KR101098946B1 - A compound for feed additive comprising novel Lactobacillus salivarius G1-1 - Google Patents

Abstract

The present invention has acid resistance, bile, and intestinal attachment ability, and Lactobacillus salivarius G1-1, a strain deposited with the accession number KACC91449P to the Korea Agricultural Microorganism Support Center (KACC) and feed containing the same as an active ingredient. It relates to an additive composition.

According to the present invention, the Lactobacillus salivarius G1-1 strain of the present invention exhibits excellent antimicrobial activity against various pathogenic bacteria, as well as excellent acid resistance and bile resistance, and thus, such as intestinal pathogenic bacterial inhibitors, digestive agents, formal drugs, etc. The biotic composition may be usefully used for feed and animal medicines, and may significantly reduce the economic damage caused by livestock diseases of livestock farmers, and may replace imported animal medicines.

Lactobacillus salivarius, antibacterial activity, bile resistance, acid resistance

Description

Novel Lactobacillus salivarius G1-1} A compound for feed additive comprising novel Lactobacillus salivarius G1-1

The present invention relates to Lactobacillus salivarius G1-1 and a feed additive composition containing the same, and more specifically, has antimicrobial activity, acid resistance, bile, and intestinal adhesion, and on February 20, 2009, Korea Agricultural Microbiological Support Center (KACC) It relates to a lactobacillus salivarius G1-1, a strain deposited with the accession number KACC91449P) and a feed additive composition containing the same as an active ingredient.

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 benefit of probiotics is that it contributes to lowering blood cholesterol levels and increasing nutrient utilization.

Lactobacillus is widely present in nature and uses carbohydrates anaerobicly to produce lactic acid. The natural environment in which lactobacillus is found is diverse, not only in the gut of humans or animals, but also in various vegetables and fruits, and plays an important role in the fermentation process in fermented foods such as yogurt, kimchi in Korea, and sauerkraut in Germany. In charge of. Such lactic acid bacteria include Streptococcus sp., Pediococcus sp., Leukonostoc sp., Lactobacillus sp., Sporolactobacillus sp. .), Bifidobacterium sp. These lactic acid bacteria enters the intestinal epithelial cells and enters the intestinal epithelial cells to prevent intestinal settlement of harmful microorganisms and secrete antimicrobial substances, thereby inhibiting the growth of harmful microorganisms and improving diarrhea and constipation, as well as enhancing immune activity and anticancer activity. It has the effect of back. As lactic acid bacteria enter the intestine and secrete through its own metabolic activity, it gives many benefits to the host, so much research is being conducted using lactic acid bacteria. Lactic acid bacteria are normally present in the intestines of animals and balance the intestinal microflora 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 form colonies in the digestive tract until reaching 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 having many functional characteristics have been developed. There are lactic acid bacteria having a function of inhibiting the proliferation of pathogens, and BSH activity (Begley et al., 2006; Pereira et al., 2003) or starch hydrolysis activity. (lactic acid bacteria with 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, when identifying a novel Lactobacillus salivarius having antimicrobial activity in pig small intestine, when used as a feed additive, Intestinal pathogenic bacterial inhibitors, digestive agents, suit preparations and other probiotic compositions were confirmed that can be usefully used in feed and animal medicines, and completed the present invention.

Therefore, the main object of the present invention is to provide Lactobacillus salivarius G1-1 strain having excellent antibacterial activity against various pathogenic bacteria as well as having acid resistance and bile resistance.

Another object of the present invention is to provide a feed and veterinary medicine by using the Lactobacillus salivarius G1-1 strain as a feed additive to prepare a probiotic composition such as intestinal pathogenic bacterial inhibitors, digestive agents, suits and the like.

According to an aspect of the present invention, the present invention provides Lactobacillus salivarius G1-1, which is a strain deposited with the accession number KACC91449P to the Korea Agricultural Microorganism Support Center (KACC).

In Lactobacillus salivarius G1-1 of the present invention, the strain has 16s rDNA of SEQ ID NO: 1.

In Lactobacillus salivarius G1-1 of the present invention, it is preferred that the strain has acid resistance, bile, and intestinal adhesion.

According to another aspect of the present invention, the present invention provides a feed additive composition containing Lactobacillus salivarius G1-1 as an active ingredient.

In the feed additive composition of the present invention, the feed additive is preferably a feed additive that inhibits 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 place in the natural world, such as seawater, soil, plant, or animal, but to identify a strain for use as a feed additive for livestock, a healthy animal digestive organ is collected. It is preferable to use.

Strains showing the characteristics of Lactobacillus were purified purely from the small intestine of the pig intestine, and the antibacterial activity against the pathogenic microorganisms was selected to select strains conjugated for use in the present invention. . In the present invention, Escherichia coli O55 , Staphylococcus aureus and Salmonella typhimurium was evaluated for the antimicrobial activity of the Lactobacillus strain of the present invention, in addition to the evaluation of the antimicrobial activity against as many pathogenic bacteria as possible using other microorganisms known as pathogenic bacteria desirable.

Lactobacillus with excellent antimicrobial activity was selected through the above investigation, and then 16s rDNA sequences were analyzed to identify them, and the Lactobacillus was identified by comparing the sequences with those of known strains through a database. Can be. In the present invention, it was confirmed that the Lactobacillus of the present invention showed 99% homology with Lactobacillus salivarius subsp. Salivarius UCC118 through the above method, and thus was named Lactobacillus salivarius G1-1.

When the lactobacillus thus identified is applied to a real animal, it is necessary to confirm whether it can exert such useful ability, that is, antibacterial activity. Since the present invention is to enable the animal to consume the Lactobacillus as a feed additive, it must be able to survive in the digestive system of the animal after ingestion, stay in the intestine for a long time, exert an effect, artificially regulate growth I tried to evaluate it because it should be possible.

The evaluation method was carried out to evaluate acid resistance, bile resistance, intestinal adhesion and antibiotic resistance. For the evaluation of acid resistance, the identified strains were cultured in a liquid medium, and then the number of bacteria was adjusted to the same, and the pH was differently adjusted. Similarly, it can be evaluated by confirming the bacterial count change with and without bile salts added to the medium.

In order to evaluate the intestinal adhesion ability, it is possible to investigate the nonpolarity of the surface of the strain cell. In general, the strain having high nonpolarity adheres well to 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.

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.

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 check the metabolic changes 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.

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

Example 1. Lactobacillus Isolation

About 1 g of small intestine derived from healthy pigs was collected and washed with sterile saline. The small intestine was put in MRS broth and incubated for 24 hours. MRS containing 0.1 ml of the culture solution containing 0.5% CaCO ₃ (in 2 grams of glucose per 100 grams, in 0.1 grams of Tween 80, in 0.2 grams of ammonium citrate, in 0.5 grams of acetate, in 0.01 grams of magnesium sulfate, manganese sulfate In 0.005 grams, 0.2 grams of dibasic potassium phosphate, 1 gram of beef extract, 0.5 grams of yeast extract, and 1 gram of proteinase-treated peptone) were streaked to agar medium and incubated at 37 ° C. for 48 hours. Single colonies with a clear ring around the colonies were inoculated in MRS agar medium for pure separation.

Example 2 Evaluation of Antimicrobial Activity of Purely Isolated Lactic Acid Bacteria

When 10 pure Lactobacillus colonies cultured in MRS agar medium were inoculated in MRS broth and incubated for 18 to 20 hours, the pH of each Lactobacillus culture was 3.79 to 3.94. Using this culture supernatant and 5N sodium hydroxide, the culture supernatant adjusted to 4.5 and 5.0, respectively, was used to control the antibacterial activity against pathogenic bacteria such as Escherichia coli O55 , Staphylococcus aureus (ATCC 25923) and Salmonella typhimurium (ST302). It was investigated (Table 1, antimicrobial activity against E. coli O55 see FIG. 1).

Table 1 Antimicrobial activity against pathogenic bacteria E. coli O55

Strain name
PH of culture Diameter of restraint ring (mm) E. coli O55 Origin (pH 3.7) pH 4.5 pH 5.0 A11-1 3.94 ± 0.01 19.7 ± 0.3 14.4 ± 0.6 11.7 ± 0.4 A2-3 3.79 ± 0.01 19.9 ± 0.2 13.2 ± 0.4 12.1 ± 0.2 B4-5 3.88 ± 0.02 17.7 ± 0.6 13.5 ± 0.5 12.0 ± 1.0 E4 3.86 ± 0.03 19.7 ± 0.3 14.5 ± 0.5 12.1 ± 0.1 G1-1 3.89 ± 0.04 20.0 ± 0.0 16.3 ± 0.3 14.8 ± 0.7 G3-4 3.86 ± 0.04 20.0 ± 0.0 16.7 ± 0.7 14.0 ± 0.2 G7-2 3.88 ± 0.01 20.0 ± 0.0 15.2 ± 0.4 13.5 ± 0.5

(Expressed as mean ± standard deviation)

Table 2 Antimicrobial activity against pathogenic bacteria S. aureus ATCC 25923

Strain name
PH of culture Diameter of restraint ring (mm) E. coli O55 Origin (pH 3.7) pH 4.5 pH 5.0 A11-1 3.94 ± 0.01 18.5 ± 0.5 14.5 ± 0.8 ± A2-3 3.79 ± 0.01 19.0 ± 1.0 13.3 ± 0.3 ± B4-5 3.88 ± 0.02 17.9 ± 0.4 13.7 ± 1.2 10.8 ± 0.3 E4 3.86 ± 0.03 20.1 ± 1.0 14.4 ± 0.6 ± G1-1 3.89 ± 0.04 19.6 ± 0.5 16.4 ± 0.4 14.1 ± 0.1 G3-4 3.86 ± 0.04 20.5 ± 0.5 15.6 ± 1.4 14.1 ± 1.0 G7-2 3.88 ± 0.01 19.0 ± 0.3 15.7 ± 0.4 12.1 ± 0.2

(Expressed as mean ± standard deviation)

Table 3 Antimicrobial activity against pathogenic bacteria S. typhimurium ST302

Strain name
PH of culture Diameter of restraint ring (mm) E. coli O55 Origin (pH 3.7) pH 4.5 pH 5.0 A11-1 3.94 ± 0.01 19.2 ± 1.1 15.1 ± 0.1 11.9 ± 0.1 A2-3 3.79 ± 0.01 19.3 ± 1.1 13.5 ± 0.4 11.3 ± 0.3 B4-5 3.88 ± 0.02 19.2 ± 0.8 13.9 ± 0.4 11.7 ± 0.3 E4 3.86 ± 0.03 19.7 ± 0.6 14.5 ± 0.6 11.5 ± 0.5 G1-1 3.89 ± 0.04 20.4 ± 0.5 15.9 ± 0.7 13.1 ± 0.1 G3-4 3.86 ± 0.04 19.1 ± 1.2 15.9 ± 0.8 13.4 ± 0.4 G7-2 3.88 ± 0.01 19.3 ± 0.6 14.7 ± 0.6 12.2 ± 0.3

(Expressed as mean ± standard deviation)

As a result, as shown in Table 1 to Table 3, it was confirmed that the novel strain of the present invention Lactobacillus G1-1 is a strain excellent in inhibitory activity against harmful pathogenic bacteria. On the other hand, when the neutralized supernatant (pH 6.5) of Lactobacillus G1-1 was treated with trypsin and α-chymiotrypsin, antimicrobial activity was observed to be completely lost, confirming that strain G1-1 of the present invention is a bacteriocin-producing strain. (FIG. 2).

Example 3. Identification of Lactobacillus G1-1

In order to identify the G1-1 strain at the species level, 16S rDNA partial sequencing method, which is a molecular genetic strain identification method, was used, and the nucleotide sequence of the 16S rDNA portion was determined.

As a result, the base sequence of SEQ ID NO: 1 was determined, and the data of the NCBI (National Center for Biotechnological Information, http://www.ncbi.nlm.nih.gov/blast) were determined for the determined base sequence. 99% homology with Salivarius ( Lactobacillus salivarius subsp. Salivarius UCC118), so it was named Lactobacillus salivarius G1-1.

On February 20, 2009, the strain was deposited with the Korea Agricultural Microorganism Support Center (KACC), which is a depository institution for Korean patent strains, and was assigned accession number KACC91449P.

Example 4 Evaluation of Acid and Bile Resistance of Lactobacillus salivarius G1-1

The strain of the present invention, which has been tested for its ability to inhibit pathogenic bacteria, must survive in the digestive system in order to exert such ability in the digestive system of animals. Therefore, in order to confirm whether the strain of the present invention can survive in the digestive tract of the animal was evaluated for acid resistance and bile resistance.

4-1. Acid resistance evaluation

The Lactobacillus salivarius G1-1 of the present invention was incubated in MRS broth for 18 hours and then centrifuged (3000 × g, 15 minutes) to wash the pellet twice with phosphate buffer (pH 7.2). The washed pellet was placed in 1 ml of suspension and dissolved in 9 ml of phosphate buffer, and the final inoculation amount was adjusted to 7.2 × log CFU / ml. Then, the pH was adjusted to 7.2, 3.0, and 2.0 using 4N hydrochloric acid, respectively. After 3 hours of incubation of each pH-controlled culture medium, the number of bacteria was increased to 7.14 ± 0.05, 7.14 ± 0.03 and 3.97 ± 0.18, respectively. It was confirmed that the strain having excellent growth resistance (Table 4).

Table 4 Acid Resistance

Strain name Acid resistance (number of strains, log ₁₀ CFU / mL) pH 7.2 pH 3.0 pH 2.0 G1-1 7.14 ± 0.05 7.14 ± 0.03 3.97 ± 0.18 *

* Student's t-test showed significant differences (P <0.05).

4-2. Bile Resistance Assessment

In the MRS medium added 0.3% bile salt (Difco, USA) did not show a significant difference in the number of bacteria (7.0 × log CFU / ㎖) confirmed that the strain of the present invention is a strain having excellent bile resistance (Table 5) .

Table 5 Bile Resistance

Strain name Bile resistance 0.3% 1.0% G1-1 + -

(-: Do not grow, +: grow)

Example 5 Antibiotic Resistance Evaluation of Lactobacillus salivarius G1-1

The strain of the present invention was used in animals, and then antibiotic resistance was checked to determine whether the growth of the strain could be controlled by the use of antibiotics. Antibiotic resistance assessments were performed using a commercially available antibiotic sensitivity kit (Sensi-Disc ^® , BD Biosciences, Sparks, MD).

The Lactobacillus salivarius G1-1 strain of the present invention was incubated in MRS liquid medium for 18 hours and then centrifuged (3000 × g, 15 min), the pellet was washed twice with saline and dissolved in saline to give a final inoculation of 10 ^8. Adjustment was made with the bacterial count of CFU / ml. Put 200 μl of this solution into 100 ml soft agar, pour 15 ml into Petri dish (9cm), harden the medium, put the disc containing antibiotics on the medium and incubate for 48 hours at 37 ℃. The susceptibility to antibiotics was confirmed by measuring. As a result of this, most antibiotics were found to be susceptible, confirming that the strain of the present invention can be usefully used as a probiotic (Table 6).

[Table 6] Antibiotic Resistance Patterns

Antibiotic G1-1 Ampicillin (10 μg) S (sensitive) Cephalothin (30 µg) S Penicillin (10 μg) S Vancomycin (30 µg) R (resistant) Amikacin (30 µg) R Chloramphenicol (30 µg) S Erythromycin (15 μg) S Rifampin (5 μg) S

Example 6 Intestinal Attachment Evaluation of Lactobacillus Salivarius G1-1

In order for the strain of the present invention to remain in the animal's digestive organs for a long time, it is preferable that the intestinal adhesion ability is high, and thus the intestinal adhesion ability of the strain was evaluated. In order to evaluate the intestinal adhesion ability, the non-polarity of the cell surface was investigated, and the non-polarity rate was evaluated by the following Equation 1.

[Equation 1]

[Non-polarity rate (H%) = (1-A1 / A0) × 100]

Hexadecane (Sigma) was used to investigate the polarity of the cell surface. The strain was incubated in MRS liquid medium for 18 hours, followed by centrifugation (3000 × g, 15 min), and the pellet was washed twice with saline and then suspended in clean saline to adjust the absorbance to 0.5 at 600 nm (A0). 1.5 ml of hexadecane was added to the test tube containing 1.5 ml of the washed strain, shaken vigorously for 2 minutes, and the test tube was left to stand for 15 minutes to separate the aqueous layer, and the lower aqueous layer was carefully removed using a sterile pipette. After collecting the absorbance at 600 nm (A1).

As a result, the Lactobacillus salivarius G1-1 of the present invention showed a nonpolarity of 90.1%, and Lactobacillus coryniformis KCTC 3159 used as a control strain showed very low polarity (5.3%) (Table 1). 7).

TABLE 7

Strain name Nonpolarity rate (%) G1-1 90.1 ± 6.5 L. coryniformis KCTC 3159 5.3 ± 0

(Mean ± standard deviation, 3 repetitions)

In general, the strains of the present invention have excellent intestinal traits because they have high apolarity and adhere well to mucus or epithelial cells of the intestine, and have a high correlation between apolarity and colonic epithelial cell adhesion. It could be seen that the adhesion ability.

Example 7 Evaluation of the Effect of Lactobacillus Salivarius G1-1 on Real Animals

When the strain of the present invention is actually applied to an animal, in order to check the effect on the metabolism of the animal and the defense ability against the infection of harmful microorganisms, the weight change of the animal, blood chemistry, feces in accordance with the use of the strain The effect on the inoculation of the pathogen was confirmed as well as the effect on the composition, pH and moisture content of the microorganism.

After incubating Lactobacillus salivarius G1-1 in MRS medium, centrifugation (3000 × g, 15 min), washed the pellet twice with saline solution, dissolved in clean saline solution, and then strained to 5 ± 10 ⁶ CFU / RED (5 ± 10 ⁷ CFU / mL in pathogenic bacteria experiment) was allowed to be ingested in red (treated group).

As a positive control group, commercially available probiotics containing L. acidophilus ( L. acidophilus ), Lactobacillus casi ( L. casei ), and Bifidobacterium bifidum ( Primarac , Bayer Animal Drug) were used. About 1 g of a commercial probiotic was inoculated in MRS liquid medium and prepared in the same manner as G1-1, and the negative control group was ingested with red distilled water using clean distilled water.

7-1. Change in weight gain

The average weight of rats before the experiment was 122 ± 6g and the experiment was performed for 17 days. Body weight and feed intake were measured on the first and 17th days. Feed efficiency is calculated by dividing feed intake by weight gain. The lower the feed efficiency, the better the feed efficiency.

[Table 8] Change in weight gain

Experimental group Weight gain (g / d) Feed Intake (g / d) Feed efficiency Drinking water intake (ml / d) Negative Control 9.04 ± 0.31 22.30 ± 0.62 2.47 ± 0.04 36.8 ± 2.7 Treated group 9.12 ± 0.37 22.17 ± 0.88 2.43 ± 0.01 ^* 35.4 ± 1.5 Positive control group 9.01 ± 0.60 21.36 ± 0.90 2.37 ± 0.06 ^* 36.6 ± 2.6

(*: P <0.05)

As a result, as shown in Table 8, the G1-1 administration group (treatment group) of the present invention showed a low feed efficiency value, and the strain of the present invention was found to have an excellent effect as a commercial probiotic when used as a feed additive.

7-2. Fecal change

On the 8th and 17th days, fresh feces were collected, diluted 10 times with saline, mixed, and made a homogeneous solution to measure pH, and moisture was measured by freeze drying. For microbial measurement, 0.1 ml of the homogenate diluted by diluting fecals 10 times stepwise was incubated in MRS agar, MacConkey agar and Brilliant Green agar, which were used for measuring the number of microorganisms.

Table 9 Changes in microbes, pH and water in feces

Experimental group
8 days LAB Coliform pH Moisture (log ₁₀ CFU / g) (log ₁₀ CFU / g) (%) Negative Control 9.30 ± 0.11 7.41 ± 0.27 6.13 ± 0.22 50.3 ± 4.1 Treated group 9.37 ± 0.05 * 7.01 ± 0.25 * 6.02 ± 0.07 * 47.4 ± 4.5 Positive control group 9.45 ± 0.26 * 7.19 ± 0.43 * 6.07 ± 0.10 * 51.3 ± 5.2
Experimental group 17 days LAB Coliform pH Moisture (log10 CFU / g) (log10 CFU / g)  (%) Negative Control 9.15 ± 0.09 7.13 ± 0.05 5.86 ± 0.02 45.3 ± 3.8 Treated group 9.19 ± 0.07 * 6.81 ± 0.25 * 5.83 ± 0.07 52.8 ± 3.8 * Positive control group 9.30 ± 0.16 * 7.05 ± 0.24 5.81 ± 0.05 52.7 ± 0.5 *

(LAB: Lactic acid bacteria, *: P <0.05)

As a result, as shown in Table 9, the harmful microbial Coliform of the treated group was confirmed to be somewhat reduced compared to the control group, it was found that inhibiting the intestinal harmful microorganisms when using the strain of the present invention as a feed additive.

7-3. Changes in blood chemistry

Blood was collected on day 17 and reacted at 37 ° C. for 1 hour, followed by centrifugation at 2,000 × g for 10 minutes to separate serum, followed by 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.

[Table 10] Changes in Blood Chemistry

Experimental group
TG TCHO BUN HDLD AMYL (Mg / dl) (Mg / dl) (Mg / dl) (Mg / dl) (U / ℓ) Negative Control 137 ± 29 81 ± 9 15.4 ± 1.1 157 ± 13 2748 ± 132 Treated group 139 ± 45 73 ± 6 * 14.8 ± 0.9 * 161 ± 14 * 2830 ± 134 Positive control group 150 ± 33 78 ± 6 * 12.4 ± 2.1 * 165 ± 18 * 2703 ± 122

(*: P <0.05)

As a result, as shown in Table 10, when the G1-1 of the present invention was administered (treated group), the total cholesterol value in serum was significantly decreased.

7-4. Protective effect against pathogenic bacteria

Salmonella typhimurium D45, which is used as a pathogenic bacterium, was isolated from diarrhea in broilers, 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.

When challenged with Salmonella strains, no rats died or showed clinical symptoms for 11 days after challenge, and as shown in Table 11, weight gain and feed intake increased significantly in the G1-1 group. The feed efficiency was also confirmed to be excellent. The test results were expressed as mean ± standard deviation, and the component difference of Lactobacillus was analyzed using a one-way ANOVA test.

[Table 11] Protective effect against Salmonella strains

Experimental group Weight gain (g / d) Feed Intake (g / d) Feed efficiency Drinking water intake (ml / d) Treated group 7.97 ± 1.25 ^* 19.44 ± 1.52 ^* 2.48 ± 0.29 ^* 55.67 ± 5.92 Negative Control 6.05 ± 0.80 16.65 ± 1.59 2.77 ± 0.29 51.77 ± 11.71

(*: P <0.05)

The effect of Lactobacillus salivarius G1-1 on the excretion of Salmonella strains was also investigated. As a result, Salmonella was detected in all 8 animals at day 1. The number of Salmonella bacteria excreted 1 day after challenge was significantly reduced in the number of Salmonella bacteria excreted in rats to which the Lactobacillus strain of the present invention was administered (Table 12). As a result of comparing the number of Salmonella detected after 3 days and 6 days, the number of Salmonella detected in the Lactobacillus strain administration group of the present invention was significantly reduced compared to the control group (Table 12). Therefore, it was found that the administration of the lactic acid bacteria of the present invention inhibits the growth of the pathogenic bacteria in the body.

[Table 12] In vitro excretion effect of Salmonella

Experimental group
Days Since Vaccination 1 day (average number of bacteria) 3 days (detection object) 6 days (detection object) Negative Control 6.92 ± 0.22 3/8 2/8 Treated group 6.00 ± 0.32 * 1/8 0/8

(*: P <0.05)

As described above, according to the present invention, the Lactobacillus salivarius G1-1 strain of the present invention exhibits excellent antibacterial activity against various pathogenic bacteria, as well as excellent acid resistance and bile resistance, and thus, enteropathogenic bacterial inhibitors and digestive agents. Probiotics composition, such as suits, can be useful for feed and animal medicines, and can significantly reduce the economic damage caused by livestock diseases of livestock farms, as well as replace imported animal medicines have.

1 is a photograph showing the evaluation of the antimicrobial activity of E. coli O55 of Lactobacillus salivarius G1-1 of the present invention (top: Lactobacillus sp. G1-1 , left: Lactobacillus sp. B4-5 , right: Lactobacillus sp. A2-3 , and below: Lactobacillus sp.E2).

Figure 2 is a photograph showing that the inhibitory ring disappears and antibacterial activity is completely lost when Lactobacillus salivarius ( Lactobacillus salivarius ) G1-1 (pH 6.5) neutralized supernatant of the present invention is treated with trypsin and α-chymiotrypsin (1: G1-1 stock solution, 2: catalse treatment, 3: trypsin treatment, 4: pepsin treatment, 5: α-chymotrypsin treatment).

<110> REPUBLIC OF KOREA (Management: Ministry for Food, Agriculture, Forestry and Fisheries.National Veterinary Research and Quarantine Service (NVRQS)) <120> A compound for feed additive comprising Lactobacillus salivarius          G1-1 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 807 <212> DNA <213> Lactobacillus salivarius G1-1 <400> 1 gtttgatcat ggctcaggac gaacgctggc ggcgtgccta atacatgcaa gtcgaacgaa 60 actttcttac accgaatgct tgtattcacc gtaagaagtt gagtggcgga cgggtgagta 120 acacgtgggt aacctgccta aaagaagggg ataacacttg gaaacaggtg ctaataccgt 180 atatctctaa gggtcgcatg atccttagat gaaagatggt tctgctatcg cttttagatg 240 gacccgcggc gtattaacta gttggtgggg taacggccta ccaaggtgat gatacgtagc 300 cgaactgaga ggttgatcgg ccacattggg actgagacac ggcccaaact cctacgggag 360 gcagcagtag ggaatcttcc acaatggacg caagtctgat ggagcaacgc cgcgtgagtg 420 aagaaggtct tcggatcgta aaactctgtt gttagagaag aacacgagtg agagtaactg 480 ttcattcgat gacgtatcta accagcaagt cacggctaac taactacgtg ccagcagccg 540 cggtaatacg taggtggcaa gcgttgtccg gatttattgg gcgtaaaggg aacgcaggcg 600 gtcttttaag tctgatgtga aagccttcgg cttaaccgga gtagtgcatt ggaaactgga 660 agacttgagt gcagaagagg agagtggaac tccatgtgta gcggtgaaat gcgtagatat 720 tatggaagaa caccagtggc gaaagcggct ctctggtctg taactgacgc tgaggttcga 780 aagcgtgggt agcaaacagg attagat 807  

Claims (5)

Lactobacillus salivarius G1-1, which has a 16s rDNA of SEQ ID NO: 1, has antibacterial activity, acid resistance, bile, and intestinal attachment ability, and has been deposited with the accession number KACC91449P to the Korea Agricultural Microorganism Support Center (KACC). delete delete A feed additive composition comprising Lactobacillus salivarius G1-1 of claim 1 as an active ingredient. 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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