KR20180098729A - Novel microorganism lactobacillus plantarum cjl-159 or additives for fish feeds containing it - Google Patents

Abstract

The present invention relates to a Lactobacillus plantarum CJL-159 strain or a feed additive for fish comprising the same. The bacterial cells of the Lactobacillus plantarum CJL-159 strain or a culture thereof have excellent resistance to Streptococcus parauberis due to salt having salt tolerance, acid resistance and bile resistance. Particularly, the addition of Cynanchi wilfordii to a medium for culturing the Lactobacillus plantarum CJL-159 strain is confirmed to further increase the antimicrobial activity against Streptococcus parauberis, thereby being easily used as a composition for controlling fish diseases or a feed composition.

Description

TECHNICAL FIELD [0001] The present invention relates to a novel microbial lactobacillus plantarum CJL-159 or a feed additive for fishes comprising the same. BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel microbial lactobacillus plantarum CJL-

The present invention relates to a novel microbial lactobacillus plantarum CJL-159 or a feed additive for fishes comprising the same.

Currently, in fish farms, problems such as increased disease caused by dense farming, nutritional imbalance and other factors are causing serious problems such as fish growth and mortality.

Additives are added to the feed to solve the above problems. Commonly used feed additives include antibiotics, vitamins, amino acid feeders, mineral feeders, enzymes, and hormones.

However, the above-mentioned feed additives may cause drug abuse due to an increase in tolerance to chemicals of cultured fish in the case of excessive feeding, so that the harmful substances accumulate in the cultured fish, The fact that indirect consumption of harmful substances has a problem in the safety of marine products.

In order to solve this problem, Korean Patent No. 10-057771 and Korean Patent No. 10-860111 and the like have been carried out. However, natural antibiotics showing the control effect against bacteria causing fish diseases, Research into feed additive compositions is still in its early stages.

Cynanchi Wilfordii Radix is the roots of a silver mocking or big mockery ( Cynanchum wilfordii ). It is also referred to as "bagasseo". It is a herbal medicine different from the lobster beef which became a problem due to toxicity in recent years. It has the effect of nourishment, tonic, blood, tincture enhancement and also acts to calm the boil. The diseases are anemia, weakness in the aftermath of the disease, lack of ovaries, neuralgia, chronic airflow (a disorder in the brain and spinal cord that causes paralysis of the body and limbs and impaired sensation and movement), pain in the back and knee, (Systemic disease, lymphadenoma, eczema, and vesicular conjunctivitis). Other early symptoms of hair whitening and ulcers are used for a long time when you are not healed.

The inventors of the present invention conducted studies on the pharmacological activity of a bacterium showing a control effect against fish microbes or a herbal medicine applicable to the culture of such a bacterium and found that a new strain of Lactobacillus plantarum and a culture medium It was confirmed that the antimicrobial activity against Streptococcus parauberis was excellent, and the present invention was completed.

Korean Patent No. 10-057771 (composition for adding fish feed containing Bacillus polyfermentus, Bacillus licheniformis, and saccharomyces cerevisiae) Korean Patent No. 10-860111 (feed additives and feedstuffs containing new microbial strains Truace, Tokyintium, KJase-1, Bacillus polyfermenticus kejeze-2 and these and Bacillus licheniformis)

In order to solve the above problems, the present invention provides a novel microorganism Lactobacillus plantarum CJL-159 strain exhibiting an antimicrobial activity against Streptococcus parauberis or a fish microorganism containing the novel microorganism And to provide feed additives.

The present invention relates to a novel microorganism Lactobacillus plantarum CJL-159 (Accession No. KCTC18550P) showing antimicrobial activity against Streptococcus parauberis .

The strain is characterized by having bile resistance, salt tolerance or acid resistance.

The present invention also relates to a composition for controlling fish diseases caused by Streptococcus parauberis , which comprises a bacterium of the above-mentioned strain or a culture thereof.

The present invention can provide a feed additive for fish characterized by comprising the above-mentioned strain or a culture thereof.

The culture medium may be cultured in a microorganism culture medium for lactic acid bacteria culture, or may be cultured in a microorganism culture medium for lactic acid bacteria culture supplemented with white water.

The present invention also relates to a pharmaceutical composition comprising 5 to 50 parts by weight of a carbon source, 5 to 50 parts by weight of peptone, 3 to 11 parts by weight of sodium acetate, 2 to 5 parts by weight of ammonium citrate, 0.5 to 4 parts by weight of polysorbate 80, 0.5 to 4 parts by weight of Polysorbate 80, 0.1 to 0.2 parts by weight of magnesium sulfate and 0.05 to 0.1 part by weight of manganese sulfate. The mixed powder may be mixed with 1000 parts by weight of water to be used for culturing the lactic acid bacteria.

Hereinafter, the present invention will be described in detail.

The present invention relates to a Lactobacillus plantarum CJL-159 strain, wherein said strain comprises the nucleotide sequence of SEQ ID NO: 1.

The culture medium may be cultured in a microorganism culture medium for lactic acid bacteria culture, or may be cultured in a microorganism culture medium for lactic acid bacteria culture supplemented with white water.

The culture medium for culturing a lactic acid bacterium used in the present invention may be any of those used for culturing microorganisms as a carbon source, but preferably one or more selected from molasses, glucose, sugar, and dextran. In addition, glucose is mainly contained in a culture medium for culturing a conventional lactic acid bacterium. When molasses is included, the growth of the strain can be further improved.

The culture medium for lactic acid bacteria may be selected from the group consisting of carbon source, peptone, sodium acetate, ammonium citrate, dipotassium phosphate, polysorbate 80, magnesium sulfate, And Manganese sulfate may be included. Preferably 5 to 50 g of carbon source, 5 to 50 g of peptone, 3 to 11 g of sodium acetate, 2 to 5 g of ammonium citrate, 2 to 5 g of dipotassium phosphate 0.5 to 4 g of polysorbate 80, 0.5 to 4 g of Polysorbate 80, 0.1 to 0.2 g of magnesium sulfate and 0.05 to 0.1 g of manganese sulfate. Each of these components may be mixed with purified water, preferably distilled water, and sterilized to prepare a culture medium for lactic acid bacteria.

The culture medium for culturing the lactic acid bacteria may have a pH adjusted to 6.8-7.2.

In order to increase the antimicrobial activity of the Lactobacillus plantarum CJL-159 culture medium, lactic acid bacteria culture medium may be added to the culture medium. Preferably, 10 to 50 g per hectare of the medium may be added. Therefore, the culture broth of Lactobacillus plantarum CJL-159 of the present invention can be said to be a fermentation broth of Lactobacillus plantarum CJL-159 strain of Bacillus subtilis as another expression.

The culture time of the strain of the present invention is preferably 12 to 48 hours, more preferably 24 to 30 hours.

In the present invention, the microbial cells or the microbial cells used in the composition for controlling fishes or feed additives for fish may be used as they are, or they may be concentrated or dried.

The term " culture medium " in the present invention refers to a culture medium obtained by inoculating a spore with a certain concentration in a medium suitable for microorganism growth, inducing the microorganism to grow for a certain period in an incubator, Is a liquid mixed with nutrients necessary for the production of a protein.

The term " control " of the present invention generally includes prevention, reduction or elimination of pathogenic infections, and the prevention or reduction in infection is statistically significant for untreated subjects (fish in the present invention) it means.

The culture broth of the present invention can be used as a microorganism preparation for controlling Streptococcus parauberis . The microorganism preparation can be prepared in the form of a wettable powder, granule or encapsulation for the purpose of stable formulation of microorganisms.

In the present invention, the disease of fish caused by Streptococcus parauberis as a cause of disease is a fish streptococcal disease, and the disease occurs in both sea water and freshwater fish. Infected individuals of this fungus are weakened, the eye protrudes, the bleeding occurs, bleeding to the fins, the body surface and the muscles, the repercussion, the nodule in the muscle, the bloating of the intestines and the fading of the liver are observed.

The fishes to be controlled by the Lactobacillus plantarum CJL-159 strain of the present invention can be any fishes for which disease is caused by Streptococcus parauberis , in particular, flounder, turbot, defense, dolomite, rainbow trout, It is effective in the control of sweetfish or rhododendron.

The composition for controlling fish diseases of the present invention can be used as a pharmaceutical composition. The pharmaceutical composition may contain various pharmaceutically acceptable carriers, diluents or excipients in conventional pharmaceutical compositions for controlling fish diseases. The pharmaceutical composition may be prepared in unit dose form by formulation or may be manufactured by intrusion into a multi-dose container. The formulations may be, but are not limited to, animal injections, powders, solutions, granules or tablets. For example, when it is prepared as an animal injectable solution or an oral solution, it can be formulated using distilled water, ethyl oleate, ethanol, propylene glycol, glycerin, etc., and antioxidants such as ascorbic acid, sodium hydrogen sulfite, sodium pyruvate, Tocopherol, and the like, but the present invention is not limited thereto. In the case of an animal powder or granule preparation, it may be formulated by using vitamins, glucose, lactose, starch, enzyme, etc., but is not limited thereto. The pharmaceutical composition may be administered orally or parenterally. In detail, in the case of oral administration, they can be mixed directly with oral or drinking water or mixed with feed, and in the case of parenteral administration, they can be administered by injection, but the present invention is not limited thereto. The dosage of the pharmaceutical preparation of the present invention can be adjusted depending on the kind of animal to be administered, body weight, age and the like, and a suitable dosage can be adjusted by those skilled in the art. The pharmaceutical composition may contain 0.1 to 30% by weight of cells or cultures of the Lactobacillus plantarum CJL-159 strain, concentrates thereof, and dried products thereof.

The feed additive composition for fish may also contain various excipients used in common feed additive fish or feed. The fish feed containing the feed additive of the present invention may be added with 0.1 to 30% by weight of cells or culture broth of Lactobacillus plantarum CJL-159 strain, concentrates thereof, and dried product thereof.

The present invention relates to a pharmaceutical composition comprising 5 to 50 parts by weight of a carbon source, 5 to 50 parts by weight of peptone, 3 to 11 parts by weight of sodium acetate, 2 to 5 parts by weight of ammonium citrate, 0.5 to 4 parts by weight of a polysorbate 80, 0.5 to 4 parts by weight of Polysorbate 80, 0.1 to 0.2 parts by weight of magnesium sulfate and 0.05 to 0.1 part by weight of manganese sulfate The mixed powder may be mixed with 1000 parts by weight of water to be used for culturing the lactic acid bacteria.

The present invention relates to a strain of Lactobacillus plantarum CJL-159 or a disease control agent and a feed additive for fishes comprising the same.

1 shows the antimicrobial activity against Streptococcus parauberis of the present invention, and includes the nucleotide sequence of SEQ ID NO: 1. The cells of the Lactobacillus plantarum CJL-159 strain or the culture thereof have excellent resistance to Streptococcus parauberis due to salt tolerance, acid resistance and bile resistance. Particularly, it has been found that the addition of hundreds of oats to the culture medium for culturing Lactobacillus plantarum CJL-159 further increases the antimicrobial activity against Streptococcus parauberis, and thus it can be easily used as a composition for controlling fish diseases or a feed composition have.

FIG. 1 is a phylogenetic tree showing phylogenic tree results of Lactobacillus plantarum CJL-159 strain.
FIG. 2 is a graph showing the number of viable cells of a culture broth of Lactobacillus plantarum CJL-159 according to the amount of white water added in the culture medium for lactic acid bacteria culture.
Fig. 3 is a graph showing the number of viable cells of a culture broth of Lactobacillus plantarum CJL-159 according to the kind of carbon source in a culture medium for lactic acid bacteria culture.
FIG. 4 shows changes in viable cell count / pH / sugar content (Brix) in the culture medium during the culturing of Lactobacillus plantarum CJL-159 strain in a 500 L-volume culture medium for lactic acid bacteria.
5 is a graph showing the survival rate of the flounder infested with Streptococcus parauberi. Negative control refers to 'feed additive group treated with strain culture solution', Positive control refers to 'feed additive group supplemented with Lactobacillus plantarum CJL-159 strain culture solution cultivated in Baishuomi supplemented medium' The group is the 'feed additive group to which Lactobacillus plantarum CJL-159 culture medium cultured in white water addition medium was added', and each numeral means the number of viable cells per ml (cfu) contained in the culture liquid.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the invention to those skilled in the art.

≪ Example 1: Isolation and growth of strain &

Example 1-1. Isolation of lactic acid bacteria strain

2 L of MRS agar (glucose 20 g, peptone # 3 10 g, beef extract 10 g, yeast extract 5 g, sodium citrate 5 g, ammonium citrate 2 g, dipotassium phosphate 2 g, Tween 80 1 g, Bromophenol blue 0.002 g, Bacto agar 17 g / Was added to 2 L of beaker, and distilled water was added thereto to prepare 2 L medium. The pH of the solution was adjusted to 7.0-7.2 by adding 3 mol of caustic soda solution with stirring. After heating to 95 ° C, it was confirmed that all the contents were melted and autoclaved. * Tween 80: Product name of polysorbate 80.

As a separation source for isolating the lactic acid bacteria, there are a variety of segregation sources for isolating lactic acid bacteria, such as heat-kimchi, shrimp sauce, salted sashimi, chopped cabbage kimchi (CJ), Jejun Onggi tasty kimchi, , Shrimp noodle, pork kimchi, dongchwon dongchimi, mung bean aged mineral spring shrimp, octopus salted fish, squid salted salted fish, salted salted salted fish and so on.

Examples 1-2. Separation procedure and strain analysis

After adding 90 ml of sterilized distilled water to 10 g of sample, the mixture was pulverized with a blender for 1 minute and serial dilution was performed in 10-fold increments. After serial dilution, 0.05 ml of the solution was spread on MRS agar using a glass spreader and cultured at 37 ° C for 24-48 hours. After observing the shape and morphology of the colonies grown here, single colonies were separated by pure platinum. After 328 colonies were obtained, MRS agar was subjected to a second culture on an MRS agar plate after pure separation by a single colony. After the second culture, colonies were inoculated into MRS broth, cultured at 37 ° C for 24-48 hours, glycerol stock was prepared by mixing 0.25 ml of glycerol 80% and 0.75 ml of culture medium. Purely isolated lactic acid bacteria were streaked on MRS agar and cultured for 48 hrs. After that, they were sent to solgent for strain analysis. As a result, these strains were found to be lactic acid bacteria such as Lactobacillus sp., Leuconostoc mesenteroides, Lactobacillus plantarum, Leuconostoc mesenteroides, Lactobacillus curvatus, Lactobacillus sakei, Leuconostoc pseudomesenteroides, Lactobacillus brevis and Lactobacillus rhamnosus .

<Example 2: Selection of lactic acid bacteria having antibacterial activity>

It was confirmed that the lactic acid bacteria isolated in Example 1 had antimicrobial activity against strains causing fish diseases. To this end, BHI broth, Streptococcus parauberis strain, which was supplied from Jeju National University, was added to the culture medium to 0.75% BHI soft agar prepared at 50 ℃ for 24 hours at 37 ℃. Separately, the isolated lactic acid bacteria were inoculated into a 10 ml test tube filled with both MRS and incubated at 37 ° C for 48 hours, then inoculated on a MRS agar using a sterilized toothpick, and then dried. After that, 0.75% soft MRS agar was coated dropwise on the dried lactic acid bacteria culture to prevent the spread of lactic acid bacteria. After agar solidification for 15 minutes, 50 ml of 0.75% BHI soft agar inoculated with the prepared Streptococcus parauberis strain was poured onto 10 ml of the agar and the mixture was incubated at 37 ℃ for 48 hours. After the culture, the diameter of the hollow zone formed around the lactic acid bacteria with antimicrobial activity was measured and used as an indicator of the antibacterial activity. As a result of testing with 281 lactic acid bacteria, three strains of CJL-153, CJL-159 and CJL-173, which have an average antibacterial ring diameter of 2 cm, among 328 lactic acid bacteria having excellent antibacterial activity, were selected as candidate strains. The results of these three strains are shown in Table 1 below (results of other strains having an antibacterial ring diameter of less than 2 cm are not shown).

Strain name Antibacterial ring diameter (cm)
1 time Antibacterial ring diameter (cm)
Episode 2 Average diameter of antibacterial rings (cm) CJL-153
( Lactobacillus sp. ) 2.0 2.2 2.1 CJL-159
( Lactobacillus plantarum ) 3.0 2.4 2.7 CJL-173
( Leuconostoc mesenteroides ) 2.0 2.0 2.0

Example 3 Artificial Gastric Resistance and Artificial Bile Acid Resistance and Salt Tolerance Test

In order for the fermented product containing the live bacteria to exhibit the efficacy as the feed composition of the fish, the survival rate in the gastric juice and bile and the salt resistance to the seawater should be excellent when the fish ingests the fish, so that the artificial gastric juice resistance And artificial bile acid tolerance and salt tolerance.

Example 3-1. Confirming Artificial Gastric Resistance

For this purpose, artificial gastric juice was added to 1000 units / ml of pepsin (Sigma, USA) in MRS broth adjusted to pH 2.5 with 1N HCl. After serial dilution, 100 μl of each was applied to the MRS agar medium, and the cells were left at 37 ° C for 24 hours to count the number of bacteria. The results are shown in Table 2, indicating that the CJL-159 strain is highly resistant to artificial gastric juice.

Condition Strain Before processing After processing increase pH 2.5 artificial gastric juice CJL-159 1.2 x 10 &lt; ⁵ &gt; cfu / ml 1.9 × 10 ⁵ cfu / ml 166%

Example  3-2. art Bile acid  Confirm tolerance

Artificial rehydration acid was prepared by adding 1%, 2%, and 3% of bile bovine filtered with 0.45μm filter to Lactobacillus MRS broth (Difco, USA). The artificial bile juice was administered to the lactic acid bacteria in an appropriate amount. And then cultured at 37 ° C for 2 hours. At this time, 100 μl of MRS agar was applied by serial dilution before and after culturing for 2 hours, and colony number was measured by culturing at 37 ° C.

As shown in Table 3, the biliary properties of all the three strains were confirmed. In particular, the bacterium CJL-159 was 207% grown at the Bile 3% condition.

Example  3-3. Identify salt resistance

1%, 3%, and 5% NaCl were added to the MRS liquid medium, followed by 1% inoculum culture at 30 ° C for 2 hours. After 2 hours of incubation, 100 μl of MRS agar was applied by serial dilution, and cultured at 37 ° C. The number of colony was counted and survival rate was calculated by comparing the number of viable cells.

Table 4 shows that the salt number was increased to 117% in the culture broth of CJL-159 under the condition of NaCl 3%, which is similar in salinity and saltiness, and thus the salt resistance is the most excellent.

Condition Strain Before treatment (cfu / ml) After treatment (cfu / ml) Remarks
NaCl 1% CJL-153 8.0 × 10 ⁵ 1.3 × 10 ⁶ 160% CJL-159 4.6 × 10 ⁴ 1.1 × 10 ⁵ 230% CJL-173 1.5 × 10 ⁶ 2.1 × 10 ⁶ 141%
NaCl 2% CJL-153 1.7 × 10 ⁶ 1.7 × 10 ⁶ 100% CJL-159 1.8 × 10 ⁵ 1.8 × 10 ⁵ 98% CJL-173 2.4 × 10 ⁶ 2.0 × 10 ⁶ 82%
NaCl 3% CJL-153 1.2 × 10 ⁶ 8.0 × 10 ⁵ 69% CJL-159 1.2 × 10 ⁵ 1.4 x 10 ⁵ 117% CJL-173 2.0 × 10 ⁶ 1.7 × 10 ⁶ 86%

< Example  4. Confirmation of antibiotic resistance>

Lactobacillus was cultured for 24 hours, and the bacterial concentration was diluted to a Mcfarland 0.5, and then the lactic acid bacteria diluted with a cotton swab was spread evenly on the MRS agar. The antibiotic disc being sold on the medium was then pressed onto the medium using tweezers. Next, the antibiotic resistance was determined by measuring the diameter (cm) of the hollow zone formed after culturing at 37 ° C for 24 hours.

As shown in Table 5, CJL-159 and CJL-173 strains are more resistant to antibiotics than CJL-153 strains.

Condition CJL-153 (cm) CJL-159 (cm) CJL-173 (cm) Ampicillin 1.7 1.3 2.5 Oxytetracycline 1.7 2.0 2.1 Florfenicol 1.8 2.0 2.1 Cephalexin 1.4 1.2 1.3 Clindamycin 1.7 2.0 2.3 Erythrpmycin 2.0 2.3 2.3 Amoxycillin 1.7 1.5 1.7

&Lt; Example 5: Selection of an antimicrobial strain &

The CJL-159 strain was finally selected as a strain for the preparation of the feed additive composition of the present invention by comparing the antibacterial activity with various experimental data.

The CJL-159 strain was identified as Lactobacillus plantarum strain in Example 1, and the phylogenic tree results for this strain are shown in FIG.

This strain was confirmed to be a Lactobacillus plantarum strain having the characteristics of 16s rRNA containing the nucleotide sequence of SEQ ID NO: 1 shown in Table 6, referenced by solgent as described in Example 1-2.

< Example  6. Set medium optimization conditions for culture culture>

Example  6-1. Baewooo Oh  Check the amount added

MRS broth (20 g of glucose, 10 g of peptone # 3, 10 g of beef extract, 5 g of sodium acetate, 2 g of ammonium citrate and 2 g of K ₂ HPO ₄₎ were added to the lactic acid bacteria culture medium of the present invention , was compared tween 80 1g, magnesium sulfate 0.1g, was added to the ^{Manganese sulfate 0.05g) 1.0 × 10 6} cfu / ml of lactic acid bacteria having a viable cell count of 1% MRS broth and incubated for 24 hours (number of viable cells initial inoculation concentration Hereinafter the same). The results of the experiment are shown in Fig.

2, it can be confirmed that the growth of the lactic acid bacteria is not limited when the amount of white water added is 1 to 3% (w / v) in the whole culture condition. In addition, although not shown in this graph, it was confirmed that the culture of the strain was performed well even when added up to 5% (w / v). In the subsequent experiments, the amount of white water added was set to 1% (w / v).

Example 6-2. Optimize carbon source addition

Experiments were conducted on molasses, brown sugar, and dextran to optimize medium for lactic acid bacteria cultivation. The basal concentration was 20g / L, and the medium composition was 10g, 10g, carbon source 20g, peptone # 3 10g, beef extract 10 g of sodium acetate, 2 g of ammonium citrate, 2 g of dipotassium phosphate, 1 g of tween 80, 0.1 g of magnesium sulfate and 0.05 g of Manganese sulfate were added to 1 L of distilled water to adjust the pH to 7.0 ± 0.2. After serial incubation at 37 ° C and 100 rpm for 24 hours, serial dilution was applied to MRS agar and counted. As a result of the experiment, it was found that the molasses was the best. Therefore, the culture composition prepared by varying the amount of molasses added in the range of 5 to 50 g / L was confirmed, and the molasses concentration was confirmed. And the viable cell count at this time was 3.82 × 10 ¹¹ cfu / ml.

Example 6-3. Optimization of nitrogen source addition

Six nitrogen sources, yeast extract, peptone, soybean meal, ammonium sulfate, ammonium phosphate and ammonium nitrate were tested for the optimization of nitrogen source. 10 g of white sugar, 15 g of molasses, 10 g of sodium acetate, 5 g of sodium acetate, 2 g of ammonium citrate, 2 g of dipotassium phosphate, 1 g of tween 80, 0.1 g of magnesium sulfate and 0.05 g of Manganese sulfate were added to 1 L of distilled water, 0.2. After serial incubation at 37 ° C and 100 rpm for 24 hours, serial dilution was applied to MRS agar and counted. Beef peptone (meat peptone) was the best. When the amount of beef peptone added was optimized, the growth of the strain was good (1 × 10 ⁹ cfu / ml or more) within the range of 5 to 50 g / L, and the number of viable cells was 1.72 × 10 ¹⁰ cfu / ml. &lt; / RTI &gt;

Example 6-4. Mineral addition optimization - Sodium acetate

In order to optimize the addition amount of sodium acetate, the concentration of sodium acetate was changed to 0g, 1g, 3g, 5g, 7g, 9g and 11g. Add 1 g of BSA, 15 g of beef peptone, 15 g of ammonium citrate, 2 g of dipotassium phosphate, 1 g of tween 80, 0.1 g of magnesium sulfate, and 0.05 g of Manganese sulfate, add 1 L of distilled water and adjust pH to 7.0 ± 0.2 . After serial incubation at 37 ° C and 100 rpm for 24 hours, serial dilution was applied to MRS agar and counted. As a result, the growth of the strain in the strain was good (1 × 10 ⁹ cfu / ml or more) at the addition amount of sodium acetate of 3 ~ 11 g / L, especially 1.16 × 10 ¹⁰ cfu / ml at the addition amount of 3 g.

Example 6-5. Mineral supplementation optimization - Ammonium citrate

The concentration of Ammonium citrate was changed to 0g, 1g, 2g, 3g, 4g, 5g, 6g and 7g in order to optimize the amount of Ammonium citrate. 10 g of beef peptone, 15 g of beef peptone, 3 g of sodium acetate, ₂ g of K ₂ HPO ₄ , 1 g of tween 80, 0.1 g of magnesium sulfate, and 0.05 g of Manganese sulfate were added to 1 L of distilled water and the pH was adjusted to 7.0 ± 0.2 Respectively. After serial incubation at 37 ° C and 100 rpm for 24 hours, serial dilution was applied to MRS agar and counted. As a result, the growth of the strain in the strain was good (1 × 10 ⁹ cfu / ml or more) at the amount of 2 to 5 g / L of Ammonium citrate, and 3.5 × 10 ⁹ cfu / ml at the addition amount of 2 g.

Examples 6-6. Mineral addition optimization - dipotassium phosphate

In order to optimize the amount of dipotassium phosphate added, the concentration of dipotassium phosphate was changed to 0 g, 0.5 g, 1 g, 1.5 g, 2.0 g, 2.5 g, 3.0 g and 3.5 g. Add 1 g of BSA, 15 g of beef peptone, 15 g of sodium acetate, 2 g of Ammonium citrate, 1 g of tween 80, 0.1 g of magnesium sulfate and 0.05 g of Manganese sulfate, add 1 L of distilled water and adjust pH to 7.0 ± 0.2 . After serial incubation at 37 ° C and 100 rpm for 24 hours, serial dilution was applied to MRS agar and counted. As a result, the growth of the strain was good (1 × 10 ⁹ cfu / ml or more) at 0.5-4 g / L of dipotassium phosphate, and 3.5 × 10 ⁹ cfu / ml at 3 g of the added amount.

Examples 6-7. Mineral supplementation optimization - Tween 80

For the optimization of the amount of Tween 80, the concentration of Tween 80 was changed to 0 g, 0.5 g, 1 g, 1.5 g, 2.0 g, 2.5 g, 3.0 g, 3.5 g and 4.0 g. Add 1 g of beef peptone, 15 g of beef peptone, 3 g of sodium acetate, 2 g of ammonium citrate, 3.0 g of dipotassium phosphate, 0.1 g of magnesium sulfate and 0.05 g of Manganese sulfate, add 1 L of distilled water and adjust pH to 7.0 ± 0.2 I did it. After serial incubation at 37 ° C and 100 rpm for 24 hours, serial dilution was applied to MRS agar and counted. As a result, the growth of the strain in the strain was good (1 × 10 ⁹ cfu / ml or more) at 0.5 ~ 4 g / L of Tween 80 added amount, especially 1.9 × 10 ⁹ cfu / ml at the addition amount of 3.5 g.

Examples 6-8. Confirmation of trace material culture condition

In the same manner as in the above experiments, culture conditions were also confirmed for magnesium sulfate and manganese sulfate, and growth of strains was good in the range of 0.1 to 0.2 g / L of magnesium sulfate and 0.05 to 0.1 g / L of manganese sulfate (1 × 10 ⁹ cfu / ml or more).

Examples 6-9. Establishment of lactic acid bacteria culture medium condition

The best culture medium conditions for culturing the strain of the present invention were set as follows by adding white ginseng through the above-mentioned culture condition experiments.

Constituents of microorganism culture medium composition Addition amount Baewooo Oh 10 to 50 g / L molasses 5 to 50 g / L peptone 5 to 50 g / L Sodium acetate 3 to 11 g / L Ammonium citrate 2 to 5 g / L dipotassium phosphate 0.5 to 4 g / L Tween 80 (polysorbate 80) 0.5 to 4 g / L Magnesium sulfate 0.1 to 0.2 g / L Manganese sulfate 0.05 to 0.1 g / L

Example 7. Confirmation of Mass Fermentation Conditions &gt;

Example 7-1. Culture conditions i

The seed culture cultured in the flask was cultured on a 3 L scale in a 5 L jar fermenter. The cultures of the flasks and the fermenter were incubated in the same manner as in Example 1 except that [10 g / L of BSA, 15 g / L of molasses, 15 g / L of Peptone, 3 g / L of sodium acetate, 2.0 g of Ammonium citrate, 3 g / L of dipotassium phosphate, L, 0.1 g / L of magnesium sulfate and 0.05 g / L of manganese sulfate. The seed culture was inoculated in 3 L medium at 1% (v: v). The cells were cultured for 24 hours at an initial pH of 7.0, a culture temperature of 37 ° C, an air supply of 1 vvm, and a rpm of 100 for other culture conditions. The degree of culture was analyzed based on the viable cell count. The number of viable cells confirmed at 24 hours was 1.61 × 10 ⁹ cfu / ml. Based on these results, the incubation time was increased to 30 hours in the next experiment.

Example 7-2. Culture conditions ii

The medium was cultured in a 1.5-fold concentrated medium (15 g / L of BSA, 22.5 g / L of molasses, 22.5 g / L of Peptone and 4.5 g / L of Peptone) using the same procedure as the above Culture Condition I, L, Ammonium citrate 3.0 g, K ₂ HPO ₄ 4.5 g, Tween 80 3.0 g, Magnesium sulfate 0.15 g, and Manganese sulfate 0.075 g).

Example 7-3. Culture conditions iii

The same procedure as in Culturing Condition I was used for the cultivation condition i, but the culture time was 30 hours. The culture was carried out in a 2-fold concentrated medium (20 g / L BaSO x, 30 g / L molasses, 30 g / L Peptone, 6 g / L Sodium acetate, 4 g Ammonium citrate / L, K ₂ HPO _{4 4} g / L, Tween 80 4 g / L, Magnesium sulfate 0.2 g / L, and Manganese sulfate 0.1 g / L).

Example  7-4. Culture conditions iv

In the culture condition iv, the same fermentation amount was used as in the culture condition I except that the total fermentation amount was 500 L, medium (20 g / L of BSA, 30 g / L of molasses, 30 g / L of Peptone, 6 g / L of sodium acetate, 4 g / L of Ammonium citrate , K ₂ HPO _{4 4} g / L, Tween 80 4 g / L, Magnesium sulfate 0.2 g / L, and Manganese sulfate 0.1 g / L) for 30 hours.

It was confirmed that the lactic acid bacterium was increased to an appropriate concentration of 10 ^{8 to 9} cfu / ml or more under the above culture conditions, and it was confirmed that the culture medium used in the present invention was useful for culturing the strain of the present invention. At this time, the culture state of Example 7-4 is represented in FIG. 4 as a representative.

< Example  8. Fish experiment>

Example 8-1. Experimental results of fish paste immobilization

Negative control, Positive control feed ( Lactobacillus plantarum CJL-159 cultured in a medium containing no feed water was diluted to a concentration of 1.0 × 10 ⁶ cfu / g of Lactobacillus plantarum in the feed, Lactobacillus plantarum CJL-159 culture medium was diluted to a concentration of 1.0 × 10 ^{6 to 8} cfu / g of live bacteria, Lactobacillus plantarum, and fed to the feed. , And each feed composition was fed to flounders of 10 mice per group (2 repeated experiments).

As shown in Table 8, it was confirmed that the lactic acid bacteria were not detected in the negative control feed during the 8 weeks of the experiment. However, in the case of positive control feed ( Lactobacillus plantarum 1.0 x 10 ⁶ cfu / g feedstuff, which was fed with culture medium supplemented with Bacillus subtilis medium), and the culture medium in which the cultured medium was sprayed with Bacillus subtilis medium ( Lactobacillus plantarum 1.0 x 10 ⁶ cfu / g feedstuff), lactic acid bacteria were observed in the intestine after feeding. In addition, it was confirmed that the number of lactic acid bacteria in the intestines of the experimental feed group injected with the culture liquid cultured with the Bacillus subtilis medium was particularly increased.

Example 8-2. Fish Challenge test

A fish aggression experiment was conducted on the 8th week after the administration of each feed composition in Example 8-1. Ten adult flounders were selected from each feed treatment area and the three-week cumulative mortality rate was confirmed by inoculation with Streptococcus parauberis adjusted to a concentration of 10 ⁶ CFU / g feed.

5, the control (control feed) flounder showed the highest mortality rate at 90% at 21 days after the infection, but it was confirmed that the flounder rate of the flounder fed with the microorganism supplemented feed decreased by 40 ~ 50%. In addition, this is more effective in the case of the flounder fed with the cultured medium supplemented with the culture medium supplemented with the culture medium of the present invention than the positive control treated with the culture of the present invention without the culture of the lactic acid bacteria culture medium . Therefore, it can be confirmed that when the strain of the present invention was supplemented with a feed additive, the mortality rate of the fish was remarkably reduced.

Korea Biotechnology Research Institute KCTC18550P 20170210

<110> Repiatec Co. Ltd. <120> NOVEL MICROORGANISM LACTOBACILLUS PLANTARUM CJL-159 OR ADDITIVES          FOR FISH FEEDS CONTAINING IT <130> 2017-0066 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 1442 <212> DNA <213> LACTOBACILLUS PLANTARUM <400> 1 tgcaagtcga acgaactctg gtattgattg gtgcttgcat catgatttac atttgagtga 60 gtgggagaact ggtgagtaac acgtgggaaa cctgcccaga agcgggggat aacacctgga 120 aacagatgct aataccgcat aacaacttgg accgcatggt ccgagtttga aagatggctt 180 cggctatcac ttttggatgg tcccgcggcg tattagctag atggtggggt aacggctcac 240 catggcaatg atacgtagcc gacctgagag ggtaatcggc cacattggga ctgagacacg 300 gcccaaactc ctacgggagg cagcagtagg gaatcttcca caatggacga aagtctgatg 360 gagcaacgcc gcgtgagtga agaagggttt cggctcgtaa aactctgttg ttaaagaaga 420 acatatctga gagtaactgt tcaggtattg acggtattta accagaaagc cacggctaac 480 tacgtgccag cagccgcggt aatacgtagg tggcaagcgt tgtccggatt tattgggcgt 540 aaagcgagcg caggcggttt tttaagtctg atgtgaaagc cttcggctca accgaagaag 600 tgcatcggaa actgggaaac ttgagtgcag aagaggacag tggaactcca tgtgtagcgg 660 tgaaatgcgt agatatatgg aagaacacca gtggcgaagg cggctgtctg gtctgtaact 720 gacgctgagg ctcgaaagta tgggtagcaa acaggattag ataccctggt agtccatacc 780 gtaaacgatg aatgctaagt gttggagggt ttccgccctt cagtgctgca gctaacgcat 840 taagcattcc gcctggggag tacggccgca aggctgaaac tcaaaggaat tgacgggggc 900 ccgcacaagc ggtggagcat gtggtttaat tcgaagctac gcgaagaacc ttaccaggtc 960 ttgacatact atgcaaatct aagagattag acgttccctt cggggacatg gatacaggtg 1020 gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca 1080 acccttatta tcagttgcca gcattaagtt gggcactctg gtgagactgc cggtgacaaa 1140 ccggaggaag gtggggatga cgtcaaatca tcatgcccct tatgacctgg gctacacacg 1200 tgctacaatg gatggtacaa cgagttgcga actcgcgaga gtaagctaat ctcttaaagc 1260 cattctcagt tcggattgta ggctgcaact cgcctacatg aagtcggaat cgctagtaat 1320 cgcggatcag catgccgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac 1380 catgagagtt tgtaacaccc aaagtcggtg gggtaacctt ttaggaacca gccgcctaag 1440 gt 1442

Claims (7)

A novel microorganism Lactobacillus plantarum CJL-159 (Accession No. KCTC18550P) strain characterized by exhibiting antibacterial activity against Streptococcus parauberis . The method according to claim 1,
The strain is Lactobacillus plantarum CJL-159 (Accession No. KCTC18550P) strain, which is characterized by having bile bite, salt tolerance or acid resistance. A composition for controlling fish diseases caused by Streptococcus parauberis , which comprises the bacterium of claim 1 or a culture thereof. The method of claim 3,
Wherein the culture medium is cultured in a culture medium for culturing a lactic acid bacteria or a culture medium for culturing a lactic acid bacterium to which white water is added. A feed additive for fish characterized by containing the strain of the strain of claim 1 or a culture thereof. 6. The method of claim 5,
Wherein the culture medium is cultured in a culture medium for culturing a lactic acid bacteria or a culture medium for culturing a lactic acid bacterium to which white water is added. 5 to 50 parts by weight of a carbon source, 5 to 50 parts by weight of peptone, 3 to 11 parts by weight of sodium acetate, 2 to 5 parts by weight of ammonium citrate and 0.5 to 5 parts by weight of dipotassium phosphate, , 0.5 to 4 parts by weight of polysorbate 80, 0.1 to 0.2 parts by weight of magnesium sulfate and 0.05 to 0.1 parts by weight of manganese sulfate.

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