U.S. patent application number 10/521947 was filed with the patent office on 2006-06-15 for composition for promoting the proliferation of lactobacillus casei subsp. casei.
Invention is credited to Masakatsu Hasegawa, Tomohiko Ogawa, Kenji Yasuda.
Application Number | 20060127378 10/521947 |
Document ID | / |
Family ID | 30767803 |
Filed Date | 2006-06-15 |
United States Patent
Application |
20060127378 |
Kind Code |
A1 |
Yasuda; Kenji ; et
al. |
June 15, 2006 |
Composition for promoting the proliferation of lactobacillus casei
subsp. casei
Abstract
The present invention relates to a composition for promoting
bacterial proliferation for selectively proliferating Lactobacillus
casei subsp. casei, which includes a dextran. According to the
present invention, a variety of biological activities originated
from L. casei subsp. casei can be sustained in a living body by
selectively growing-proliferating and colonizing L. casei subsp.
casei in the intestine of a human being, animal, or the like or by
selectively growing-proliferating L. casei subsp. casei in the
intestine, without supplying L. casei subsp. casei at all
times.
Inventors: |
Yasuda; Kenji; (Ogaki-shi,
JP) ; Ogawa; Tomohiko; (Toyonaka-shi, JP) ;
Hasegawa; Masakatsu; (Nagoya-shi, JP) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
30767803 |
Appl. No.: |
10/521947 |
Filed: |
July 22, 2003 |
PCT Filed: |
July 22, 2003 |
PCT NO: |
PCT/JP03/09272 |
371 Date: |
February 24, 2005 |
Current U.S.
Class: |
424/93.45 ;
514/59 |
Current CPC
Class: |
A61P 1/00 20180101; A61P
37/00 20180101; C12N 1/38 20130101; A61P 1/16 20180101; A61P 15/00
20180101; A23K 20/163 20160501; A23V 2002/00 20130101; A61P 13/02
20180101; A61P 37/06 20180101; A23V 2002/00 20130101; A61P 37/08
20180101; A61P 3/12 20180101; A23V 2002/00 20130101; A61P 7/00
20180101; A61P 29/00 20180101; A23L 29/273 20160801; A61P 9/12
20180101; A61P 37/04 20180101; A61P 35/00 20180101; A61P 43/00
20180101; A23L 33/135 20160801; A61P 1/04 20180101; A61P 31/18
20180101; A61P 37/02 20180101; A61P 3/06 20180101; A61P 17/02
20180101; A61P 39/00 20180101; A61P 1/14 20180101; A61P 1/10
20180101; C12N 1/20 20130101; A61P 25/00 20180101; A61P 3/00
20180101; A61P 39/06 20180101; A61P 3/10 20180101; A61P 31/00
20180101; A23V 2200/3202 20130101; A23V 2200/3204 20130101; A23V
2250/5042 20130101; A23V 2250/5042 20130101 |
Class at
Publication: |
424/093.45 ;
514/059 |
International
Class: |
A61K 35/74 20060101
A61K035/74; A61K 31/721 20060101 A61K031/721 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 22, 2002 |
JP |
2002-212336 |
Claims
1. A composition for promoting bacterial proliferation for
selectively proliferating Lactobacillus casei subsp. casei,
comprising a dextran.
2. The composition for promoting bacterial proliferation according
to claim 1, further comprising Lactobacillus casei subsp.
casei.
3. The composition for promoting bacterial proliferation according
to claim 1, wherein the dextran has a molecular weight of 2,000 to
40,000,000.
4. A pharmaceutical composition comprising the composition for
promoting bacterial proliferation according to claim 2.
5. A health food comprising the composition for promoting bacterial
proliferation according to claim 2.
6. A feed comprising the composition for promoting bacterial
proliferation according to claim 2.
Description
TECHNICAL FIELD
[0001] The present invention relates to: a dextran-comprising
composition for promoting bacterial proliferation for selectively
proliferating Lactobacillus casei subsp. casei (hereinafter,
abbreviated as "L. casei subsp. casei") which is one species of
lactic acid bacteria having a variety of biological activities; and
the composition for promoting bacterial proliferation, which
further comprises L. casei subsp. casei. More specifically, the
present invention relates to: a pharmaceutical composition for
enhancing biological activities, which comprises the composition
for promoting bacterial proliferation; and a health food and feed
for enhancing biological activities, which comprise the composition
for promoting bacterial proliferation.
BACKGROUND ART
[0002] Recently, ingestion of "microorganism formulations" which
are referred to as probiotics and consist of useful microorganisms
such as living lactic acid bacteria for the purpose of effect of
controlling intestinal function, prevention of infectious diseases,
immunostimulation, or the like has been attracting attraction.
However, there is a doubt whether the useful microorganisms can
grow and colonize in the intestine of a human being, animal, or the
like. If the useful microorganisms cannot grow and colonize in the
intestine, expected biological activities of the useful
microorganisms cannot be obtained permanently. In order to solve
such a problem, the fact is that supplying the microorganism
formulation at all times is proposed and performed. On the other
hand, oligosaccharides or the like which can be utilized by useful
microorganisms in probiotics are referred to as prebiotics, and
ones prepared by combining prebiotics and probiotics are referred
to as symbiotics. Those are also administered to a human being,
animal, or the like for the aforementioned purpose. However, when
the prebiotics are utilized by great many enteric bacteria other
than intended bacteria, those bacteria scramble for the prebiotics
in the intestine, so it is difficult to grow and colonize intended
useful microorganisms selectively.
DISCLOSURE OF THE INVENTION
[0003] An object of the present invention is, after ingestion of
useful microorganisms referred to as probiotics to a human being,
animal, or the like, to sustain a variety of biological activities
originated from the useful microorganisms in a living body by
selectively growing-proliferating and colonizing the microorganisms
in the intestine or by selectively growing-proliferating useful
indigenous microorganisms in the intestine without supplying those
useful microorganisms at all times.
[0004] The inventors of the present invention have found out
through an in vitro test that, among many useful microorganisms, L.
casei subsp. casei is a species of bacteria capable of
growing-proliferating by utilizing a dextran. L. casei subsp. casei
is classified and named in Approved Lists (1980). The bacterium is
Gram positive, indigenous to the intestine of a human being,
animal, or the like, and a facultative anaerobic bacillus. The
bacterium forms the intestinal bacterial flora together with other
bacteria.
[0005] Thus, at first, it was confirmed whether or not the
bacterium itself has biological activities, for example, an
immunostimulating effect in the case of subcutaneous immunization.
As a result, the bacterium has been found to enhance and maintain
humoral immunity and cellular immunity. Next, L. casei subsp. casei
has been administered to a mouse only in the initial
administration, and a dextran was then orally ingested permanently,
in order to confirm whether L. casei subsp. casei can selectively
grow-proliferate and colonize in the intestine of a human being,
animal, or the like, and the biological activities of the bacterium
can be sustained or not by oral ingestion of a dextran. As a
result, the inventors of the present invention have found through
an in vivo test that the bacterium grows-proliferates and colonizes
in the intestine by utilizing the dextran, and the biological
activities of the bacterium such as humoral immunity and cellular
immunity are enhanced and maintained.
[0006] On the other hand, a dextran itself is also known to have a
variety of biological activities, and it has been confirmed that,
in the case of administration of a dextran together with L. casei
subsp. casei, a higher effect is maintained than that in the case
of administration of only a dextran, and a synergistic effect is
large.
[0007] The present invention has been accomplished as described
above. The gist of the present invention is as follows.
(1) A composition for promoting bacterial proliferation for
selectively proliferating Lactobacillus casei subsp. casei,
comprising a dextran.
(2) The composition for promoting bacterial proliferation according
to (1), further comprising Lactobacillus casei subsp. casei.
(3) The composition for promoting bacterial proliferation according
to (1), wherein the dextran has a molecular weight of 2,000 to
40,000,000.
(4) A pharmaceutical composition comprising the composition for
promoting bacterial proliferation according to any one of (1) to
(3).
(5) A health food comprising the composition for promoting
bacterial proliferation according to any one of (1) to (3).
(6) A feed comprising the composition for promoting bacterial
proliferation according to any one of (1) to (3).
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 is a drawing showing an effect of a dextran on the
bacterial number of L. casei subsp. casei in mouse feces.
[0009] FIG. 2 is a drawing showing the results of enhancing humoral
immunity by a dextran in the case of orally administering an
antigen.
[0010] FIG. 3 is a drawing showing the results of enhancing
cellular immunity by a dextran in the case of orally administering
the antigen.
[0011] FIG. 4 is a drawing showing the results of enhancing humoral
immunity by a dextran in the case of subcutaneously administering
the antigen.
[0012] FIG. 5 is a drawing showing the results of enhancing
cellular immunity by a dextran in the case of subcutaneously
administering the antigen.
BEST MODE FOR CARRYING OUT THE INVENTION
[0013] The composition of the present invention is a
dextran-comprising composition for promoting bacterial
proliferation for selectively proliferating L. casei subsp. casei
and the composition for promoting bacterial proliferation, which
further comprises L. casei subsp. casei. Dextrans to be used in the
composition of the present invention can be synthesized, prepared,
or the like according to a general method that is well known to a
person skilled in the art. Moreover, culture of L. casei subsp.
casei, methods of preparing and drying of bacterial cells, gene
recombination, and the like can be performed according to a general
method that is well known to a person skilled in the art.
(Description of Dextran to be Used in the Present Invention)
[0014] A dextran is a polysaccharide which consists of glucose,
which are mainly bonded with an .alpha.-1,6 bond. The dextrans to
be used in the present invention may be commercially available
dextrans or dextrans obtained by culturing bacteria selected from
the group consisting of genus Leuconostoc and genus Streptococcus,
for example, Leuconostoc mesenteroides, under a general condition,
for example, in a sucrose-containing enriched medium.
(Molecular Weight of Dextran to be Used in the Present
Invention)
[0015] Each of the molecular weights of dextrans to be used in the
present invention is 2,000 to 40,000,000, preferably 4,000 to
5,000,000, more preferably 4,000 to 2,000,000. Note that a dextran
having a molecular weight of less than 2,000 is not preferable
because bacteria other than L. casei subsp. casei may proliferate.
Examples of the dextrans to be used in the present invention
include a dextran obtained by culturing the aforementioned bacteria
without any treatment, and a dextran having an appropriate
molecular weight obtained by further partial hydrolysis treatment
of such a dextran.
(L. casei subsp. casei to be used in the Present Invention)
[0016] In the present invention, L. casei subsp. casei can be used
in the form of viable cells, dried cells capable of
growing-proliferating, or the like. As L. casei subsp. casei to be
used in the present invention, there can be used a recombinant of
L. casei subsp. casei which has at least a utilizing ability for a
dextran in the composition for promoting bacterial proliferation of
the present invention, in addition to a wild type. There can also
be used a recombinant obtained by transducing a gene which
participates in a dextran-utilizing ability originated from L.
casei subsp. casei to another bacterium. Preferably, the
recombinant is a novel bacterium in which a new biological activity
is expressed by gene recombination of L. casei subsp. casei or
another novel bacterium obtained by transducing a gene which
participates in a dextran-utilizing ability originated from L.
casei subsp. casei to another bacterium.
[0017] The composition of the present invention is a
dextran-comprising composition for promoting bacterial
proliferation for selectively proliferating L. casei subsp. casei
and the composition for promoting bacterial proliferation which
further comprises L. casei subsp. casei. The composition of the
present invention can be prepared, administered, or the like
according to a general method which is well known to a person
skilled in the art except that the composition comprises a dextran
or further comprises L. casei subsp. casei.
(Dosage Form of Composition of the Present Invention)
[0018] The composition of the present invention may be a dextran
only or a mixture of a dextran and L. casei subsp. casei.
Preferably, the composition of the present invention can be
provided as a composition in the form of solid, semi-solid, or
liquid containing a dextran in combination with a diluent, carrier,
or the like which is pharmaceutically well known and
pharmaceutically acceptable, or a composition in the form of solid,
semi-solid, or liquid containing a dextran and L. casei subsp.
casei. Note that the composition of the present invention includes:
a form in which the dextran of the present invention and L. casei
subsp. casei have already been combined; and a form in which the
dextran of the present invention and L. casei subsp. casei have
been separately processed, and are combined when the composition is
used. The amount of a diluent, carrier, or the like in the
composition of the present invention is appropriately varied in
accordance with a purpose or usage, and is not particularly
limited. In general, the amount is, for example, about 0.1 to about
99.9% by weight, preferably about 1 to about 99% by weight, more
preferably about 5 to about 95% by weight based on the weight of
the composition.
[0019] Note that, when a dextran and L. casei subsp. casei are
simultaneously present in the form of liquid, the dextran content
reduces gradually due to bacterial utilization of the dextran, so
simultaneous presence of them for a long time is preferably
avoided. So, it is desirable that those be separately processed and
packed, and the resultant products be mixed when they are ingested,
or be separately ingested.
[0020] The pharmaceutical composition of the present invention may
be the composition of the present invention itself without other
components. However, preferably, the composition may be one
prepared in combination with a diluent, carrier, or the like, which
is pharmaceutically well known and pharmaceutically acceptable,
into a dosage form such as a soft capsule, hard capsule, granule,
pill, powder, tablet, syrup, troche, or elixir. A method of
preparing the composition into such a dosage form is well known to
a person skilled in the art. When the composition is prepared into
such a dosage form, a diluent, carrier, or the like which is
suitable to each dosage form, pharmaceutically well known, and
pharmaceutically acceptable may further be contained.
[0021] The pharmaceutical composition of the present invention can
be used as a drug which aims at sustaining a variety of biological
activities originated from L. casei subsp. casei in a living body
by: selectively growing-proliferating and colonizing L. casei
subsp. casei in the intestine of a human being, animal, or the
like; or selective growth-proliferation of L. casei subsp. casei in
the intestine, which is indigenous to the intestine.
[0022] When the pharmaceutical composition of the present invention
is prepared, the addition amount of the composition of the present
invention may be appropriately determined with reference to the
description which relates to the dose and dosage cycle of the
composition of the present invention described below.
[0023] The health food of the present invention is one containing
the composition of the present invention and a composition such as
a food or beverage. Note that the health food of the present
invention includes: a form in which the composition of the present
invention and a composition such as a food or beverage have already
been combined; and a form in which the composition of the present
invention and a composition such as a food or beverage have been
separately processed, and are combined when the composition is
ingested. Examples of such a food or beverage include:
confectioneries such as candy, chocolate, and biscuit; breads;
noodles; beverages in the form of powder, liquid, and the like;
dairy products such as yogurt. In particular, in the case of
yogurt, yogurt can be produced using such L. casei subsp. casei,
and it is desirable that a dextran be mixed when they are ingested
or be individually ingested.
[0024] The feed of the present invention contains the composition
of the present invention and formula feed of livestocks, fowls,
fishes, pets, or the like. Note that the feed of the present
invention includes: a form in which the composition of the present
invention and a feed composition have already been combined; and a
form in which the composition of the present invention and a feed
composition have been separately processed, and are combined when
the composition is ingested.
[0025] The health food or feed of the present invention can be used
for sustaining a variety of biological activities originated from
L. casei subsp. casei in a living body by: selective
growth-proliferation and colonization of L. casei subsp. casei in
the intestine of a human being, animal, or the like; or selective
growth-proliferation of L. casei subsp. casei in the intestine,
which is indigenous to the intestine.
[0026] When the health food or feed of the present invention is
prepared, the addition amount of the composition of the present
invention may be appropriately determined with reference to the
description which relates to the dose and dosage cycle of the
composition of the present invention described below.
(Dose and Dosage Cycle of Composition of the Present Invention)
[0027] The dose of the composition of the present invention is, in
terms of the dose of a dextran in the composition, 0.01 mg to 2
g/kg weightday, preferably 0.1 mg to 1 g/kg weightday, more
preferably 1 mg to 100 mg/kg weightday, and it is preferable that
at least a composition comprising the dextran of the present
invention be generally administered every day. Note that L. casei
subsp. casei is originally a bacterium which is indigenous to the
intestine of a human being, animal, or the like, and in order to
induce the reliable effects of the biological activities of L.
casei subsp. casei, viable cells or dried cells capable of
growing-proliferating are administered at least once, preferably at
an appropriate interval. In addition, in order to induce more
reliable effects, those cells may be administered every day.
(Applicable Target of Composition of the Present Invention)
[0028] The composition of the present invention can be targeted for
all living beings such as mammals including a human being, aves,
reptiles, fishes, crustaceans, and insects.
(Biological Activity Expected to be Imparted and/or Improved by
Composition of the Present Invention)
[0029] The following effects are expected: in addition to an
immunostimulating effect, generally mentioned biological activities
of lactic acid bacteria such as antitumor, antihypertension,
antioxidation, antiulcer, anti-HIV, improvement of lipid
metabolism, suppression of recurrent malignant tumor,
antiinflammation, prevention and suppression of autoimmune disease,
decrease of serum cholesterol, decrease of blood glucose,
suppression of producing IgE antibody, antiallergy, promotion of
producing interleukin 12, promotion of absorbing mineral,
enrichment of mineral, regulation of alcohol absorption and
metabolism, prevention and treatment of urinary tract infection,
suppression of producing harmful matter, control of intestinal
function, prevention of constipation, beauty, treatment and
prevention of hyperammonemia and hepatic encephalopathy, or the
like, prevention of infection, suppression of feces malodor,
decrease of intestinal pH of livestocks, fowls, improvement of
growth, promotion of fattening, and help of egg laying.
EXAMPLES
[0030] Hereinafter, the present invention is described in more
detail by way of examples, but the present invention is not limited
thereto.
Example 1
Dextran Preparation Method Using Sucrose as Principal Material
[0031] Leuconostoc mesenteroides, which is one species of lactic
acid bacteria, was inoculated in a medium containing sucrose as a
principal material (sucrose-containing enriched medium), and
cultured anaerobically at 25.degree. C. for 24 hours. Subsequently,
methanol was added to the culture supernatant, and the resultant
precipitates were dissolved in distilled water. After the
insolubles had been removed, methanol was added thereto again, and
the resultant precipitates were redissolved in distilled water. The
mixture was subjected to spray drying, to thereby yield a dextran.
The dextran was further partially hydrolyzed, and the resultant
products were subjected to methanol fractionation and spray drying,
to thereby yield dextrans having a variety of molecular
weights.
Example 2
Test of Dextran-Utilizing Ability of Enteric Bacteria
[0032] A variety of related enteric bacteria were inoculated in a
PYF medium (Peptone yeast extract Fildes solution broth) to which a
dextran having a molecular weight (number average) of 10,000 (in
the following examples, a dextran having such a molecular weight
was used unless otherwise defined) was added to 0.5%, and the
dextran-utilizing ability was determined. As a result, two strains
of L. casei subsp. casei (JCM 1134 and JCM 8129) were confirmed to
have the dextran-utilizing ability. However, no dextran-utilizing
ability was observed for other lactic acid bacteria such as
bacteria of the genus Lactobacillus or Bifidobacterium, and other
related enteric bacteria. (See Table 1) TABLE-US-00001 TABLE 1-1
Dextran-utilizing ability of related enteric bacteria Bacterial
Dextran- strain utilizing Bacterial name name ability Lactobacillus
casei subsp. casei JCM 1134 + casei subsp. casei JCM 8129 +
acidophilus #527 - acidophilus JCM 1132 - agilis JCM 1187 -
amylovorus JCM 1126 - aviarius subsp. aviarius JCM 5666 - brevis
JCM 1059 - buchneri JCM 1115 - casei subsp. alactosus JCM 1133 -
casei subsp. fusiforxnis JCM 1177 - casei subsp. plantarurn JCM
8136 - casei subsp. pseudoplantarum JCM 1161 - casei subsp.
rhamnosus JCM 1136 - crispatus JCM 1185 - curvatus JCM 1096 -
deibrueckii subsp. JCM 1002 - bulgaricus - deibrueckli subsp.
lactis JCM 1248 - ferrnentum JCM 1173 - gallinarum JCM 2011 -
gasseri JCM 1131 - johnsonii JCM 2012 - oris JCM 11028 - paracasei
subsp. paracasei JCM 1053 - plantarum IFO 3070 - reuteri JCM 1112 -
salivarius subsp. salicinius JCM 1150 - salivarius subsp.
salivarius JCM 1231 - Clostridium butyricum JCM 1391 - perfringens
JCM 1290 - Klebsiella pneumoniae subsp. pneumoniae JCM 1662 -
Fusobacterium varium JCM 3722 - Enterococcus faecalis JCM 5803 -
faecium JCM 5804 - Eubacterium limosum JCM 6421 - Propionibacterium
acnes JCM 6425 - Bacteroides fragills ATCC 25285 - Escherichia coil
IFO 3972 - Bacillus subtilis subsp. subtilis IFO 3134 -
[0033] TABLE-US-00002 TABLE 1-2 Dextran-utilizing ability of
related enteric bacteria Bacterial Dextran- strain utilizing
Bacterial name name ability Bifidobacterium adolescentis JCM 1275 -
angulatum JCM 7096 - animalis JCM 1190 - asteroides JCM 8230 -
bifidum JCM 1209 - boum JCM 1211 - breve JCM 1192 - catenulatum JCM
1194 - choerinum JCM 1212 - coryneforme JCM 5819 - cuniculi JCM
1213 - dentium JCM 1195 - gallicum JCM 8224 - gallinarum JCM 6291 -
indicum JCM 1302 - infantis JCM 1222 - longum JCM 1217 - magnum JCM
1218 - merycicum JCM 8219 - minimum JCM 5821 - pseudocatenulatum
JCM 1200 - pseudolongum subsp. JCM 5820 - globosum pseudolongum
subsp. JCM 1205 - pseudolongum pullorum JCM 1214 - ruminantium JCM
8222 - saeculare JCM 8223 - subtile JCM 5822 - suis JCM 1269 -
thermophilum JCM 1207 -
[0034] Moreover, using two strains of L. casei subsp. casei, a
difference of the utilizing abilities according to dextrans having
different molecular weights was determined. As a result, for all
tested dextrans, the dextran-utilizing abilities were confirmed.
However, a dextran having a smaller molecular weight has a tendency
to be more utilized. (See Table 2) TABLE-US-00003 TABLE 2
Difference of utilizing ability according to dextran molecular
weight Dextran L. casei subsp. case: Molecular weight JCM 1134 JCM
8129 2,000 +++ +++ 4,000 +++ +++ 7,000 ++ ++ 10,000 ++ ++ 14,000 ++
++ 40,000 ++ ++ 200,000 + + 2,000,000 + +
Example 3
Effect of dextran on bacterial number of L. casei subsp. casei in
Mouse Feces
[0035] 12 eight-week-old BALB/c male mice were divided into two
groups of six each. Those groups were referred to as (1) control
group and (2) dextran group. Over the test period from the
beginning of the test, a conventional feed was given to (1), while
a dextran-added conventional feed (75 mg dextran/kg conventional
feed) was given to (2). Over the period of three days from the
beginning of the test, L. casei subsp. casei cells were orally
inoculated to both groups (10.sup.6 cells/mouse). On 28th day, each
mouse was dissected, and the feces were collected from the large
intestine. The feces were serially diluted with phosphate buffered
saline (PBS), and the resultant mixture was smeared on a
dextran-added LB medium, followed by culture at 37.degree. C. for
48 hours. After colonies had been stained by Gram staining and
observed, the bacterial number of L. casei subsp. casei in feces
was counted. As a result, the bacterial number of L. casei subsp.
casei in the dextran group was significantly larger than that in
the control group. (See FIG. 1)
Example 4
Enhancement of Humoral Immunity by Dextran in Oral Administration
of Antigen
[0036] 30 eight-week-old BALB/c male mice were divided into five
groups of six each. Those groups were referred to as (1) control
group, (2) BSA (bovine serum albumin) group, (3) BSA+L. casei
subsp. casei group, (4) BSA+dextran group, and (5) BSA+L. casei
subsp. casei+dextran group. Over the test period from the beginning
of the test, a conventional feed was given to each of (1), (2), and
(3), while a dextran-added conventional feed (75 mg dextran/kg
conventional feed) was given to each of (4) and (5). Over the
period of three days from the beginning of the test, L. casei
subsp. casei cells were orally inoculated to (3) and (5) (10.sup.6
cells/mouse). On third and forth days, BSA was dissolved in PBS at
a concentration of 10 mg/ml, and the solution was orally
administered (1 mg (100 .mu.l)/mouse) to (2) to (5). On 33rd and
34th days, in a manner similar to that on the third and forth days,
BSA was orally administered (1 mg/mouse). Five days after the
administration, the blood was drawn from the suborbital vein, and
the anti-BSA antibody titer was determined by ELISA. As a result,
in (5) BSA+L. casei subsp. casei+dextran group, the specific
antibody titer was significantly high. (See FIG. 2)
Example 5
Enhancement of Cellular Immunity by Dextran in Oral Administration
of Antigen
[0037] 30 eight-week-old BALB/c male mice were divided into five
groups of six each. Those groups were referred to as (1) control
group, (2) BSA group, (3) BSA+L. casei subsp. casei group, (4)
BSA+dextran group, and (5) BSA+L. casei subsp. casei+dextran group.
Over the test period from the beginning of the test, a conventional
feed was given to each of (1), (2), and (3), while a dextran-added
conventional feed (75 mg dextran/kg conventional feed) was given to
each of (4) and (5). Over the period of three days from the
beginning of the test, L. casei subsp. casei cells were orally
inoculated to (3) and (5) (10.sup.6 cells/mouse). On third and
forth days, BSA was dissolved in PBS at a concentration of 10
mg/ml, and the solution was orally administered (1 mg (100
.mu.l)/mouse) to (2) to (5). On 33rd and 34th days, in a manner
similar to that on the third and forth days, BSA was orally
administered (1 mg/mouse). Five days after the administration, BSA
was dissolved in PBS, and the solution was intradermally
administered to the earlobe, followed by measuring the thickness of
the mouse earlobe after 24, 48, and 72 hours. As a result, in (5)
BSA+L. casei subsp. casei+dextran group, the thickness of the
earlobe significantly increased. (See FIG. 3)
Example 6
Enhancement of Humoral Immunity by Dextran in Subcutaneous
Administration of Antigen
[0038] 30 eight-week-old BALB/c male mice were divided into five
groups of six each. Those groups were referred to as (1) control
group, (2) BSA group, (3) BSA+L. casei subsp. casei group, (4)
BSA+dextran group, and (5) BSA+L. casei subsp. casei+dextran group.
Over the test period from the beginning of the test, a conventional
feed was given to each of (1), (2), and (3), while a dextran-added
conventional feed (75 mg dextran/kg conventional feed) and the
resultant feed was given to each of (4) and (5). Over the period of
three days from the beginning of the test, L. casei subsp. casei
bacteria cells were orally inoculated to (3) and (5) (10.sup.6
cells/mouse). On a third day, using FIA (Freund's incomplete
adjuvant), PBS (phosphate buffered saline) (PBS+FIA) or BSA (5
mg/ml) (BSA+FIA) was emulsified at a ratio of 1:1, and the emulsion
of PBS+FIA was subcutaneously administered to the footpads of the
(1) and the emulsion of BSA+FIA was subcutaneously administered to
those of the (2) to (5), respectively (200 .mu.l/mouse). On a 35th
day, booster was performed in a manner similar to that on the third
day. Five days after the booster, the blood was drawn from the
suborbital vein, and the anti-BSA antibody titer was determined by
ELISA. As a result, in (5) BSA+L. casei subsp. casei+dextran group,
the specific antibody titer was significantly high. (See FIG.
4)
Example 7
Enhancement of Cellular Immunity by Dextran in Subcutaneous
Administration of Antigen
[0039] 30 eight-week-old BALB/c male mice were divided into five
groups of six each. Those groups were referred to as (1) control
group, (2) BSA group, (3) BSA+L. casei subsp. casei group, (4)
BSA+dextran group, and (5) BSA+L. casei subsp. casei+dextran group.
Over the test period from the beginning of the test, a conventional
feed was given to each of (1), (2), and (3), while a dextran-added
conventional feed (75 mg dextran/kg conventional feed) was given to
each of (4) and (5). Over the period of three days from the
beginning of the test, L. casei subsp. casei bacteria cells were
orally inoculated to (3) and (5) (10.sup.6 cells/mouse). On a third
day, using FIA, PBS (PBS+FIA) or BSA (5 mg/ml) (BSA+FIA) was
emulsified at a ratio of 1:1, and the emulsion of PBS+FIA was
subcutaneously administered to the footpads of the (1) and the
emulsion of BSA+FIA was subcutaneously administered to those of the
(2) to (5), respectively (200 .mu.l/mouse). Ona35th day, booster
was performed in a manner similar to that on the third day. Five
days after the booster, BSA was dissolved in PBS, and the solution
was intradermally administered to the mouse earlobe, followed by
measuring the thickness of the earlobe after 24, 48, and 72 hours.
As a result, in (5) BSA+L. casei subsp. casei+dextran group, the
thickness of the earlobe significantly increased. (See FIG. 5)
INDUSTRIAL APPLICABILITY
[0040] According to the present invention, a variety of biological
activities originated from L. casei subsp. casei can be sustained
in a living body by selectively growing-proliferating and
colonizing L. casei subsp. casei in the intestine of a human being,
animal, or the like or by selectively growing-proliferating L.
casei subsp. casei in the intestine, without supplying L. casei
subsp. casei at all times.
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