[DESCRIPTION]
[invention Title]
NOVEL SEPARATED BACILLUS LICHENIFORMIS AND PROBIOTICS
USING SAME
[Technical Field]
[01] The present invention relates to a novel probiotics
and a use thereof.
[02]
[Background Art]
[03] In accordance with an increase in world population
and improvement of a standard of living, there has been an
increased interest in public hygiene and stability of food.
Subsequently, a residue of an antibiotic feed additive,
which added in order to promote growth of livestock and
improve efficiency of feed, and a antibiotics-resistant
bacteria have.been continuously controversial. As a result,
the additions of antibiotics and antimicrobial agents in
feed have been entirely prohibited on and after July 1,
2011.
[04] As the addition of antibiotics in feed has been
entirely prohibited, research into materials replacing the
antibiotics has been actively conducted.
[05] Among them, probiotics has been spotlighted as an
immunne stimulating agent. The term "probiotics", which

has an etymological meaning opposed to antibiotics
indicating an antibiotic material, is defined as a
microbial agent or microbial component assisting in a
balance of intestinal microflora. Species called Lactic
acid bacteria such as Lactobacillus, Bifidobacteria, and
the like, are representative. In addition, the probiotics
are classified as microorganisms granted generally
recognized as safe (GRAS) and do not have a virulence gene
for humans and animals nor produce a pathogenic material.
In addition, it should be confirmed that a microorganism
has an effect of improving productivity in a livestock
industry.
[06] The probiotics have effects of being attached to
intestinal mucous membrane to competitively drop pathogenic
bacteria causing enteropathy so as to be excreted to the
outside, and rapidly recovering intestinal microflora
disrupted -by administration of antiboitics. In addition,
the probiotics prevent infection of pathogenic bacteria,
suppresses propagation of the pathogenic bacteria, composes
an environment optimal for growth of beneficial intestinal
bacteria, promotes growth of the beneficial intestinal
bacteria, produces lactic acid or acetic acid, which is a
important component of intestinal organic acids, and
decreases a pH in intestine to suppress growth of harmful
pathogenic bacteria. Therefore, the probiotics may

maintain normal intestinal bacterial flora and promote
improvement of productivity in the livestock industry by
various complex actions.
[07] As mainly used probiotics, there are lactic acid
bacteria such as Lactobacillus, Enterococcus, and the like,
and, Bacillus. Bacillus is Gram-positive bacillus, forms
endospores, and has a unique shape among the bacteria used
as the probiotics. Bacillus has excellent heat resistance
as compared to Lactobacillus that does not produce the
endospores. In addition, since Bacillus may survive at a
low pH in stomach walls, most of the administered Bacillus
may arrive in the small intestine (Barbosa, T.M. et al.f
Appl. Environ. Microbiol., 71(2005)968-978; Spinosa, M. R.
et al., Res. Microbiol., 151(2000) 361-368).
[08] As the related prior art associated with probiotics
isolated for boosting immunity of livestock, which is one
of the main functions of the probiotics, a novel strain
mixture of Bacillus sp. CMB LI and Lactobacillus sp. CMB201,
food composition for increasing immunity and anticancer
activity containing the same, and a microbial agent having
an antibiotic activity have been disclosed in Korean Patent
Laid-Open Publication No. 10-2011-035554. In addition, an
immune boosting agent for animal, a vaccine adjuvant
additive, an auxiliary drug, and a feed additive containing
a bacteriolysis extract of Zygosaccharomyces bailii have

been disclosed in Korean Patent Registration No. 10-
0977407.The Zygosaccharomyces bailii is capable of
increasing various activities of neutrophils, which are the
main macrophages of animals, and improving non-specific
protection effect against challenge of pathogenic bacteria.
However, research into an immune strengthen effect of
Bacillus is insufficient.
[09] Further, it has been known that among the probiotics,
Lactobacillus has a important feature of producing lactic
acid, but a paper has been reported that a Bacillus
polyfermenticus KJS-2 strain produces lactic acid (Kim, K.
M et al., J. Microbiol. Biotechnol., 19(2009) 1013-1018).
[10] Therefore, the present inventors isolated a bacillus
strain producing digestive enzymes and lactic acid from
soybean paste, which is a traditionally fermented Korean
food, and confirmed morphological, biochemical, and genetic
properties of thereof. As a result, the present inventors
confirmed that the bacillus strain was probiotics having an
excellent heat resistance, an excellent bone marrow cell
proliferating effect in a bone marrow transplanted mouse,
an immune boosting effect of proliferating spleen cells and
bone marrow cells of an immunized mouse, and a vaccine
adjuvant effect of increasing antibody production.
[11]
[Disclosure]

[Technical Problem]
[12] As an embodiment of the present invention, it
provides a novel Bacillus licheniformis CJMPB361 producing
digestive enzymes and lactic acid, and having an immune
boosting activity.
[13] In addition, as an embodiment of the present
invention, it provides a culture product comprising a
culture solution of the Bacillus licheniformis CJMPB361, a
concentrate solution thereof, or a dried material thereof.
[14] Further, as an embodiment of the present invention,
it provides a probiotic formulation containing the Bacillus
licheniformis CJMPB361 or the culture product.
[15] Further, as an embodiment of the present invention,
it provides a feed additive containing the probiotic
formulation.
[16] Furthermore, as an embodiment of the present
invention, it provides a feed containing the feed additive.
[17] In addition, as an embodiment of the present
invention, it provides an immune boosting agent or vaccine
adjuvant containing the probiotic formulation.
[18] In addition, as an embodiment of the present
invention, it provides a health functional food for
boosting immune system containing the probioticformulation.
[Technical Solution]
[19] According to an embodiment of the present invention,

there is provided a novel Bacillus licheniformis CJMPB361
producing digestive enzymes and lactic acid.
[20] In detail, the Bacillus licheniformls CJMPB361
according to the present invention is isolated from soybean
paste, which is a traditional Korean food. As
morphological properties of the strain according to the
present invention, the strain is a Gram-positive bacillus
and has a 16s rDNA nucleotide sequence represented by SEQ
ID No: 1. As a result of analyzing the nucleotide sequence,
the strain has homology of 99% with Bacillus licheniformis.
Therefore, the present inventors deposited the novel
isolated Bacillus licheniformis CJMPB361 in Korean Culture
Center of Microorganisms (361-221, Hongje 1-dong,
Seodaemun-gu, Seoul) under accession number KCCM11269P on
March 22, 2012.
[21] In detail, the strain from soy bean paste after 70 to
80 days which is a traditional Korean food, isolated in a
brain heart infusion (BHI) agar medium. The isolated
strain has an excellent digestive enzyme activity such as
amylase, cellulase, mannanase, and xylanase and produces
lactic acid when being cultured for 24 hours or more under
facultative anaerobic or anaerobic conditions. As a
culture supernatant of the strain increases activities of
non-T cell in the spleen of a mouse, mouse bone marrow, and
peripheral blood of a human, the culture supernatant has a

strong effect to boosting the immune system specific to
mouse B-cells.In the case of injecting a fraction of the
culture supernatant having a molecular weight of 1,000 kDa
or more together with an antigen, the culture supernatant
had an adjuvant effect on producing antibody against the
antigen.
[22] In addition, when a fraction of the culture
supernatant of the strain having a molecular weight of
1,000 kDa or more was administered to an irradiated mouse
after bone marrow transplantation, the fraction had an
effect of increasing a propagation response of transplanted
bone marrow cells to increase a survival rate of the mouse.
[23]
[24] According to another embodiment of the present
invention, there is provided a culture product comprising a
culture solution of a novel isolated strain according to
the present invention, a concentrated solution thereof, or
a dried material thereof.
[25] The novelisolated strain according to the present
invention may be cultured by a general method for Bacillus
strains. As a medium, a natural medium or synthetic medium
may be used. As a carbon source of the medium, for example,
glucose, sucrose, dextrin, glycerol, starch, or the like,
may be used, and as a nitrogen source,- peptone, a. meat
extract, a yeast extract, dried yeast, a soybean, an

ammonium salt, nitrate, other organic or inorganic
nitrogen-containing compounds may be used. However, the
sources of the present invention are not limited thereto.
As an inorganic salt contained in the medium, magnesium,
manganese, calcium, iron, potassium, or the like, may be
used, but the inorganic salt is not limited thereto. In
addition to the carbon source, the nitrogen source, and the
inorganic salt, amino acid, vitamin, nucleic acid, and
compounds related thereto, may be added to the medium. The
novel isolated strain according to the present invention
may be cultured in a culture temperature range of 20 to 400
for 12 hours to 4 days.
[26] In detail, the culture solution of the novel isolated
strain may be a culture solution containing the microbial -
cell, or microbial cells obtained by removing a culture
supernatant or concentrating the culture solution. A
composition of the culture solution may additionally
contain components having a synergy effect on growth of
Bacillus as well as components required for generally
culturing Bacillus, and this composition may be easily
composed by those skilled in the art.
[27] In addition, the strain may be a liquid state or
dried state. A drying method may be an air drying method, a
natural drying method, a spray drying method, and a freeze-
drying methodbut, is not limited thereto.

[28]
[29] According to another embodiment of the present
invention, there is provided a probiotic formulation
containing a novel isolated strain according to the present
invention or a culture product thereof.
[30] The probiotics settles in the intestinal digestive
tract wall to prevent harmful bacteria from staying and
suppresses propagation of pathogenic bacteria. In addition,
beneficial digestive enzymes produced by the probiotics
support absorption and use of nutrients, thereby improving
feed efficiency.
[31] The probiotics formulation according to the present
invention contains Bacillus licheniformis CJMPB361 in an
amount of 5 x 104 CFU/ml to 5 x 1010 CFU/ml, specifically 1
x 106 CFU/ml to 1 x 109 CFU/ml.
[32] The probiotic formulation according to the present
invention may additionally contain a pharmaceutically
" acceptable carrier and be formulated together with the
carrier, thereby being provided as a food and feed additive.
[33] The term "pharmaceutically acceptable carrier" as
used herein means a carrier or a diluent that does not
stimulate living organism nor inhibit biological activities
and properties of an administered compound.
[34] As a pharmaceutically acceptable carrier, which is
sterilized or suitable for a living body, in the liquid

formulation of probiotics, normal saline, sterile water,
buffered saline, an albumin injection solution, a dextrose
solution, a maltodextrin solution, glycerol, and a mixture
of at least one thereof may be used. Further, if necessary,
other general additives such as an antioxidant, a buffer
solution, a bacteriostatic agent, or the like, may be added.
In addition, a diluent, a 'dispersant, a surfactant, a
binder, and a lubricant are additionally added thereto, so
that the probiotic formulation may be formulated into an
injection such as an aqueous solution, a suspension, an
emulsion, or the like, a pill, a capsule, a granule, or a
tablet- Further, a binder, an emulsifier, a preservative,
and the like, may be added in order to maintain the quality
of the probiotic formulation. An amino acid, a vitamin, an
enzyme, a flavoring agent, a non-protein nitrogen compound,
silicate, a buffering agent, an extractant, oligosaccharide,
and the like, may be added to increase 'the efficiency of
the probiotic formulation. In addition, the probiotic
formulation may further contain a feed mixture, or the like,
but is not limited thereto.
[35] As an example of the oral formulation, containing the
probiotics formulation as an active component, there are
tablets, troches, lozenges, aqueous or oily suspensions,
prepared powders or granules, emulsions, hard or soft
capsules, or syrups, and elixirs. In order to formulate

the probiotics into a formulation such as a tablet, a
capsule, or the like, the formulation may further contain a
binder such as lactose, saccharose, sorbitol, mannitol,
starch, amylopectin, cellulose, or gelatin, an excipient
such as dicalcium phosphate, a disintegrant such as corn
starch or sweet potato starch, a lubricant such as
magnesium stearate, calcium stearate, sodium stearyl
fumarate, or polyethylene glycol wax. In the case of the
capsule formulation, the formulation may additionally
contain a liquid carrier such as fatty oil.
[36]
[37] According to another embodiment of the present
invention, there is provided a feed additive, containing
the probiotics formulation as an active component.
[38] Generally, endospores forming Bacillus species has a
significantly stable property against heat. Therefore, the
novel isolated Bacillus licheniformis CJMPB361 may be
prepared in a form of the feed additive and then mixed with
feed, or directly added to feed. The Bacillus
licheniformis CJMPB361 contained in the feed according to
the present invention may be in a liquid state or dried
state, and specifically, in a dried powder form. A drying
method may be an air drying method, a natural drying method,
a spray drying method, and a freeze-drying method, but is
not limited thereto. The Bacillus licheniformis CJMPB361

according to the present invention may be mixed in the
powder form at a content ratio of 0.05 wt% to 10wt%,
specifically 0.1 wt% to lwt% based on a weight of the feed.
In addition, the feed may further contain general additives
capable of improving preservability of the feed.
[39] The feed additive according to the present invention
may be provided in a form selected from a group consisting
of a concentrated solution, powders, and granules. The
concentrated. solution may contain the Bacillus
licheniformis CJMPB361 strain according to the present
invention, the culture solution thereof, the dried material
thereof, or the probiotic formulation at a content of 20
wt% to 90wt% based on the total weight of the feed additive.
[40] The feed additive according to the present invention
may be immersed, sprayed, or mixed to be added to feed for
animals, and then used.
[41] An example of animals that be feeded containing the
feed additive according to the present invention may be
livestock such as pigs, cows, sheep, goats, horses, rabbits,
dogs, cats, and the like, and poultry such as chickens,
ducks, geese, turkeys, quails, or the like, but are not
limited thereto.
[42] In a method of mixing the feed for animals containing,
the feed additive according to the present invention, the
feed additive may be mixed at a content of 0.05 wt% to

0.5wt% based on a dried weight of the feed for animals.
The term "%" as used herein means a weight% (wt%) unless
otherwise defined.
[43]
[44] According to another embodiment of the present
invention, there is provided a feed containing the feed
additive according to the present invention.
[45] In the present invention, an example of the feed
containing the Bacillus licheniformis CJMPB361 according to
the present invention may include plant feed such as grains,
roots and fruits, food processing byproducts, algaes,
fibers, fats, starches, cucurbitaceaes, grain byproducts,
or the like, and animal feed such as proteins, inorganic
materials, fats, minerals, single cell proteins,
fats, animal planktons, fish meal, or the like, but the
present invention is not limited thereto.
[46]
[47] According to another embodiment of the present
invention, there is provided an immune boosting agent or
vaccine adjuvant containing the novel strain, a culture
solution thereof, a concentrated solution thereof, or a
dried material thereof as an active component.
[48] The culture solution , the concentrated solution
thereof, or the dried material thereof contains a fraction
of a molecular weight of 100 kDa to 300 kDa or 1,000 kDa or

more, having an immune boosting activity.
[49] In a specific embodiment, after the culture
supernatant of the Bacillus lichen iformis CJMPB361 was
separated by molecular weight, a lymphocyte proliferating
activity was measured. As a result, in a fraction having a
molecular weight of 1,000 kDa or more and 100 to 300 kDa,
proliferation of spleen cells of a mouse was excellent (FIG.
3). Particularly, proliferation response of B cells
depending on a concentration of the culture supernatant was
observed, and proliferation response of non-T cells and
non-B cells was increased as well as the B cells. A
proliferation effect was higher in a culture supernatant
having a molecular weight of 1,000 kDa or more (FIGS. 5 and
6). Particularly, it was confirmed that the culture
supernatant having a molecular weight of 1,000 kDa or more
had an adjuvant effect on producing an antibody against an
antigen.
[50] Further, among mice irradiated with 7.5 Gy, a group
treated with bone marrow cells and the culture supernatant
(1,000 kDa or more) showed 100% of survival rate (FIG. 8A).
In mice irradiated with 9 Gy, a group treated with bone
marrow cells and the culture supernatant (1,000 kDa or
more) showed 66% of survival rate (FIG. 8B). The reason of
the results as described above may be that the fraction of
the culture supernatant (1,000 kDa or more) induces

proliferation of the transplanted bone marrow cells to
increase the survival rate of the irradiated mouse.
Therefore, the immune boosting agent or vaccine adjuvant
according to the present invention may be used for
stimulating proliferation response of transplanted bone
marrow cells in leukemia patients.
[51] The immune boosting agent or vaccine adjuvant
according to the present invention may contain the strain,
the culture solution thereof, the concentrated solution
thereof, or the dried material thereof at a content of 0.1
wt% to 50wt% based on a total weight of a composition.
[52] The immune boosting agent according to the present
invention may be used as an adjuvant to prevent or treat an
immune-compromised disease that may occur in animals or
humans. In addition, the vaccine adjuvant according to the
present invention may be used as an adjuvant to induce a
systemic immune response against the antigen by
administering with an antigen in a body of an animal. As
the antigen, one or a mixture of two or more antigens
selected from a group consisting of a fungus, actinomyces,
bacteria, virus, protozoa, and a component, an organ, a
cell component, an antigen protein, and an antigen peptide
of these microbes may be used.
[53] When preparing a pharmaceutical composition of the
immune boosting agent and the vaccine adjuvant containing

the strain according to the present invention, the culture
solution thereof, the concentrate solution thereof, or the
dried material thereof, it may further contain a suitable
carrier, a suitable excipient, and a suitable diluent that
are generally used.
[54] The immune boosting agent or the vaccine adjuvant
according to the present invention may be formulated into
an oral formulation, such as dispersants, granules, tablets,
capsules, suspensions, emulsions, syrups, aerosols, or the
like, a formulation for external application, suppository,
and sterile injection solutions by a general method,
respectively. The carrier, the excipient, and the diluent
for the pharmaceutical composition may include lactose,
dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol,
maltitol, starch, acacia rubber, alginate, gelatin, calcium
phosphate, calcium silicate, cellulose, methyl cellulose, '
microcrystalline cellulose, polyvinylpyrrolidone, water,
and mineral oil. In the case in which the pharmaceutical
composition is formulated, generally used diluents or
excipients such as fillers, extenders, binders, wetting
agents, disintegrants, surfactants, or the like, may be
used. Solid formulations for oral administration include
tablets, pills, powders, granules, capsules, and the like,
and these solid formulations are prepared by mixing at
least one excipient with the culture solution, the

concentrated solution thereof, or the dried material
thereof. Liquid formulations for oral administration
include suspensions, solutions, emulsions, syrups, and the
like, and these liquid formulations may containvarious
excipierits as well as water, liquid and paraffin that are
generally used simple diluents. Formulations for
parenteral administration include sterile aqueous solutions,
non-aqueous solvents, suspensions, emulsions, and freeze-
dried formulations. In the non-aqueous solvent or the
suspensions, propylene glycol, polyethylene glycol,
vegetable oil such as olive oil, injectable esters such as
ethyl oleate, or the like, may be used. As a base for
suppositories, witepsol, macrogol, Tween 61, cacao butter,
sevum laurinum, glycerogelatin, or the like, may be used.
[55] A preferable dosage of the immune boosting agent and
the vaccine adjuvant according to the present invention
depends on the state and weight of a patient, the degree of
disease, the formulations, the administration route, and
duration, but be appropriately selected by those skilled in
the art. However, in order to obtain a preferable effect,
the immune boosting agent and the vaccine adjuvant
according to the present invention may be administered at a
daily dose of 0.001 mg/kg to 100 mg/kg, specifically 0.001
mg/kg to 10 mg/kg. Administration may be once a day, or
divided into several times. The scope of the present

invention is not limited to the administration dose.
[56]
[57] The immune boosting agent and the vaccine adjuvant
according to the present invention may be administered to
livestock, poultry, human, or mammals through various
routes. The immune boosting agent and the vaccine adjuvant
according to the present invention may be administered by
oral administration or intramuscular injection,
subcutaneous injection, or intravenous injection.
[58]
[59] According to another embodiment of the present
invention, there is provided a health functional food for
boosting immune system containing the novel strain, the
probiotic formulation, or the immune boosting agent as an
active component.
[60] A food to which the probiotic formulation or the
immune boosting agent according to the present invention
may be various types of food, drinks, gum, teas, or health
functional food.
[61] In this case, the novel strain, the probiotic agent,
or the immune boosting agent according to the present
invention may be added to the food in an amount of 0.01 wt%
to 50wt% based on a total weight of the food, and added to
the drink in an amount of 0.05 wb% to 5wt% based on 100ml
of a health drink composition. A form of the health

functional food according to the present invention may
include tablets, capsules, solutions, or the like.
[62] The health drink composition according to the present
invention may have other components without a particular .
limitation except that the ncvel strain, the probiotic
agent, or the immune boosting agent according to the
present invention may be contained in the above-mentioned
range as an essential component. Further various flavoring
agents or natural carbohydrates may be contained as
additional components, like general drinks. The natural
carbohydrates mean general sugars such as glucose, fructose,
maltose, sucrose, dextrin, cyclodextrin, and the like, and
sugar alcohols such as xylitol, sorbitol, erythritol, and
the like. In addition, as the flavoring agent, a natural
flavoring agent such as taurine, stevia extracts, and the
like, and a synthetic flavoring agent such as saccharine,
aspartame, and the like, may be used.
[63] Besides the-above mentioned components, the probiotic
formulation or the immune strengthen agent according to the
present invention may contain various nutrients, vitamins,
minerals (electrolytes) , flavoring agents, colorants and
enhancers (cheese, chocolate, or the like), pectic acid and
salts thereof, alginic acid and salts thereof, organic
acids, protective colloid thickeners, pH control agents,
stabilizers, preservatives, glycerin, alcohols, carbonating

agents used in a carbonated beverage, or the like. In
addition, according to the present invention may contain
flesh fruit or natural fruit juice for fruit drink, or
vegetable drink. These ingredients may be used
independently or in combination.
[Advantageous Effects]
[64] According to the present invention, a novelisolated
Bacillus licheniforntis CJMPB361 may have an excellent
effect of producing digestive enzymes such as amylase,
cellulase, mannanase, and xylanase and lactic acid, an
excellent immune boosting effect in animals, an excellent
adjuvant effect on antibody production, and an induction
effect on proliferation response of transplanted bone
marrow cells in a leukemia patient.
[65] Therefore, the novel isolated Bacillus licheniformis
CJMPB361 may be used as an immune boosting agent for
animals and a vaccine adjuvant as well as probiotics.
[Description of Drawingsl
[66] FIG. 1 shows an electron microscope photograph
representing a Bacillus licheniformis CJMPB361 strain
according to the present invention.
[67] FIG. 2 shows a view representing the presence or
absence of hemolysis of the Bacillus licheniformis CJMPB361
according to the present invention.
[68] FIG. 3 shows a graph representing proliferation

response of lymphocytes of a mouse depending on molecular
weight fractions of a culture supernatant of the Bacillus
licheniformis CJMPB361 according to the present invention.
[69] FIG. 4 shows a graph representing proliferation
response of spleen cells" (lymphocytes), bone marrow cells,
and thymocytes of a mouse depending on molecular weight
fractions of a culture supernatant of the Bacillus
licheniformis CJMPB361 according to the present invention.
[70] FIG. 5 shows a graph representing proliferation
response of spleen B cells of a mouse under in vitro
conditions depending on molecular weight fractions of a
culture supernatant of the Bacillus licheniformis CJMPB361
according to the present invention.
[71] FIGS. 6A to 6E show fluorescence activated cell
sorter (FACS) results representing in vivo proliferation
response of lymphocytes of a mouse at the time of
administering a fraction of a culture supernatant (1,000
kDa or more) of the Bacillus licheniformis CJMPB361
according to the present invention to the abdominal cavity
of a mouse. FIG. 6A shows .a FACS result of an isotype
control group, and FIGS. 6B to 6E show FACS results in the
case of administering 0.67mg, 1.35mg, 2.7mg, and 5.4mg of
the culture supernatant (100 kDa or more), respectively.
[72] FIG. 7 shows a graph representing results obtained by
evaluating an adjuvant effect on antibody production at the

time of administering the fraction of the culture
supernatant (1,000 kDa or more) of the Bacillus
licheniformis CJMPB361 according to the present invention
together with an antigen.
[73] FIGS. 8A and 8B show graphs representing mortality
rates obtained by evaluating an effect of the fraction of
the culture supernatant (1,000 kDa or more)-of the Bacillus
licheniformis CJMPB361 according to the present invention
on cell proliferation after bone marrow transplantation in
irradiated mice. FIG. 8A shows a graph representing a
result evaluated in mice irradiated with 7.5 Gy, and FIG.
8B shows a graph representing a result evaluated in mice
irradiated with 9 Gy.
[74]
[Best Mode)
[75] Hereinafter, the present invention will be described
in detail through Examples. However, these Examples are
only to illustrate the present invention, and a scope of
the present invention is not limited to these Examples.
[76]
.[77] Example 1: Isolation and Identification of Bacillus
licheniformis CJMPB361 Strain
[78] (1) Preparation of Sample and Isolation of Strain
[79] After samples derived from soybean paste, which is a
traditional Korean food, after 70 to 80 days of preparation,

and the prepared samples were diluted step by step, spread
on a brain heart infusion (BHI) agar medium (Difco, USA)
containing 3% sodium. chloride, and cultured at 370 for 24
hours. Then predominant strains were selected from the
samples. The selected colony was re-isolated by a method
of transferring and culturing the selected colony in a new
medium three times, and purely cultured strains were put
into a medium containing 20% glycerol and preserved at -70D
or less.
[80] (2) Investigation of Morphological and Biochemical
Properties
[81] In order to identify the isolated strain,
morphological and biochemical investigation was performed.
The morphological properties were as follows. The strain
was a Gram-positive bacillus, and it was confirmed that the
strain was a bacillus from an electron microscope
photograph (FIG. 1) . In order to analyze the biochemical
properties, a sugar fermentation pattern of .the strain was
analyzed using API 50 CHB system (Biomerieux, France)
(Table 1) . It was confirmed from the results shown in
Table 1 that the isolated strain had homology of 99% with
Bacillus licheniformis according to API 50 CHB system
[82] [Table 1]

[84] +: positive, -: negative
[85]
[86] (3) Identification of Strain
[87] In order to accurately identify the strain, a
molecular phylogenetic method using a DNA nucleotide
sequence was performed. For nucleotide sequence analysis,
a 16s rDNA gene was amplified using a PCR premix (Bioneer,
Korea) and universal primers 27F (5'
AGAGTTTGATCMTGGCTCAG3': SEQ ID No: 2) and 1492R
(5'GGTTACCTTGTTACGACTT3': SEQ ID No: 3). A total amount of
reaction solution was set to 20|i£. Polymerase chain
reaction (PCR) was performed for 30 cycles at 94D for 1
minute, 56D for 1 minuter and 720 for 1 minute.. Then, the

amplified DNA nucleotide sequence was analyzed. The
analyzed 16s rDNA nucleotide sequence of the isolated
strain is represented by a sequence of SEQ ID No: 1. As a
result of the analysis, the strain has homology of 99%
with Bacillus licheniformis. Therefore, the isolated
strain was named Bacillus licheniformis CJMPB361 and the
novel strain according to the present invention was
deposited in Korean Culture Center of Microorganisms
(accession number KCCM11269) on March 22, 2012.
[88]
[89] Example 2: Analysis of Probiotic properties of
Bacillus lich&niformls CJMPB361
[90] (1) Digestive Enzyme Activity of isolated Strain
[91] 1) Extraction of Crude Enzyme Solution
[92] After culturing the isolated strain in a BHI liquid
medium for 24 hours, a degree of decomposition of a
substrate was analyzed by using the culture solution as a
crude enzyme solution and a medium containing each of the
substrates corresponding to each of the enzymes.
[93] 2) Cellulase Activity
[94] A YM{Yeast Mold) medium containing 1% carboxyl methyl
cellulose (CMC) substrate was prepared. After spotting 2\x£
of the extracted crude enzyme solution onto the substrate
medium, a reaction was performed at 370 for 15 hours. Then,
the reactant was stained using 0.2% Congo red aqueous

solution for 30 minutes, and then decolorized using 1M NaCl
aqueous solution. The enzyme activity was measured by a
degree of formation of a clear zone generated by
decomposition of the substrate around the crude enzyme.
[95] 3) Amylase Activity
[96] A YM medium containing 1% soluble starch substrate
was prepared. After spotting 2[i£ of the extracted crude
enzyme solution onto the substrate medium, a reaction was
performed at 37D for 15 hours. The reactant was stained
using an aqueous solution containing 0.1% I2 and 2% KI, and
then the enzyme activity was measured by a degree of
formation of a clear zone generated by decomposition of the
substrate around the crude enzyme.
[97] 4) Xylanase Activity
[98] A YM medium containing 1% xylan was prepared. After
spotting 2|i£ of the culture solution onto the substrate
medium, a reaction was performed at 37D for 15 hours. Then,
the reactant was stained using 0.2% Congo red aqueous
solution for 30 minutes, and then decolorized using 1M NaCl
aqueous solution. The enzyme activity was measured by a
degree of formation of a clear zone.
[99] 5) Mannahase Activity
[100] A substrate medium (Yeast extract 3g/f, Peptone 5q/t,
KH2P04 lg/(, Agar 20g/f, pH 5) containing 1% mannan (logust
bean gum, Sigma, USA) was prepared. After spotting 2|i£ of

the culture solution onto the substrate medium, a reaction
was performed at 37D for 15 hours. Then, the enzyme
activity was measured by a degree of formation of a clear
zone.
[101] The novel isolated Bacillus licheniformis CJMPB361
has digestive enzyme activities for cellulase, amylase,
xylanase, and mannanase as shown in Table 2. Particularly,
activities for xylanase and mannanase were high.
[102] [Table 2]

[103]
[104] -: absence of activity, +: presence of Activity, ++:
excellent activity, +++: significantly excellent activity
[105] (2) Lactic acid productivity
[106] In order to evaluate lactic acid productivity of the
isolated strain, the isolated strain (1%) was inoculated
into a 100ml of PSG liquid medium (polypeptone lOg/L, yeast
extract lOg/L, glucose 20g/L, potassium phosphate dibasic
35g/L) in a 250ml flask with a stopper and cultured at 37D
and 200 rpm for 24 hours. After the culture was terminated,
a lactic acid production amount in the culture supernatant
was quantified using high pressure liquid chromatography
(HPLC). A lactic acid conversion rate was calculated as
follows.
[107] Lactic acid conversion rate = (lactic acid production

rate/ initial sugar concentration) x 100
[108] The lactic acid conversion rate of the strain was
93.5%. It was reported that a lactic acid conversion rate
of Bacillus polyfermenticus producing lactic acid was 60.7%
(Kim, K. M et al., J. Microbiol. Biotechnol., 19(2009)
1013-1018) . It was confirmed that in the case of Bacillus
licheniformis CJMPB361, a larger amount of lactic acid was
converted. Therefore, it was appreciated that the Bacillus
licheniformis CJMPB361, which is the novelisolated strain
in the present invention, produces lactic acid.
[109] (3) Endospore Formation Capacity
[110] Bacillus forms endospores for survival under stress
such as depletion of one or more of necessary nutrients, or
the like. Since the endospores has resistance against
extreme conditions such as ultraviolet rays, a high
temperature, low temperature drying and high pressure, or
the like, formation of the endospores . is important for
maintaining a survival rate of bacillus. .Therefore, the
Bacillus licheniformis CJMPB361 was cultured for a long
period, and endospore formation capacity thereof was
confirmed.
[Ill] The strain (0.1%) was inoculated in a BHI liquid
medium and cultured at 37D and 200 rpm for 24 and 48 hours.
The culture solution at each time was smeared on a BHI
solid medium, and a total cell was counted. In addition, a

culture solution heat-treated at 950 for 10 minutes was
spread on a BHI solid medium, and the number of endospores
was counted.
[112] As shown in Table 3, it may be appreciated that when
the Bacillus licheniformis CJMPB361 was cultured for 24
hours, an endospore formation rate was about 0.3%, and when
the Bacillus licheniformis CJMPB361 was cultured for 48
hours, the endospore formation rate was 78%. The Bacillus
licheniformis CJMPB361 according to the present invention
has excellent endospore formation capacity when cultured
for 48 hours or more, so that the Bacillus licheniformis
CJMPB361 as probiotics may maintain a high survival rate.
[113] [Table 3]

[114]
[115] {4) (3-Hemolysis
[116] p-hemolysis is an action of hemolyzing red blood
cells by producing phospholipase in harmful bacterial to
hydrolyze phospholipids supplied by the red blood cells.
[117] In order to confirm hemolysis by the isolated strain,
tryptic soy agar (TSA, Difco, USA) containing 5% sheep
blood (Kisan Biotech, Korea) was prepared. The strain was
streaked on the prepared blood agar medium and then

cultured at 37 for 24 hours. Then, as a result of
confirming the presence or absence of hemolysis, it was
confirmed that hemolysis did not occur as shown in FIG. 2.
[118] ■
[119] Example 3: Immune boosting Activity of Bacillus
Izcheniformls CJMPB361
[120] (1) Lymphocyte Proliferation Response Test
[121] After a BALB/c mouse (6 to 7 weeks old, Orient Corp.,
Korea) was sacrificed by an inhalation anesthesia apparatus,
the spleen was collected and slightly pressed using two
sheets of sterilized slide glass, thereby isolating
lymphocytes. The isolated lymphocytes were washed with a
RPMI 1640 (Invitrogen, USA), which is a cell culture medium,
three times. In addition, after removing red blood cells
in a cell suspension, the resultant was suspended at 2xl06
cell/ml in Roswell Park Memorial Institute (RPMI) 1640
medium containing 10% fetal bovine serum. A culture
supernatant■ obtained by culturing Bacillus lichenlformis
CJMPB361, which is a test sample, for 24 hours was
primarily diluted at 1:20 using the PRMI 1640 medium, and
then, two-fold serial dilution was performed.
[122] After spotting 100|i£ of the suspended lymphocytes
onto each well of a 96-well plate, 100^ of the prepared
sample was added thereto. After the plate was cultured at
37D in a 5% C02 incubator for 2 days, 10fi£ of titrated

thymidine (3H TdR) (New England Nuclear, Boston) was added
to each well and further cultured for 6 hours. After the
cultured cells were adsorbed using a glass fiber filter and
dried at room temperature for 24 hours, Melt-on
scintillator sheet (MeltLexTM A, Wallac) was put on a
filtermat and melted .on a constant temperature oven for 1
to 4 minutes. As a result of measuring an amount of 3H TdR
absorbed in cells using a liquid scintillation counter
(Microbeta 1450, Tailux), it was confirmed that the
Bacillus licheniformis CJMPB361 proliferated lymphocytes.
Therefore, after separating the culture supernatant of the
strain by molecular weight, the lymphocyte proliferation
activity was measured by the same method as described above.
As a result, in fractions of a molecular weight of 1,000
kDa or more and 100. kDa to 300 kDa, proliferation of the
spleen cells of the mouse was excellent as shown in FIG. 3.
[123] (2) Bone Marrow Cell and Thymocyte proliferation -
Response
[124] When isolating bone marrow cells, both ends of a
thigh bone of the BALB/c mouse were cut with scissors, and
a needle of a 10ml syringe filled with a RPMI 1640 medium
was put into the cut. thigh, thereby isolating bone marrow
cells. In addition, isolation of thymocytes was performed
by the same method as that in isolation of the spleen cells,
thereby isolating thymocytes from the thymus.

Proliferation response of the bone marrow cells and the
thymocytes was performed by the same method as that in the
proliferation response test of the spleen cells.
[125]
[126] As a result, fractions of the culture supernatant of
the Bacillus licheniformis CJMPB361 having a molecular
weight of 1,000 kDa or more and 100 kDa to 300 kDa
proliferated the spleen lymphocytes and the bone marrow
cells, and the proliferation response was not observed in
the thymocytes. Therefore, it was comfirmed that the
fractions did not affect proliferation of T cells (FIG. 4).
[127] (3) In-Vitro Effect on Spleen B cell of Mouse
[128] In order to confirm that which fractional material is
capable of inducing proliferation response of spleen cells
among fractions of the culture supernatant of the Bacillus
licheniformis CJMPB361 separated by • molecular weight, B
cells and non-B cells were separated from spleen
lymphocytes of a mouse. A general ratio between T cells, B
cells, non-T cells, and non-B cells in spleen cells of a
normal mouse was analyzed and investigated, and the result
was shown in Table 4. The B cells were isolated from the
spleen lymphocytes using a magnetic cell selection method
and analyzed using FACS. As a result, ratios of T cells, B
cells, and non-B and non-T cells were 0.1%, 94.6%, and 4.7%
respectively, so that the isolated B cells had a

significantly high purity. In addition, ratios of T cells,
B cells, non-B and non-T cells in the other non-B cells
obtained by separating the B cells from the spleen
lymphocytes were 46.8%, 22.3%, and 27%, respectively (Table
4).
[129] [Table 4]

[130]
[131] Three fractions of the culture supernatant of the
Bacillus licheniformis CJMPB361 having different molecular
weights were added to the isolated B cells and non-B cells,
and cultured by the same method as in the proliferation
response of the spleen cells. As a result, in the fractions
of molecular weights of 1,000 kDa or more and 100 kDa to
300 kDa, proliferation response of the B cells was observed
as shown in FIG. 5. Besides, proliferation response of non-
T and non-B cells was increased. A proliferation effect
was higher in the fraction of a molecular weight of 1,000
kDa or more (FIG. 5}.
[1-32] (4) In-Vivo Effect on Spleen B cell of Mouse
[133] In order to confirm an in-vivo effect of an immune
boosting material on proliferation of lymphocyte and bone
marrow cell of a mouse, and immune response, a fraction of

a culture supernatant of Bacillus licheniformis CJMPB361
having a molecular weight of 1,000 kDa or more was
administered to the abdominal cavities of mice. After 2
and 3 days of administration, the mice were killed and then
spleens were isolated. A weight of the isolated spleen was
measured, and a change in the number of spleen lymphocytes
was measured using FACS. In the case of administering
0.67mg, 1.35 mg, 2.7 mg, and 5.4 mg of the immune
strengthen material in the abdominal cavities of the mice,
sizes of the spleens were increased by 15%, 20%, 98%, and
51%, respectively. In the FACS analysis of the spleen
lymphocyte,. in the 0.67mg administration group, there was
no change in the ratio of the B cells. However, in the
1.35mg administration group, the ratio of the B cells was
increased from 41% to 45.3%, in the 2.7mg administration
group, the ratio of the B cells was increased to 47.6%, and
in the 5.4mg administration group, the ratio of the B cells
was increased to 49.7%. Therefore, the B cells were
proliferated depending on the concentration of the fraction
of the culture supernatant of the Bacillus licheniformis
CJMPB361 having a molecular weight of 1,000 kDa or more
(FIG. 6).
[134] (5) Adjuvant effect on increase in antibody
production
[135] It was investigated whether or not the fraction of

the culture supernatant of the Bacillus licheniformis
CJMPB361{1,000 kDa or more) functions as an adjuvant
producing an antibody against an antigen. BALB/c mice
were divided into three groups, and ovalbumin (OVA) and the
fraction of the culture supernatant of the Bacillus
licheniformis CJMPB361 (1,000 kDa or more) were
administered as follows. In A group, natural saline was
subcutaneously injected, in B group, 100|ig of OVA was
subcutaneously injected into backs of the mice, and in C
group, after subcutaneously injecting 100|ig of OVA into
backs of the mice, the fraction of the culture supernatant
of the Bacillus licheniformis CJMPB361 (1,000 kDa or more)
was injected into abdominal cavity every other day. lOOfig
(lmg/ml) of OVA, which was an antigen in immune response,
was ' subcutaneously injected into the mice and 100pi (2.7mg)
of the fraction of the culture supernatant of the strain
(1,000 kDa or more) was injected into the abdominal cavity.
In order to measure an antibody, after 3 days of final
injection, blood was extracted from inferior vena cava of
the mice and serum was isolated, and the antibody specific
to OVA was measured by an enzyme-linked immunosorbent assay
(ELISA) method. First, OVA was diluted at a concentration
of 2jig/ml in a coating buffer (0.03M Na?C03, 0.068M NaHC03,
pH9.4~9.8), added at an amount of 100fi£ in a 96-well plate,
and reacted at 40 for 15 hours. The plate was washed with

phosphate buffered saline (washing buffer) containing 0.1%
Tween 20 three times. The washed plate was reacted for 1
hour using a solution containing 1% bovine serum albumin in
order to block non-specific binding. After washing the
plate using the washing buffer five times, the prepared
serum of the mouse was serially diluted and added thereto,
and followed by a reaction for 2 hours. After the reaction
was terminated, the plate was washed with the washing
buffer five times. Then, a second antibody (goat anti-
mouse polyvalent immunoglobulins peroxidase conjugate) was
diluted and added, followed by reaction for 1 hour. The
plate was washed five times and a TMB substrate was added
thereto. After coloring for 30 minutes, 0.12M H2S04
solution was put into there to stop the reaction, and
absorbance was measured at 4 50nm.
[136] As shown in FIG. 7, in the control treated only with
normal saline, the antibody specific to OVA was not
measured, and the group treated only with OVA, an antibody
specific to IBA was produced, but an antibody titer was low.
It was confirmed that in the group treated with both the
OVA and the fraction of the culture supernatant of the
strain (1,000 kDa or more), the antibody titer was
significantly increased as compared to the group treated
only with OVA. Therefore, it means that the fraction of
the culture supernatant of the isolated strain (1,000 kDa

or more)in the present invention had an adjuvant effect on
antibody production against.antigen.
[137] (6) Effect on Cell Proliferation after Bone Marrow
Transplantation
[138] Based on the fact that the fraction of the culture
supernatant of the Bacillus licheniformis CJMPB361 (1,000
kDa or more) increases proliferation response of bone
marrow cells and spleen non-T and non-B cells as well as B-
cells, it was evaluated whether or not bone marrow cells
transplanted in a irradiated mouse by this immune material
proliferated. In order to damage bone marrow cells of the
mouse, the mouse was irradiated. In order to investigate a
minimum cobalt gamma irradiation dose capable of killing a
normal mouse within 10 days, after the mice were irradiated
at an irradiation dose of 7.5, 9, 10, and 12 Gy, and
survival rates were compared. It was shown that in the
case of irradiation with 7.5 Gy, an average lifetime was
about 9 to 10 days, in the case of irradiation with 9 Gy,
an average lifetime was 8 to 9 days, in the case of
irradiation with 10 Gy, an average lifetime was 7 days, and
in the case of irradiation with 12 Gy, an average lifetime
was 6 days. Based on the above-mentioned results,
allergenic bone marrow cells were transplanted into
irradiated mice, and the fraction of the culture
supernatant of the isolated strain (100 kDa or more) was

administered or was not administered thereto. Thereafter,
survival rates were compared. BALB/c mice were irradiated
with 7.5 Gy or 9 Gy, and the irradiated mice were divided
into three groups. In Group 1, normal saline was
administered, and in Group 2, allergenic bone marrow cells
(lxlO7 cell/mouse) obtained from a normal mouse were
transplanted. In Group 3, allogeneic bone marrow cells
were transplanted by the same method, and 2. 7mg of the
fraction of the culture supernatant' of the strain (1,000
kDa or more) was administered into the abdominal cavity.
Both of the normal saline and bone marrow cells were
administered into mouse tail veins, and the fraction of the
culture supernatant of the strain (1,000 kDa or more) was
administered into the abdominal cavity. The administration
as described above was entirely performed within 2 hours
after irradiation. The survival rate of the mice after the
experiment was observed for 30 days. As shown in FIG. 8A,
among the mice irradiated with 7.5 Gy, in. the group in
which normal saline was administered, all of the mice died
within 8 to 11 days. In the group in which only the bone
marrow cells were transplanted, the survival rate was 66%.
On the contrary, the group treated with both the bone
marrow cells and the fraction of the culture supernatant of
the strain (1,000 kDa or more), the survival rate was 100%
(FIG. 8A) . Meanwhile, in the case in which the irradiation

dose was increased to 9 Gy, in the group in which only
normal saline was administered, all of the mice died within
6 to 9 days, and in the group in which only the bone marrow
cells were transplanted, the survival rate was 33%.
However, in the group in which the fraction of the culture
supernatant of the strain (1,000 kDa or more) was
administered with bone marrow cell transplantation, the
survival rate was 66% (FIG. 8B) . In the case of the mice
that was survived until 13 days after, all of them were
alive for 30 days, , and it took about 12 days to reach a
normal weight. The reason of the results as described
above is that the fraction of the culture supernatant of
the isolated strain (1,000 kDa or more) induces
proliferation of the transplanted bone marrow cells to
increase the survival rate of the irradiated mouse.
Therefore, the immune strengthen agent or vaccine adjuvant
according to the present invention may be used for
stimulating proliferation response of transplanted bone
marrow cells in leukemia patients.
[139]
[140] According to the present invention, a novel Bacillus
licheniformis CJMPB361 may have an excellent effect of
producing digestive enzymes such as amylase, cellulase,
mannanase, and lipase and lactic acid, an excellent immune
strengthen effect, an excellent adjuvant effect to promote

production of an antibody in animals, and an induction
effect to enhance proliferation response of transplanted
bone marrow cells in a leukemia patient. Therefore, the
novel isolated Bacillus licheniformis CJMPB361 may be used
as an immune strengthen agent for animals and a vaccine
adjuvant as well as probiotics.
[141]

[CLAIMS]
[Claim 1]
A Bacillus licheniformis CJMPB361 producing digestive
enzymes and lactic acid, and having an immune boosting
activity, which is deposited under accession number
KCCM11269.
[Claim 2]
A culture product of Bacillus licheniformis CJMPB361
comprising a culture solution of Bacillus licheniformis
CJMPB361 (accession number KCCM11269), a concentrate
solution thereof, or a dried material thereof.
[Claim 3]
A probiotic formulation "comprising the Bacillus
licheniformis CJMPB361 (accession number KCCM11269) of
claim 1 or the culture product of claim 2.
[Claim 4]
A feed additive comprising the probiotic formulation
of claim 3.
[Claim 5]
The feed additive according to claim 4, wherein it is
in a form selected from a group consisting of a highly

concentrated solution (20 to 90%), powders, and granules.
[ciaim 6]
A feed comprising the feed additive of claim 4 or 5.
[Claim 7]
An immune boosting agent or vaccine adjuvant
comprising Bacillus licheniformis CJMPB361 (accession
number KCCM11269), a culture solution thereof, a
concentrated solution thereof, a dried material thereof, or
containing a probiotic formulation thereof as an active
component.
[Claim 8]
The immune boosting agent or vaccine adjuvant
according to claim 7, wherein the Bacillus licheniformis
CJMPB361 (accession number KCCM11269), the culture solution
thereof, the concentrate solution thereof, or the dried
material thereof contains a fraction having a molecular
weight of 100 to 300 kDa or 1,000 kDa or more, having an
immune strengthen activity.
[Claim 9]
A health functional food for boosting immune system
comprising Bacillus licheniformis CJMPB361 (accession

number KCCM11269), a culture solution thereof, a
concentrate solution thereof, a dried material thereof, or
containing a probiotic formulation thereof.
[Claim 10]
The health functional food according to claim 9,
wherein the food is in a form selected from a group
consisting of powders, granules, tablets, capsules, and
drinks.