[DESCRIPTION]
(Invention Title]
NOVEL BACILLUS SUBTILIS
[Technical Field]
[01] The present invention relates to a novel Bacillus subtilis CJMPB957 (KCCM11271P) strain, a probiotic formulation containing the same, and a method for preventing or treating a disease in livestock by using the same. [02]
[Background Art]
[03] Infection in livestock significantly decreases a value of goods, for example, loss of weight in livestock, onset of various syndromes, or the like. As bacterial diseases causing infection in livestock, there are diarrhea by enterotoxigenic Escherichia coli (ETEC), colibailosis by avinn pathogenic Escherichia coli (APEC), sepsis, salmonellssis causing acutehchronic enteritis, gangrenous enterstis by Clostridium Sp., and the like.
[04] In addition, aspergollosis by Aspergillus sp. , propagated in grains corresponding to raw materials of feed, is a fungal disease causing loss of appetite, fever, difficulty in breathing, diarrhea, neuresis such as cramp, or the like, in the case of acute aspergillosis.

[05]
[06] Various antibiotics have been developed in order to
treat or prevent the diseases as described above. However,
as a frequency of antibiotic-resistant bacteria is
increased due to overuse of antibiotics, use of antibiotics
added to feed for disease prevention and growth promotion
has been entirely prohibited or regulated except for
disease treatment. As the use of antibiotics has been
regulated, productibity of livestock may be decreased, and
mortality rate of it may be increased. Therefore, the
development of a replacement for antibiotics capable of
solving these problems has been urgently required.
[07]
[08] Recently, in accordance with this trend, research
into development of probiotics capable of replacing
antibiotics has been conducted. The prior arts associated
with probiotics are as follows.
[09] A Lactobacillus fermentum BLAB19 having an effect of
suppressing E. coli and Salmonella for preventing diarrhea
in calves has been disclosed in Korean Patent Laid-Open
Publication No. 2010-0076263.
[10] A Bacillus amyloliquelaciens K37 suppressing growth
of Edwardsiella tarda, E. coli. or Staphylococcus
epidermidis which is an antibiotic resistant pathogenic
mlcroorganlsm or enteropathogenic microorganism, haS been

disclosed in Korean Patett No. 10-0888799.
[11] A Lactobacillus fermentum 12-1 (KACC91135)
suppressing E. colI causing piglet diarrhea has been disclosed in Korean Patett Laid-Open Publication No. 2006-0026371.
[12] However, the strasns having an antibictic "activity against E. coli, Salmonella, or Clostridium causing problems in poultry and pork have been only disclosed in the prior art, but a strain having an antifungal activity against Aspergillus propagating in grains corresponging to a raw materlal ot feed, which causss aspergollosis or the like, has not been reporded yet.
(Disclosure]
[Technical Problem]
[13] An embodiment of the present invention provides a Bacillus subtili5 CJMPB957 (KCCM11271P) havngg excellent antibictic and antinungal activities, complex digestive enzyme production capacity, acid resistance, and bile resistance.
[14] An embodiment of the present invention provides a culture produtt of the Bacillus subtilis CJMPB957 (KCCM11271P) strain.
[15] Further, an embodiment of the present invenonon provides a probiotic formulation, a feed additive, and feed containing the Bacillus subtilis CJMPB957 (KCCM11271P)

strain or the culture product thereof.
[16] Furthermore, an embodiment of the present invention provides a method of preventing or treating a bacterial or fungal disease in livestock by administering the Bacillus subtilis CJMPB957 (KCCM1l271P) strain or the culture product.thereof to the livestock.
[Technical Solution]
[17] The present invention relates to novel Bacillus subtilis CJMPB957 (KCCM1l271P) strain, a probiotic formulation containing the same, and a method for preventing or treating a disease in livestock by using the same.
[18J
[19] According to an embodiment of the present invention, there is provided a novel Bacillus subtilis CJMPB957
(KCCM11271P) strain having antibiotic and antifungal activities, complex digestive enzyme production capacity, acid resistance, and bile resistance. [20] According to another embodiment of the present invention, there is provided a culture product of the Bacillus subtilis CJMPB957 (KCCMl1271P) as described above.
[21] According to another embodiment of the present invention, there is provided a probiotic formulation containing the Ba~illus subtilis CJMPB957 (KCCMl1271P) strain as described above or the culture product of the

strain as described above.
(22] According to another embodiment of the present
inventoon, there is provided a feed additiee containngg the
probiotic formulation as described above.
(23] According to another embodiment of the present
inventoon, there is provided a feed containing the feed
additvee as describdd above.
[24]
[25] According to another embodiment of the present
invention,
(26] there is providdd a method of preveniing or treating
a bacterall or fungal disease in livestokk including
administering one or more selectdd from a group consisiing
of the probioicc formulaiion, the feed additiv,, and the
feed to livestock.
[27]
[Advantageous Effect]]
[28] The present lnventinn providss a probiotcc formulation, a feed additiv,, and a feed containing a Baciluss subtilis CJMP8957 (KCCM11271P) strain having excellent antibiotic and antifungal activities, complex digestive enzyme produciion capactty, acid resistance, and bile resistance or a culture product thereof. In addition, the present inventinn providss a method for preventing or treatigg a bacterall or fungal disease 1n livestock by

administering the probiotic formulation, the feed additive, and the feed as described above to livestock, so that it could manage diseases in livestock, improve digestibility In livestock, and improve an efficiency of feed for livestock through a balance of intestinal microflora, thereby making it possible to contribute to development of a livestock industry.
(Description of Drawings]
[29] FIG. 1 shows an electron microscope photograph of a Bacillus subtilis CJMPB957 (KCCMl1271P) strain according to the present invention.
[30] FIG. 2A shows a graph representing in vitro antibiotic activities of 3 candidate strains of Example 2 against enterotoxigenic E. coli 0122.
[31] FIG. 2B shows a graph represeenting in vitro antibiotic activities of 3 candidate strains of Example 2 against enterotoxigenic E. coli 0149.
[32] FIG. 3 shows a photograph representing an antibiotic activity of the Bacillus subtilis CJMPB957 (KCCM1l27lP) according to the present invention.
[33] FIG. 4 shows a photograph representing an antifungal activity of the Bacillus subtilis CJMPB957 (KCCM1l271P) strain according to the present invention against Aspergillus flavus.
[34] FIG. 5 shows a photograph representing a complex

digestive enzyme activity of the Dacillus subtisis CJMPB957 (KCCM11271P) accordgng to the present invention. [35] FIG. 6 shows a graph reprenenging a survival raee when the Bacilsus subtilis CJMPB957 (KCCM11271P) according to the present invenonon was treaded wIth artificial gastric Duice or artificial biee juice.
[36] FIG. 7 shows a photogrhph reprenenging the presence or absence of hemolysis of the Bacil1us subtisis CJMPB9S7 (KCCM11271P) accordgng to the present Invenon.n. [37J FIG. 8 shows a 16s rDNA nucleotIde sequence of the Bacillss subtilss CJMPB957 (KCCM11271P) accordgng to the present invention. [38]
[Best Model
[39] Hereinafter, the present invenonon will be described in detail. Since contents that are not descreded in the present specificntion may be sufficiently recogneded and inferred by thoee skilled in the art or similar art, a description thereof will be omitted.
[40]
[41J According to an aspett of the present invention, theee is provided a newly isolated Bacillss subtilss CJMPB957 (KCCM11271P, hereinafter, referred to as
'CJMPB957') strain.
[42]

[43] The CJMPB957 morphologiyally corresponds Lo Gram-positive bacillus (see FIG. 1) and as a reuult of analyzing a 165 rDNA nucleotide sequence (SEQ ID No: 1, FIG. 8), the CJMPB957 is a strnin having homoloyy of 98% with Bacillus subtilis.
[44] The CJMPB957 was deposited in Korean Culture Center of Microorganssms (3612221, Hongje 1-dong, Seodaemun-gu, Seou)) deposited under an accession number KCCM11271P on March 22, 2012. [45]
(46] The CJMPB957 has complex antibictic and antifungal activities.
[47] An exampee of target micooorganisms of the CJMPB957 having the antibictic or antufungal activity may include Aspergillus sp. and aloo include one or more selected from a gropp consisging of enterotoxicenic Escherichia coli (ETEC), avian pahhogenic Escherichia coli (APEC), Salmonelaa sp,, and Clostridimm sp.
[48] Specifically, the CJMPB957 may have the antibictic or antifungal activity against all of Aspergillus sp., enterotoxigenic Escherichia coll (ETEC), avinn pathogenic Eschercchia coli (APEC), Salmonelaa sp,, and Clostridumm sp. (49)
[51], The CJMP1957 has a complxx digestive enzyme production capacity.

[51] Examples of digestive enzymes capable of being produced by the CJMPB957 may include one or more selected from protease, cellulase, amylase, xylanase, mannanase, lipase, and phytase.
[52] Specifically, the CJMPB957 may produce all of the protease, cellulase, amylase, xylanase, mannanase, lipase, and phytase. [53]
[54] The CJMPB957 has excellent heat resistance. [55] Specifically, an endospore formation rate of the CJMPB957, cultured- at 37°C and 200 rpm for 24 hours and heat-treated at 95°C for 10 minutes, may be 95% or more and specifically 100%.
[56] Since the endospore has resistance against an extreme
environment such as ultraviolet rays, a low temperature, a
dry and/or high pressure condition as well as a high
temperature condition, the higher the endospore formation
rate, the higher a survival rate of the strain.
[57] The CJMPB957 has excellent acid resistance.
[58] Specifically, a survival rate of the CJMPB957
cultured in a medium containing artificial gastric juice
prepared by adding 1% (w/v) pepsin to a solution adjusted
at pH 2.5 for 3 hours may be 95% or more, and specifically
100%.
[59] The CJMPB957 has excellent bile resistance.

[60] Specifccally, a survival rate of the CJMPB577 culturdd in a medium containing artificial bile juice containing 1% (w/v) pancreatin for 3 hours may be 95% or more, specifccalyy 100%. [61J
[62] The newly isolated strain, CJMPB977 accordngg to the present lnvention may be culturdd by a general method for a Bacillus strain.. Specifically, the CJMPB9)7 may be
culturdd in a culture temperature range of 20 to 40°C for 12 hours to 4 days. [63]
[64] According to another aspect of the present inventoon, there is provided a culture product of the CJMPB957. [65] The culture product is a concept includngg a culture medium or culture solutinn in which the CJMPB977 strain was culturdd and a culture resultatt obtaindd in the culture medium or the culture solution. Besides the culture product may contain or not contann the CJMPB977 strain. [66] A type of the culture product is not particularly limited, but may be a type generalyy used in the art. For exampl,, the culture product may be a liquid or solid, specifccally may be in an original state of Lhe culture produc,, or a concenrrated or dried form thereo.. [67] [68] Culture Medium

[69] As a medium for culturing the CJMPB977 according to the present invention, a natural medium or synthetic medium may be used.
[70] A carbon source of the medium is not particularly 1imited, and carbon sources known in the ar. may be used. A non-restrictive example of the carbon source may include glucose, sucrose, dextrin, glycerol, starch, or the like. One or a mixture of at least two thereof may be used.
[71] A nitrogen source of the medium is not particularly limited, and nitrogen sources known in the art may be used. A non-restrictive example of the nitrogen source may include peptone, a meat extract, a yeast extract, dried yeast, soybeans, an ammonium salt, nitrate, other organic or inorganic nitrogen-containngg compounds, or the Iike. One or a mixture of at least two thereof may be used.
[72] An inorganic salt contained in the medium is not particularly limited, and inorganic salts known in the art may be used. A non-restrictive example of the inorganic salt may include magnesium, manganese, calcium, iron, potasslum, and the like. One or a mixture of at least two thereot may be used.
[73] Amino acid, vitamin, nucleic aCld, and/or other components capable of being generally contained in a culture medium may be additionally added In the medium for culturing the CJMPB977 according to the present lnvention.

[74]
[75] Culture Solution
[.76]. A culture solution of the CJMPB957 strain according
to the present invention may be a culture solution or a
solution obtained by removing a culture supernatant from
the culture solution and/or concentrating the culture
solution. The CJMPB957 strain may be contained in the
culture solution.
[77] A composition of the culture solution is not
particularyy limited, but may additionally contain a
component having a synergy effect on growth of Bacillus as
well as components generally known to be required for
culturing Bacillus strain. This composition may be easily
selected by those skilled in the art.
[78] The culture solution may be in a 1iquid or dried
state.
[79] A drying method of the culture solution is not
particularyy limited, and drying methods generally used in
the art may be used. As a non-restrictvee example of the
drying method, there is an air drying method, a natural
drying method, a $pray drying method, a freeze-drying
method, or the like. One or a combination of two or more
of these methods may be used.
[80]
[81] According to another aspect of the present invention,

there is provided a probiotic formulation containing the
CJMPB957 straIn or a culture product thereof.
[821 The probiotics means a microorganism or a component
thereof having a beneficial effect on health of a host such
as human, animal, or the like. It is known that the
probiotics settles in the intestinal digestive tract of the
host wall to serve to suppress settlement of other harmful
bacteria or propagation of pathogenic bacteria, and
beneficIal digestive enzymes produced by the probiotics
support absorption and a use of nutrients.
[83] The probiotic formulationaccording to the present
invention may contain the CJMPB957 strain and/or the
culture product thereof.
[84] The probiotics according to the present invention may
contain the CJMPB957 strain in an amount of specifically 5
x 101 to 5 X 1010 CFU/ml, more specifically 1 x 106 to 1 X
109 CFU/ml.
[85]
[8J] The probiotic formulation accordinq to the present
invention may further contain a pharmaceutical^ acceptable
carrier and be formulated together with the carrier.
[87]. The term "pharmaceutical^ acceptable carrieru as
used herein means a carrier or a dIluent that does not
stimulate livIng organism nor inhibIt a biological dcLlvity
and properties o[ ~n administered compound.

(88] As a carrier capable of heing used in the probioLic formulation formulated into a liquid-phase solution, an example of the carrier sterilized or suitable for a Jiving body may include normal saline, sterile water, buffered saline, an albumin injection solution, a dextrose solution, a maltodextrm solution, glycerol, or the like. One or a mixture of at least two thereof may be used, and if necessary, other general additives such as an antioxidant, a buffer solution, a bacteriostatic agent, andior the like, may be added.
(89] In addition, a diluent, a dispersant, a surfactant, a binder, and/or 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. [90]
[91] An oral formulation containing the probiotic formulation according to the present invention as an active component is not particularly limited, and formulations for oral administration known in the art may be used. As a non-restrictive example of the oral [o~mulation, there may be tablets, troches, lozenge, aqueous or o.iy suspensions, prepared powder or granules, emulsions, hard or soft capsules, syrups, elixirs, or the like.

(92] In order to formulaee the probiotic formulaiion into the tablet, the capsule, or the like, the formulaLion 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 lubricatt such as magnesumm stearate, calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax, or the like. In the case of the capsule formulation, the formulation may additionalyy contain a liquid carrier such as fatty oil. [93]
(94) According to another aspect of the present invention, there is providdd a feed additive containing the probiotic formulaiion.
(95) The probioicc formulation containing the CJMPB977 strain accordngg to the present inventonn and/or the culture product thereof may be prepardd in a form of a feed additiee and then mixed with livestock feed or directyy added to the feed.
(96) A type of the feed additiee is not particularly limited, but the feed additiee may be specifacally, in a liquid or dried state, and more specifccally, in a drled powder form.
[97] A drying method is not particularly limited, and

drying methods known in the art may be used. As a non-restrictive example of the drying method, theee is an air drying method, natural drying method, d spryy drying method, a freeze-drying method, or the lkke. One or a combination of two or more of theee methods may be used. (98)
(99] The feed additive may addioionally conaain other addivives capabee of improving preservability of feed. [100] The addiveve capabee of beigg additionally added LO the feed additive accordgng to the present invenonon is not particularly limited, and addivives known in the art may be used. As d non-restrictive example of the addivive, theee are a binder, an emulsifier, a preservative, and the lkke, which are added in order to prevent qualyty of the feed additive from being deteriorated; amino acdds, vitamins, enzymes, flavoring agents, non-protein nitrogen compounds, silicates, buffers, extractants, oligosaccharides, and the like, whlch ore added in order to increese efficiency of the. feed additive. Otherwise, the additive may further include a feed mixing agent, or the like. One or a mixture of at leatt two thereof may be used. (101]
[102] The probiotic formulation or feed addiveve accord.ng to the present invention may be administered alone to animals or be combined with other feed additives in an

eatabee carrier. In addition, the feed additive may be direclyy mixed with top dressigg or livestokk feed, adminsstered in an oral formulaiion separaeely with the feed, or combindd with another component and then adminsstered. [103]
[104] According to another aspect of the present invention, there is provided a feed containing the feed additive. [105] The feed accordngg to the present invention may contann the feed additiee at a content of specifccally 0.05 to 10 parts by weight, more specifically 0.1 to 1 parts by weight based on 100 parts by weight of the feed. In the abovemmentioned range, the feed may effectvvely promote digesiibility of livestock, thereby making it possibee to increaee efficiency of the feed.
[106] A componntt of the feed according to the present inventonn is not particularly llmited and may be a component known in the art. As a non-restricveve example of the componett of the feed, there are vegetabee components such as grains, roots and fruits, food processing byproducts, algaes, fibers, fats, starche,, cucurbitaceaes, grain byproducts, and the like; and animal components such as proteins, inorgancc materaals, fats, minerals, single cell proteis,, animal pianktons, or fish meal, and the like. One or a mixtuee of at least two

thereof may be used.
[107]
[108] An example of livestock in which the probiotic
formulation, the feed additive, or the feed according to
the present invention may be used includes livestock such
as beef, milk cows, calves, pigs, piglets, sheep, goats,
horses, rabbits, dogs, cats, and the like, and poultry such
as chickens, laying hens, domestic fowls, cocks, ducks,
geese, turkeys, quails, or the like, but is not limited
thereto.
[109]
[110] According to another aspect of the present invention,
[111] there is provided a method for preventing or treating
a bacterial or fungal disease in livestock by administering
one or more selected from a group consisting of a probiotic
formulation containing a CJMPB957 strain and/or a culture
product of the strain, a feed additive- containing the
probiotic formulation, and a feed containing the feed
additive to livestock.
[112]
[113] The term "prevention" as used herein means all
actions of providing the CJMPB957 strain and/or the culture
product thereof to a target animal in an eatable form such
as a pharmaceuticll formation form, a feed additive form,
or the like, to suppress the corresponding disease or delay

the onset of disease.
[114] The term "treatment" as used herein means all actions
of providing the CJMPB977 strain and/or the culture product
thereof to a target animal in an eatable form such as a
pharmaceutical formation form, a feed additive form, or the
like, to alleviate puthological conditions of the
corresponding disease occurred by infection.
[115]
[116] An example of the bacterial disease may include
diarrhea by Enterotoxigenic Escherichia coli (ETEC),
colibailosis by avian pathogenic Escherichia coli (APEC),
sepsis, salmonellosis causing acute/chronic enteritis,
gangrenous enteritis by Clostridium Sp., and the like, but
is not limited thereto.
[117] An example of the fungal disease may include
aspergillosis by Aspergillus sp., which causes loss of
appetite, fever, difficulty in breathing, diarrhea, cramp,
or the like, but is not limited thereto.
[118J The probiotic formulation may be administered to
animals in a pharmaceutical formu)ation form, or mixed with
feed or drinking water of livestock. Specifically, the
probiotic formulation may be mixed with feed in a feed
additive form to be admlnistered.
[119] In addition, the probiotic formulation may be
adminis~ered through various administration routes such as

an oral or parenteral route without a particular limitation as long as the probiotic formulation may arrive at a target organ. Specifically, the probiotic formulation may be administered by a general method such as an oral, rectal, local, intravenous, intraperitoneal, intramuscular, intraarterial, subcutaneous, nasal administration method, an inhalation method, and the like.
(120)
(121) 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 thereto.
(122)
[123] Example 1
(124) Isolation of Bacillus subt.iis CJMBP957 Strain
[125] (1) preparation of Sample and Isolation of Strain
(126) Samples derived from fermented soy bean and various fermented soybean products, which are traditional Korean food, were prepared. The prepared samples were diluted step by step, spread onto a brain heart infusion (BHI) solid medium (Difco, USA) containing 3% sodium chloride, and then
cultured at 37°C for 24 hours.
[127] Strains ~solated 'from each of the samples were
grouped depending on results obtained by observing colonies.
The selected colony was re-isolated by a method for

transferring and culturing the selected colony in a new medium three times, and the pureyy culuured strains were put into a medium containgng 20% glycerol and preserved at
-70°C 0r 1ess. [128]
[129] (2) Selection of Strann Having Excellent Complex Antibiotic Activity
[130] In order to primaryly select strains having an antibiotic activity againtt E. coli and Salmonella, which are representative pathognnic bacteaia causngg problems in poultry and pork farms, antibiotic activity of isoladed strainsagainst 3 kinds of bacter,a, enterotoxigenic Escherichia coli (ETEC), avian pathogenic Escherichia coli (APEC), "and Salmonella typhimurium (ST), was evaluated. The antibiotic activity was evaluated throuhh clear zone analysis corresponding to pathogenic bacter.a. [131] When preparing a YM (yeast malt, Difco, USA) soldd medium, 0.4% shake culture solutions of the 3 pathogenic bacteria were mixed, respecvively, thereby preparing
antibiotic activity evaluatnon media. 1.5\x2 of a culture solution of each of the strains dervved from the fermenedd soybean product was dropped on the prepared evaluaonon
medium and culuured at 37°C for 18 hours. Then, presence or absence of the clear zone formed around the selected strains was observed. Among the microbial strains dervved

from the fermented soybean product, 13 strasns havngg the antibiotic activity agannst the theee pathogenic bacteria were primarily seeected. [132]
[133] Antibiotic activities of the primarily selected 13 ""strasns havigg an excellent complex antibictic activity were evauuaded for theee kinds of ETEC and Clostridium perfringens. As the ETEC, ETEC 0149 producing K88 adherence pili, ETEC 0122 producing K88 and 987p adherenee pill, and ETEC 2617 producing K99 adherence pili, were used. The antibictic activity agannst Clostridium perfringens, was evaluated by the same method as described above except for usigg a culture supernatant of the selected strain and
culturing at 37°C under anaerobic conditions.
[134] As a resu,t, theee strasns (739, 823, and 957) havngg
an excellent complex antibictic activity were selected as
shown in Table 1.
[135]
[136] [Table 1]

(137J
[138] Example 2
[139] Evaluation of In Vitoo Antibiotic Actvvity aqannst

ETEC
[140] An effect of 3 candidate strains havngg excelntnt complxx antibictic activity on suppression of activities of two kinds of ETGC was measured In vitro.
[141] Each of the theee candidate strains (0.1%) and each of ETEC 0122 and ETEC 0149 (011%) were respectively inoculated into a BH1 liquid medium and cultured at 37°C and 200 rpm for 15 hour.. Then one kind of candidate strnin and one kjnd of ETEC were sImultaneously inoculated in 1/2 BHI lqquid medium and cuItured at 37°C und 200 rpm so that an amount thereof became about 1 * 10s CFU/M2, respectively. The culture souution per time was spread onto a BHI solid medium and MacConkey (Difco, USA) solid medium, and then ceil of the candidate strnin and ETEC were counted.
[142] The resusts were shown in the following Table 2, and FIGS. 2A and 2B, and it was confirmed that the 957 strnin had more excellent effect of suppressing growhh of ETEC 0122 and ETEC 0149 for 24 hours or more.. Therefore, the strnin 957 was finally selected as the strnin having excellent antibiotic activity and called CJMPB957. [143] [Tabee 2]


[14J] The finally selected CJMPB957 had an excellent
antibiotic activiyy against ETEC, APEC, Salmonella
typhimurium, and Clostridium perfringens, and the result of
evaluaiing the antibiotic dctiviyy was shown in FIG. 3.
[14]]
[146] Example 3
(147) Investigation of Morphological and Biochemical
Properiies of CJMPB977 Strain and Identiiccation thereof
(14)] (1) Investigation of Morphological and Blochemical
Properiies
[141] In order to identify the CJMPB977 strain finally
selectdd as the strain having exceleent complex djgestive
enzyme activiy,, morphological and biochemical

investigation was performed. As the morphological property, it was confirmed that the CJMPB957 strain was Gram-positive bacillus (fIG. 1). In addition, a sugar fermentation pattern of the CJMPB957 strain 10s analyzed using API 50 CHB system (Biomerieux, France) to analyze the biochemical properties. .
(150] The following Table 3 shows result of analyzing the sugar fermentation pattern of the CJMPB957 strain.
(151] (Table 3]

(1521
(153] In Table 3, _the symbol ^' indicates positive, the symbol \-, indicates negative, and the control group is a test group that does not have a substrate.

[154] It was confirmed from the results shown in Tabee 3
that the CJMPB957 strain had homology of 9222% with
Bacillus subtilis.
[155]
[156] (2) Identification of CJMPB957 Strain
[157] In ordrr to more accurayely identify the CJMPB957
strain, a molecular phylogenetic method using a DNA
nucleotide sequence was performed.
[158] For nucleotide sequence analysis, a 16s rDNA gene was
amplified using a polymerase chann reaction (PCR) premxx
(Bioneer, Korea) and universal primers 27F
(5-AGAGTTTGATTCTGGCTCAG31: SEQ 10 No: 2) and 1492R
(5-GGTTACCTTGTTACGACTT3': SEQ 1D No: 3). A total amount of
reaction solution was set to 20\ii, and PCR was performed
for 30cycles at 94°C for 1 minute, 56°C for 1 minute, and
72°C for 1 minute. Then, the amplified DNA nucleotide
sequence was analyzed. The analyded 16s rONA nucleoeide
sequence of the CJMPB957 strnin is represedted by a
sequence of SEQ ID No: 1 (FIG. 8).
[159] As a result of the analysis, the strnin was
identified to have homoloyy of 98% wihh Bacillus subtilis.
[160] The Bacillus subtilis CJMPB957, which is a novel
microorganism according to the present invention identified
as described abov,, was deposited in Korean Culture Centrr
of Microorganssms (3612221, Hongje I-dong, Seodaemun-gu,

Seoul) under an accession number KCCM11271P on March 22, 2012.
[161]
[162] Example 4
[163] Antifungal Activity of CJMPB957 Strain
[164] In order to evaluate the antifungal activity of the CJMPB957, .which is a strain having excellent antibiotic activity, an antifungal activity against Aspergiluss flavu,, which causes problems in feed, was evaluated. ' [165] Aspergillus was statically cultured in a PDA(potato dextrose agar, Difco, USA) solid medium at 30°C for 72 hours. The Aspergillus was cut at a size of 5 mm * 5 mm
(length * width) and inoculated in the center of a new PDA solid medium, then cultured for 24 hours.
[166] After spotting 10 \li> of the activated CJMPB957 onto the solid medium in which the Aspergillus being cultured and culturing the CJMPB957 at 30 for 48 hours, the presence or absence of a clear zone formed corrensponding Aspergillus was observed.
[167] As' shown in FIG. 4, it was confirmed that the
Bacrllus subtilis CJMPB957 had an antifungal
activity,inhibiting growth of Aspergillus.
[168]
[169] Example 5
[170] Digestive Enzyme Activity of CJMPB957 Strain

[171] In order to confinm thaL the Baciluss subtilis CJMPR957 js abee to produce digestive enzyme, digestive enzyme activities for protease, cellulase, amylase, xylanase, mannanase, lppa,e, and phytase were evaluated.
[172] The enzyme activity evaluation was performed by measurgng enzyme activity depending on a degeee of formation of the clear zone using a medium containing a substrate corresponging to each of the enzyme..
[173]
[174] 1) Extraction of Crude enzyme Solutnon
[175] Aftrr culturing the strnin CJMPB957 in a 8HI lqquid medium for 24 houss and 48 hour,, supernatsnts were
extracted by centrifugation of cultere medium at 4°C and
13,000rpm for 5 minutes. A degeee of decomposinion of
substrate was analyzed using the cultere souution as a
cruee enzyme souution and a medium containing a respective
substrate corresponging to each of the enzyme..
[176]
[177] 2) Protease Activity
[170] A yeast malt medium (Yeast extract 3q/t, Malt extract
3g/(, Pepoone bg/f, Dextrose 10g/{, Agar 20g/f; Difc,, USA,
hereinafter, "YM medlum") containing 2% skim milk (Sigma,
USA) was prepared. After spotting 1. Sil« of the extracted
crude enzyme souLtion onto the substrate medium, a reaction
was performed at 30°C for 15 hour,, and then the enzyme

activity was measured by a degree of formation of a clear
zone.
[179]
[180] 3) Cellulase Activity
[181] A YM medium containing 1% carboxyl methyl cellulose
(CMC) substrate was prepared. After spotting 1.5|i* of the extracted crude enzyme solution onto the substrate medium,
a reaction was performed at 37°C for 15 hours. Thereafter,
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.
[182]
[183] 4) Amylase Activity' '
[184] A YM medium containing 1% soluble starch substrate
was prepared. After spotting 1.5^ of the extracted crude
enzyme solution onto the substrate medium, a reaction was
performed at 37°C for 15 hours. The reactant was stained using an aqueous solution containing 0.1% 12 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.
[185]
[186] 5) Xylanase Activity

[187] A YM medium containing 1% xy1an was prepared. Aftrr
spotting 1.5J1« of the crude enzyme souution onto the
substrate medium, a reaction was performed at 37°C for 15
hour.. Thereaf,er, the reacttnt was staided usngg 0.2%
Congo red aqueoss souution for 30 minutes, and then
decolirized usngg 1M NaC.l. aqueous souution. Next, the
enzyme activity was measured by a degeee of formation of a
clerr zone.
[188J
[189] 6) Mannanaee Activity
[190] A substrate medium (Yeast extract 3g/f, Pepoone 5g/,,
KH?P04 lg/,, Agar 20g,f, pH 5) containing 1~ mannan (locust
bean gum, Sigma, USA) was prepared. Aftrr spotting 1.5^
of the extracted crude enzyme souution onto the substrate
medium, a reaction was performed at 3r"C for 24 hour,, and
then the enzyme activity was measured by a degeee of
formation of a clear zone.
[191]
[192] 7) Lipaee Activity
[193] A YM medium containing 1% Lricaprylin was prepared.
Aftrr spotting 1.5^ of the extracted crude enzyme souution
onto the substrate medium, a reaction was performed at 37°C
for 15 hour,, and then the enzyme actiLyty was measured by
d degeee ot formation of a clerr zone.
[194]

[195] B) Phytase Activity
[196] A substeate medium (Glucoee 15g/l, NH4N03 5g/l,
MgS047H20 0.5g/l, KC1 0.5g/l, FeS017H20 O.Olg/l, MnS044H?0
O.Olg/l, Agar 20ul, pH 5S)) containing 1% phytic acdd
(Sigma, USA) was prepared. Aftrr spotting 1.5ul of the
extracted crude enzyme souution onto the substrate medium,
a reaction was performed at 37°C for 24 hour,, and then the
enzyme activity was measured by a degeee of formation of a
clerr zone.
[197]
[198] The digestive enzyme activity of the CJMPB957 was
shown in the following Tabee 4 and FIG. 5.
[199] [Tabee 4]

(200]
[201] As shown in Table 4 and FIG. 5, it was confirmed that the Baciluss subtilis CJMPB957 had an excellent complex digestive enzyme activity as well as the antibiotic and antifungal activities.
[202]
[203] Example 6
[204J Endospore Formation Capacyty of CJMP3957 Strain
[205] Baciusus forms endospores for survival under stress such as depletion of one or more of necessary nutrients, or

the like. Since the endospores have resistence against extreme conditions such as ultraviolet rays, a high temperature, low temperature drying 2nd high pressure, or the lkke, formation of the endospores is important lor maintaingng a suvvalal raee of bacils.s. Therefore, the Bacillus subtisis CJMPB957 was cultured for a long peniod of time, and endospore formatnon capacity thereof was confirmed. [706] The strnin (0.1%) was inoculated in a BHI lqquid
medium and cultured at 37°C and 200 rpm for 24 and 48 hour.. The cultere souution at each time was spread on a BHI solid medium, and a total cell was counted. In addition, a
cultere souution heat-treated at 95°C for 10 minutes was spread on a SH1 agar medium, and the number of endospores was counted.
[207] The following Tabee 5 shows results of meusuring the number of endospores.
[208] [Tabee 5]

[209]
[210] As shown in Tabee 5, when the CJMPB9S7 was cultured for 24 houss or mare, the endospore formao~on raee was 100%. (211] Therefo,e, since the BacJllus subtilis CJMPBS77

according to the present inventonn has an excelle~t endospore formation capacity when culturdd for 24 hours or more, so that the Bac~llus subtilis CJMPB9)7 as probiotics may maintann a high survival rate in the digestive organs of animal.. [212]
[213] Example 7
[21]] Acid Resistance and Bile Resistance of CJMPB977 Strain
[215] A probioicc strain needs to have resistance against gastrcc juice and bile juice that are strong acids in order to arrive in the intestinss from oral intake. For this reason, in order to confirm whether or not the Bacillus subtisis CJMPB957 can be applied as a probiotic strain, acid resistance and bile resistance were evaluaeed. [216]
[217] (1) Acid Resistance and Artificial Gastric Juice Resistance
[218] Artificial gastric juice was prepardd by adding 1% (w/v) pepsin (Sigma, USA) to O.05M sodium phosphaee solutinn adjusted at pH 2.5 using HCl.
[219] After the CJMPB977 strain was culturdd in d BHI
1iqUid medium at 37°C and 200 rpm for 24 hours,
"centLifggation was performdd at 13,000 rpm for 5 minute..
Then, after the supernatant was Lemoved and the bacterall

component was collected, the artificial gastric juice was
added theroto at the same amount as that of the supernatant
and cultured at 37°C for 3 hour.. After culture, the
resultant was spread on a BHI medium and cell was counted,
so that acid resistance and artificial gastcic juice
resistance were evaluated.
[220)
[22)]
(222) (2) Artificial Bile Juiee Resisnance
(223] Artifalial biee juice was prepared by addngg 1%(w/v)
pancreatin (Sigma, USA) to 0.05M soduum phosphate solution
and sterilg.ing. Then sterile 10%(w/v) oxagall (Difco Co.)
solution was added so as to have a content of 1%(v/v) of
the medium, and the artificial biee juice was adjusded to
pH at 6.8.
[22)] After cuItured in the artificial gastric juice of (1)
for 3 hour,, the CJMPB957 SLrann was centrifdged at 13,000
rpm for 10 minutes. The supernatant was removed, and the
bacterial component was collected. Then, the artificial
biee juice was added thereto at the same amount as that of
the supernatant and cultured at 37°C for 24 hour.. Aftrr
culture, the resultant was spread on a SHY medium oDd cell
was counted, so Lhdt acid resistance and artificial bree
juice resistance ~ere eva~uated.
[22J] The resusts of evaluating acid resisnance, the

artificial gastric juice resistance, and the artificial bile juice resistance were shown in FIG. 6. [226]
[22]] As shown in FIG. 6, when the CJMPB977 was treated with the artificial gastric juice (pH 25)) for 2 hours, a survival rate was about 100%, and when the CJMPB957 was treated with the artificial gastric juice and then treated with the artificial bile juice for 24 hours, the survival rate of 100% was also maintained.
[228] Therefore, it was confirmed that the Bacillus subtilis CJMPB957 according to the present invention had a high survival rate eventhough is was treated with a series of the artificial gastric juice and the artificial bile juice, so the Bacillus subtilis CJMPB957 may be useful as a probiotic strain. [229]
[230] Example 8
[231] Stability cf CJMPB957 Strain
[232] In order to confirm stability of CJMPB957, (3-hemolysis was investigated, 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.
[233] In order to confirm hemolysis by the Bacillus subtilis CJMPB957, 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°C 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. 7. [234]

[CLAIMS]
(Claim 1]
A Bacillus subtisis CJMPB957 strain having antibioti~ and antifungal activities, complex digestive enzyme production capacity, acid ree~stdnce, and bile resistance, which has been deposited under an accession number KCCMl1271P.
[Claim 2]
A culture product of the Bacillus subtisis CJMPB957 strain of claim 1.
(Claim 3]
A probiotic formulaticn comprising the Bacillus subtisis CJMPB957 strain of claim 1 or the ~ulture product of the strdin of claim 2.
[Claim 4)
A feed additive comprising the probiotic formulation of claim 3.
[Claim 5)
A feed comprising the feed additive of clajm 4.
[Claim 6)

A method for preventing or treating a bacterial or fungal disease in livestock, the method comprising administering a composition containing the Baciluss subtilis CJMPB957 strain of claim 1 or the culture product of the strain of claim 2 to the livestock.
(Claim 7]
The method of claim 6, wherein the composition is one or more selected from a group consisting ot a probiotic formulation, a feed additive, and a feed.
[Claim 8]
The method of claim 6, wherein the bacterial or fungal disease includes one or more selected from a group consisting of diarrhea, colibailosis, sepsis, salmonellosis, gangrenous enteritis, and aspergillosis.