U.S. patent application number 14/759124 was filed with the patent office on 2016-04-28 for lactic acid bacterium, drug, food or drink, and feed which contain the lactic acid bacterium.
This patent application is currently assigned to MORINAGA MILK INDUSTRY CO., LTD.. The applicant listed for this patent is MORINAGA MILK INDUSTRY CO., LTD.. Invention is credited to Noriyuki Iwabuchi, Toshitaka Odamaki, Yohei Sato, Kanetada Shimizu, Tomohiro Tanaka.
Application Number | 20160114024 14/759124 |
Document ID | / |
Family ID | 52279648 |
Filed Date | 2016-04-28 |
United States Patent
Application |
20160114024 |
Kind Code |
A1 |
Tanaka; Tomohiro ; et
al. |
April 28, 2016 |
LACTIC ACID BACTERIUM, DRUG, FOOD OR DRINK, AND FEED WHICH CONTAIN
THE LACTIC ACID BACTERIUM
Abstract
The Lactobacillus paracasei MCC1849 (NITE BP-01633) strain,
which has a high IL-12 production-promoting action, is used as an
ingredient of a drug, food or drink, or feed used for promotion of
IL-12 production, immunostimulation, antivirus, or the like.
Inventors: |
Tanaka; Tomohiro; (Zama-shi,
JP) ; Iwabuchi; Noriyuki; (Zama-shi, JP) ;
Sato; Yohei; (Zama-shi, JP) ; Shimizu; Kanetada;
(Zama-shi, JP) ; Odamaki; Toshitaka; (Zama-shi,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MORINAGA MILK INDUSTRY CO., LTD. |
Tokyo |
|
JP |
|
|
Assignee: |
MORINAGA MILK INDUSTRY CO.,
LTD.
Tokyo
JP
|
Family ID: |
52279648 |
Appl. No.: |
14/759124 |
Filed: |
March 18, 2014 |
PCT Filed: |
March 18, 2014 |
PCT NO: |
PCT/JP2014/057319 |
371 Date: |
July 2, 2015 |
Current U.S.
Class: |
435/252.9 |
Current CPC
Class: |
A23L 2/52 20130101; A23K
10/16 20160501; C12R 1/225 20130101; A23V 2002/00 20130101; A61K
35/74 20130101; A61K 35/747 20130101; A61K 39/145 20130101; A61K
2039/523 20130101; A23Y 2220/63 20130101; A23K 10/18 20160501; A23K
10/12 20160501; A23L 33/135 20160801; A61P 31/16 20180101; A61P
31/12 20180101; A61K 2035/11 20130101; A61P 37/04 20180101 |
International
Class: |
A61K 39/145 20060101
A61K039/145; A23L 2/52 20060101 A23L002/52; A61K 35/74 20060101
A61K035/74 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 12, 2013 |
JP |
2013-146319 |
Claims
1. (canceled)
2. A drug which contains Lactobacillus paracasei MCC1849 (NITE
BP-01633).
3. The drug according to claim 2, which is for
immunostimulation.
4. The drug according to claim 2, which is for antivirus.
5. The drug according to claim 4, wherein the virus is an influenza
virus.
6. A food or drink which contains Lactobacillus paracasei MCC1849
(NITE BP-01633).
7. A feed which contains Lactobacillus paracasei MCC1849 (NITE
BP-01633).
8. An IL-12 production-promoting agent which contains Lactobacillus
paracasei MCC1849 (NITE BP-01633).
9. The IL-12 production-promoting agent according to claim 8, which
is in the form of food or drink.
Description
TECHNICAL FIELD
[0001] The present invention relates to a novel lactic acid
bacterium belonging to Lactobacillus paracasei, a drug, food or
drink, and feed which contain the lactic acid bacterium.
BACKGROUND ART
[0002] It has been reported that some lactic acid bacteria show
prophylactic action and defensive action against various infectious
diseases (Non-patent document 1). It has also been reported that
these actions of lactic acid bacteria are based on activation of
cell-mediated host immunity, promotion of IgA secretion from
mucosae, such as those of intestinal tract and respiratory organs
(Non-patent document 1), and so forth. For example, it has been
reported that lactic acid bacteria belonging to Lactobacillus casei
induce production of cytokines such as IL-12 (interleukin-12) and
IFN-.gamma. (interferon-.gamma.) by immunocompetent cells of hosts
to activate cell-mediated host immunity and thereby defend the
hosts from infection by influenza virus, and so forth (Non-patent
documents 2 to 4).
[0003] IL-12 and IFN-.gamma. are cytokines having an action of
inducing differentiation of naive helper T cells into type 1 helper
T cells (Th1), an action of activating natural killer cells (NK
cells), and an action of promoting phagocytosis of cells such as
macrophages, and are involved in defensive actions against
infections by viruses or bacteria, and antitumor effect of hosts.
Therefore, in order to acquire high prophylactic action and
defensive action of lactic acid bacteria against infectious
diseases, it is important to use a lactic acid bacterium having a
potent IL-12 production-inducing ability. As such lactic acid
bacteria, there is known the Lactobacillus paracasei FERM BP-11313
strain, which shows high survivability under acidic conditions, and
superior IL-12 production-inducing ability (Patent document 1, in
this reference, this strain is also referred to as MCC1375
strain).
[0004] There has also been suggested involvement of cell walls of
lactic acid bacteria in the induction of IL-12 production by lactic
acid bacteria (Patent document 2), and it has been reported that if
lactic acid bacteria are treated with a cell wall-digesting enzyme
(N-acetyl muramidase), the IL-12 production-inducing ability is
spoiled (Non-patent document 5). It has been also suggested that
RNAs contained in lactic acid bacteria participate in the induction
of IL-12 production by lactic acid bacteria, and it has been
reported that if dead cells of lactic acid bacteria killed by
heating are treated with RNase, the IL-12 production-inducing
ability is markedly spoiled (Non-patent document 6). There have so
far been also provided immunostimulants utilizing RNA of lactic
acid bacteria itself (Patent documents 3 and 4).
[0005] Since human bodies contain cell wall-digesting enzymes such
as lysozyme having the N-acetyl muramidase activity against
bacteria, it is considered that lactic acid bacteria taken into the
bodies are influenced by those enzymes. Further, RNases exist
everywhere in the environment, and they are extremely stable
against heat. Therefore, they easily contaminate products, and they
are not inactivated by sterilization or the like, and may remain in
the products. Moreover, since RNases also exist in human bodies,
for example, in saliva and digestive juices, it is considered that
lactic acid bacteria are also influenced by RNases after they are
orally taken into the bodies.
[0006] Even if lactic acid bacteria inherently have high
IL-12-inducing ability, they may not maintain and exhibit
sufficient IL-12-inducing ability in living bodies, because they
are influenced by the cell wall-digesting enzymes (N-acetyl
muramidase) and RNases existing in saliva or digestive juices. It
is considered that secretion amounts of these cell wall-digesting
enzymes and RNases differ among individuals, and it is strongly
considered that such difference possibly provides differences of
the effect observed among individuals.
PRIOR ART REFERENCES
Patent Documents
[0007] Patent document 1: International Patent Publication
WO2012/133827 [0008] Patent document 2: Japanese Patent Laid-open
(Kokai) No. 2009-155221 [0009] Patent document 3: International
Patent Publication WO2009/005124 [0010] Patent document 4:
International Patent Publication WO2011/027829
Non-Patent Documents
[0010] [0011] Non-patent document 1: Delcenserie, V. et al., Curr.
Issues Mol. Biol. (2008) 10:37-54 [0012] Non-patent document 2:
Hori T. et al., Clin. Diagn. Lab. Immunol. (2002) 9:105-108 [0013]
Non-patent document 3: Takeda, K. et al., Clin. Exp. Immunol.
(2006) 146:109-115 [0014] Non-patent document 4: Ogawa, T., Clin.
Exp. Immunol. (2006) 143:103-109 [0015] Non-patent document 5:
Shida, K. et al., J. Dairy Sci. (2006) 89:3306-3317 [0016]
Non-patent document 6: Inoue, R. et al., FEMS Immnol. Med.
Microbiol. (2011) 61:94-102
SUMMARY OF THE INVENTION
Object to be Achieved by the Invention
[0017] An object of the present invention is to provide a lactic
acid bacterium useful for prophylaxis and defense against various
infections, i.e., a lactic acid bacterium showing high IL-12
production-promoting action or immunostimulation action, which
actions are preferably not easily reduced in human bodies etc.
Means for Achieving the Object
[0018] In order to achieve the aforementioned object, the inventors
of the present invention assiduously searched for such target
lactic acid bacteria, and found a novel strain of Lactobacillus
paracasei having a high IL-12 production-promoting action. Thus,
they accomplished the present invention.
[0019] That is, the present invention provides the Lactobacillus
paracasei MCC1849 (NITE BP-01633) strain.
[0020] The present invention also provides a drug which contains
the strain.
[0021] In a preferred embodiment of the aforementioned drug, the
drug is for immunostimulation.
[0022] In a preferred embodiment of the aforementioned drug, the
drug is for antivirus.
[0023] In a preferred embodiment of the aforementioned drug for
antivirus, the drug is for anti-influenza virus.
[0024] The present invention also provides a food or drink which
contains the strain.
[0025] The present invention also provides a feed which contains
the strain.
[0026] The present invention also provides an IL-12
production-promoting agent which contains the strain.
[0027] In a preferred embodiment of the aforementioned IL-12
production-promoting agent, the agent is in the form of food or
drink.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1 shows amounts of IL-12 produced from mouse spleen
cells in the presence of lactic acid bacteria. The lactic acid
bacterium strains are indicated on the horizontal axis, and the
vertical axis indicates average and standard deviation (S.D.) of
the IL-12 production amount.
[0029] FIG. 2 shows influenza virus infection-preventing action of
the MCC1849 strain.
[0030] FIG. 2, (a) shows symptom scores determined after infection.
The symbol * indicates presence of a statistically significant
difference between the two groups on the observation day.
[0031] FIG. 2 (b) shows virus concentrations in the lungs. The
symbol * indicates presence of a statistically significant
difference between the two groups. The statistical analysis was
performed by using the Dunnett test.
EMBODIMENTS FOR CARRYING OUT THE INVENTION
[0032] Hereafter, the present invention will be explained in
detail.
[0033] The present invention relates to the Lactobacillus paracasei
MCC1849 (NITE BP-01633) strain, which is a novel strain of a lactic
acid bacterium belonging to Lactobacillus paracasei. This strain is
henceforth also referred to as the "lactic acid bacterium of the
present invention", or the "strain of the present invention", or
simply as the MCC1849 strain.
[0034] The lactic acid bacterium of the present invention was
isolated from human feces as an isolation source. The
bacteriological characteristics of this strain will be shown in
Example 1 described later. This strain was deposited on Jun. 6,
2013 at the independent administrative agency, National Institute
of Technology and Evaluation, NITE Patent Microorganisms Depositary
(#122, 2-5-8, Kazusakamatari, Kisarazu-shi, Chiba, 292-0818, Japan)
with an accession number of NITE P-01633, and the deposit was
converted to an international deposit under the provisions of the
Budapest Treaty on Jan. 31, 2014, and given an accession number of
NITE BP-01633.
[0035] The lactic acid bacterium of the present invention is not
limited to the aforementioned deposited strain, and it may be a
strain substantially equivalent to the deposited strain. Such a
substantially equivalent strain is a strain belonging to
Lactobacillus paracasei, exhibiting an IL-12 production-promoting
action at a level comparable to that exhibited by the deposited
strain, and preferably exhibiting reduction of the IL-12
production-promoting action at a low level comparable to that
exhibited by the deposited strain even after a treatment with a
cell wall-digesting enzyme or RNase. Moreover, the substantially
equivalent strain further shows a homology of the nucleotide
sequence of the 16S rRNA gene of 98% or more, preferably 99% or
more, more preferably 100%, with respect to the nucleotide sequence
of the 16S rRNA gene of the aforementioned deposited strain, and
preferably has the same bacteriological characteristics as those of
the aforementioned deposited strain. Furthermore, the lactic acid
bacterium of the present invention may be a strain bred from the
deposited strain or a strain substantially equivalent to the
deposited strain by a mutation treatment, gene recombination,
selection of a naturally mutated strain, or the like, so long as
the effect of the present invention is not degraded.
[0036] The MCC1849 strain has a higher IL-12 (interleukin-12)
production-promoting activity as compared with known lactic acid
bacteria. Although the IL-12 production-promoting activity of
lactic acid bacteria is generally markedly reduced by a treatment
with a cell wall-digesting enzyme such as N-acetyl muramidase or
RNase, the MCC1849 strain shows less reduction of the IL-12
production-promoting activity even after a treatment with a cell
wall-digesting enzyme or RNase. The IL-12 production-promoting
activity can be measured by the method described in the Example
section.
[0037] The MCC1849 strain can be easily proliferated by, for
example, culturing the strain. The culture method is not
particularly limited so long as the MCC1849 strain can be
proliferated, and a method usually used for culture of lactic acid
bacteria can be appropriately modified as required, and used. For
example, the culture temperature may be 25 to 50.degree. C., and is
preferably 35 to 42.degree. C. Although the culture may be
performed under aerobic conditions, or anaerobic conditions, the
culture is preferably performed under anaerobic conditions, for
example, with supplying an anaerobic gas such as carbon dioxide
gas. The culture may also be performed under microaerobic
conditions as liquid stationary culture, or the like.
[0038] The medium for culturing the MCC1849 strain is not
particularly limited, and a medium usually used for culture of
lactic acid bacteria can be appropriately modified as required, and
used. That is, as a carbon source, for example, saccharides such as
galactose, glucose, fructose, mannose, cellobiose, maltose,
lactose, sucrose, trehalose, starch, starch hydrolysate, and
blackstrap molasses can be used according to the assimilation
characteristics. As a nitrogen source, for example, ammonia,
ammonium salts such as ammonium sulfate, ammonium chloride, and
ammonium nitrate, and nitrates can be used. Further, as inorganic
salts, for example, sodium chloride, potassium chloride, potassium
phosphate, magnesium sulfate, calcium chloride, calcium nitrate,
manganese chloride, ferrous sulfate, and so forth can be used.
Furthermore, organic components such as peptone, soybean flour,
defatted soybean meal, meat extract, and yeast extract may also be
used. Further, as a ready-made medium, for example, the MRS medium
can be preferably used.
[0039] As the MCC1849 strain, culture obtained by culturing the
strain may be used as it is, or may be used after dilution or
concentration, or cells collected from the culture may also be
used. Further, so long as the effect of the present invention is
not degraded, various additional operations such as heating and
lyophilization can be performed after the culture. Such additional
operations are preferably those providing high live cell
survivability. In the drug, food or drink, and feed of the present
invention, the cells of the MCC1849 strain are preferably live
cells, but they may be dead cells.
[0040] The MCC1849 strain can be used as an IL-12
production-promoting agent. The MCC1849 strain, an IL-12
production-promoting agent containing the strain, or a composition
containing either one of them can be used for a wide rage of uses,
for example, as a drug, food or drink, and feed. For example, a
drug for promoting IL-12 production, a food or drink for promoting
IL-12 production, and a feed for promoting IL-12 production can be
provided.
[0041] Further, the IL-12 production-promoting agent of the present
invention can be used as an immunostimulant or an antiviral agent.
The virus as the target of the antiviral agent is not particularly
limited so long as the disease caused by the virus can be prevented
or treated by promotion of the IL-12 production or
immunostimulation, and examples include, for example, influenza
viruses.
[0042] The drug of the present invention is not particularly
limited so long as the MCC1849 strain is contained. As the drug of
the present invention, the MCC1849 strain per se may be used, or a
pharmaceutical preparation thereof may be prepared by adding a
physiologically acceptable liquid or solid carrier, and used.
[0043] The dosage form of the drug of the present invention is not
particularly limited, and specific examples include tablet, pill,
powder, solution, suspension, emulsion, granule, capsule, syrup,
suppository, injection, ointment, patch, eye drop, nose drop, and
so forth. Further, for preparation of a pharmaceutical preparation,
there can be used additives such as excipient usually used as a
carrier of pharmaceutical preparation, binder, disintegrating
agent, lubricant, stabilizer, flavor, diluent, surfactant, and
solvent for injection.
[0044] Although the content of the MCC1849 strain in the drug of
the present invention is appropriately determined depending on the
dosage form, direction for use, patient's age, sex, type of
disease, severity of disease, and other conditions, it is usually
preferably in the range of 1.times.10.sup.6 to 1.times.10.sup.12
cfu/g, or 1.times.10.sup.6 to 1.times.10.sup.12 cfu/ml, more
preferably in the range of 1.times.10.sup.7 to 1.times.10.sup.11
cfu/g, or 1.times.10.sup.7 to 1.times.10.sup.11 cfu/ml. When the
cells of the MCC1849 strain are dead cells, the unit of cfu/g or
cfu/ml can be replaced with number of cells/g or number of
cells/ml.
[0045] Further, so long as the effect of the present invention is
not degraded, the drug containing the MCC1849 strain and another
drug, for example, an IL-12 production-promoting agent,
immunostimulant, antiviral agent, anti-inflammatory agent,
antiulcer agent, or the like other than the MCC1849 strain can be
used in combination.
[0046] The administration time of the drug of the present invention
is not particularly limited, and it can be appropriately chosen
according to the type of therapy for the objective disease. The
drug of the present invention may be prophylactically administered,
or used for maintenance therapy. The administration route is
preferably determined according to the dosage form, patient's age,
sex, other conditions, severity of symptoms of patient, and so
forth. In any case, the drug of the present invention can be
administered once or two or more times a day, or it may be
administered once in several days or several weeks.
[0047] The IL-12 production-promoting agent of the present
invention or a composition containing it comprises cells of the
MCC1849 strain as an active ingredient, and can markedly promote
production of IL-12 in a living body. The increased IL-12 activates
the cell-mediated immunity. Therefore, the IL-12
production-promoting agent of the present invention can enhance the
cell-mediated immunity, and thus it can be used also as an
immunostimulant. The term "immunostimulation" means to stimulate
various immunoreactions, and is synonymous with "immune
activation". Further, the IL-12 production-promoting agent can also
be used as an IL-12 production-inducing agent.
[0048] The drug or IL-12 production-promoting agent of the present
invention has an IL-12 production-promoting action or an
immunostimulation action based on the foregoing action, and can be
used for a prophylactic or therapeutic treatment of a disease that
can be prevented or cured by promotion of IL-12 production, or
immunostimulation. The term "cure" also means to ameliorate a
disease.
[0049] Specific examples of the application of the drug of the
present invention include, for example, ameliorations of allergies
such as food allergies, bronchial asthma, urticaria, rhinitis,
pollinosis, and anaphylactic shock, as well as enhancement of
resistance to infectious diseases, prophylaxis of cancer,
prevention of advance of cancer, and so forth. Further, the drug or
IL-12 production-promoting agent of the present invention can be
widely used as an antitumor agent, a prophylactic or therapeutic
agent for an opportunistic infection, or a prophylactic or
therapeutic agent for an allergic disease.
[0050] The immunosuppresant of the present invention may be
independently administered, or may be used together with another
drug such as another immunosuppresant.
[0051] Another aspect of the present invention is use of the
MCC1849 strain in manufacture of an IL-12 production-promoting
agent or an immunostimulant. A further aspect of the present
invention is a method for prophylactic or therapeutic treatment of
a disease which is preventable or curable by promotion of IL-12
production, which comprises the step of administrating the MCC1849
strain or the drug of the present invention to an object of
application.
[0052] As another embodiment of the drug of the present invention,
the drug of the present invention can be used as an antiviral
agent, and in particular, it can be used as an anti-influenza virus
agent. The anti-influenza virus agent of the present invention can
be used for prophylactic or therapeutic treatment of a disease
caused by an influenza virus. The anti-influenza virus agent of the
present invention can reduce infection or proliferation of an
influenza virus in a living body. The virus as an object of the
antiviral agent is not particularly limited, so long as the disease
caused by the virus can be prevented or cured by promotion of IL-12
production or immunostimulation.
[0053] Another aspect of the present invention is use of the
MCC1849 strain in manufacture of an antiviral agent, for example,
an anti-influenza virus agent. A further another aspect of the
present invention is a method for prophylactic or therapeutic
treatment of a disease caused by a virus, for example, influenza,
which comprises the step of administrating the MCC1849 strain or
the drug of the present invention to an object of application.
[0054] The food or drink of the present invention is not
particularly limited so long as the MCC1849 strain is contained,
and examples of the food or drink include drinks such as soft
drinks, carbonated drinks, nutritious drinks, fruit-juice drinks,
and lactic acid bacteria beverages (including concentrated
undiluted solutions or powders for preparation of these drinks);
frozen deserts such as ice cream, sherbet and chipped ice;
confectioneries such as hard candy, chewing gum, candy, gum,
chocolate, tablet confectioneries, snack, biscuit, jelly, jam,
cream, and baked confectioneries; dairy products such as processed
milk, milk beverages, fermented milk, drinkable yogurt, and butter;
breads; foods for enteral feeding, liquid foods, infant formulas,
sport drinks; other functional foods, and so forth. The food may be
a supplement in the form of, for example, a tablet. When the food
is a supplement, the MCC1849 strain can be taken without being
influenced by other foods concerning daily amount of meals and
ingested calories.
[0055] The food or drink of the present invention can be produced
by adding the MCC1849 strain to a raw material of the food or
drink, and can be produced in the same manner as those for usual
foods or drinks except that the MCC1849 strain is added. The
MCC1849 strain may be added at any stage of the manufacturing
process of the food or drink. The food or drink may also be
produced through a fermentation process advanced by the MCC1849
strain. Examples of such a food or drink include lactic acid
bacteria beverages, fermented milks, and so forth.
[0056] As the raw material of the food or drink, raw materials used
for producing usual drinks and foods can be used. The produced food
or drink can be orally taken.
[0057] The food or drink of the present invention includes raw
materials for food or drink production and those to be added to
foods or drinks in the course of the food or drink production
process or after the production, such as food additives. For
example, the lactic acid bacterium of the present invention can be
used as a starter for production of fermented milk. The lactic acid
bacterium of the present invention can also be added to a produced
fermented milk afterward.
[0058] Although the content of the MCC1849 strain in the food or
drink of the present invention is appropriately determined
according to the form of the food or drink, it is usually
preferably in the range of 1.times.10.sup.6 to 1.times.10.sup.12
cfu/g, or 1.times.10.sup.6 to 1.times.10.sup.12 cfu/ml, more
preferably in the range of 1.times.10.sup.7 to 1.times.10.sup.11
cfu/g, or 1.times.10.sup.7 to 1.times.10.sup.11 cfu/ml, of the food
or drink.
[0059] The food or drink of the present invention can be used for
various uses utilizing effects of IL-12 production promotion,
immunostimulation, antivirus, and so forth. That is, there can be
provided an IL-12 production-promoting agent, immunostimulant, and
antiviral agent that contain the lactic acid bacterium of the
present invention and are in the form of food or drink. The IL-12
production-promoting agent, immunostimulant, and antiviral agent in
the form of a food or drink are synonymous with an IL-12
production-promoting agent containing the food or drink of the
present invention, immunostimulant containing the food or drink of
the present invention, and antiviral agent containing the food or
drink of the present invention, respectively.
[0060] The food or drink of the present invention can be sold as a
food or drink with indication of use, i.e., for promotion of IL-12
production, for immunostimulation, or for antivirus. Further, the
food or drink of the present invention may have an indication such
as "For promotion of IL-12 production", "For immunostimulation",
"For immune activation", "For antivirus", "For anti-influenza
virus", and "For prophylaxis of infectious disease". Further,
indications other than those described above expressing an effect
secondarily obtainable by promotion of IL-12 production can of
course be used.
[0061] The aforementioned term "indication" includes all actions
for informing consumers of the aforementioned use, and any
indications reminding or analogizing the aforementioned use fall
within the scope of the "indication" of the present invention
regardless of purpose, content, objective article, medium etc. of
the indication. However, the indication is preferably made with an
expression that allows consumers to directly recognize the
aforementioned use.
[0062] Specific examples include actions of indicating the
aforementioned use on goods or packages of goods relating to the
food or drink of the present invention, actions of assigning,
delivering, displaying for the purpose of assigning or delivering,
or importing such goods or packages of goods on which the
aforementioned use is indicated, displaying or distributing
advertisements, price lists or business papers relating the goods
on which the aforementioned use is indicated, or providing
information including those as contents with indicating the
aforementioned use by an electromagnetic method (Internet etc.) and
so forth. Indications on packages, containers, catalogues,
pamphlets, advertisement materials used at the sales spots such as
POPs, and other papers are especially preferred.
[0063] The indication is preferably an indication approved by the
administration etc. (for example, an indication in a form based on
an approval, which is qualified on the basis of any of various
legal systems provided by the administration). Examples of the
indication further include, for example, indications as health
food, functional food, enteric nutritive food, food for special
dietary uses, food with nutrient function claims, quasi-drug and so
forth as well as indications approved by the Ministry of Health,
Labor and Welfare, for example, indications approved on the basis
of the system of food for specified health uses and similar
systems. Examples of the latter include indications as food for
specified health uses, indications as food for specified health
uses with qualified health claims, indications of influence on body
structures and functions, indications of reduction of disease risk
claims and so forth, and more specifically, typical examples
include indications as food for specified health uses (especially
indications of use for health) provided in the enforcement
regulations of Health Promotion Law (Japanese Ministry of Health,
Labor and Welfare, Ministerial ordinance No. 86, Apr. 30, 2003) and
similar indications.
[0064] Examples of the feed of the present invention include pet
foods, feeds for farm animals, feeds for fish culture, and so
forth. The feed of the present invention can be produced by mixing
the MCC1849 strain with a common feed material, for example,
cereals, lees, bran, fish meal, bone meal, oil and fat, skim milk
powder, whey, mineral feed, and yeast. Further, the feed may be
produced through a fermentation process advanced by the MCC1849
strain as in the case of, for example, silage. The produced feed
can be orally given to common mammals, livestock, bred fish, pets,
and so forth.
[0065] Although the content of the MCC1849 strain in the feed of
the present invention is appropriately determined according to the
form of the feed, and object to which the feed is given, it is
usually preferably in the range of 1.times.10.sup.6 to
1.times.10.sup.12 cfu/g, or 1.times.10.sup.6 to 1.times.10.sup.12
cfu/ml, more preferably in the range of 1.times.10.sup.7 to
1.times.10.sup.11 cfu/g, or 1.times.10.sup.7 to 1.times.10.sup.11
cfu/ml.
EXAMPLES
[0066] Hereafter, the present invention will be further
specifically explained with reference to examples. However, the
present invention is not limited by the following examples.
Example 1
Bacteriological Characteristics of the MCC1849 Strain
[0067] The MCC1849 strain was isolated from a human feces sample.
As the bacteriological characteristics of the strain, those
concerning cell morphology, mobility, sporulation, Gram staining,
catalase, gas production from glucose, and sugar fermentability are
shown in Table 1. The sugar fermentability was investigated by
using a bacterium identification kit, API 50CH (Sysmex Biomerieux
Co., Ltd.). That is, according to the method described in the
manual attached to the kit, the bacterium was cultured overnight,
the bacterium suspension was inoculated on a medium containing an
objective substrate, the bacterium was cultured in an incubator at
37.degree. C., and fermentation state of the substrate was
evaluated on days 1 and 2 of the culture.
[0068] The bacteriological characteristics are shown in Table 1. On
the basis of these results, the MCC1849 strain was determined to be
Lactobacillus paracasei.
TABLE-US-00001 TABLE 1 Bacteriological characteristics of
Lactobacillus paracasei MCC1849 strain 1 Cell morphology Rod 2
Mobility None 3 Sporulation None 4 Gram staining + 5 Catalase - 6
Gas production from glucose - 7 Sugar fermentability (1) Control -
(2) Glycerol - (3) Erythritol - (4) D-Arabinose - (5) L-Arabinose -
(6) D-Ribose + (7) D-Xylose - (8) L-Xylose - (9) D-Adonitol - (10)
Metyl-.beta.-D-Xylopyranoside - (11) D-Galactose + (12) D-Glucose +
(13) D-Fructose + (14) D-Mannose + (15) L-Sorbose - (16) L-Rhamnose
- (17) Dulcitol - (18) Inositol - (19) D-Mannitol + (20) D-Sorbitol
+ (21) Metyl-.alpha.-D-Mannopyranoside - (22)
Methyl-.alpha.-D-glucopyranoside .+-. (23) N-acetylglucosamine .+-.
(24) Amygdalin .+-. (25) Arbutin + (26) Esculine ferric citrate +
(27) Salicin + (28) D-Cellobiose + (29) D-Maltose + (30) D-Lactose
+ (31) D-Melibiose - (32) D-Sucrose + (33) D-Trehalose + (34)
Inulin + (35) D-Melezitose + (36) D-Raffinose - (37) Starch - (38)
Glycogen - (39) Xylitol - (40) Gentiobiose .+-. (41) D-Turanose +
(42) D-Lyxose - (43) D-Tagatose + (44) D-Fucose - (45) L-Fucose -
(46) D-Arabitol - (47) L-Arabitol - (48) Gluconate .+-. (49)
2-Ketogluconate - (50) 5-Ketogluconate - +: Positive; -: Negative;
.+-.: False Positive
[0069] Further, on the basis of the analysis of 16S rRNA gene
nucleotide sequence of the MCC1849 strain, the MCC1849 strain was
determined to be Lactobacillus paracasei or Lactobacillus casei.
Lactobacillus paracasei and Lactobacillus casei are closely related
species, and it has been demonstrated that the homology of the 16S
rRNA gene nucleotide sequences of the standard strains of them is
99% or higher (Huang C. H., and Lee F. L., Antonie Van Leeuwenhoek,
2011, 99(2):319-27). The genome sequence of the MCC1849 strain was
determined, and the homology was confirmed by mapping the read
using the genome of the Lactobacillus paracasei standard strain
(ATCC 25302), or the Lactobacillus casei standard strain (ATCC 393)
as the reference genome.
[0070] As a result, the MCC1849 strain showed a homology of 85%
with respect to the Lactobacillus paracasei standard strain,
whereas it showed a homology of 45% with respect to the
Lactobacillus casei standard strain.
[0071] Therefore, it was confirmed that the MCC1849 strain is
Lactobacillus paracasei.
Example 2
Evaluation of IL-12 Production-Inducing Ability Using Mouse Spleen
Cells
[0072] The effect of an RNase treatment or cell wall-digesting
enzyme treatment on the IL-12 production-inducing ability of lactic
acid bacteria was evaluated by using mouse spleen cells, for the
Lactobacillus paracasei MCC1849 strain described in Example 1, and
the Lactobacillus paracasei FERM BP-11313 strain (International
Patent Publication WO2012/133827), which is known to have high
IL-12 production-inducing ability, as well as other lactic acid
bacterium strains, the Lactobacillus rhamnosus ATCC 53103 strain,
Lactbacillus johnsonii JCM 2012 strain, Lactobacillus plantarum JCM
1149 strain, and Lactobacillus bulgaricus ATCC 11842 strain.
[0073] The FERM BP-11313 strain was deposited as an international
deposit at the National Institute of Technology and Evaluation,
NITE Patent Microorganisms Depositary. The JCM 2012 strain and the
JCM 1149 strain are available from the independent administrative
agency, Institute of Physical and Chemical Research, Japan
Collection of Microorganisms (JCM) (2-1, Hirosawa, Wako-shi,
Saitama-ken, 351-0198). Further, the ATCC 53103 strain and the ATCC
11842 strain are available from the American Type Culture
Collection (address, 12301 Parklawn Drive, Rockville, Md. 20852,
United States of America).
[0074] Each of the aforementioned lactic acid bacterial strains was
cultured in the MRS (de Man Rogasa Sharpe) medium (BD Company) for
16 hours, and the cells were washed 3 times by centrifugation and
re-suspension in distilled water, then suspended in distilled water
at a density of 10 mg (in terms of dry weight of cells)/ml, and
subjected to a heat treatment at 100.degree. C. for 15 minutes to
obtain a lactic acid bacterium dead cell suspension ("enzyme
untreated").
[0075] Each lactic acid bacterium dead cell suspension was
centrifuged, the supernatant was removed, the precipitates were
suspended in a 50 mM Tris-malate buffer (pH 7.0) containing 4 mM
magnesium chloride at a concentration of 10 mg (in terms of dry
weight of cells)/ml, N-acetyl muramidase (N-Acetylmuramidase SG,
Seikagaku Corporation) was added to the suspension at a
concentration of 5 .mu.g/ml, and the reaction was allowed at
37.degree. C. for 120 minutes. Then, the enzyme was inactivated by
a heat treatment at 100.degree. C. for 5 minutes to obtain a cell
wall-digesting enzyme-treated product ("N-acetyl muramidase
treated").
[0076] Further, 0.1 mg/ml of ribonuclease A (RNase A, Life
Technologies) was added to each lactic acid bacterium dead cell
suspension, and an enzyme treatment was performed at 37.degree. C.
for 30 minutes. After the treatment, the precipitates were washed 3
times by centrifugation and re-suspension in distilled water, and
then suspended again in distilled water at a concentration of 10 mg
(in terms of dry weight of cells)/ml to obtain an RNase-treated
product ("RNase treated").
[0077] As the experimental animals, 7-week old male BALB/c mice
(Japan SLC) were used, and dissected at 7 to 9 weeks old to extract
spleens. Spleen cells were collected from the extracted spleens,
treated with an erythrocyte lysis solution (0.144 M ammonium
chloride, 17 mM trishydroxymethylaminomethane, pH 7.65) for 2
minutes, and centrifuged to obtain spleen cells free from the
erythrocyte fraction. To these spleen cells, and each of the lactic
acid bacterium dead cell suspension (enzyme untreated), the cell
wall-digesting enzyme treated product (N-acetyl muramidase
treated), and the RNase treated product (RNase treated), which were
prepared above, a medium obtained by adding 10% FBS (fetal bovine
serum, Life Technologies), 100 IU/ml penicillin, and 0.1 mg/ml
streptomycin to RPMI1640 (SIGMA-ALDRICH) was added to prepare test
solutions containing 2.5.times.10.sup.6 spleen cells/ml and 5 .mu.g
(in terms of dry weight of cells)/ml as the final concentration of
the lactic acid bacterium dead cell suspension, the cell
wall-digesting enzyme treated product, or the RNase treated
product. Each test solution was applied in a volume of 200 .mu.l to
a 96-well microplate (BD Company), and incubation was performed at
37.degree. C. in the presence of 5% CO.sub.2.
[0078] After 2 days, the culture supernatant was collected, and the
concentration of IL-12 p70 (p40-p35 heterodimer) in the culture
supernatant was measured by using a measurement kit (Mouse IL-12
p70 DuoSet, R&D Systems).
[0079] The test was performed in triplicate, and the results are
shown in Table 2 and FIG. 1.
TABLE-US-00002 TABLE 2 Amount of IL-12 prodced by mouse spleen
cells with lactic acid bacteria IL-12 production amount (pg/ml)
(Average .+-. S.D.) N-Acetyl Strain Enzyme untreated muramidase
treated RNase treated MCC1849 407.6 .+-. 35.3 436.7 .+-. 49.1 252.8
.+-. 25.0 FERM BP- 291.8 .+-. 42.6 15.7 .+-. 1.5 9.1 .+-. 1.8 11313
ATCC 53103 148.5 .+-. 38.7 22.1 .+-. 2.9 8.5 .+-. 3.2 JCM 2012
101.3 .+-. 6.7 19.7 .+-. 6.8 17.5 .+-. 2.4 JCM 1149 63.3 .+-. 15.2
20.7 .+-. 2.2 14.5 .+-. 3.4 ATCC 11842 47.1 .+-. 1.0 8.7 .+-. 2.8
11.6 .+-. 5.2
[0080] The Lactobacillus paracasei MCC1849 strain showed an IL-12
production-inducing ability higher than that of the FERM BP-11313
strain, which is known to have high IL-12 production-inducing
ability. The IL-12 production-inducing abilities of the other
lactic acid bacterium strains were lower than those of the
aforementioned strains.
[0081] By the N-acetyl muramidase treatment or RNase treatment, the
IL-12 production-inducing abilities of the lactic acid bacterium
strains other than the MCC1849 strain were markedly reduced.
However, the MCC1849 strain maintained the high IL-12
production-inducing ability even after it was subjected to the
aforementioned treatments. Since the IL-12 production-inducing
activity of the FERM BP-11313 strain, which is a strain of the same
species as the MCC1849 strain, was degraded by the N-acetyl
muramidase treatment or RNase treatment, it was demonstrated that
the stability of the IL-12 production-inducing ability of the
MCC1849 strain is not species-dependent property, but a
strain-specific property.
Example 3
Influenza Virus Infection-Preventing Action of MCC1849 Strain
[0082] The influenza virus infection-preventing action by
administration of the MCC1849 strain was investigated by using
mice.
[0083] The MCC1849 strain was cultured in the MRS medium for 16
hours, the cells were washed twice with distilled water, and then
suspended in distilled water, sucrose and sodium glutamate were
added to the suspension, and the mixture was lyophilized. The
lyophilized cells were suspended in physiological saline at a
density of 1.times.10.sup.10 cfu/ml to prepare an MCC1849 strain
live cell suspension.
[0084] The MCC1849 strain was cultured in the MRS medium for 16
hours, the cells were washed twice with PBS, and once with
distilled water, then suspended in distilled water, and subjected
to a heat treatment at 100.degree. C. for 30 minutes. After the
heat treatment, the cells were washed twice with distilled water,
suspended in distilled water, and lyophilized. The lyophilized
cells were suspended in physiological saline at a concentration of
5 mg/ml to prepare an MCC1849 strain dead cell suspension.
[0085] Each of the live cell suspension (Live group) of the MCC1849
strain, the dead cell suspension (HK group) of the same, and
physiological saline (Control group) was orally administered in a
volume of 0.2 ml to BALB/c mice for 14 days, and then the mice were
infected with an influenza virus (A/PR8/34(H1N1) strain) from the
nasal cavity. Six days after the infection, general statuses of the
eyes (degree of opening of eyelid and state of eyelid), hair (lie
of hair and piloerection), respiration (irregular respiration), and
behavior (degree of spontaneous behavior) of the mice were
observed, and the results of those items were represented with
scores according to the following criteria to evaluate the onset
state of the disease.
[0086] Normal state: 0
[0087] Slight infection: 1
[0088] Moderate infection: 2
[0089] Severe infection: 3
[0090] Death: 4
[0091] The average of the scores for the items was used as a
symptom score for each mouse individual.
[0092] Further, 6 days after the infection, the lungs were
extracted, and the virus concentrations in the lungs were measured
by the plaque assay method using the MDCK cells (canine-derived
kidney cells).
[0093] The test results are shown in Table 3 and FIG. 2.
[0094] Compared with the MCC1849 strain non-administration group
(Control group), the symptom score was significantly reduced, and
the virus concentration in the lungs (virus titer) was also
significantly reduced by administration of either one of the
MCC1849 strain live cell suspension (Live group) and the MCC1849
strain dead cell suspension (HK group). On the basis of these
results, it was demonstrated that administration of the MCC1849
strain both in the live cell state and the dead cell state is
effective for the prophylaxis of influenza virus infection.
TABLE-US-00003 TABLE 3 Influenza virus infection-preventing action
of MCC1849 Virus concentration Symptom score in lung
(.times.10.sup.3 PFU) (Average .+-. S.D.) (Average .+-. S.D.)
Pysiological saline 1.31 .+-. 1.47 215.6 .+-. 109.4 (Control group)
Live cell suspension 0.26 .+-. 0.41 47.7 .+-. 62.6 (Live group)
Dead cell suspension 0.26 .+-. 0.41 24.8 .+-. 44.9 (HK group)
INDUSTRIAL APPLICABILITY
[0095] The strain of the present invention, the Lactobacillus
paracasei MCC1849 strain, has high IL-12 production-promoting
activity. The IL-12 production-promoting activity of the strain is
hardly reduced by a cell wall-digesting enzyme treatment or an
RNase treatment. Therefore, it is thought that individual
differences of the IL-12 production-promoting activity observed
among objects of the application thereof would be small, and thus
the strain is useful as an immunostimulant. Further, the strain can
reduce infection or proliferation of influenza virus etc., and can
be used for prophylactic and therapeutic treatments of infectious
diseases, and so forth.
* * * * *