KR101201420B1 - A feed additive containing novel Lactobacillus jonhsonnii - Google Patents

Abstract

The present invention is a novel Lactobacillus Johnson, which has excellent acid resistance, bile resistance, intestinal adhesion ability, bile acid hydrolysis ability, susceptibility to antibiotics, and shows excellent effects such as weight gain and pathogenic bacterial defense effects when ingested by animals. It's about you.
When the novel Lactobacillus Johnson ni of the present invention is used as an animal feed or a medicine, it can continuously exhibit the intestinal pathogenic bacteria suppression and intestinal effects of the animal, thereby greatly reducing the economic damage caused by the livestock disease of the livestock farms, It can also be used to replace imported veterinary medicine.

Description

Novel Lactobacillus jonhsonnii and Lactobacillus jonhsonnii

The present invention relates to a novel Lactobacillus Johnson nee and a feed additive composition comprising the same, more specifically, excellent in acid resistance, bile resistance, intestinal adhesion ability, bile acid hydrolysis ability, susceptibility to antibiotics, and ingestion by animals The present invention relates to a novel Lactobacillus Johnson knee which exhibits excellent effects such as weight gain and pathogenic bacterial defense effects.

Probiotics, living microorganisms, promote the health of animals by activating their function in the gut (Fuller, 1989). Many researchers have reported that probiotics such as Lactobacillus and Bifidobacterium reduce or protect against symptoms such as diarrhea (Gilliland, 1990). Another advantage of probiotics is that it contributes to lowering blood cholesterol levels or increasing nutrient utilization (du Toit et al., 1998; Fukushima et al., 2007; Gilliland, 1990; Lee et al., 2001; Torres Rodriguez et al., 2007).

Lactic acid bacteria (LAB) are the most studied probiotics (Chang et al., 2001; De Angelis et al., 2006; Geier et al., 2007). Lactic acid bacteria are normally present in the intestines of most animals, and balance the intestinal microorganisms and have a beneficial effect on the health of the host. Therefore, to select useful probiotics in the gut, several important features are identified and selected (Fuller, 1989; Garriga et al., 1998; Martin et al., 2006; Mishra and Prasad, 2005). That is, lactic acid bacteria must survive in the digestive tract, so they must have acid or bile resistance, and colonies form in the digestive tract until they reach the intestine. It should also be safe for food or feed or clinical use and at the same time have a beneficial effect on the host. Recently, lactic acid bacteria have been developed that have many functional properties, one of which is to inhibit the growth of pathogens (Pascual et al., 1999; Tsai et al., 2005) or BSH activity (Begley et al., 2006; Pereira et al., 2003) or amylolytic activity (Lee et al., 2001; Vizoso Pinto et al., 2006).

Thus, the inventors of the present invention, as a result of earnest research to find a probiotic strain that can be used as a feed additive with the above advantages, as a result of separating the new Lactobacillus Johnson ny from pig small intestine, the isolated strain is bile salt hydrolytic ability, acid resistance And excellent bile resistance, showed susceptibility to antibiotics can be used as a probiotic, excellent intestinal adhesion, confirmed that the animal shows an excellent effect, such as weight gain, pathogenic bacterial defense effect when ingested to complete the present invention .

Therefore, the main object of the present invention is to provide a microorganism of the genus Lactobacillus beneficial to animals.

Another object of the present invention is to provide a feed and veterinary medicine comprising the Lactobacillus microorganism and exhibiting the intestinal pathogenic bacteria inhibition and intestinal effect of the animal.

According to one aspect of the present invention, the present invention provides Lactobacillus Johnson Knee deposited with the Korea Agricultural Microbial Resources Center (KACC) under accession number KACC 91458.

The 16s rDNA of the Lactobacillus Johnson nee of the present invention is characterized by comprising the nucleotide sequence of SEQ ID NO: 1.

The Lactobacillus Johnson Knee of the present invention is characterized by having acid resistance, bile resistance, and enteric adhesion.

According to another aspect of the present invention, the present invention provides a feed additive composition comprising the Lactobacillus Johnson.

Feed additive composition of the present invention is characterized by inhibiting the growth of pathogenic bacteria in the body.

Hereinafter, the present invention will be described in more detail step by step.

The present invention is to identify a new lactobacillus exhibiting antimicrobial activity against pathogenic microorganisms for the purpose of helping the healthy growth of animals and apply it to feed additives.

In the present invention, the small intestine of healthy pigs was collected and used in order to obtain a strain that can be used as the above useful Lactobacillus, that is, probiotics (probiotics). In order to obtain such probiotic strains, it is possible to use a sample obtained from any part of the natural world such as seawater, soil, plants, or animals.However, to identify strains for use as feed additives for livestock, the digestive organs of healthy animals are collected and It is preferable.

In order to isolate the Lactobacillus microorganisms of the present invention, first, the small pig small intestine is mixed with MRS medium to incubate the microorganisms of the small intestine, and then the microorganisms in the MRS solid medium containing calcium carbonate (CaCO ₃ ) Colonies that produce transparent rings were selected when cultured to form colonies. Then, Gram staining and catalase activity experiments were performed on the selected strains to select gram positive and catalase negative strains. As a result, the strain G22-2 of the present invention could be selected.

In order to identify the selected G22-2 strain, the DNA of the strain was extracted, and then DNA fragments for 16S rRNA were amplified by PCR, and its nucleotide sequence (nucleotide sequence of SEQ ID NO: 1) was analyzed and compared with the database of NCBI. . The results showed a 99% homology with Lactobacillus Johnson.

In order to achieve the object of the present invention, the microorganism of the present invention must survive while passing through the digestive organs of animals, must be able to stably settle in the intestine, and artificial growth can be controlled to evaluate the evaluation .

Since strains with bile salt hydrolase activity are involved in the detoxification of bile salts or increase intestinal viability, this activity of selected strains was investigated. As a result, as shown in Figure 1, a very large precipitate ring was produced, which means that the strain of the present invention has a very high bile salt hydrolase activity.

The microorganisms must survive low pH in the gastric acid in order to survive the passage of the animal's digestive tract and bile secreted from the digestive tract. Thus, we investigated the acid and bile resistance of the selected strains.

As a result of confirming acid resistance of the G22-2 strain of the present invention, pH7.2. When cultured at 3.0 and 2.0 for 3 hours, the bacterial counts showed excellent acid resistance of 7.82 ± 0.04, 7.84 ± 0.03, and 4.12 ± 0.04 (unit: log CFU / mL), respectively, and showed good growth in medium containing 0.3% bile salt. Bile resistance was also shown to be good (see Table 1 and Table 2).

For antibiotic resistance evaluation, various antibiotics used in the past may be evaluated by checking the sensitivity of the strain by a method using a disk or adding an antibiotic to a medium. As a result of the antibiotic resistance evaluation of the G22-2 strain of the present invention, it was found to be susceptible to most antibiotics, confirming its applicability to probiotics (see Table 3).

In order to evaluate the intestinal adhesion ability, it is possible to investigate the nonpolarity of the cell surface of the strain. In general, the strain having high nonpolarity adheres well to the mucus or epithelial cells of the intestine, and the nonpolar rate and colonic epithelial cell adhesion. This is because there is a high correlation between them. As shown in Table 4, Lactobacillus coryniformis KCTC 3159, which was used as a control strain, showed a very low polarity of 5.3%, whereas the G22-2 strain of the present invention showed an excellent intestinal attachment ability of 86.8%. It is expected to indicate.

Although the usefulness of the probiotic strain can be confirmed through in vitro experiments as described above, it is necessary to confirm whether the effect can be obtained when applied to an animal. Therefore, after applying the strain using the actual animal model it is good to confirm the change in the metabolism of the animal accordingly. At this time, any animal that can be used for the purpose of experiment can be used as an animal model, but it is better to use an experimental red that is relatively easy to breed.

In order to evaluate the metabolic changes in animals according to the application of the strain, the animals are ingested by drinking water or feed, and then examined for changes in weight gain, blood chemistry, microbial composition in feces, pH and water content. Can be used. In addition, by inoculating the pathogen under the same conditions, by monitoring the metabolic changes as described above, it is possible to confirm the antimicrobial activity of the strain of the present invention in maintaining the physical health of the animal.

As a result of confirming the change in the weight gain of the red when ingested the G22-2 strain of the present invention, as shown in Table 5, it was confirmed that the feed efficiency of the G22-2 administration group was as good as the commercial probiotics (positive control), As a result of the investigation, the harmful microorganism Coliform was slightly decreased on the 8th and 17th days compared to the negative control group (see Table 6).

Total cholesterol (TCHO), total glyceride (TG), bilirubin (BUN), high density lipoprotein (HDLD) in serum of red when ingested G22-2 strain of the present invention As a result, the levels of total cholesterol, bilirubin, and high-density lipoprotein were significantly decreased in serum of the G22-2 group.

When challenged with Salmonella typhimurium , there were no rats that died or showed clinical symptoms for 11 days after challenge, and as shown in Table 8, the weight gain, feed intake and feed efficiency increased significantly in the G22-2 group. The feed efficiency was also confirmed to be excellent.

As a result of investigating the effect on the excretion of Salmonella strains, Salmonella excretion was significantly decreased in the G22-2 group after 1 day of challenge, and the number of Salmonella strains excreted on the 6th day of challenge was significantly reduced compared to the control group. (See Table 9). This means that the G8-5 strain of the present invention inhibits the growth of pathogenic bacteria in the body.

As described above, when the novel Lactobacillus Johnson nee of the present invention is used as an animal feed or medicine, it is possible to continuously suppress the intestinal pathogenic bacteria and intestinal effects of the animal, thereby greatly reducing the economic damage caused by the livestock disease of the livestock farms. It can be reduced, as well as used to replace imported veterinary medicine.

1 is a photograph showing the bile salt hydrolase activity of the novel Lactobacillus Johnson teeth of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Isolation and Identification of Strains of the Invention

1 g of small intestine of healthy pigs were collected, washed with sterile saline, and sliced. The sliced small intestine was treated with MRS liquid medium (2 g of glucose per 100 g, Tween 80 0.1 g, ammonium citrate 0.2 g, 0.5 g acetate, magnesium sulfate 0.01 mg). g, anhydrous manganese sulfate 0.005g, dibasic potassium phosphate 0.2g, beef extract 1g, yeast extract 0.5g, proteinase-treated peptone 1g) and incubated for 24 hours. 0.1 ml of the culture solution was taken and inoculated in MRS agar medium containing 0.5% CaCO ₃ and incubated for 48 hours at 37 ° C. A single colony having a transparent ring around the colonies was inoculated again on MRS agar medium for pure separation.

Gram staining and catalase activity were examined for purely isolated strains, and strains that were gram positive and catalase negative were selected.

DNA of the purely isolated strain was extracted and amplified DNA fragments for 16S rRNA by PCR, and the nucleotide sequence of the amplified fragment was analyzed and compared with the database of NCBI.

Example 2. Investigation of Bile Salt Hydrolase Activity

50 μl of the strain of the present invention (G22-2) was inoculated onto a sterile 8 mm paper disk, followed by 0.5% (w / v) sodium salt of taurodeoxycholic acid (TDCA); Sigma) and 0.37% g / L of calcium chloride was added to the MRS medium added to culture the size of the resulting ring was observed (see Figure 1).

Example 3 Acid and Bile Resistance Investigations

After culturing the strain of the present invention (G22-2) in an MRS liquid medium for 18 hours, centrifugation (3000 × g, 15 minutes), washing the pellet twice with phosphate buffer (pH 7.2), and washing the pellet suspension 1 ml 9 ml of phosphate buffer was mixed to prepare a sample having a bacterial count of 7.2 × log CFU / ml. Using 4N hydrochloric acid, the pH of the sample was adjusted to 3.0 and 2.0, respectively, and cultured for 3 hours, and then the survival rate was confirmed (see Table 1). In addition, the survival rate was confirmed after culturing for 24 hours in each MRS medium containing 0.3% or 1.0% of bile salts (Difco, USA) (see Table 2).

Strain name
Number of cells survived at adjusted pH (log CFU / mL) pH 7.2 pH 3.0 pH 2.0 G22-2 7.82 ± 0.04 7.84 ± 0.03 4.12 ± 0.04

(Expressed as mean ± standard deviation)

Strain name
My bile 0.3% 1.0% G22-2 ++ ++

(-: Do not grow, +: grow)

Example 4. Antibiotic Resistance Survey

Commercially available antibiotic sensitivity kits (Sensi-Disc®, BD Biosciences, Sparks, MD) were used.

The strain (G22-2) of the present invention was incubated, suspended in a buffer solution, adjusted to 10 ⁸ CFU / mL bacterial count, and then 200 µl of the adjusted culture solution was added to 100 ml soft agar and mixed. Each mL was poured into Petri dishes (9 cm) and hardened. Here, the susceptibility was confirmed by placing a paper disc containing each antibiotic on the paper disc and measuring the inhibitory ring produced (see Table 3).

Antibiotic G22-2 Ampicillin S Cephalothin S Penicillin S Vancomycin S Amikacin R Chloramphenicol S Erythromycin S Rifampin S

[S: sensitivity, R: resistance]

Example 5. Nonpolar Investigation of Cell Surface

The strain (G22-2) of the present invention was incubated in MRS liquid medium for 16-18 hours, followed by centrifugation (3000 × g, 15 minutes), washing the pellet twice, suspended in saline solution, and absorbance at a wavelength of 600 nm. Was set at 0.5 to 0.7 (A ₀ ). 1.5 ml of hexadecane was added to a test tube containing 1.5 ml of the suspension, followed by vigorous mixing for 2 minutes. After leaving the test tube for 15 minutes, the aqueous layer of the lower layer was carefully collected using a sterile pipette, and the absorbance was measured at a wavelength of 600 nm (A ₁ ). Based on the measured absorbance results, the specific polarity was calculated using Equation 1 below. Lactobacillus coriniformis ( L. coryniformis KCTC 3159) was used as a control, and the nonpolar ratio was calculated in the same manner as described above (see Table 4).

[Equation 1]

Specific Polarity (H%) = (1-A ₁ / A ₀ ) × 100

Strain Nonpolarity rate (%) G22-2 86.8 ± 13.7 L. coryniformis KCTC 3159 5.3 ± 0.

(Expressed as mean ± standard deviation)

Example 6. Animal Experiment

After culturing the strain of the present invention (G22-2) in MRS medium and centrifuged (3000 × g, 15 minutes) to wash the pellet twice with saline and dissolved in clean saline, the number of strains 5 × 10 ⁶ CFU Negative water was prepared to be / ml (5 × 10 ⁷ CFU / ml in pathogenic bacterial experiments) to be consumed by the red (experimental group).

Positive controls include commercial probiotics (Primarac , Bayer veterinary drug) containing L. acidophilus , L. casei and Bifidobacterium bifidum . Used. About 1 g of commercial probiotics were inoculated in MRS liquid medium and prepared in the same manner as the experimental group, and the negative control group was ingested with red distilled water using clean distilled water.

6-1. Weight gain survey

Body weight, feed intake and negative intake were measured at the start and day 17 of the experiment, and feed efficiency was calculated by dividing feed intake by weight gain (see Table 5).

Weight gain (g / d) Feed intake
(g / d) Feed efficiency Negative water intake
(Ml / d) Negative control group 9.04 ± 0.31 22.30 ± 0.62 2.47 ± 0.04 36.8 ± 2.7 G22-2 9.18 ± 0.14 22.35 ± 0.56 2.43 ± 0.03 ^* 35.1 ± 0.4 Positive control group 9.01 ± 0.60 21.36 ± 0.90 2.37 ± 0.06 ^* 36.6 ± 2.6

(Mean ± standard deviation, *: P <0.05)

6-2. Investigation of the condition of feces

On the 8th and 17th day of the experiment, fresh feces were collected, diluted and mixed 10 times with saline, and then homogeneous solution was prepared to measure pH, and moisture was measured by freeze drying. 0.1 ml of the homogenate in which the feces were diluted in 10-fold steps was incubated in MRS solid medium, MacConkey agar and Brilliant Green agar. Coliform) was measured (see Table 6).

group

8 days 17 days LAB Coliform pH moisture LAB Coliform pH moisture (log10
CFU / g) (log10
CFU / g)  (%) (log10
CFU / g) (log10
CFU / g)  (%) voice
Control group 9.30 ×
0.11 7.41 ×
0.27 6.13 ×
0.22 50.3 ×
4.1 9.15 ×
0.09 7.13 ×
0.05 5.86 ×
0.02 * 45.3 ×
3.8 G22-2 9.56 ×
0.26 * 7.36 ×
0.14a 6.09 ×
0.24 * 49.0 ×
7.6 9.31 ×
0.17 * 6.97 ×
0.34 * 5.94 ×
0.09 50.2 ×
4.8 * positivity
Control group 9.45 ×
0.26 * 7.19 ×
0.43 * 6.07 ×
0.10 * 51.3 ×
5.2 9.30 ×
0.16 * 7.05 ×
0.24 5.81 ×
0.05 * 52.7 ×
0.5 *

(LAB: Lactobacillus, Coliform: Coliform)

(Mean ± standard deviation, *: P <0.05)

6-3. Blood Chemistry Survey

On the 17th day of the experiment, blood of each red blood was collected and reacted for 1 hour at 37 ° C, and then serum was separated by centrifugation at 2,000 × g for 10 minutes, and total cholesterol (TCHO), total glyceride (TG), and bilirubin (Bilirubin, BUN), lipoprotein (High density lipoprotein (HDLD)) and amylase levels (using enzymatic diagnostic kits, Sigma co, USA) were measured (see Table 7).

group
TG TCHO BUN HDLD AMYL (Mg / dl) (Mg / dl) (Mg / dl) (Mg / dl) (U / ℓ) Negative control group 137 ± 29 81 ± 9 15.4 ± 1.1 157 ± 13 2748 ± 132 G22-2 148 ± 43 75 ± 5 * 13.1 ± 0.6 * 188 ± 12 * 2830 ± 31 Positive control group 150 ± 33 78 ± 6 * 12.4 ± 2.1 * 165 ± 18 * 2703 ± 122

(Mean ± standard deviation, *: P <0.05)

6-4. Investigation of protective effect of pathogenic bacteria

Salmonella typhimurium D45, which is used as a pathogenic bacterium, was isolated from diarrhea in broilers and incubated at 37 ° C for 24 hours using BHI liquid medium (Difco), and the number of bacteria was repeated three or more times using dilution method in BHI solid medium. Tested and measured.

The experimental group and negative control group ingested the strain of the present invention for 5 days were challenged with 1 ml of the diluted solution of Salmonella to 1 × 10 ¹⁰ CFU / ml of bacterial cells, and then confirmed that the reddish cells died or showed clinical symptoms. The weight gain, feed intake, negative intake, and feed efficiency were calculated in the same manner as in Example 6-1 (see Table 8).

group Weight gain (g / d) Feed Intake (g / d) Feed efficiency Drinking water intake (ml / d) G22-2 7.84 ± 0.72 ^* 18.85 ± 1.86 ^* 2.40 ± 0.06 ^* 49.68 ± 5.18 Negative control group 6.05 ± 0.80 16.65 ± 1.59 2.77 ± 0.29 51.77 ± 11.71

(Mean ± standard deviation, *: P <0.05)

In addition, feces were collected on days 1, 3, and 6 after challenge inoculation, suspended in ringer solution, and the number of Salmonella bacteria in feces was measured in a brilliant green agar (see Table 9).

group
Days Since Vaccination 1 day 3 days 6 days Negative control group 6.92 ± 0.22 3/8 2/8 G22-2 6.37 ± 0.33 * ND ND

(Mean ± standard deviation, *: P <0.05)

Attach an electronic file to a sequence list

Claims (5)

Lactobacillus Johnson, a strain deposited under the accession number KACC 91458 with the Korea Agricultural Microbiological Resources Center (KACC), which has 16s rDNA comprising the nucleotide sequence of SEQ ID NO: 1 and has acid resistance, bile resistance, and intestinal attachment ability ( Lactobacillus johnsonnii ). delete delete A feed additive composition comprising the Lactobacillus johnsonnii of claim 1 ( Lactobacillus johnsonnii ). The feed additive composition according to claim 4, wherein the feed additive inhibits the growth of pathogenic bacteria in the body.

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