KR101073791B1 - Lactobacillus pentosus PL-11 having the dietary enzyme activities, the resistance of bile acid and acid, and the probiotics for fishes using thereof - Google Patents

Abstract

The present invention relates to a novel Lactobacillus pentosus PL-11 strain having excellent intestinal enzyme activity, acid resistance, and bile resistance, and a probiotic for fish using the same, wherein the Lactobacillus pentosus PL-11 strain is It has physiological activities such as anti-bile and acid resistance, and has anti-inflammatory effect and enzyme degrading ability. Therefore, when used as probiotics for fish, it can protect fish from bacteria that are not useful for fish farming and improve survival rate. In addition, it has the effect of improving the fish growth rate and feed efficiency.

Lactobacillus pentosus, Lactobacillus pentosus, Probiotics, Intestinal enzyme activity, Acid resistance, Bile resistance

Description

Lactobacillus pentosus PL-11 having the dietary enzyme activities, the resistance of bile acid and acid, and the probiotics for intestinal enzyme activity, acid resistance, and bile resistance fishes using pretty}

The present invention relates to a novel Lactobacillus pentosus PL-11 strain having excellent intestinal enzyme activity, acid resistance, and bile resistance, and to fish probiotics using the same, and more specifically, to Lactobacillus pentosus . Since PL-11 has physiological activities such as bile and acid resistance, and anti-inflammatory effect and enzyme degrading ability, when used as probiotics for fish, it protects fish from bacteria that are not useful for fish farming. The present invention relates to a novel Lactobacillus pentosus PL-11 strain having an effect of improving survival rate as well as improving fish growth rate and feed efficiency and fish probiotics using the same.

Probiotics (probiotics) are known to have antimicrobial activity that inhibits the growth of pathogenic bacteria in various types of normal intestinal flora, and plays an important role in maintaining the balance of normal intestinal flora and suppressing the growth of pathogenic bacteria. Fuller, R. (1989) Probiotics in man and animal. J. Appl. Bacteriol ., 66, 365-378, Shahani, KM and AD Ayebo. 1980.Role of dietary lactobacilli in gastrointestinal microecology. Am. J. Clin. Nutr . 33: 2448-2457. However, the useful effects of these probiotics are mainly experimental reports on humans and livestock, and the developed probiotics are also applied to humans and livestock [Tournut, J. 1989. Applications of probiotics to animal husbundary. Rev. Sci. Tech. Off. Int. Epiz . 8: 551-566.] There are problems in using these products as they are in saltwater fish, especially eels. Probiotics used in humans and livestock have an optimal growth temperature of 37 ° C., which is inappropriate for farmed fish growing at 15 to 25 ° C., and particularly probiotic that can withstand high salt concentrations is essential for sea fish.

With the rapidly growing fish aquaculture, the quality of the water environment deteriorates and the frequency of fish disease increases, so probiotics that can replace antibiotics are essential.

In general, lactic acid bacteria used in probiotics are Gram-positive bacillus, non-motility, non-spore, and catalase-negative bacteria, which are widely distributed in nature and are easily found in animal intestines and fermented foods [Ringo, E. and FJ Gatesoupe. Lactic acid bacteria in fish: a review. Aquaculture . 160: 177-203. It is well known that these lactic acid bacteria are important microorganisms to improve the intestinal flora by antimicrobial activity. The antimicrobial activity factors of these lactic acid bacteria are organic acid, hydrogen peroxide and bacteriocin [Piard, JC and M. Desmazeaud. 1992. Inhibiting factors produced by lactic acid bacteria. 2. Bacteriocins and other antibacterial substances. Lait . 72: 113-142. In addition, lactic acid bacteria adhere to and settle intestinal epithelial cells, thereby inhibiting infection and growth of pathogens. Studies on the antimicrobial activity of lactic acid bacteria against pathogenic bacteria include Listeria monocytogenes , Escherichia coli , Salmonella typhimurium , and Vibrio anguillarum . It has been reported to have antimicrobial activity. However, while most fish pathogens and pathogenic bacteria are Gram-negative, lactic acid bacteria as probiotics have limitations in that they show antimicrobial activity only against Gram-positive bacteria whose systemic activity is similar. K., MC Misra, and MC Varadaraj. Plantaricin LP84, a broad spectrum heat-stable bactriocin of L. plantarum NCIM 2084 produced in a simple glucose broth medium. Int. J. Food Microbiol. 40: 17-25.]. Therefore, isolates for industrial application as probiotic strains must have a broad antimicrobial spectrum that can inhibit the growth of Gram-negative bacteria as well as Gram-negative bacteria.

In addition, in order to have a prolonged antimicrobial activity and beneficial effects of probiotics, it must survive in environmental conditions such as low pH, bile and various digestive enzymes. In other words, another important condition for probiotic strains is that they must have acid and bile resistance and adequate resistance to antibiotics [Verschuere L., G. Rombaut, P. Sorgeloos, and W. Verstraete. 2000. Probiotic bacteria as biological control agents in aquaculture. Microbiol. Mole. Biol. Rev. 64: 655-656.

Therefore, the development of fishes, especially eel-specific probiotics that overcome the above problems are required in the country and the world.

Therefore, an object of the present invention has a broad antimicrobial spectrum and has a pH resistance, storage resistance and antibiotic resistance, and can be used as a probiotic, and novel Lactobacillus pentosus having excellent intestinal enzyme activity, acid resistance, and bile resistance. To provide a strain of PL-11.

Another object of the present invention is to increase the efficiency of the aquaculture industry and improve the efficiency of fish growth and feed efficiency by preventing or treating bacterial diseases of fish using the novel Lactobacillus pentosus PL-11 strain of the above object It's about providing probiotics that work.

In order to achieve the above objects as well as other objects that are easily expressed in the present invention, blood agar (Blood agar), Trypticase soy agar (TSA), MRS agar (MRS) from the Japanese eel Anguilla japonica species , Difco, Detroit, USA) and MRS agar, adjusted to pH 4.2, were incubated for 24 hours in an incubator at 37 ° C, aerobic conditions, and adjusted to anaerobic conditions using BBL Gaspak plus (Becton Dickinson, USA). Incubated for more than 48 hours in an incubator at 30 ℃, has a broad antimicrobial spectrum, pH resistance, storage and antibiotic resistance, can be used as a probiotic, new Lactobacillus pentosus (excellent intestinal enzyme activity, acid resistance, and bile resistance) Lactobacillus pentosus) separating the strain and PL-11, this fish used as probiotics by bacterial disease prevention of lump fish for Increasing the efficiency of the treatment by the aquaculture industry and could improve the growth rate and feed efficiency of the fish.

The novel Lactobacillus pentosus PL-11 strain according to the present invention has a broad antimicrobial spectrum and has shelf life and antibiotic resistance, and is excellent in intestinal enzyme activity, acid resistance, and bile salts, and it is a probiotic for fish. By using it as a preventive or therapeutic agent for the bacterial disease of fish, it is possible to increase the efficiency of the aquaculture industry and improve the fish growth rate and feed efficiency.

The novel Lactobacillus pentosus PL-11 strain according to the present invention has a broad antimicrobial spectrum and has shelf life and antibiotic resistance, and thus can be used as a probiotic, and has excellent intestinal enzyme activity, acid resistance, and bile salts. , Gram-positive bacteria, Facultative anaerobes, rod-shaped, do not form spores, Catalase test results are negative, produce lactic acid and organic acid It is characterized as being.

In addition, the probiotics according to the present invention is characterized as containing Lactobacillus pentosus PL-11 strain as an active ingredient.

The isolation and identification of Lactobacillus pentosus PL-11 strains according to the present invention is as follows.

The eel was selected as Anguilla japonica , a Japanese eel, and the eel was rapidly frozen at the eel farm in Hwasun, Jeonnam, and brought to the laboratory for experiment. Since the number of lactic acid bacteria isolated from the frozen eel and the living eel is known to be about the same, the frozen eel was used. Eel strain culture medium is aerobic conditions using blood agar medium (Blood agar), Trypticase soy agar (TSA), MRS agar (MRS, Difco, Detroit, USA) and MRS agar adjusted to pH 4.2 The cells were incubated at 37 ° C. for at least 24 hours, adjusted to anaerobic conditions using BBL Gaspak plus (Becton Dickinson, USA), and then incubated at 30 ° C. for at least 48 hours.

* Blood agar composition

15 g of Pancreatic Digest of Casein Digested with Pancreatin

5g of Papaya Digest of Soybean Meal

Sodium Chloride 5g

Agar 15g

Blood 50ml

* Trypticase soy agar (TSA) medium composition

Tryptone 17g

Soytone (enzymatic digest of soybean meal) 3g

Dextrose 2.5g

Sodium Chloride 5g

K ₂ HPO ₄ 2.5 g

Agar 15g

* MRS agar badge composition

10 g peptone

Meat extract 8g

Yeast extract 4g

20 g of glucose

5 g of sodium acetate 3H ₂ O

Tween 80 1 g

Dipotassium hydrogen phosphate 2g

Triammonium citrate 2g

0.2 g of magnesium sulfate 7H ₂ O

0.05 g magnesium sulfate tetrahydrate (Manganese sulfate 4H ₂ O)

Agar 10g

As a Gram-positive bacterium characteristic of lactic acid bacteria, 16 strains which finally selected the strain which catalase was negative were isolated and selected.

* Morphology and culture characteristics of isolated strains

Gram staining was performed for strains cultured in optimal growth medium (MRS agar), which was Gram-positive bacteria. The cultural feature is facultative anaerobes, rod-shaped, as shown in Figure 1 did not form a spore, the catalase test results showed a negative response, Lactic acid) and organic acid production characteristics, it can be seen that it has the morphology and culture characteristics of typical Lactobacillus.

Identification and naming of isolated strains

As a result of analyzing the morphological and culture characteristics, the strain isolated in the present invention is a microorganism belonging to the Lactobacillus, and the strain was finally identified by analyzing the 16S rRNA gene sequence of the isolated strain. Genomic DNA is extracted from the isolate using a genomic DNA extraction kit (Qiagen) and then forward primer (5'-AGA GTT) for cloning of eubacterial 16S rDNA. Perform polymerase chain reaction (PCR) using a set of TGA TCC TGG CTC AG-3 ') and a reverse primer (5'-AAG GAG GTG ATC CAG CC-3') It was. PCR mixture containing 50 pmole primer, 50 ng template DNA, 5 μL of 10 ㅧ Tay DNA polymerase buffer and 1U of Taq DNA polymerase (Takara, Japen) was prepared at 95 ° C. After denatured for 5 minutes at 1 ℃ at 95 ℃, 1 minute at 55 ~ 60 ℃, 1 minute at 72 ℃ was repeated 30 times to perform a PCR. The PCR product was cloned into pSTBlue-1 (Novagen, USA), followed by sequencing with the BigDyeTM-terminator sequencing kit and ABI PRISM377 sequencer (Perkin-Elmer, USA). Lactobacillus pentosus and 99.93 % Turned out to be similar.

Among the isolates, they have a broad antimicrobial spectrum and can be used as probiotics because they have pH resistance, storage resistance, and antibiotic resistance. Lactobacillus pentosus PL- It was named 11, and it was deposited with the Korea Microorganism Conservation Center, which is a Korean patent strain depositing institution, on July 03, 2008 and was given an accession number (KFCC11427P).

Hereinafter, the present invention will be described in detail by way of examples, and the present invention is not limited by the following examples.

Example 1 Enzyme Analysis of Isolated Strains

Enzyme resolution experiments were conducted to determine whether or not lactic acid bacteria having enzymes capable of degrading carbohydrates, proteins and phosphorus. To determine whether or not there is protease activity, 0.5% of skim milk was added to the MRS agar medium, and methyl cellulose was added to the MRS agar medium to determine whether the cellulase activity was present. was added to 0.2%, α - amylase (α -amylase) corn starch and the MRS agar in order to know whether the activity (corn starch) was put in a 0.2% phytase (Phytase) pie calcium lactate to see the activity The medium to which 0.5% of salt (Ca-phytate) was added was prepared, respectively.

The strains isolated on each medium prepared above were streaking and observed after culturing for 24 hours.

α -amylase and cellulase activity was treated with 2% Congo red reagent in culture medium for 24 hours, washed with 1M sodium chloride (Nacl), and then degraded by color. Confirmed. In addition, the determination of protease and phytase activity was confirmed by the presence of a transparent ring, and the results are shown in Table 1 below.

Enzyme Activity of Isolated Strains Strain number Protease Cellulase α -amylase Faitase PL1 - + + - PL2 - - + - PL3 + - + + PL4 - - - - PL5 + - - + PL6 - - - + PL7 + - + + PL8 + - - - PL9 + - + - PL10 - - + - PL11 + + + + PL12 + - + - PL13 + + + + PL14 + + + - PL15 + + - + PL16 + + + +

Example 2: Confirmation of anti-inflammatory effect

The anti-inflammatory effect was confirmed using NO assay method (NO production quantitative analysis: quantifying the amount of NO dissolved in the cell supernatant in vitro). In the normal NO assay method, NO produced from Raw 264.7 cell line (prepared from Korea Cell Line Bank) is present in the cell culture as NO ² form and is Griess (1% sulfanilamide in 5% phosphoric acid + 1% N- (1). Measure the amount of NO using -naphthyl) -ethylenediamine in H ₂ O) reagent.

In this embodiment, Lactobacillus pentosus PL-11, 13, 16 cell culture solution (sample 1 (pH 3.5), sample 2 (pH 7.0), sample 3 (LPS (lipopolysaccharide) treatment), sample 4 ( 100 μl of sample 1 + LPS), sample 5 (sample 2 + LPS), control, and 100 μl of Griess reagent were mixed for 10 minutes in a 96 well plate, and then absorbed at 540 nm using an ELISA reader. Measured. The concentration of NO ^2- was measured by measuring the absorbance by diluting sodium nitrate and drawing the standard curve.

Using the three lactic acid bacteria candidate groups, the NO test was performed to determine the immunological activity. As a result, the P11 strain showing the best NO inhibition when the supernatant of the P11 strain was treated with LPS was selected as the final candidate group.

Example 3 Experiment of Inhibitory Activity against Fish Pathogenic Microorganisms

In order to confirm the effect of the inhibitory effect on the fish pathogenic microorganism strain, the experiment was performed using the Lactobacillus pentosus PL-11 strain finally selected in the above example.

Minimum inhibition concentration (MIC) of Lactobacillus Pantosus PL11 was determined by microtitire plate assay. As the bacteria, Escherichia coli, Edwardsiella tarda, Streptococcus iniae, Vibrio anguillarum, Vibrio alginolyticus, Vibrio parahaemolyticus, and Vibrio harveyi were used. After mixing the medium (BHI, LB broth) and the supernatant (100%, 50%, 25%, 12.5%, 6%, 3%, 1.5%) of Lactobacillus pentosus PL11 ( pH3.5 , pH7), Inoculate 50 μl at a concentration of 10 ⁵ CFU / ml, incubate at 37 ° C. for 24 hours, dilute the MRS solid medium through a microtitire plate assay method, smear and incubate at 37 ° C. for 24 hours, and measure the bacterial count.

It showed a growth inhibitory action or bactericidal action against the test bacteria, which are described in Table 3 below. These results indicate that the Lactobacillus pentosus PL-11 strain is caused by the lactic acid bacteria produced, showing that very useful results when applied as a probiotic.

Antimicrobial Activity of Lactobacillus pentosus PL-11 Against Fish Pathogenic Microorganisms strains Supernatant concentration (%) 100 50 25 12.5 6.2 3.1 1.5 0.7 V. anguillarum --- - - - ++ ++ +++ +++ V. alginolyticus --- --- - + ++ +++ +++ +++ V. parahaemolyticus --- --- - + ++ +++ +++ +++ V. harveyi - - - ++ +++ +++ +++ +++ E. coli --- - - + + +++ +++ +++ E. tarda --- - - - ++ ++ +++ +++ S. iniae - - - ++ +++ +++ +++ +++ ---: No growth,-: <10 ² CFU / ml,-: <10 ³ CFU / ml, +: <10 ⁵ CFU / ml, ++: <10 ⁷ CFU / ml, +++:> 10 ⁸ CFU / ml

Example 4

After adjusting the pH 2,3,4,5, control (pH 7.0) by the addition of 1N HCl in 5ml of MRS medium to determine whether a suitable acid-resistant strain as a probiotic, 10 ⁷ cfu / ml or greater L. pentosus strain PL11 0.1% of L. pentosus KCCM 35472 strain was added and growth was observed at 0, 0.5, 1, 2, 3, and 8 hour intervals.

In order for L. pentosus PL11 to function as a probiotic, it must pass through the stomach at pH 3 or below. After inoculating L. pentosus PL11 strain into MRS liquid medium adjusted to pH 2, 3, 4, 5, and control group (pH 7.0) with 1N HCl, the number of viable cells was incubated at 37 ° C. At pH 4, 5, and 7, the incubation time was increased to more than 10 ⁵ when the incubation time was 8 hours, but the bacteria were not proliferated at pH 3, but were maintained. At pH 2, which was strongly acidic, the bacteria survived for more than 3 hours. . This may vary depending on food intake or type, but it takes two to three hours for food to pass through the stomach, and L. pentosus PL11, which has acid resistance, actually takes up the stomach when the pH of the stomach acid is diluted to 2-3. The survival rate after passing cannot be ruled out higher than the experimental value.

Resistance test to acid Lactobacillus
pentosusPL11 0 o'clock 0.5 o'clock 1 o'clock 3 o'clock 8 o'clock Blank 2.5X10 ⁷ cells / ml 5X10 ⁷ cells / ml 2X10 ⁸ cells / ml 2.5X10 ⁸ cells / ml 7.5X10 ¹⁰ cells / ml pH 5 2.15X10 ⁷ cells / ml 4X10 ⁷ cells / ml 1X10 ⁸ cells / ml 5.5X10 ⁸ cells / ml 2.95X10 ¹⁰ cells / ml pH 4 2X10 ⁷ cells / ml 5.5X10 ⁷ cells / ml 7.15X10 ⁷ cells / ml 3.5X10 ⁸ cells / ml 4X10 ¹⁰ cells / ml pH 3 2.35X10 ⁷ cells / ml 5X10 ⁷ cells / ml 6X10 ⁷ cells / ml 1X10 ⁸ cells / ml 4X10 ⁸ cells / ml pH 2 3.5X10 ⁷ cells / ml 1X10 ⁶ cells / ml 2X10 ⁵ cells / ml 5X10 ³ cells / ml - Lactobacillus
pentosusKCCM
35472
0 o'clock
0.5 o'clock
1 o'clock
3 o'clock
8 o'clock Blank 3.55X10 ⁷ cells / ml 7.5X10 ⁷ cells / ml 2X10 ⁸ cells / ml 6X10 ⁸ cells / ml 2.35X10 ¹⁰ cells / ml pH 5 3.8X10 ⁷ cells / ml 7X10 ⁷ cells / ml 1X10 ⁸ cells / ml 2.5X10 ⁸ cells / ml 1.8X10 ⁹ cells / ml pH 4 3.7X10 ⁷ cells / ml 5X10 ⁷ cells / ml 7.15X10 ⁷ cells / ml 5.5X10 ⁸ cells / ml 2X10 ⁹ cells / ml pH 3 3.5X10 ⁷ cells / ml 1X10 ⁷ cells / ml 2X10 ⁷ cells / ml 4.75X10 ⁷ cells / ml 1.4X10 ⁸ cells / ml pH 2 2.95X10 ⁷ cells / ml 1.5X10 ³ cells / ml - - -

Example 5

The most important factor as a condition of probiotic is resistance to bile acids. To this end, 0%, 0.1%, 0.3%, and 1% of bile acids were added to ⁵ ml of MRS liquid medium, and 0.1% of L. pentosus PL11 strain diluted to 10 ⁵ CFU / ml was added at 0, 1, 5, 12, Growth was confirmed by measuring the number of bacteria at intervals of 24 hours and 48 hours.

After incubating at 37 ° C. for 24 hours, the result of measuring the number of bacteria is shown in Table 4 below. As can be seen from Table 4, even in 1% of bile acids, the bacterium is maintained without being inhibited, and thus it can be judged that they are resistant to bile acids.

Resistance test for bile acids Lactobacillus
pentosus PL11 0 o'clock 3 o'clock 6 o'clock 12 o'clock 24 hours 48 o'clock Blank 5.5X10 ⁵ cells / ml 6X10 ⁶ cells / ml 8.2X10 ⁸ cells / ml 2X10 ⁹ cells / ml 2.3X10 ¹⁰ cells / ml 3X10 ⁸ cells / ml 0.1% Oxgall 8X10 ⁵ cells / ml 8.1X10 ⁶ cells / ml 9X10 ⁸ cells / ml 4.7X10 ⁹ cells / ml 3.5X10 ¹⁰ cells / ml 4X10 ⁸ cells / ml 0.3% Oxgall 4.5X10 ⁵ cells / ml 1X10 ⁶ cells / ml 2.5X10 ⁶ cells / ml 3X10 ⁷ cells / ml 3X10 ⁸ cells / ml 4.23X10 ⁶ cells / ml 1% Oxgall 5X10 ⁵ cells / ml 5.5X10 ⁵ cells / ml 6X10 ⁵ cells / ml 6X10 ⁵ cells / ml 7X10 ⁵ cells / ml 7X10 ⁵ cells / ml Lactobacillus
pentosus KCCM 35472 0 o'clock 3 o'clock 6 o'clock 12 o'clock 24 hours 48 o'clock Blank 7X10 ⁵ cells / ml 3.5X10 ⁶ cells / ml 4X10 ⁶ cells / ml 9X10 ⁹ cells / ml 7.5X10 ¹⁰ cells / ml 1.75X10 ⁸ cells / ml 0.1% Oxgall 4.5X10 ⁵ cells / ml 5X10 ⁶ cells / ml 5X10 ⁶ cells / ml 7X10 ⁸ cells / ml 4X10 ⁷ cells / ml 5X10 ⁸ cells / ml 0.3% Oxgall 6X10 ⁵ cells / ml 7X10 ⁵ cells / ml 8X10 ⁵ cells / ml 8.5X10 ⁵ cells / ml 3X10 ⁴ cells / ml 1X10 ⁴ cells / ml 1% Oxgall 6X10 ⁵ cells / ml 2.5X10 ⁴ cells / ml 5X10 ³ cells / ml 5X10 ³ cells / ml - -

Example 6

Antibiotic Resistance Test In order to investigate the resistance of antibiotics, which are widely used for feed and treatment of fish and animals, the overnight culture of Lactobacillus pentosus PL-11 was adjusted to 10 ⁶ CFU / ml, and then 100 µl was placed on a 96well plate. 100 μl of the antibiotics to be diluted beforehand were added, and then placed in a 37 ° C. incubator and examined for growth of bacteria after 24 hours to determine the minimum growth inhibition concentration and the lowest bactericidal concentration, and the results are shown in Table 5 below. It was found to be resistant to most antibiotics, and compared with conventional lactic acid bacteria and antibiotic resistance was found to be equal or better. These results confirm that L. pentosus PL11 strain can be used as a probiotic even when used in feed containing antibiotics.

Comparison of Minimum Growth Inhibition Concentration and Minimum Sterilization Concentration Antibiotic Lactobacillus pentosus MIC (µg / ml) MBC (μg / ml) Cephalexin 4 8 Colistin sulfate > 256 > 256 Enrofloxacin 8 16 Cefalonium 16 16 Amoxicillin trihydrate <0.5 2 Penicillin G procaine <0.5 2 Norfloxacin > 256 > 256 Spectinomycin 128 > 256 Tylosin base > 256 > 256 Cefuroxine sodium One 16 Florfenicol 4 32 Penicillin G Benzathine 4 8 Gentamicin sulfate 128 128 Streptomycin sulfate 128 > 256 Dihydrostreptomycin 256 > 256

Example 7 Lactobacillus Pentosus ( Lactobacillus pentosus Acute Toxicity Test of PL-11 Strain

Acute toxicity test was performed to evaluate the safety of Lactobacillus pentosus PL-11 strain.

The test group consisted of a control group (control, 0.85% NaCl), a low dose group (10 ⁷ CFU / ml), a medium dose group (10 ⁹ CFU / ml) and a high dose group (10 ¹¹ CFU / ml), and 7-week-old Sprague-Dawley. Specific pathogen free (SPF) rats of the strain were fasted overnight before administration, and then orally administered once using a large-adhered injection tube for rats, followed by observation for 14 days, and the results are shown in Table 6 below. It was.

No mortality due to L. pentosus PL11 was observed during the test period in all male and female test groups, including the high dose group of 10 ¹¹ CFU / ml when orally administered L. pentosus PL11. In addition, there were no toxic symptoms or unusual clinical symptoms caused by L. pentosus PL11. As a result, LD ₅₀ in both sexes of L. pentosus PL11 is estimated to be 10 ¹¹ CFU / ml or more.

Mortality in Rats After Oral Administration of Lactobacillus pentosus PL-11 gender Dose
(Mg / kg) Days after dosing Mortality rate
(%) LD ₅₀ One 2 3 4 5 6 7
Number
High dose group (10 ¹¹ CFU / ml) 0 0 0 0 0 0 0 0
> 10 ¹¹
CFU / mL
Medium dose group (10 ⁹ CFU / ml) 0 0 0 0 0 0 0 0 Low dose group (10 ⁷ CFU / ml) 0 0 0 0 0 0 0 0 Control group 0 0 0 0 0 0 0 0
cancer
High dose group (10 ¹¹ CFU / ml) 0 0 0 0 0 0 0 0
> 10 ¹¹
CFU / mL
Medium dose group (10 ⁹ CFU / ml) 0 0 0 0 0 0 0 0 Low dose group (10 ⁷ CFU / ml) 0 0 0 0 0 0 0 0 Control group 0 0 0 0 0 0 0 0

gender Dose
(Mg / kg) Days after dosing Mortality rate
(%) LD ₅₀ 8 9 10 11 12 13 14
Number
High dose group (10 ¹¹ CFU / ml) 0 0 0 0 0 0 0 0
> 10 ¹¹
CFU / mL
Medium dose group (10 ⁹ CFU / ml) 0 0 0 0 0 0 0 0 Low dose group (10 ⁷ CFU / ml) 0 0 0 0 0 0 0 0 Control group 0 0 0 0 0 0 0 0
cancer
High dose group (10 ¹¹ CFU / ml) 0 0 0 0 0 0 0 0
> 10 ¹¹
CFU / mL
Medium dose group (10 ⁹ CFU / ml) 0 0 0 0 0 0 0 0 Low dose group (10 ⁷ CFU / ml) 0 0 0 0 0 0 0 0 Control group 0 0 0 0 0 0 0 0

Example 8 Lactobacillus Pentosus ( Lactobacillus pentosus Effect of PL-11 on the Eel Growth

1 month of feeding Lactobacillus pentosus PL-11 at 1% (10 ⁸ CFU / g) in eel feed and feed without added strain to eel with an average weight of 200-300 g After that, the growth rate and feed efficiency of the eel were examined, and the results are shown in Table 7.

Weight Gain and Feed Efficiency of Lactobacillus pentosus PL-11 division Individual weight gain (g) Feed efficiency (%) Treatment (1% treatment) 84.37 1.56 No treatment 75.91 2.55

Figure 1 is an electron micrograph of the strain Lactobacillus pentosus ( Lactobacillus pentosus ) PL-11 according to the present invention.

Claims (5)

It has a broad antibacterial spectrum, pH resistance, storage resistance and antibiotic resistance, so it can be used as a probiotic, has excellent intestinal enzyme activity, acid resistance and bile resistance, is Gram-positive bacteria, facultative anaerobes, and rods A novel Lactobacillus pentosus , characterized in that it does not form spores in the form of rods, and catalase test results are negative, and have the property of producing lactic acid and organic acid. PL-11 strain (KFCC11427P). The novel Lactobacillus pentosus PL-11 strain (KFCC11427P) according to claim 1, which has acid resistance which can be grown for 2 hours at pH 2.0. The novel Lactobacillus pentosus PL-11 strain (KFCC11427P) according to claim 1, which can be grown under conditions of 1% bile acid. Probiotics for fish, characterized in that Lactobacillus pentosus PL-11 (KFCC11427P) is contained as an active ingredient. The probiotics of claim 4, wherein the fish is an eel.

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