PH12014502245B1

PH12014502245B1 - Novel separated bacillus licheniformis and probiotics using same

Info

Publication number: PH12014502245B1
Application number: PH12014502245A
Authority: PH
Inventors: Seung Hee Back; Si Yong Yang; Seo Hyung Woo; Hyo Seel Seo
Original assignee: Cj Cheiljedang Corp
Priority date: 2012-04-05
Filing date: 2014-10-03
Publication date: 2014-12-15
Also published as: MY174997A; JP6129294B2; JP2015517807A; KR20130113108A; WO2013151364A1; PH12014502245A1; KR101370943B1

Abstract

The present invention relates to a novel Bacillus licheniformis CJMPB283 (accession number KCCM11270P) producing digestive enzymes and having oxidative capacity for ammonia and nitrous acid, and a use thereof.

Description

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 product. In addition, the probiotic product may further contain a feed mixture, or the like, but is not limited thereto.

As an example of the oral formulation containing the probiotics as an active component, there are tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, and soft capsules. In order to formulate the probiotics into 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.

According to another embodiment of the present invention, there is provided a feed additive containing the probiotic product as an active component.

Generally, Bacillus forms endospores, thereby be significantly stable against heat. Therefore, the novel isolated Bacillus licheniformis CJMPB283 may be prepared in a form of the feed additive and then mixed with feed, or directly added to feed. Bacillus licheniformis CJMPB283 contained in feed according to the present invention may be in a liquid state or dried state, and specifically, in a dried powder form. As a drying method, an air drying method, a natural drying method, a spray drying method, and a freeze-drying method may be used, but the present invention is not limited thereto. Bacillus licheniformis

CJMPB283 according to the present invention may be mixed in the powder form at a content ratio of 0.05 wt% to 10 wt%, specifically 0.1 wt% to 1 wt% based on a weight of the feed.

In addition, the feed additive for aquaculture may further contain general additives capable of improving preservability of feed.

In the present invention, an example of the feed 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, animal planktons, fish meal, or the like, but the present invention is not limited thereto.

The feed additive for aquaculture according to the present invention may be immersed, sprayed, or mixed, thereby be added to the feed for aquaculture and then used.

Fish species, being fed the feed containing the feed additive according to the present invention, may include farmed fish or crustaceans, and more specifically, flatfish, sea breams, shrimp, tilapias, salmon, eels, trout, or the like, but are not limited thereto.

In a composition of the feed for aquaculture containing the feed additive according to the present invention, the feed additive may be mixed at a content of 0.05 wt% to 0.5 wt% based on a dried weight of animal feed.

According to another embodiment of the present invention, there are provided a water depruative for aquaculture, containing the Bacillus licheniformis CJMPB283 as an active component, and a method of improving water quality of a fish farm by spraying the water deprurative before or during farming in the fish farm.

The novel isolated Bacillus licheniformis CJMPB283 according to the present invention may be used to decrease contents of ammonia and nitrous acid existing in an aquaculture environment.

Fish species, for which the water depurative according to the present invention may be used in the fish farm, may include fish or crustaceans, specifically, flatfish, sea breams, shrimp, tilapias, salmon, eels, trout, or the like, but are not limited thereto.

As the water depurative according to the present invention, the novel isolated Bacillus licheniformis

CJMPB283 may be prepared in a form of the water depurative, or the strain and/or the probiotic product may be directly sprayed. Bacillus licheniformis CJMPB283 in the water depurative according to the present invention may be in a liquid or dried state, and specifically, in a dried powder form. As a drying method, an air drying method, a natural drying method, a spray drying method, and a freeze-drying method may be used, but the present invention is not limited thereto. The Bacillus licheniformis CJMPB283 according to the present invention may be mixed in a powder form at a content ratio of 0.05 wt% to 10 wt$%, specifically 0.1 wt% to 1 wt% based on a weight of feed.

As an acceptable carrier, which is sterilized or suitable for a living body, in the water depurative, 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, such that the probiotic product 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.

Advantageous Effects

According to the present invention, a novel isolated

Bacillus licheniformis CJMPB283 may have an excellent digestive enzyme activity like amylase, cellulase, and protease and excellent oxidative capacity for ammonia and nitrous acid, so that the novel isolated Bacillus licheniformis CJMPB283 may improve water quality while farming fish and shrimp.

Therefore, the novel Bacillus licheniformis CJMPB283 may be used as a water depurative as well as probiotics.

Description of Drawings

FIG. 1 is a graph showing results obtained by quantitatively evaluating ammonia consumption amounts of primarily isolated strains according to the present invention in a medium containing ammonia.

FIG. 2 is a graph showing results obtained by quantitatively evaluating nitrous acid consumption amounts of the primarily isolated strains according to the present invention in a medium containing nitrous acid.

FIG. 3 1s a graph showing results obtained by evaluating growth capacity of secondarily isolated strains according to the present invention under facultative anaerobic and anaerobic conditions.

FIG. 4 1s a graph showing results obtained by evaluating salt resistance of the secondarily isolated strains according to the present invention.

FIG. 5 1s a graph showing results obtained by ee ————————— = 14 evaluating ammonia decreasing efficacy of the secondarily isolated strains according to the present invention, depending on water quality improvement evaluation in a water tank of young flatfish.

FIG. 6 1s an electron microscope photograph of the

Bacillus licheniformis CJMPB283, which is a finally isolated strain according to the present invention.

Best Mode

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.

Example 1: Isolation of Strain Oxidizing Ammonia and

Nitrous Acid (1) Preparation of Sample and Isolation of Strain

Strains derived from good fermented-soybean products such as soy bean paste, fermented soy bean, soy sauce, or the like, which are traditional Korean food were isolated as samples. The samples were spread on BHI agar medium (Difco, USA) with 3% NaCl and then cultured at 37°C for 24 hours. Strains isolated from each of the samples were grouped depending on results obtained by observing colonies.

The isolated colony was re-isolated by a method of transferring and culturing the selected colony in a new medium three times, and the purely cultured strains were put into a medium containing 20% glycerol and preserved at -70°C or less. (2) Evaluation of Oxidizing Ammonia and Nitrous Acid

In order to primarily isolate strains oxidizing ammonia and nitrous acid, which are main causes of water pollution, oxidation of ammonia and nitrous acid was evaluated.

In order to evaluate oxidation of ammonia, an ion medium containing ammonia ((NH4),SOs 4.95g/L, K,HPO, 8.82g/L,

MgSO4 (1M solution) 1.1ml/L, CaCl,(1M solution) 0.3ml/L,

FeSO4(30mM solution) 0.5ml/L, CuSO,(50mM solution) 0.04ml/L,

NaH,POs 0.7g/L, 5% (W/V) NayC0Os3 anhydrous 12ml/L) and an ionic medium containing nitrous acid (CaCl, 0.01g/L,

MgSO4- 7TH20 0.1g/L, EDTA 1.4mg/L, FeSO4* 7H0 5mg/L, H2S04 0.5

W/L, NaMoO4 + 2H,0 0.05mg/L, MnCl, * 4H,0 0.1lmg/L, CoCl;* 6H20 0.00Img/L, ZnSO4* 7H,0 0.05mg/L, CuS04 :5H,0 0.01lmg/L, NaNO, 0.21g/L, KHPO, 0.3mg/L) were prepared. The isolated strains (0.01%) were each inoculated into 5ml of two media prepared as described above and then cultured at 30°C for 14 days. After the cultured solutions were centrifuged and

Iml of each of the culture supernatants was extracted, an indicator (sulanilic acid, N, N-Dimethyl-l-naphylamine) allowing a color to be changed when nitrous acid exists was added by 1004. The culture supernatant to which the indicator was added was reacted at 25°C for 10 minutes, and a —————————————————————————————— rr 16 whether the color changed or not was visually observed. In the case in which nitrous acid existed or was formed, the color of the culture solution was changed into dark purple, and in the case in which nitrous acid did not exist or was consumed, a change of the color was not observed.

About 1000 isolated strains were evaluated, 7 strains oxidizing both ammonia and nitrous acid, 2 strains having excellent ammonia decomposition efficacy, and 1 strain having nitrous acid decomposition efficacy were selected as shown in Table 1. [Table 1]

Strain Oxidation of Oxidation of

Number Ammonia Nitrous Acid

Example 2: In vitro Evaluation of Oxidizing Ammonia and Nitrous Acid (1) Quantitative Evaluation of Ammonia for Primarily

Selected Strain

In order to evaluate an effect of decreasing ammonia depending on oxidation of ammonia by the primarily isolated strains, a consumption amount of ammonia was quantified in a medium containing ammonia.

The 10 primarily isolated strains (0.1%) were each inoculated into a BHI liquid medium and then cultured at 37°C and 200 rpm for 15 hours, thereby activating the strains. The activated strains (1%) were each inoculated into a medium containing ammonia ((NHg),S0; 0.5g/L, NaH,PO, 13.5g/L, KpHPO, 0.7g/L, MgSO4-° 7H,0 0.1g/L, CaCl," 2H,0 0.18g/L, NaHCO; 0.5g/L, FeCls- 6H,O 0.014g/L, glucose 0.5g/L) and cultured at 37°C and 200 rpm. During the culturing process, culture solutions cultured for 6, 12, 24, and 36 hours were extracted and centrifuged. Then, only culture supernatants were recovered. An amount of ammonia remaining in the recovered culture supernatant was quantified.

As a result, it was confirmed that a content of ammonia in the culture supernatant was decreased for all of the 10 isolated strains as shown in FIG. 1. Particularly, in the cases of strains Nos. 283, 296, 303, and 396, the content of ammonia was rapidly decreased after 6 hours, corresponding to an initial culture step. In order to confirm that ammonia was oxidized and formed nitrous acid, the indicator allowing a color change when nitrous acid exists was added and the color reaction was observed. As a result, a change in the color was not observed in an initial stepe, but in each of the samples extracted when the culture was terminated, colors of all of the culture

Supernatants were changed into dark purple. Therefore, it is appreciated that all of the primarily isolated strains oxidize ammonia into nitrous acid. (2) Quantitative Evaluation of Nitrous Acid for

Primarily Isolated Strains.

In order to evaluate an effect of decreasing nitrous acid depending on oxidation of nitrous acid by the primarily isolated strains, a consumption amount of nitrous acid was quantified in a medium containing nitrous acid.

The 10 primarily isolated strains (0.1%) were each inoculated into a BHI liquid medium and then cultured at 37°C and 200 rpm for 15 hours, thereby activating the strains. The activated strains (1%) were each inoculated into a medium containing nitrous acid (NaNO; 0.5g/L, NaH,PO, 13.5g/L, KHPOs; 0.7g/L, MgSO,+7H,0 0.1g/L, CaCl, * 2H,0 0.18g/L, NaHCO; 0.5g/L, FeCls* 6H,0 0.014g/L, glucose 0.5g/L) and cultured at 37°C and 200 rpm. During the culturing process, culture solutions cultured for 6, 9, 12, 24, and 30 hours were extracted and centrifuged. Then, only culture supernatants were recovered. An amount of nitrous acid remaining in the recovered culture supernatant was quantified.

As a result, it may be confirmed that a content of nitrous acid in the culture supernatant was decreased for ¢5 all of the 10 selected strains as shown in FIG. 2.

Particularly, in the cases of strain Nos. 102, 109, 251,

NOVEL SEPARATED BACILLUS LICHENIFORMIS AND PROBIQTLICS | Lins

USING SAME

Technical Field

The present invention relates to a novel probiotics and a use thereof.

Background Art

An aquaculture industry has become an important economic means in various countries, and consequently, generation of diseases in farmed fish has a large influence on economic development (Jose Luis Balcazar et al.,

Veter.Microbio., 114(2006):173-186).

In general, in order to treat diseases generated in farmed fish and improve water quality, antibiotics, antimicrobial agents, chemicals have been used. However due to excessive uses thereof, problems such as an increase of resistant bacteria, residues in the farmed fish, water pollution in the surroundings, and the like, were generated, therefore a use thereof has been restricted. In addition, as the people’s interest in whether or not the antibiotics have used while aquaculturing was increased, an alternative material such as an organic acid, probiotics, a non- specific immune stimulator, a natural material, or the like, has been urgently developed in order to basically block the antibiotics (Fish Disease Diagnosis and Treatment

253, and 268, the content of nitrous acid was rapidly decreased after 6 hours, corresponding to an initial culture step. In order to judge whether or not nitrous acid was oxidized, the indicator allowing a color change when nitrous acid exists was added, and the color reaction was observed. As a result, a dark purple color was observed in initial medium, but in each of the samples extracted when the culture was terminated, a color of the culture supernatant was not changed. Therefore, it is appreciated that the primarily isolated strains oxidized nitrous acid, so there was no nitrous acid. (3) Evaluation of Growth Capacity

As a water quality environment, aerobic, facultative anaerobic, and anaerobic conditions co-exist, and particularly, since an anammox process (process in which ammonia is oxidized into nitric acid under anaerobic condition) proceeds under the anaerobic condition, it is an important that the isolated strains can grow under various conditions. Therefore, growth capacity of the 10 primarily isolated strains was evaluated under facultative anaerobic and anaerobic conditions.

The 10 primarily isolated strains(0.1%) were each inoculated into a BHI liquid medium, and then cultured at 37°C under the facultative anaerobic and anaerobic conditions for 24 hours. Most of the isolated strains grew under both of the facultative anaerobic and anaerobic conditions, and particularly, in the cases of strain Nos. 253 and 268, growth capacity under the anaerobic condition was excellent, and in the case of strain No. 283, growth capacity under the facultative anaerobic condition was most excellent (FIG. 3). Therefore, strain Nos. 253, 268, and 283 having excellent growth capacity under facultative anaerobic and anaerobic conditions were secondarily isolated. (4) Evaluation of Salt Resistance

In order to evaluate an activity of the strain in fresh water and sea water, growth capacity of the secondarily isolated strains was evaluated in a medium containing sodium chloride.

The three isolated candidate strains (0.1%) were each inoculated into BHI liquid media containing 0%, 3%, 5%, and 10% sodium chloride and cultured at 37°C and 200 rpm.

During the culture, each of the samples cultured for 8, 10, and 22 hours was extracted, and absorbance thereof was measured.

As a result, it was confirmed that as shown in FIG. 4, all of the three strains grew in the medium containing 3% sodium chloride. Particularly, in the case of strain No. 283, growth capacity in the medium containing 5% sodium chloride was excellent as compared to other strains.

Therefore, it is appreciated that the three secondarily isolated strains, strains Nos. 253, 268, and 283, may be applied under the sea water condition as well as the fresh water condition.

Example 3: In vivo Evaluation and Final Isolation of

Strains

Evaluation of water quality improvement capacity of the 3 secondarily isolated strains was performed in a fishfarm of young flatfish. The young flatfish were fed with a commercial feed for satiation and randomly distributed in experimental water tanks (64L). After distributing experimental fish, farming water in all of the experimental water tanks were all recovered. Then 50ml of the farming water was sampled in each of the water tanks after 0, 24, 48, 72, 92, and 102 hours. The secondarily selected strain was diluted to about 1x10° CFU/3ml and then put into the water tank every 24 hours for 5 days. In centrol groups, the same amount of sea water was put thereinto. After extracting a sample, a concentration of ammonia was analyzed, and the remained sea water sample after analysis was stored at -20°C. Thereafter, the stored sea water sample was used to analyze nitrite and nitrate.

For analyzing water quality, three categories of ammonia, nitrite, and nitrate were analyzed. The analysis was performed using a water quality analyzer (RQflex 10, Merck,

Germany) after reacting the sea water samples with a reagent of a kit, respectively.

As a result, after 72 hours, an effect of removing ammonia was shown (FIG. 5), and after 120 hours, the effect was further increased. After 140 hours, the flatfish started dying due to toxicity of ammonia in all of the water tanks. Among the three secondarily selected strains, the strains Nos. 253 and 283 had significantly high ammonia removing efficacy and also decreased mortality as compared to the control groups. Accordingly, it may be appreciated that the addition of strain oxidizing ammonia and nitrous acid to farming water (water used to farm fish (flatfish)) may increase a survival rate of the fish. Finally, the strain No. 283 having the excellent ammonia removing efficacy and growth capacity under facultative anaerobic and anaerobic conditions was isolated.

Example 4: Identification of Isolated Strain and

Investigation of Morphological and Biochemical Properties thereof (1) Investigation of Morphological and Biochemical

Properties

In order to identify the finally isolated strain, morphological and biochemical investigation was performed.

The morphological properties were as follows. The strain was a Gram-positive strain, and it was confirmed that the strain was a bacillus from an electron microscope photograph (FIG. 6). In order to analyze the biochemical properties, a sugar fermentation pattern of the strain was analyzed using API 50 CHB System (Biomerieux, France). [Table 2]

Control Group | - | ~~ Esculine [| +

Glycerol

Erythritol = Cellobiose

D-Arabinose | - | ~~ Maltose | +

L-Arabinose | + | Lactose | - [Ribose | + | Welibiose | _-

D-Xylose

L-Xylose | - | Trehalose | +

Adonitol - | ~~ Inuline | +

B-Methyl-Xyloside | - [ = Melezitose | -

D-Raffinose

DGlucose | - [Amidon | +

D-Fructose | ~~ | Glycogen =

Xylitol = _ L-Sorbose | - | p-Gentiobiose

Rhamnose [| - | = D-Turamose | =

Dulcitol | - | = D-Lyxose | -

D-Tagatose =

Mannitol | + | = D-Fucose | =

N-Acetyl Glucosamine | - [| ~~ Gluconate [| -

Amygdalin | - | 2-ceto-gluconate =

Arbutin | - | S-ceto-gluconate | - +: positive, -: negative (2) Identification of bacteria

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 polymerase chain reaction (PCR) premix (Bioneer, Korea), universal primers 27F (5'AGAGTTTGATCMTGGCTCAG3': SEQ ID No: 2) and 1492R

(3'GGYTACCTTGTTACGACTT3': SEQ ID No: 3). A total amount of reaction solution was set to 2044, and PCR was performed for 30cyclesat 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 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 96% with Bacillus licheniformis. Therefore, the isolated strain was named Bacillus licheniformis CJMPB283 and the novel strain according to the present invention was deposited in Korean Culture Center of Microorganisms (accession number KCCM11270P) on March 22, 2012.

Example 5: Analysis of Probiotic properties of

Bacillus licheniformis CJMPB283 (1) Digestive Enzyme Activity 1) Extraction of Crude Enzyme Solution

After culturing the isolated strain in a BHI liquid medium for 24 hours, a degree of decomposition of substrate was analyzed by using the culture solution as a crude enzyme solution and a medium containing a respective substrate corresponding to each of the enzymes. 2) Protease Activity

A yeast mold (YM) medium (Difco, USA) containing 2% skim milk (Sigma, USA) was prepared. After spotting 24 of each of the extracted crude enzyme solutions onto the substrate medium, a reaction was performed at 37°C for 15 hours, and then the enzyme activity was measured by a degree of formation of a clear zone. 3) Cellulase Activity

A YM medium containing 1% carboxyl methyl cellulose (CMC) substrate was prepared. After spotting 2ul of each of the extracted crude enzyme solutions onto each of the substrate media, a reaction was performed at 37°C for 15 hours. Thereafter, the reactant was dyed 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. 4) Amylase Activity

A YM medium containing 1% soluble starch substrate was prepared. After spotting 3ul of each of the extracted crude enzyme solutions onto the substrate medium, a reaction was performed at 37°C for 15 hours. The reactant was dyed using an aqueous solution containing 0.1% I, 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.

The finally selected Bacillus licheniformis CJIMPB283 strain had activities of the digestive enzymes (protease, amylase, and cellulase) as shown in Table 3. Particularly, the Bacillus licheniformis CJMPB283 strain had an excellent protease activity. [Table 3] [protease [| Amylase [| Cellulase] -: absence of activity, +: presence of Activity, ++:

So excellent activity, +++: significantly excellent activity (2) Endospore Formation Capacity

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 1s important for maintaining a survival rate of bacillus. Therefore, the

Bacillus licheniformis CJMPB283 was cultured for a long period, and endospore formation capacity thereof was confirmed.

The strain (0.1%) was inoculated into a BHI liquid medium and cultured at 37°C and 200 rpm for 24 and 48 hours.

The culture solution at each time was spread on a BHI agar medium, and a total cell was counted. In addition, a culture solution heat-treated at 95°C for 10 minutes was spread on a BHI agar medium, and the number of endospores was counted.

As shown in Table 4, it may be appreciated that when the Bacillus licheniformis CJMPB283 was cultured for 24 hours, an endospore formation rate was about 24.5%, and when the Bacillus licheniformis CJMPB283 was cultured for 48 hours, the endospore formation rate was 100%. The

Bacillus licheniformis CJMPB283 according to the present invention has an excellent endospore formation capacity when cultured for 48 hours or more, so that the Bacillus licheniformis CJMPB283 as probiotics may maintain a high survival rate. [Table 4] (CFU/ml) Rate (%)

The novel Bacillus licheniformis CJMPB283 according to the present invention has an excellent effect of producing digestive enzymes such as amylase, cellulase, and protease and excellent oxidation capacity for ammonia and nitrous acid, so that the novel Bacillus licheniformis CJMPB283 may have the probiotic effect and improve water quality when farming fish or crustacean. Therefore, the novel Bacillus licheniformis CJMPB283 may be used as the water depurative as well as the probiotics.

Countermeasures, Ministry of Oceans and Fisheries, 2001).

Probiotics are added to feed or additionally input to water in aquaculture industry. Recently, research on an effect of stimulating immunity against pathogen and improving water quality to alleviate disease by providing probiotics to farmed fish and shrimp has been actively conducted (Jigiu Li et al., Aquaculture 291(2009) 35-40).

The term “probiotics”, which has an etymological meaning opposed to antibiotics, is defined as a microbial material or microbial component assisting in a balance of intestinal microflora. Lactic acid bacteria such as

Lactobacillus, Bifidobacteria, and the like, are representative.

Bacillus used as probiotics, which is Gram-positive bacillus, forms endospores and has a unique shape among the bacteria used as probiotics. The Bacillus has excellent heat resistance as compared to Lactobacillus that does not form endospores. In addition, since Bacillus can survive at a low pH of stomach walls, most of the administrated

Bacillus may arrive at small intestine (Barbosa,T.M. et al.,

Appl. Environ. Microbiol., 71(2005)968-978; Spinosa, M. R. et al., Res. Microbiol., 151(2000) 361-368).

As prior art related to a material and microbe used for improving water quality, a method of preventing or alleviating nitrite accumulation in aquatic environments using nitrite-oxidizing bacteria has been disclosed in acctgtcact ctgcccccga aggggacgte ctatctctag gattgtcaga ggatgtcaag 480 acctggtaag gttcttcgeg ttgcttcgaa ttaaaccaca tgctccaccg cttgtgeggg 540 ccceegteaa ttectttgag tttcagtctt gegaccgtac tccccaggeg gagtgcttaa 600 tgcgttaget gcagcactaa ggggeggaaa ceccctaaca cttagcacte atcgtttacg 660 gcgtggacta ccagggtatc taatcctgtt cgetceccac getttegete ctcagegtea 720 gttacagacc agagagtcgc cttegecact ggtgttecte cacatctcta cgcatttcac 780 cgctacacgt ggaattccac tetectctte tgcactcaag ttccccagtt tccaatgace 840

Cteceeggtt gagecgggog ctttcacate agacttaaga aaccgectge gageccttta 900 cgcccaataa ttccggacaa cgcttgecac ctacgtatta ccgeggcetge tggeacgtag 960 ttagccgigg ctttctggtt aggtaccgtc aaggtaccge cctattcgaa cggtacttnt 1020

Cttccctaac anagagcttt acgatccgaa aaccttcatc actcacge 1068 <210> 2 - <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer 27F <400> 2 agagtttgat cmtggctcag 20

Nn iy rity ”.

GL ce $s

SEQUENCE LISTING RY Fir 2: 36 <110> CJ CHEILJEDANG CORPORATION <120> NOVEL SEPARATED BACILLUS LICHENIFORMIS AND PROBIOTICS USING SAME <130> P13-5035 <150> KR 10-2012-0035434 <151> 2012-04-05 : <160> 3 <170> Kopatentln 2.0 <210> 1 <211> 1068 <212> DNA <213> Bacillus licheniformis <400> 1 cttcggeggce tggctectaa aggttaccte accgacttcg ggtgttacaa actctcgtgg 60 tgtgacgggc ggtgtgtaca aggcccggga acgtattcac cgcggcatge tgatccgega 120 ttactagcga ttccagctte acgcagtcga gttgcagact gcgatccgaa ctgagaacag 180 atttgtggga ttggcttaac ctcgeggttt cgctgecctt tgttctgtee attgtagcac 240 gtgtgtagce caggtcataa ggggcatgat gatttgacgt catccccace ttectceggt 300 ttgtcaccgg cagtcacctt agagtgccca actgaatgct ggcaactaag atcaagggtt 360 gcgctegttg cgggacttaa cccaacatct cacgacacga gctgacgaca accatgcacc 420

<210> 3

<211> 19

<212> DNA

<213> Artificial Sequence

<220> <223> primer 1492R

<400> 3 ggytaccttg ttacgactt 19

Korean Patent Laid-Open Publication No. 2007-0036016. The nitrite-oxidizing bacteria mean Nitrococcus and Nitrospira.

In addition, a method of removing carbon and nitrogen pollution materials from waste water using heterotrophic ammonia-oxidizing bacteria has been disclosed in Korean

Patent Laid-Open Publication No. 2010-0040960, and the heterotrophic ammonia-oxidizing bacteria mean Bacillus pseudofimus NH-2 and Arthrobacter globiformis WR-2.

However, since Nitrococcus and Nitrospira disclosed in the documents are not strains granted GRAS (generally recognized as safe), it is impossible to use the Nitrococcus and

Nitrospira as probiotics. Further since Nitrococcus and

Nitrospira belong to archaebacteria, production thereof is significantly difficult, so that it is impossible to industrially use the Nitrococcus and Nitrospira. In addition, Bacillus pseudofimus NH-2 known as a strain granted GRAS that is not registered as a probiotic.

The present inventors isolated a strain having digestive enzyme activity and oxidizing ammonia and nitrous acid from soybean paste, which is a traditionally fermented

Korean food, and confirmed morphological, biochemical, and genetic properties of this strain, thereby completing the present invention.

Disclosure Technical Problem

As an embodiment of the present invention, it provides a novel Bacillus licheniformis CJMPB283 producing digestive enzymes and having oxidative capacity for ammonia and nitrous acid.

As an embodiment of the the present invention, it

O provides a culture product comprising a culture solution of the Bacillus licheniformis CJMPB283, a concentrated solution thereof, or a dried material thereof.

Further, as an embodiment of the present invention, it provides a probiotic product containing Bacillus licheniformis CJMPB283 or a culture product thereof.

Furthermore, the present invention provides a feed additive containing the probiotic product.

In addition, as an embodiment of the present invention, it provides a water depurative for aquaculture, containing

Bacillus licheniformis CJMPB283, a culture solution thereof, a concentrated solution thereof, or a dried material thereof.

Further, as an embodiment of the present invention, it provides a method of improving water quality of a fish farm by spraying the water depurative in the fish farm before or during a farming process.

Technical Solution

According to an embodiment of the present invention, there is provided novel Bacillus licheniformis CJMPB283 producing digestive enzymes and having oxidative capacity for ammonia and nitrous acid.

In detail, Bacillus licheniformis CJMPB283 according to the present invention is isolated from fermented soybean, which 1s a traditional Korean food. As morphological 5 properties of the strain according to the present invention, the strain is a Gram-positive bacillus (FIG. 6) and has a les rDNA nucleotide sequence represented by SEQ ID No: 1.

As a result of analyzing the nucleotide sequence, the strain has homology of 97% with Bacillus licheniformis.

Therefore, the present inventors deposited a novel Bacillus licheniformis CJMPB283 in Korean Culture Center of

Microorganisms (361-221, Hongje l-dong, Seodamun-gu, Seoul), and assigned as accession number KCCM11270P on March 22, 2012.

In detail, the strain was founded from fermented soybean in a curing process for 40 days, which is a traditional Korean food, and isolated in a brain heart infusion (BHI) agar medium. The isolated strain has an excellent digestive enzyme activity such as amylase, cellulase, and protease and a property of oxidizing ammonia and nitrous acid. In addition, since the strain may grow under facultative anaerobic and anaerobic conditions and has salt resistance against 10% sodium chloride, the strain may grow under the environments having various water qualities. In addition, water purification capacity was evaluated on young of flatfish, and as a result, ammonia controlling efficacy in farming tank of flatfish was excellent, and a mortality rate was low.

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.

The novel isolated 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 present invention is not limited thereto. As an inorganic salt contained in the medium, magnesium, manganese, calcium, iron, phosphorus, 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 °C to 40°C for 12 hours to 4 days.

In detail, the culture solution of the novel isolated strain may be a culture solution containing the microbial cell, or microbial cell 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. Therefore, this composition may be easily composed by those skilled in the art.

In addition, the strain may be a liquid state or dried state. As a drying method, an air drying method, a natural drying method, a spray drying method, and a freeze-drying method may be used. However, the present invention is not limited thereto.

According to another embodiment of the present invention, there is provided a probiotic product containing a novel isolated strain according to the present invention

Or a culture product thereof.

The probiotics settles in the digestive tract wall to prevent settlement of harmful bacteria and suppresses propagation of pathogenic bacteria. In addition, beneficial digestive enzymes produced by the probiotics support absorption and a use of nutrients, thereby improving feed efficiency.

The probiotic product according to the present invention may contain Bacillus licheniformis CJMPB283 in an amount of 5 x 10° CFU/ml to 5 x 10° CFU/ml, specifically, 1 x 10° CFU/ml to 1 x 10° CFU/ml.

The probiotic product according to the present invention may additionally contain a pharmaceutically acceptable carrier and be formulated together with the carrier, thereby be provided as a food and feed additive.

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. 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 product 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. In the present invention, a binder, an emulsifier, a preservative, and the like, may be added in order to maintain the quality of the probiotic product. An amino acid, a vitamin, an

Claims

28 Ces CLAIMS Collis, . ~~ Claim 1 ’ Tig 3s A Bacillus licheniformis CJMPB283 (accession number KCCM11270P) producing digestive enzymes and having oxidative capacity for ammonia and nitrous acid.

Claim 2 A culture product of Bacillus licheniformis CJMPB283 comprising a culture solution of Bacillus licheniformis CJMPB283 (accession number KCCM11270P), a concentrated solution thereof, or a dried product thereof.

Claim 3 A probiotic product comprising the Bacillus licheniformis CJMPB283 (accession number KCCM11270P) of claim 1. Claim 4 A probiotic product comprising the culture product of claim 2. Claim 5 A feed additive for aquaculture comprising the Probiotic product of claim 3 or claim 4.

Claim 6 A water depurative for aquaculture comprising Bacillus licheniformis CJMPB283 (accession number KXCCM11270P), a culture solution thereof, a concentrate solution thereof, or a dried product thereof.

Claim 7 A method for improving water quality of a fish farm by spraying the water depurative of claim 6 before or during farming in the fish farm.