KR101201337B1 - A feed additive containig novel Lactobacillus spp. complex - Google Patents

Abstract

Lactobacillus johnsonnii G22-2 (KACC91458P), Lactobacillus reuteri ( Lactobacillus reuteri ) G8-5 (KACC91450P) and Lactobacillus salivarius ( Lactobacillus salivarius ) G1-1 (KACC91449P) It relates to a feed additive containing the strain as an active ingredient.
The lactic acid bacteria complex strain of the present invention is excellent in antimicrobial activity, starch resolution, acid resistance, bile resistance and intestinal adhesion ability against various pathogenic bacteria, is not antibiotic resistance, safe and does not have a mutual inhibitory effect and is suitable for use as a feed additive. The lactic acid bacteria complex strain feed additive of the present invention can protect against infection by various pathogenic bacteria and increase the utilization efficiency of the feed as well as replace animal medicines.

Description

A feed additive containing a novel Lactobacillus spp. complex}

The present invention relates to a feed additive comprising a novel Lactobacillus complex strain of the genus Lactobacillus, and more specifically, Lactobacillus Johnsonni G22- deposited with the Korean Agricultural Microbiology Support Center (KACC) under the deposit number KACC91458P on March 19, 2009. 2, It includes a lactic acid bacteria complex strain consisting of Lactobacillus luteri G8-5 deposited under the accession number KACC91450P and Lactobacillus salivarius G1-1 deposited under the accession number KACC91449P on February 20, 2009, and has antimicrobial activity against pathogenic bacteria, starch It relates to a feed additive with resolution, acid resistance, bile resistance and intestinal adhesion.

Since 2003, interest in probiotics beneficial to the intestinal microflora and the health of the host has been increasing (Reid et al, 2006). Among probiotics, especially Lactobacillus and Bifidobacterium are It is known to be beneficial to health. In the case of animals, probiotics can be expected to improve body weight gain, increase nutrient utilization in feed, reduce antibiotic use, and contribute to the production of good quality meat (Gilliland, 1990; du Toit et al., 1998; Lee et al., 2001; Torres-Rodriguez et al., 2007).

Lactobacillus is a major strain of probiotics, and can help normal bacteria function or serve as an antibiotic substitute. Many Lactobacillus used as probiotics have been used after confirming their functional properties in a laboratory and verifying their effects in animals, and excellent effects as probiotics can be expected by mixing and using strains with excellent antibacterial activity or nutrient utilization.

Accordingly, the present inventors have made intensive research efforts to find a new probiotic strain that can be used as a feed additive with the above advantages, and as a result, new Lactobacillus Johnson ni G22-2 (KACC91458P), Lactobacillus luteri G8-5 (KACC91450P) and lacto When Bacillus salivarius G1-1 (KACC91449P) is identified and a complex strain consisting of these lactic acid bacteria is used as a feed additive, it is prepared with probiotic compositions such as inhibitors of pathogenic bacteria in the intestine, digestive agents, and intestinal agents, so that it is useful for feed and veterinary medicine. It was confirmed that it can be utilized, and the present invention was completed.

Accordingly, the main object of the present invention is to provide a feed additive comprising a novel Lactobacillus lactic acid bacteria complex strain having excellent antimicrobial activity against various pathogenic bacteria, starch resolution, acid resistance, bile resistance and intestinal adhesion.

According to one aspect of the present invention, the present invention is Lactobacillus luteri G8-5, which is a strain deposited with the Korean Agricultural Microbiological Resource Center (KACC) under the accession number KACC91458P, Lactobacillus Johnsonni G22-2, and the strain deposited under the accession number KACC91450P And Lactobacillus salivarius G1-1, which is a strain deposited with accession number KACC91449P.

* In the Lactobacillus Johnsonny G22-2, Lactobacillus luteri G8-5 and Lactobacillus salivaryus G1-1 of the present invention, these lactic acid bacteria each have a 16s rDNA nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 .

According to another aspect of the present invention, the present invention provides a feed additive containing as an active ingredient a lactic acid bacteria complex strain consisting of Lactobacillus Johnsonny G22-2, Lactobacillus luteri G8-5 and Lactobacillus salivarian G1-1.

The feed additive of the present invention has antibacterial activity, acid resistance, bile resistance, and intestinal adhesion against pathogenic bacteria.

The feed additive of the present invention inhibits the proliferation of various pathogenic bacteria such as Salmonella typhimurium in the body.

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

The present invention relates to three novel lactobacilli isolated purely and to a feed additive using the same, wherein the novel lactobacilli were isolated from pig small intestine.

In the present invention, an attempt was made to isolate lactic acid bacteria that can protect animals from pathogenic bacteria, can survive in the intestine of animals, and aid in digestion of animals, and for this purpose, pathogenic bacteria are targeted for Lactobacillus strains isolated from pig small intestine. It was attempted to select a strain that satisfies the above conditions by examining antibacterial activity against bacteria, measuring bile salt hydrolase activity, or measuring starch degradation activity.

Through the above-described effect investigation, Lactobacillus excellent in antibacterial activity against pathogenic bacteria, Lactobacillus excellent in bile salt hydrolase activity, and Lactobacillus excellent in starch decomposition activity could be selected, and after confirming their 16s rDNA nucleotide sequence. The Lactobacillus could be identified by comparing it with the nucleotide sequences of previously known strains through the database.

Since the present invention makes it possible to provide beneficial effects to animals by using each of the useful properties of Lactobacillus as described above, it is necessary to confirm whether the three strains can be used as complex probiotics that can be added to feed.

Whether or not it can be used as a complex probiotic is whether the three strains are used together, whether they do not inhibit the growth of each other, whether they can survive together in the stomach and intestines, whether they can artificially control growth, and whether they can settle in the intestine for a long period of time. It can be judged by investigating whether it is possible.

In the present invention, when three strains are cultivated together to determine whether they can be used as a complex probiotic as described above, the acid resistance, bile resistance (viability in the digestive organs), and resistance to antibiotics (potential for controlling strain growth) of the strains , The non-polarity of the cell surface (fixation power in the intestine) and the inhibitory effect of the strains were investigated.

In order to find out whether the strains that have confirmed the potential as complex probiotics that can exhibit useful effects by adding to feed by the above methods can actually increase the gain rate and antibacterial activity of animals, after applying the feed additives, the growth rate of animals and pathogenic bacteria The antibacterial activity was investigated, and it was confirmed whether the feed additive of the present invention can increase the weight gain and antibacterial activity of the animals.

As described above, according to the present invention, the novel Lactobacillus strains of the present invention each exhibit excellent antibacterial activity, bile hydrolase activity, and starch degradation activity against various pathogenic bacteria, as well as complex strains consisting of these strains. It has excellent acid resistance, bile resistance, and intestinal adhesion, is safe because it is not resistant to antibiotics, and has no mutual inhibitory effect, so it is suitable for use as a feed additive. The complex strain feed additive of the present invention protects from infection by various pathogenic bacteria in breeding animals, increases the efficiency of use of feed, and can replace veterinary drugs.

1 is a photograph showing the evaluation of the antibacterial activity of Lactobacillus salivarius G1-1 of the present invention against E. coli O55 (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 when the neutralized supernatant of Lactobacillus salivarius G1-1 (pH 6.5) of the present invention is treated with trypsin and α-chymiotrypsin, the inhibitory ring disappears and the antibacterial activity is completely lost (1: G 1-1 Stock solution, 2: catalse treatment, 3: trypsin treatment, 4: pepsin treatment, 5: α-chymotrypsin treatment).
3 is a photograph showing the bile salt hydrolase activity of the Lactobacillus strains of the present invention. A ring is formed around the active strain.
4 is a graph showing the increase in hydrolysis of starch as the strain is cultured as a result of investigating the correlation between the growth of Lactobacillus strain and amylolytic activity in MRS broth containing 1% water-soluble starch.

Hereinafter, the present invention will be described in more detail through examples. Since these examples are for illustrative purposes only, the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Isolation and identification of lactic acid bacteria

1-1. Isolation of lactic acid bacteria

About 1 g of small intestine derived from a healthy pig was collected, washed with sterile saline, and minced. Shredded small intestine per 100 g of MRS (glucose 2g, Tween 80 0.1g, ammonium citrate 0.2g, acetate 0.5g, hydromagnesium sulfate 0.01g, anhydrous manganese sulfate 0.005g, dibasic potassium phosphate 0.2g, beef extract 1g, yeast 0.5 g of extract, 1 g of protein enzyme-treated peptone) was placed in a liquid medium and cultured for 24 hours. 0.1 ml of the culture solution was taken _{, streaked on MRS agar medium containing 0.5% CaCO 3} , and incubated at 37° C. for 48 hours. Colonies of several species with transparent rings around the colonies were inoculated on MRS agar medium and cultured repeatedly until a single strain was generated. Pure separation was performed through several inoculations, and each strain was named using alphabets and numbers.

1-2. Investigation of antibacterial activity against pathogenic bacteria

When several types of Lactobacillus colonies cultured on MRS agar medium were inoculated into MRS liquid medium and cultured for 18 to 20 hours, the pH of the Lactobacillus culture medium was 3.7. After adjusting the pH of the culture supernatant to 4.5 and 5.0 using 5N sodium hydroxide, the antibacterial activity against pathogenic bacteria such as Escherichia coli O55 , Staphylococcus aureus ATCC 25923, and Salmonella typhimurium ST302 was investigated by inoculating on a sterilized paper disk ( Fig. 1). Antimicrobial activity against these pathogenic bacteria is shown in Tables 1 to 3 in terms of the diameter of the inhibitory ring.

Antibacterial activity against pathogenic bacteria E. coli O55
Strain name
PH of the culture medium Diameter of suppression 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

(Indicated as mean ± standard deviation)

Antibacterial activity against pathogenic bacteria S. aureus ATCC 25923
Strain name
PH of the culture medium Diameter of suppression 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

(Indicated as mean ± standard deviation)

Antibacterial activity against pathogenic bacteria S. typhimurium ST302
Strain name
PH of the culture medium Diameter of suppression 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

(Indicated as mean ± standard deviation)

As shown in Tables 1 to 3, the G1-1 strain was confirmed to be a strain having excellent inhibitory activity against pathogenic bacteria. On the other hand, as shown in Figure 2, when the neutralized (pH 6.5) supernatant of the G1-1 strain was treated with trypsin and α-chymiotrypsin, the antibacterial activity was completely lost, and it was confirmed that the G1-1 strain is a strain that produces bacteriocin (Fig. 2).

1-3. Investigation of bile salt hydrolase (BSH) activity

After inoculating 50 µl of strain culture solution on a sterilized 8 mm paper disc, put it on MRS agar medium containing 0.5% (w/v) sodium salt of taurodeoxycholic acid (TDCA; Sigma) and 0.37% calcium chloride, and then incubate to internal bile. It was checked if there was activity.

As shown in FIG. 3, the strain having high BSH activity had a sediment ring around it, and the G22-2 strain showed very high activity with a sediment ring of 21.6±0.4, and this strain was selected (Table 4).

Bile salt hydrolase activity of each strain Strain name Size of settling ring (mm) B21 21.5±0.6 E1-1 20.7±0.6 G1-3 20.1±0.2 G17-2 20.1±0.4 G22-2 21.6±0.4

(Indicated as mean ± standard deviation)

1-4. Starch degradation (amylolytic) activity investigation

Starch decomposition activity related to carbohydrate digestion was confirmed through two steps. 5 µl of the culture supernatant of each strain was added to the modified MRS agar medium (1 g of starch per 100 g, 0.1 g of Tween 80, 0.2 g of ammonium citrate, 0.5 of acetate. g, 0.01 g of hydrous magnesium sulfate, 0.005 g of anhydrous manganese sulfate, 0.2 g of dibasic potassium phosphate, 0.5 g of yeast extract, 1 g of protein enzyme-treated peptone) incubated at 37°C for 72 hours, and then the medium was mixed with iodine solution (5 mM I _{2 ).} , 5 mM KI) to confirm the ring around the colony. The strains that confirmed the surrounding rings were re-modified MRS liquid medium (1 g of starch per 100 g, 0.1 g of Tween 80, 0.2 g of ammonium citrate, 0.5 g of acetate, 0.01 g of hydrous magnesium sulfate, 0.005 g of anhydrous manganese sulfate, and dibasic potassium phosphate. 0.2g, yeast extract 0.5g, protein enzyme-treated peptone 1g) was inoculated and incubated for 24 hours, and the amount of remaining starch was measured. Through this method, strains having excellent starch decomposition activity were identified, and the 24-hour culture solution showed a clear color of the starch-iodine solution. As shown in FIG. 4, it was confirmed that the decomposition of soluble starch was increased as the lactobacillus proliferated. The proliferation of Lactobacillus increased more rapidly when 1% starch was added to the MRS medium (maximal OD ₆₀₀ nm <1) (FIG. 4B). No residual starch was detected in all three strains after 15 hours of incubation, but a small amount of starch (0.3 mg/ml) was detected in the modified MRS medium.

1-5. Identification of selected Lactobacillus

The strains were identified at the species level through 16s rDNA partial sequencing, which is a molecular genetic strain identification method of the strains selected from Examples 2 to 4. The genomic DNA of the strains was extracted, and the DNA fragment for 16s rRNA was amplified through polymerase chain reaction (PCR), and the sequence was analyzed and compared with the database of NCBI. The other strain showed 99% homology with Lactobacillus salivarius.

From the above results, each strain was identified and named as Lactobacillus Johnsonny G22-2, Lactobacillus luteri G8-5, and Lactobacillus salivarius G1-1, and these were identified at the Korea Agricultural Microbial Resource Center in Suwon, Gyeonggi-do in March 2009. Deposits were completed on the 19th and February 20th, and the accession numbers KACC91458P, KACC91450P, and KACC91449P were assigned in order.

Experimental Example 1. Measurement of acid resistance and bile resistance

In order to confirm the acid resistance and bile resistance of the strains selected from Examples 2 to 4, incubation in MRS liquid medium for 18 hours and centrifugation (3000×g, 15 minutes) to convert the pellet into a phosphate buffer (pH 7.2). Washed twice. In 1 ml of washed pellet suspension, 9 ml of phosphate buffer was added and dissolved. The final inoculum was adjusted to 7.2 x log CFU/ml, and then the pH was adjusted to 4.0 and 3.5 with 4N hydrochloric acid, respectively, and the survival rate was checked after 3 hours incubation. In addition, when these strains were cultured in a medium containing 0.3% and 1.0% bile salt (Difco, USA) for 24 hours, the number of bacteria also showed a significant difference in MRS medium supplemented with 0.3% bile salt (Difco, USA). Not (7.0 × log CFU/ml) showed excellent bile resistance (Table 5).

Strains showing acid resistance and bile resistance Selected strain group
Strain name
Acid resistance Bile resistance pH 4.0 pH 3.5 0.30% 1.00%

Selection group of strains with excellent antibacterial activity A2-3 + +/- +++ +++ A11-1 + +/- +++ +++ B4-5 + - +++ +++ E4 + +/- +++ +++ G1-1 + - + - G3-4 + - + - G7-2 - - ++ + Selection group of strains with excellent bile salt hydrolytic enzyme activity B21 + - ++ ++ E1-1 + - ++ ++ G1-3 + - ++ ++ G17-2 + - ++ ++ G22-2 - - ++ ++ Selected strains with excellent starch decomposition activity G8-5 + - +++ +++ G23-2 + - +++ +++ I9-1 + +/- ++ ++

(-: Not growing, +: growing)

In the experiment, the strain having excellent acid resistance was cultured in MRS liquid medium for 18 hours and then centrifuged (3000×g, 15 minutes), and the pellet was washed twice with a phosphate buffer (pH 7.2). Add 9 ml of phosphate buffer to 1 ml of washed pellet suspension, dissolve, adjust the final inoculum to 7.2 x log CFU/ml, and adjust the pH to 7.2, 3.0 and 2.0 with 4N hydrochloric acid, respectively, and each culture solution with adjusted pH After incubating for 3 hours, the number of bacteria was expressed as log CFU/ml. All strains of the present invention were confirmed to be strains having excellent acid resistance even in a low pH environment (Table 6).

Viability of each strain according to pH Strain name The number of cells surviving at the controlled pH (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 G22-2 7.82±0.04 7.84±0.03 4.12±0.04 G8-5 7.23±0.01 7.22±0.03 4.13±0.12

(Indicated as mean ± standard deviation)

Experimental Example 2. Evaluation of antibiotic resistance

Was used as the antibiotic sensitivity kit ^{(Sensi-Disc ®, BD Biosciences} , Sparks, MD) that are commercially available to determine the antibiotic resistance patterns of the selected strain from Example 2 to Example 4 as a target for antibiotics used in animals and man . After culturing the strain, ^{adjust the number of bacteria to 10 8} CFU/ml, add 200 µl to 100 ㎖ soft agar, put 15 ㎖ each into Petri dish (9 cm) to harden the medium, inoculate a disc containing antibiotics, and measure the inhibitory ring. To confirm the sensitivity. As a result of examining the antibiotic resistance pattern of the strains, it was found that they were susceptible to most antibiotics, confirming the possibility of application as a probiotic (Table 7).

Antibiotic Resistance Patterns of Isolated Lactobacillus Strains Antibiotic G1-1 G8-5 G22-2 Ampicillin (AM10) S S S Cephalothin(CF30) S S S Penicillin(P10) S S S Vancomycin (VA30) R S S Amikacin(AN30) R I R Chloramphenicol(C30) S S S Erythromycin (E15) S S S Rifampin (RA5) S S S

(S: sensitive, R: resistant, I: medium sensitivity)

Experimental Example 3. Measurement of non-polarity of cell surface

The non-polarity measurement of the cell surface to determine the intestinal adhesion ability of the strains selected from Examples 2 to 4 was performed using hexadecane recommended by other researchers, and the strain was cultured in MRS liquid medium for 16 to 18 hours. The pellet was washed twice by centrifugation (3000×g, 15 min) and suspended in saline. The strain was adjusted to 0.5 to 0.7 at 600 nm of absorbance (A ₀ ), 1.5 ml of hexadecane was added to a test tube containing 1.5 ml of the washed strain, and then shaken vigorously for 2 minutes. After allowing the test tube to stand for 15 minutes, the lower aqueous solution layer was carefully collected using a sterile pipette, and the absorbance was measured at 600 nm (A ₁ ).

Non-polarity (H%) = (1-A ₁ /A ₀ )×100

The results of measuring the degree of nonpolarity on the cell surface are shown in Table 8, and the cell surface nonpolarity of the strains was 86.8 to 90.1%, and L. coryniformis KCTC 3159 used as a control strain showed very low nonpolarity at 5.3%. Therefore, the strains of the present invention are expected to exhibit excellent intestinal adhesion.

Cell surface nonpolarity of strains Strain name Non-polarity (%) G1-1 90.1±6.5 G22-2 86.8±13.7 G8-5 91.0±3.0 L. coryniformis KCTC 3159 5.3±0

(Indicated as mean ± standard deviation)

Experimental Example 4. Investigation of Mutual Inhibitory Effect

In order to investigate the mutual inhibitory effect of the strains selected from Examples 2 to 4, the isolated strains were inoculated into MRS medium to cross each other, cultured at 37°C for 48 hours, and then examined to see if they exhibited mutual inhibitory effects. It was confirmed that it could be used as a feed additive for complex strains because it did not show any effect.

Experimental Example 5. Rat weight gain, blood chemistry, fecal microbial composition, fecal pH and fecal moisture content

In the rat weight gain experiment with the feed additive of the present invention, a commercially available probiotic containing L. acidophilus , L. casei, and Lactobacillus bifidum (Primarak, Bayer Animal Pharmaceuticals) was used as a positive control. About 1 g of a commercial probiotic was inoculated into MRS liquid medium and cultured at 37° C. for 24 hours to be used in the experiment. The culture solution of a commercial probiotic, which is a positive control, was washed twice by centrifugation, concentrated, and then suspended in saline, and the final inoculum concentration was 5×10 ⁶ CFU/ml and applied to negative water. Was the complex strains of the invention be suspended for 24 hours the culture by centrifugation and then the pellet in MRS broth with brine measure the number of bacteria, three strains 1 to ensure that the bacterial count of 5 × 10 ⁶ CFU / ㎖: 1: 1 Mixed and applied to negative water.

Rat weight gain, blood chemistry, fecal microbial composition, fecal pH and fecal water content were investigated for 17 days by the complex strain feed additive, and the average weight of the rat was 122±6g. Body weight and feed intake were measured on the first and 17th days, and fresh feces were collected on the 0, 8, and 17 days to measure microorganisms, pH, and moisture.

As for the change in weight gain of rats, it was confirmed that the group to which the compound strain feed additive of the present invention was applied showed superior weight gain compared to other groups, and feed efficiency was also excellent as shown in Table 9.

Changes in weight gain in rats Application 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 Complex probiotics 9.29±0.16 21.92±0.33 2.36±0.03 ^* 34.8±0.6 Positive control 9.01±0.60 21.36±0.90 2.37±0.06 ^* 36.6±2.6

(Indicated as mean ± standard deviation) (*: P <0.05)

As a result of investigating the effect of the complex strain feed additive on the feces of rats, it was found that the intestinal lactobacilli significantly increased compared to the control, and the harmful microorganism Coliform significantly decreased compared to the control (Table 10).

Effects on microorganisms, pH and moisture in rat feces
Application group
Feces collected on the 8th day Lactobacillus Coliform pH Moisture content (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 Complex probiotics 9.7±0.09* 6.91±0.37* 5.93±0.09 50.3±3.0 Positive control 9.45±0.26 7.19±0.43 6.07±0.10 51.3±5.2
Application group
Feces collected on day 17 Lactobacillus Coliform pH Moisture content (log ₁₀ CFU/g) (log ₁₀ CFU/g) (%) Negative control 9.15±0.09 7.13±0.05 5.86±0.02 45.3±3.8 Complex probiotics 9.34±0.08* 6.73±0.40* 5.84±0.17 52.8±3.8 Positive control 9.30±0.16 7.05±0.24 5.81±0.05 52.7±0.5*

(Indicated as mean ± standard deviation) (*: P <0.05)

To investigate the effect of multi-strain feed additives on blood chemistry in rats, serum total cholesterol (TCHO), total glyceride (TG), bilirubin (BUN), and lipoprotein (high density) were investigated. The levels of lipoprotein, HDLD) and amylase (amylase, AMYL) were investigated, and the results are shown in Table 11. When the combination probiotic was administered, serum bilirubin and HDLD significantly decreased.

Effects on blood chemistry in rats Application group
TG TCHO BUN HDLD AMYL (Mg/㎗) (Mg/㎗) (Mg/㎗) (Mg/㎗) (U/l) Negative control 137±29 81±9 15.4±1.1 157±13 2748±132 Complex probiotics 170±64 80±4 12.4±1.9* 180±18* 2775±147 Positive control 150±33 78±6 12.4±2.1* 165±18 2703±122

(Indicated as mean ± standard deviation) (*: P <0.05)

Experimental Example 6. Changes in defense ability of pathogenic bacteria in rat

A commercially available probiotic containing L. acidophilus, L. casei, and Lactobacillus bifidum (Primalac) was used as a positive control for the pathogenic bacteria defense effect test. . About 1 g of a commercial probiotic was inoculated into MRS liquid medium and cultured at 37° C. for 24 hours to be used in the experiment. The culture solution was washed twice by centrifugation, concentrated, and then suspended in saline, and the final inoculation concentration was 5×10 ⁷ CFU/ml and applied to negative water. The complex strain of the present invention was also cultured in MRS broth for 24 hours, centrifuged, and then the pellet was suspended with saline and the number of bacteria was measured, and then the three strains were mixed at 1:1:1 with a number of bacteria of ^{5×10 7 CFU/ml.} It was applied to negative numbers.

Salmonella strain ( Salmonella typhimurium ), no rats died or showed clinical symptoms for 11 days after the challenge vaccination, and as shown in Table 12, the weight gain and feed intake significantly increased in the group applied with the complex strain feed additive, and feed efficiency was also excellent. Confirmed. The difference in composition of Lactobacillus was analyzed using one-way ANOVA.

Influence of Salmonella Attack on Weight Gain in Vaccination Application group Weight gain (g/d) Feed intake (g/d) Feed efficiency Drinking water intake (ml/d) Complex probiotics 8.16±10.67 ^* 19.47±1.42 ^* 2.39±0.06 ^* 51.78±3.31 Positive control 6.05±0.80 16.65±1.59 2.77±0.29 51.77±11.71

(Indicated as mean ± standard deviation) (*: P <0.05)

As a result of investigating the effect on the excretion of Salmonella strains, excretion of Salmonella was significantly reduced in rats to which the complex strain feed additive was applied 1 day after challenge vaccination (Table 13). In addition, the number of Salmonella strains excreted from the lactic acid bacteria administration group on the 6th day of challenge vaccination was significantly reduced compared to the control group. Therefore, it can be expected that the application of the feed additive of the complex strain of the present invention will inhibit the proliferation of pathogenic bacteria in the body.

Changes in the amount of Salmonella cells discharged from the body Application group
Cell mass according to the number of days elapsed after challenge inoculation (log ₁₀ CFU/g) 1 day 3 days 6 days Negative control 6.92±0.22 3/8 2/8 Complex probiotics 6.34±0.12* ND ND

(Indicated as mean ± standard deviation, ND: not detect) (*: P <0.05)

National Institute of Agricultural Sciences Agricultural Genetic Resource Center KACC91449 20090220 National Institute of Agricultural Sciences Agricultural Genetic Resource Center KACC91450 20090220 National Institute of Agricultural Sciences Agricultural Genetic Resource Center KACC91458 20090220

Attach electronic file of sequence list

Claims (4)

Lactobacillus johnsonnii G22-2 KACC91458P having a 16s rDNA sequence of SEQ ID NO: 1, Lactobacillus reuteri G8-5 KACC91450P having a 16s rDNA sequence of SEQ ID NO: 2, and 16s of SEQ ID NO: 3 Lactobacillus salivarius (Ractobacillus salivarius ) having a rDNA sequence of Lactobacillus salivarius G1-1 KACC91449P containing a lactic acid bacteria complex strain as an active ingredient, characterized in that it suppresses the growth of pathogenic bacteria in the body. The feed additive according to claim 1, wherein the lactic acid bacteria complex strain is a strain having antimicrobial activity, starch resolution, acid resistance, bile resistance and enteric attachment ability against pathogenic bacteria. delete According to claim 1, wherein the pathogenic bacteria Salmonella typhimurium ( Salmonella typhimurium ) characterized in that the feed additive.

Images