07 204 28 152 11 392 27 3 miso
.largecircle. .largecircle. .largecircle. Bacillus coagulans sc-08
444 104 284 60 385 17 5 miso .largecircle. .largecircle.
.largecircle. Bacillus coagulans sc-09 1,062 158 643 60 501 53 7
miso .largecircle. .largecircle. .largecircle. Bacillus coagulans
sc-10 338 75 264 38 400 8 5 miso .largecircle. .largecircle.
.largecircle. Bacillus coagulans sc-11 253 16 172 15 417 27 3 miso
.largecircle. .largecircle. .largecircle. Bacillus coagulans sc-12
419 90 263 47 371 18 5 miso .largecircle. .largecircle.
.largecircle. Bacillus coagulans sc-13 143 19 141 15 364 27 3 miso
.largecircle. .largecircle. .largecircle. Bacillus coagulans sc-14
332 54 234 39 343 31 5 miso .largecircle. .largecircle.
.largecircle. Bacillus coagulans sc-15 309 41 218 37 353 17 5 miso
.largecircle. .largecircle. .largecircle. Bacillus coagulans sc-16
249 29 213 25 330 20 5 miso .largecircle. .largecircle.
.largecircle. Bacillus coagulans sc-17 131 10 120 11 182 58 3 miso
.largecircle. .largecircle. .largecircle. Bacillus coagulans sc-18
376 35 282 15 493 25 3 miso .largecircle. .largecircle.
.largecircle. Bacillus coagulans sc-19 349 31 265 20 474 28 3 miso
.largecircle. .largecircle. .largecircle. Bacillus coagulans sc-20
480 100 283 64 415 4 5 miso .largecircle. .largecircle.
.largecircle. Bacillus amyloliquefaciens bi-01 369 -- 270 -- 499 --
1 miso .largecircle. .largecircle. X Bacillus amyloliquefaciens
bi-02 213 -- 188 -- 422 -- 1 miso .largecircle. .largecircle. X
Bacillus benzoevorans bi-03 348 -- 223 -- 473 -- 1 miso
.largecircle. X X Bacillus benzoevorans bi-04 228 -- 188 -- 304 --
1 miso .largecircle. X X Bacillus firmus bi-05 161 -- 149 -- 189 --
1 miso .largecircle. X X Bacillus megaterium bi-06 148 -- 115 --
209 -- 1 miso .largecircle. .largecircle. X Bacillus megaterium
bi-07 176 -- 161 -- 412 -- 1 miso .largecircle. .largecircle. X
Bacillus megaterium bi-08 267 -- 209 -- 434 -- 1 miso .largecircle.
.largecircle. X Bacillus megaterium bi-09 264 -- 219 -- 270 -- 1
miso .largecircle. .largecircle. X Bacillus novalis bi-10 351 --
206 -- 507 -- 1 miso .largecircle. .largecircle. X Bacillus pumilus
bi-11 264 -- 223 -- 522 -- 1 miso .largecircle. X X Bacillus
tequilensis bi-12 219 -- 185 -- 378 -- 1 miso .largecircle. X X
[0102] (5) Screening Results of IL-22-Producing Inducer:
[0103] The results of screening for each bacterial species were as
follows (see Table 1, Table 2, FIGS. 1-6).
[0104] IL-22 production increased (more than 1.1 times) in all
strains (72 strains). Among these, Bacillus bacteria derived from
miso (for example, shown as the strain name of "bs-30", "bs-34" and
"sc-09" in Table 1) showed 7-10 times the amount of IL-22-producing
cells among B cells compared to the control, and the results for
total spleen cell showed about 6 times the amount of
IL-22-producing cells compared to the control. Strains with
particularly large amount of IL-22-producing cells are shown in
Table 3.
TABLE-US-00003 TABLE 3 IL-22 IL-22 production production inducing
inducing Activation potency potency potency (spleen B cells) (total
spleen cells) of B cell Isolated Strain name X.sup.- S.E. X.sup.-
S.E. X.sup.- S.E. from Bacillus subtilis bs-30 766 55 605 72 430 96
miso Bacillus subtilis bs-34 971 53 638 60 495 38 miso Bacillus
coagulans sc-09 1,062 158 643 60 501 53 miso
[0105] (Summary)
[0106] It was found that IL-22 was induced by stimulation of
bacterial cell (sterilized bacterium) of Bacillus bacterium, and
the degree of induction varied depending on the strains. Bacillus
bacterium was considered to have IL-22 production inducing
potency.
[0107] Although it is known that CD4.sup.+ T cells and NK cells
produce IL-22, it has not been reported that IL-22 is produced in B
cells. In addition, from the above results, when the Bacillus
bacteria of the present invention were used, IL-22 production in
spleen cells, in particular, from B cells was increased by
stimulation of the bacteria.
[0108] FIG. 7 is an example of measurements in the flow cytometry
in Example 1, in which the vertical axis represents the expression
of B220, the horizontal axis represents the expression of
interleukin-22, the control, the case of adding the strain of
"bs-34" (Bacillus subtilis bs-34) (referred to as "+bs-34"), and
the case of adding the strain of "sc-09" (Bacillus coagulans sc-09)
(referred to as "+sc-09") are shown in order from the left
side.
[0109] In addition, Bacillus bacteria (strains) with high potency
to induce the production of IL-22 tended to have high potency to
activate B cells. On the other hand, the bacteria with higher B
cell activation potency did not necessarily have higher
production-inducing capacity for IL-22.
[0110] From this fact, it was proven that some Bacillus bacteria
which can activate B cell by the stimulation of the bacterial cell
also possessed IL-22 production inducing potency. If the B cells
are in an activated state, an effect of activating immunity can be
expected.
[0111] In the present embodiment, the B cells were analyzed
focusing on B220 positive cells, and similar results were obtained
when lactobacillus with a higher potency to induce IL-22 production
were analyzed with CD19 positive cells (violetFluor450-labeled
anti-B220 antibody (manufactured by TONBO Biosciences)) instead of
B220 positive cells. From this, it was confirmed that IL-22
production inducing potency from B cells was improved by a
predetermined strain, and the activation potency of B cells was
further improved.
Example 2
[0112] (Measurement Test for Cell Viability and Cell Activation
Potency)
[0113] With respect to Bacillus subtilis bs-30, Bacillus subtilis
bs-34, and Bacillus coagulans sc-09, which exhibited high IL-22
production inducing potency in Example 1, bacterial cells after
sterilization were "co-cultured with spleen cells" of experimental
mouse (C57BL/6) to investigate the viability of whole spleen cells,
the viability of spleen B cells and spleen T cells, and the
activation potency of spleen B cells and spleen T cells.
Hereinafter, the test content will be described in detail.
[0114] (1) Preparation of Bacterial Cell Suspension:
[0115] A suspension similar to the suspension of Bacillus bacterium
prepared in Example 1 was used.
[0116] (2) Preparation of Cell Suspension:
[0117] Prepared in the same manner as in Example 1.
[0118] (3) Cell Culture:
[0119] The cell suspension was adjusted with the basic medium so as
to be 2.times.10.sup.6 cells/mL, and 0.5 mL of the adjusted cell
suspension was seeded in 48-well microplate (manufactured by
FALCON) to obtain 1.times.10 cells/0.5 mL/well.
[0120] After that, 5 .mu.L of each bacterial cell suspension (1
mg/mL) was added to each cell suspension and cultured for 2 days
under the conditions of 37.degree. C. and 5% CO.sub.2. A control
was prepared by culturing cell for 2 days without adding the
bacterial cell suspension to the adjusted cell suspension under the
same conditions (conditions of 37.degree. C. and 5% CO.sub.2) as
those in which the bacterial cells were added.
[0121] (4) Measurement of Cell Viability and Cell Activation
Potency:
[0122] The cell culture liquid cultured in a 48-well microplate was
transferred to a 1.5 mL reaction tube (manufactured by Greiner
Bio-One), centrifuged at 1200 rpm for 5 minutes, and the cells were
collected. Thereafter, the collected cells were suspended in 0.2 mL
of phosphate buffer solution (PBS) of pH6.8, and the following 4
antibodies were added in 1 .mu.L portions and left to stand for 60
minutes in a refrigerator (5.degree. C.).
[0123] The 4 antibodies added were violetFluor450-labeled anti-B220
antibody (manufactured by TONBO Biosciences), APC-labeled anti-CD86
antibody (manufactured by TONBO Biosciences), Brilliant
Violet510-labeled anti-CD4 antibody (manufactured by BioLegend,
Inc.), and PE-labeled anti-CD69 antibody (manufactured by
BioLegend, Inc.)
[0124] After standing, the cells were centrifuged at 1200 rpm for 5
min, and the cells were collected and suspended in 0.5 mL of
phosphate buffer solution (PBS) of pH6.8. Thereafter, 0.5 .mu.L of
Propidium Iodide (PI) nucleus staining liquid (manufactured by
Cosmo Bio Co., Ltd.) was added to obtain a measuring sample.
Measurements were performed on this measuring sample using flow
cytometry (MACSQuant Analyzer, manufactured by Milteny Biotec) For
the analysis, FCS data analysis software FlowJo (manufactured by
FlowJo, LLC) was used.
[0125] (Cell Viability)
[0126] Among the measuring samples, PI-detected cells (cells
stained with PI nucleus staining liquid) were regarded as dead
cells, and the difference from the counted number of cells (total
number of cells) was regarded as the number of living cells. Then,
the ratio of living cells in the total cells (the number of living
cells/the number of total cells.times.100) was calculated.
Similarly, the ratio of living cells in total cells in the control
(to which no bacterial cell suspension was added) was calculated.
Thereafter, these values were compared, and the value of the ratio
when the group was taken as the reference (100) was calculated to
be the value of the viability of the cell (cell viability). The
test was repeated to obtain the mean value (X.sup.-) and the
standard error (S.E.). The results were shown in Table 4. In this
embodiment, "mean value (X.sup.-)" is the mean value of six tests
(n=6).
[0127] (Viability of B Cell)
[0128] B cells were detected with violetFluor450-labeled anti-B220
antibody (manufactured by TONBO Biosciences) which is a cell
surface marker of B cells. The quotient of the number of B cells
among living cells (B220 positive cells among those not detected
PI) and the number of total cells (the ratio of the number of
viable B cells to the total number of cells) was calculated.
Similarly, the ratio of the number of viable B cells to the total
number of cells in the control (to which no bacterial cell
suspension was added) was calculated. Thereafter, these values were
compared, and the value of the ratio when the control was taken as
the reference (100) was calculated to be the value of viability of
the B cells. The test was repeated to obtain the mean value
(X.sup.-) and the standard error (S.E.). The results were shown in
Table 4.
[0129] (Viability of T Cell)
[0130] T cells were detected with Brilliant Violet510-labeled
anti-CD4 antibody (manufactured by BioLegend, Inc.) which is a cell
surface marker of T cells. The quotient of the number of T cells
among living cells (CD4 positive cells among those not detected PI)
and the number of total cells (the ratio of the number of viable T
cells to the number of total cells) was calculated. Similarly, the
ratio of the number of viable T cells to the total number of cells
in the control (to which no bacterial cell suspension was added)
was calculated. Thereafter, these values were compared, and the
value of the ratio when the control was taken as the reference
(100) was calculated to be the value of viability of the T cell.
The test was repeated to obtain the mean value (X.sup.-) and the
standard error (S.E.). The results were shown in Table 4.
[0131] (Activation Potency of B Cell)
[0132] B cells expressing B220 and CD86 were detected with
violetFluor450-labeled anti-B220 antibody which is a cell surface
marker of B cells, and APC-labeled anti-CD86 antibody which is an
activation marker of B cells, and the number of B cells was
counted. In addition, the quotient of activated B cells
(CD86.sup.+, B220.sup.+) and non-activated B cells (CD86.sup.-,
B220.sup.+) (the value of the ratio of activated B cells to
non-activated 13 cells) among the B cells (B220 positive cells) was
calculated. Similarly, the quotient of activated 13 cells
(CD86.sup.+, B220.sup.+) and non-activated B cells (CD86.sup.-,
B220.sup.+) in control (to which no bacterial cell suspension was
added) was calculated. Thereafter, these values were compared, and
the value of the ratio when the control was taken as the reference
(100) was calculated to be the value of the activation potency of
the B cell. The test was repeated to obtain the mean value
(X.sup.-) and the standard error (S.E.). The results were shown in
Table 4.
[0133] (Activation Potency of T Cell)
[0134] Cells expressing CD4 and CD69 were detected with Brilliant
Violet510-labeled anti-CD4 antibody (manufactured by BioLegend,
Inc.) which was a cell surface marker of T cells and PE-labeled
anti-CD69 antibody (manufactured by BioLegend, Inc.) which was an
activation marker of T cells, and the number of cells was counted.
In addition, the quotient of activated T cells (CD69.sup.+,
CD4.sup.+) and non-activated T cells (CD69.sup.-, CD4.sup.+) (the
value of the ratio of activated T cells to non-activated T cells)
among T cells (CD4 positive cells), was calculated. Similarly, the
quotient of activated T cells (CD69.sup.+, CD4.sup.+) and
non-activated T cells (CD69.sup.-, CD4.sup.+) in control (to which
no bacterial cell suspension was added) was calculated. Thereafter,
these values were compared, and the value of the ratio when the
control was taken as the reference (100) was calculated to be the
value of the activation potency of the T cell. The test was
repeated to obtain the mean value (X.sup.-) and the standard error
(S.E.). The results were shown in Table 4.
TABLE-US-00004 TABLE 4 Activation Activation Cell Viability
Viability potency potency viability of B cell of T cell of B cell
of T cell Strain name X.sup.- S.E. X.sup.- S.E. X.sup.- S.E.
X.sup.- S.E. X.sup.- S.E. control 100 -- 100 -- 100 -- 100 -- 100
-- Bacillus subtilis bs-30 161 9 149 12 182 8 512 28 208 12
Bacillus subtilis bs-34 161 5 151 6 194 11 506 25 210 12 Bacillus
coagulans sc-09 139 7 132 9 164 11 442 31 199 7
[0135] (Results)
[0136] According to the results of this example, it was found that
the strain of Bacillus bacterium having high IL-22 production
inducing potency selected in Example 1 not only has a high
activation potency of B cells but also has a high potency to
improve the viability of B cells. Furthermore, it was found that
the potency to improve the viability of T cells and the activation
potency of T cells were also high.
[0137] In this embodiment, B cells were analyzed focusing on B220
positive cells, and similar results were obtained when CD19
positive cells (violetFluor450-labeled anti-B220 antibody
(manufactured by TONBO Biosciences)) were used instead of B220
positive cells. From this fact, it was confirmed that the viability
of the B cell can be improved by the predetermined strain, and the
B cell can be further activated.
Example 3
[0138] (Measuring Test of Viability of B Cell and Activation
Potency of B Cell)
[0139] With respect to Bacillus subtilis bs-30, Bacillus subtilis
bs-34, and Bacillus coagulans sc-09, which exhibited high IL-22
production inducing potency in Example 1, bacterial cells after
sterilization were "co-cultured with spleen-derived B cells (B220
positive cells)" of experimental mouse (C57BL/6), and the potency
to improve the viability of spleen B cells and the activation
potency of spleen B cells (potency to activate B cells) were
investigated. Hereinafter, the measurement test will be described
in detail.
[0140] (1) Preparation of Bacterial Cell Suspension:
[0141] A suspension similar to the suspension of Bacillus bacterium
prepared in Example 1 was used.
[0142] (2) Preparation of B Cell Suspension:
[0143] Cells collected from the spleens of experimental mouse
(C57BL/6) were collected in 50 mL conical tubes (manufactured by
FALCON), and 5 mL of erythrocyte lysis buffer (0.155M NH.sub.4Cl,
0.01M Tris-HCl, pH7.5) was added to suspend the cells. Thereafter,
5 mL of phosphate buffer solution (PBS) of pH6.8 was added to this
and the mixture was centrifuged at 1200 rpm for 5 minutes.
Thereafter, cell suspension was prepared by washing twice with
phosphate buffer solution (PBS) of pH6.8.
[0144] After suspension in basic medium, a biotin-anti B220
antibody (manufactured by TONBO Biosciences) was added and left to
stand for 30 minutes in a refrigerator (5.degree. C.).
[0145] After standing, the mixture was centrifuged at 1200 rpm for
5 minutes, washed 2 times with phosphate buffer solution (PBS) of
pH6.8, and then suspended in phosphate buffer solution (PBS) of
pH6.8. Thereafter, Streptavidin Particles Plus DM, which is a
magnetic bead (manufactured by Nippon BD Company, Ltd.) was added,
and left to stand for 30 minutes in a refrigerator (5.degree.
C.).
[0146] Thereafter, the mixture was centrifuged at 1200 rpm for 5
minutes, washed once with phosphate buffer solution (PBS) of pH6.8,
and then suspended again in phosphate buffer solution (PBS) of
pH6.8 and transferred to a round tube.
[0147] Thereafter, cell separation was then carried out using BD
IMag Cell Separation System (manufactured by Nippon BD Company,
Ltd.), and the cells attracted to the magnet were collected as "B
cells (B220 positive cells)" (positive cell fractionation). The
collected cells were suspended in basic medium to prepare B cell
suspension. The number of cells in the obtained B cell suspension
was measured using a hemocytometer.
[0148] (3) Cell Culture:
[0149] B cell suspension was adjusted in the basic medium so as to
be 2.times.10.sup.6 cells/mL, and 1 mL of the adjusted B cell
suspension was seeded in a 24-well microplate (manufactured by
FALCON) to obtain 2.times.10 cells/1 mL/well. Thereafter, 10 .mu.l
of each bacterial cell suspension was added to each cell
suspension, and cultured for 2 days under the condition of
37.degree. C. and 5% CO.sub.2. In addition, a control was prepared
by culturing cells for 2 days without adding a bacterial cell
(bacterial cell suspension) to the adjusted 13 cell suspension
under the same conditions (conditions of 37.degree. C. and 5%
CO.sub.2) as the level at which the bacterial cell was added.
[0150] (4) Measurement of Viability of B Cell and Activation
Potency of B Cell:
[0151] After culturing, the viability of B cells (the amount of
viable B cells) and the activation potency of B cells (the amount
of activated 13 cells) were measured for the respective samples
(cell culture liquid) using flow cytometry (MACSQuant Analyzer
manufactured by Milteny Biotec).
[0152] First, the cell culture liquid that was cultured in a
24-well microplate was transferred to a 1.5 mL reaction tube
(manufactured by Greiner Bio-One), centrifuged at 1200 rpm for 5
minutes, and cells were collected. Thereafter, the collected cells
were suspended in 0.1 mL of phosphate buffer solution (PBS) of
pH6.8, and 0.5 .mu.L of violetFluor450-labeled anti-B220 antibody
(manufactured by TONBO Biosciences) and 0.5 .mu.L of APC-labeled
anti CD86 antibody (manufactured by TONBO Biosciences) were added,
and the mixture was left to stand for 60 minutes in a refrigerator
(5.degree. C.)
[0153] After standing, the mixture was centrifuged at 1200 rpm for
5 min, and the cells were collected and suspended in 0.5 mL of
phosphate buffer solution (PBS) of pH6.8. Thereafter, 0.5 .mu.L of
Propidium Iodide (PI) nucleus staining liquid (manufactured by
Cosmo Bio Co., Ltd.) was added to obtain a measuring sample. This
measuring sample was subjected to measurement using flow cytometry.
For the analysis, FCS data analysis software FlowJo (manufactured
by FlowJo, LLC) was used.
[0154] (Viability of B Cell)
[0155] Among the measuring samples, PI detected cells (cells
stained with PI nucleus staining liquid) were regarded as dead
cells, and the difference from the counted number of cells (total
number of cells) was regarded as the number of living cells. Then,
the ratio of living cells in the total cells (the number of living
cells/the number of total cells.times.100) was calculated.
Similarly, the ratio of living cells in total cells in the control
(to which no suspension of Bacillus bacterium was added) was
calculated. Thereafter, these values were compared, and the value
of the ratio when the control was taken as the reference (100) was
calculated to be the value of the viability of B cells (cell
viability). The test was repeated to obtain the mean value
(X.sup.-) and the standard error (S.E.). The results are shown in
"Viability of B cell" in Table 5. In this embodiment, "mean value
(X.sup.-)" is the mean value of four tests (n=4).
[0156] (Activation Potency of B Cell)
[0157] B cells expressing B220 and CD86 were detected with
violetFluor450-labeled anti-B220 antibody which is a cell surface
marker of B cells, and APC-labeled anti-CD86 antibody which is an
activation marker of B cells, and the number of B cells was
counted. In addition, the quotient of activated B cells
(CD86.sup.+, B220.sup.+) and non-activated B cells (CD86.sup.-,
B220.sup.+) (the ratio of the number of activated B cells to the
number of non-activated B cells) among B cells (B220 positive
cells), was calculated. Similarly, the quotient of activated B
cells (CD86.sup.+, B220.sup.+) and non-activated B cells
(CD86.sup.-, B220.sup.+) in control (to which no bacterial cell
suspension was added) was calculated. Thereafter, these values were
compared, and the value of the ratio when the control was taken as
the reference (100) was calculated to be the value of the
activation potency of the B cell. The test was repeated to obtain
the mean value (X.sup.-) and the standard error (S.E.). The results
are shown in "Activation potency of B cell" in Table 5.
TABLE-US-00005 TABLE 5 IL-22 Activation production Viability
potency inducing of B cell of B cell potency Strain name X.sup.-
S.E. X.sup.- S.E. X.sup.- S.E. control 100 -- 100 -- 100 --
Bacillus subtilis bs-30 370 38 217 26 734 49 Bacillus subtilis
bs-34 340 57 234 24 745 10 Bacillus coagulans sc-09 500 27 220 6
1,089 183
[0158] (Results)
[0159] According to the results of this example, it was further
confirmed that the "strain having high IL-22 production inducing
potency" selected in Example 1 was able to directly act on B cells
to improve the viability of B cells and activate B cells.
Example 4
[0160] (Measuring Test for IL-22 Production Inducing Potency of B
Cell)
[0161] With respect to Bacillus subtilis bs-30, Bacillus subtilis
bs-34, and Bacillus coagulans sc-09, which exhibited high IL-22
production inducing potency in Example 1, bacterial cells after
sterilization were "co-cultured with B cells (B220 positive cells)
derived from spleens" of experimental mouse (C57BL/6), to
investigate IL-22 production inducing potency. Hereinafter, the
measurement test will be described in detail.
[0162] (1) Preparation of Bacterial Cell Suspension:
[0163] A suspension similar to the suspension of Bacillus bacterium
prepared in Example 1 was used.
[0164] (2) Preparation of B Cell Suspension: Prepared as in Example
3
[0165] (3) Cell Culture: Cultured in the Same Manner as in Example
3
[0166] (4) Measurements of IL-22:
[0167] After 42 hours of cultivation, 0.67 .mu.L of BD GolgiStop
(manufactured by BD) was added to the each culture liquid and
mixed. Thereafter, the culture liquids were cultured at 37.degree.
C. and 5% CO.sub.2 for 6 hours.
[0168] Thereafter, the cell culture liquid cultured in a 24-well
microplate (manufactured by FALCON) was transferred to a 1.5 mL
reaction tube (manufactured by Greiner Bio-One), centrifuged at
1200 rpm for 5 minutes, and cells were collected. After that, the
collected cells were fixed and permeated by using BD
Cytoflx/Cytoperm.TM. Fixation/Permeabilization Kit (manufactured by
BD). This procedure followed the instructions attached to
Fixation/Permeabilization Kit.
[0169] For staining of B cells, violetFluor450-labeled anti-B220
antibody (manufactured by TONBO Biosciences) was used. In addition,
for staining of IL-22, PE-labeled anti-IL-22 antibody (manufactured
by affymetrix eBioscience) was used.
[0170] Staining reaction was performed by being left to stand for
60 minutes in a refrigerator (5.degree. C.). Thereafter, the
mixture was centrifuged at 1200 rpm for 5 minutes, and the cells
were collected and suspended in 0.5 mL of phosphate buffer solution
(PBS) of pH6.8 to obtain a measuring sample. This measuring sample
was subjected to measurement using flow cytometry. For the
analysis, FCS data analysis software FlowJo (manufactured by
FlowJo, LLC) was used.
[0171] The ratio of IL-22 positive cells in B cells (IL-22.sup.+,
B220.sup.+/B220.sup.+) was determined for respective measuring
sample, and the ratio of IL-22 positive cells in B cells in the
control (to which no bacterialcell suspension was added) was taken
as the standard (100), and the relative value of respective
measuring sample was calculated to be the value of IL-22 production
cell amount of B cells. The test was repeated to obtain the mean
value (X.sup.-) and the standard error (S.E.). The results are
shown in "IL-22 production inducing potency" in Table 5. In this
embodiment, "mean value (X.sup.-)" is the mean value of four tests
(n=4).
[0172] (Results)
[0173] According to the results of this example, it was found that
the "strain having a high IL-22 production inducing potency"
selected in Example 1 directly acted on B cells and increased B
cells producing IL-22.
Example 5
[0174] (Feeding Test (Normal Mouse))
[0175] Experimental mouse was ingested with a bacterial cell after
sterilization of "Bacillus coagulans sc-09," which had a high IL-22
production inducing potency, and then the skin condition was
measured (TransEpidermal Water Loss(TEWL)). In addition, a group to
which IL-22 was administered ("IL-22 administered group" in Table
11) and a group to which IL-22 neutralizing antibody was
administered ("Bacterial cell ingested/Anti-IL-22 antibody
administered group" in Table 11) were also prepared, and skin
changes due to IL-22 inoculation were also confirmed.
[0176] (1) Preparation of Bacillus Bacterium Mixed Feed:
[0177] A feed (Bacillus bacterium mixed feed) was prepared by
containing 1 w/w % bacterial cells of Bacillus coagulans sc-09,
which were sterilized and then lyophilized, in a general feed for
mouse. As a general feed for mouse, a mouse breeding feed CE-2
(manufactured by CLEA Japan, Inc.) was used.
[0178] (2) Feeding Breeding:
[0179] Twelve general experimental mice (C57BL/6) (8-week-old
females) were divided into four groups (three per group), wherein
two groups were fed with the Bacillus bacterium mixed feed and the
other two groups were fed with general feed for mouse not
containing bacterial cells of Bacillus bacterium, and they were
bred for 21 days.
[0180] One of the two groups fed with general feed for mouse not
containing bacterial cells of Bacillus bacterium was injected IL-22
recombinant proteins "Recombinant IL-22 (Recombinant Mouse IL-22
manufactured by TONBO Biosciences)" into the tail vein (2
.mu.g/mouse each) after 14 days and 17 days from the start of the
feeding, respectively. Among the above two groups, the group in
which the "Recombinant IL-22" was injected into the tail vein was
referred to as the "IL-22 administered group", and the group in
which the "Recombinant IL-22" was not injected (administered) into
the tail vein was referred to as the "Control group".
[0181] One of the two groups fed with the Bacillus bacterium mixed
feed was injected "Anti-IL-22 Antibody (IL-22 Monoclonal Antibody
manufactured by Thermo Fisher)" as a IL-22 neutralizing antibody
into the tail vein (20 .mu.g/mouse each) after 14 days and 17 days
from the start of the feeding, respectively. Among the above two
groups, the group in which "Anti-IL-22 antibody" was injected into
the tail vein was referred to as the "Bacterial cell
ingested/Anti-IL-22 antibody administered group" and the group in
which "Anti-IL-22 antibody" was not injected (administered) into
the tail vein was referred to as the "Bacterial cell ingested
group".
[0182] (3) Measurements of Transepidermal Water Loss (TEWL):
[0183] After 21 days from the start of the feeding, the
transepidermal water loss (TEWL) of the skin on the back of the
mouse in each group was measured. In this measurement, the back of
the mouse was shaved the day before (day 20). TEWL was measured by
a skin measuring device "DermaLab.RTM." manufactured by CORTEX
TECHNOLOGY. TEWL was measured three times in each mouse, the mean
value was obtained, and the mean value and the standard deviation
of each group were obtained. The results of transepidermal water
loss (TEWL) were shown in Table 6 and FIG. 8. The numerical values
of the control group and each other group were subjected to F-test
to confirm whether or not there was a significant difference in
variance. A Student's t-test, which is a two-sample test assuming
equal variance, was then performed.
TABLE-US-00006 TABLE 6 IL-22 Bacterial cell ingested/ Control
Bacterial cell administered Anti-IL-22 antibody TEWL (g/m.sup.2/h)
group ingested group group administered group Mean value 63.5 32.2
40.6 82.8 Standard deviation 2.3 4.3 5.5 4.6 p = 0.0004 p = 0.003 p
= 0.003
[0184] As is clear from the results of Table 6 and FIG. 8, TEWL was
lower in the bacterial cell ingested group (the group ingesting
"Bacillus coagulans sc-09" and not administering "Anti-IL-22
antibody") than in the control group, and p<0.01 (p=0.0004) was
obtained as a result of Student's t-test, which was significantly
different at the significance level of 1%.
[0185] As can be seen from Table 6, TEWL was lower in IL-22
administered group (the group administering IL-22 without
lactobacilli mixed feed) than in the control group, and p<0.01
(p=0.003) was obtained as a result of Student's t-test, which was
significantly different at the significance level of 1%. In
addition, TEWL was higher (i.e., the transpiration rate of water
from the skin was higher) in the "Bacterial cell
ingested/Anti-IL-22 Antibody administered group" than in the
bacterial cell ingested group, and in the control group, and
p<0.01 (p=0.003) was obtained as a result of Student's t-test,
which was significantly different at the significance level of 1%.
No changes in skin conditions were visually observed in the mice of
each group.
[0186] Thus, according to this embodiment, it was found that TEWL
was lowered in the bacterial cell ingested group, and that the
barrier function of the skin was enhanced by ingestion of the
Bacillus bacterium of the present invention.
[0187] While TEWL was decreased by the administration of IL-22, it
was confirmed that IL-22 enhanced the barrier function of the skin
because TEWL was increased by the administration of neutralizing
antibody of IL-22 (injected into the tail vein) (see Table 6 and
FIG. 8). And, the improvement of the barrier function of the skin
by the ingestion of bacterial cell is based on the stimulation
caused by the ingestion of the bacterial cell, and it may be based
on the production inducing of IL-22 by the bacterial cell.
Example 6
[0188] (Feeding Test (Sterile Mouse))
[0189] Sterile mouse was ingested with a bacterial cell after
sterilization of "Bacillus subtilis bs-34" which had high IL-22
production inducing potency, and then the skin condition was
measured (TransEpidermal Water Loss(TEWL)).
[0190] (1) Preparation of Bacterial Cell Mixed Feed:
[0191] A feed (Bacillus bacterium mixed feed) was prepared by
containing 1 w/w % bacterial cells of Bacillus subtilis bs-34,
which were sterilized and then lyophilized, in a general feed for
mouse. As a general feed for mouse, a mouse breeding feed CE-2
(manufactured by CLEA Japan, Inc.) was used.
[0192] (2) Feeding Breeding:
[0193] Ten sterile mice (C57BL/6NJcl [Gf]) (8-week-old females)
were divided into the groups of five, wherein one group was fed
with the Bacillus bacterium mixed feed (Bacterial cell ingested
group) and another group was fed with the general feed for mouse
not containing bacterial cells (Control group), and they were bred
in a sterile environment for one month.
[0194] (3) Measurements of Transepidermal Water Loss (TEWL):
[0195] After 1 month from the start of the feeding, TEWL in the
skin of the back was measured. Immediately before the measurement,
the back of the mouse was shaved with a hair clipper for a small
animal, and then the measurement was performed. TEWL was measured
by a skin measuring device "DermaLab.RTM." manufactured by CORTEX
TECHNOLOGY. TEWL was measured three times in each mouse and the
mean value and standard deviations were determined. The results are
shown in Table 7 and FIG. 9. The numerical values of the control
group and the bacterial cell ingested group were subjected to
F-test to confirm whether or not there was a significant difference
in variance. Thereafter, a Student's t-test was performed.
TABLE-US-00007 TABLE 7 Control Bacterial cell TEWL (g/m.sup.2/h)
group ingested group Mean value 6.2 4.3 Standard deviation 0.7 0.5
p = 0.001
[0196] The bacterial cell ingested group had significantly lower
TEWL than the control group (group not ingested Bacillus bacterium
mixed feed), and the results of Student's t-test showed p<0.01
(p=0.001), with significant differences at the significance level
of 1%. As described above, it was also found from this embodiment
that TEWL was low in the bacterial cell ingested group, and that
the barrier function of the skin was enhanced by ingestion of the
Bacillus bacterium of the present invention.
[0197] As described above, it was found that the Bacillus bacterium
of the present invention has a high IL-22 production inducing
potency. Furthermore, it has been found that the Bacillus bacterium
of the present invention have the potency to improve the viability
of B cells by directly acting on B cells, and have the activation
potency of B cells. From this result, it can be seen that the
Bacillus bacterium of the present invention has an
immunostimulatory action. It has also been found that Bacillus
bacterium of the present invention enhance (potentiate) the barrier
function of the skin.
INDUSTRIAL APPLICABILITY
[0198] The Bacillus bacterium of the present invention can be
employed as an active ingredient of a IL-22 production inducing
agent (further, an active ingredient of a skin barrier function
enhancing agent) by being added to food and drink, supplement,
medicine, or the like to produce IL-22, or the Bacillus bacterium
of the present invention may be used itself as a food and drink, a
supplement, a medicine, or the like. Examples of food and drinks
include miso, instant miso soup, cooked miso (processed miso
products), name-miso such as Kinzanji miso, soy sauce, soup, season
sauce, seasoning sauce, seasoning for rice, side dishes, and sweet
rice drinks (yeast drinks).
ACCESSION NUMBER
[0199] Accession number NITE BP-02583
Date of Accession: Dec. 5, 2017
Accession Institution: NITE Patent Microorganisms Depositary,
National Institute of
Technology and Evaluation
Accession Institution Address: Room122, 2-5-8 Kazusakamatari,
Kisarazu-shi, Chiba 292-818, Japan
[0200] Accession number NITE BP-02584 Date of accession: Dec. 5,
2017
Accession Institution: NITE Patent Microorganisms Depositary,
National Institute of
Technology and Evaluation
Accession Institution Address: Room122, 2-5-8 Kazusakamatari,
Kisarazu-shi, Chiba 292-818, Japan
[0201] Accession number NITE BP-02590 Date of accession: Dec. 5,
2017
Accession Institution: NITE Patent Microorganisms Depositary,
National Institute of
Technology and Evaluation
Accession Institution Address: Room122, 2-5-8 Kazusakamatari,
Kisarazu-shi, Chiba 292-818, Japan
* * * * *