KR20200043736A - Antimicrobial composition - Google Patents

Abstract

The present invention provides an antimicrobial composition having antibacterial activity on Salmonella spp., which contains one or more selected from the group consisting of Pediococcus acidilactici, Lactobacillus pentosus, Lactobacillus sakei, and Bacillus coagulans, as an active component.

Description

Antibacterial composition {Antimicrobial composition}

It relates to a poultry antimicrobial composition comprising at least one lactic acid bacteria as an active ingredient.

Salmonella is not only pathogenic in animals, but can also cause food poisoning in humans, and has attracted worldwide attention in the fields of public health, food safety and livestock hygiene. Among them, Salmonella enterica Serovar Enteritidis (S.ENTERITIDIS) can infect humans through feces of chickens, the highest rate of food poisoning among various Salmonella serotypes, and other food poisoning-causing bacteria. Compared to this, it is identified as a causative agent having a higher mortality rate. It can also cause fatal diseases in the elderly, patients and children. Therefore, in most countries, various efforts are being made to prevent and eradicate S.ENTERITIDIS infections, but it is a reality that they are experiencing many difficulties.

Poultry products, including eggs and poultry, are known to be the leading cause of food poisoning in Salmonella. In particular, bacteria that have grown in the intestine of chickens infected with S.ENTERITIDIS are discharged in large quantities through feces, contaminating the surrounding environment and re-infecting poultry, causing food poisoning. Infection passes through the yolks to the later chicks. In this case, horizontal infection of the infected chicks in the incubator or other chicks in the flock causes mortality, leading to direct farm loss in addition to public health loss. However, the regulation of antibiotic use is increasing due to the residual and transfer of antibiotics from livestock products, the emergence of antibiotic-resistant bacteria, and increased consumer demand for non-antibiotic broilers, making it difficult to control bacterial diseases in farms. In Korea, the use of antibiotics for feed additives is completely banned from 2012, and as the protective efficacy of salmonella vaccines, competitive exclusion agents, and antibacterial substances is incomplete, new antibiotic substitutes are needed.

One aspect comprises one or more selected from the group consisting of Pediococcus acidilactici, Lactobacillus pentosus, Lactobacillus sakei, and Bacillus coagulans. Provided is an antimicrobial composition having antibacterial activity against Salmonella spp., Which is included as an active ingredient.

Other aspects include one or more selected from the group consisting of Pediococcus acidilactici, Lactobacillus pentosus, Lactobacillus sakei, and Bacillus coagulans. Provided is a composition having a protective effect against Salmonella enteritidis comprising as an active ingredient.

Other aspects include one or more selected from the group consisting of Pediococcus acidilactici, Lactobacillus pentosus, Lactobacillus sakei, and Bacillus coagulans. Provided is a composition for preventing and treating Salmonella enteritidis comprising as an active ingredient.

Another aspect is one selected from the group consisting of Pediococcus acidilactici, Lactobacillus pentosus, Lactobacillus sakei, and Bacillus coagulans in poultry. Provided is a method for improving or treating Salmonella enteritidis-causing diseases of poultry, comprising administering a composition for preventing and treating Salmonella enteritidis comprising the above as an active ingredient.

Other aspects include one or more selected from the group consisting of Pediococcus acidilactici, Lactobacillus pentosus, Lactobacillus sakei, and Bacillus coagulans. It provides an additive for poultry feed containing as an active ingredient.

One aspect comprises one or more selected from the group consisting of Pediococcus acidilactici, Lactobacillus pentosus, Lactobacillus sakei, and Bacillus coagulans. It provides an antibacterial composition comprising as an active ingredient. The antimicrobial composition is to include a combination of Pediococcus acidilactici, Lactobacillus pentosus, Lactobacillus sakei, and Bacillus coagulans You can.

The antibacterial composition may have antibacterial activity against Salmonella spp., For example, Salmonella Enteritidis.

The antimicrobial composition may be acidic and anti-biliary. The acid resistance can be referred to as acid resistance. The anti-biliary properties may be referred to as anti-biliary properties.

The antimicrobial composition may have resistance to antibiotics. The antibiotic may be at least one selected from the group consisting of vancomycin, kanamycin, streptomycin, erythromycin, clofamfeticol, and clindamycin.

The antimicrobial composition may be for the prevention or treatment of Salmonella infection disease.

The antimicrobial composition may be administered to poultry. The poultry can be, for example, chicken, duck, goose, turkey or quail. The poultry can be broiler or broiler. The broiler can be poultry immediately after hatching, for example chicks.

    The antimicrobial composition may be a composition for oral administration. The antimicrobial composition may be a composition for nasal administration.

Pharmaceutical or veterinary agents of the antimicrobial composition can be made in a manner common in the art. Suitable solid or liquid herbal formulations are, for example, granules, powders, dragees, tablets, (micro) capsules, suppositories, syrups, juices, suspensions or emulsions, for the preparation of carrier substances, explosives, binders, coatings, expanders, lubricants Conventional means such as, taste, sweeteners and solution mediators are used. Animal and vegetable oils such as magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, starch, cellulose and derivatives, cod liver oil, sunflower oil, peanut oil or sesame oil, polyethylene glycol And solvents such as sterile water and monovalent or polyhydric alcohols such as glycerin. The composition is prepared by mixing a combination of one or more of the aforementioned lactic acid bacteria with a pharmaceutically or veterinary-compatible and physiologically resistant carrier and optionally additional suitable activity, additions or auxiliary substances in a predetermined amount and formulated into a desired dosage form. can do.

One or more selected from the group consisting of Pediococcus acidilactici, Lactobacillus pentosus, Lactobacillus sakei, and Bacillus coagulans as active ingredients It provides a composition having a protective effect against Salmonella enteritidis containing.

It provides a poultry feed composition comprising the above-described antimicrobial composition.

Provided is a method for improving or treating Salmonella enteritidis-causing diseases of poultry, wherein the composition for antibacterials is administered to poultry.

One selected from the group consisting of Pediococcus acidilactici, Lactobacillus pentosus, Lactobacillus sakei, and Bacillus coagulans showing antibacterial effects Provided is an antimicrobial composition having antibacterial activity against Salmonella spp. Containing the above strain or a culture medium thereof as an active ingredient. According to an example, the antimicrobial composition exhibits a bactericidal discharge suppression effect on digestive pathogens such as Salmonella. The antimicrobial composition has a competitive exclusion effect that prevents digestive pathogens, such as Salmonella, from settling in the intestine by forming a normal microflora in the intestine of poultry.

Figure 1 shows the mean results of Salmonella enteritidis re-separation in appendix stool according to administration of a mixture of four lactic acid bacteria (referred to as 'KCAV', test group) and the positive control PoultryStar.

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are intended to illustrate one or more embodiments by way of example, and the scope of the present invention is not limited to these examples.

Example 1: Preparation of four lactic acid bacteria mixed formulation

1.1 Animals used

One-day-old Ross broiler (Samhwawon species, Korea) was used. All experiments were performed according to the protocol with the approval of the Animal Experimental Ethics Committee (IACUC) of Konkuk University.

1.2 Isolation of lactic acid bacteria

Lactic acid bacteria were isolated from kimchi (cabbage, kkakdugi and dongchimi) and miso collected from various parts of Korea. For lactic acid bacteria separation, 1 ml fermentation soup was first added to 30 ml MRS liquid medium and incubated at 37 ° C. for 24 hours. One loop of cultured liquid medium was streaked on Rogosa solid medium and incubated at 37 ° C for 48 hours. Each grown colony was further streaked in MRS solid medium and incubated at 37 ° C. for 24 hours. For identification of lactic acid bacteria strains, a single colony isolated from each sample was sequenced and LAB was identified by 16s rRNA gene sequence analysis. The identified lactic acid bacteria were cultured in MRS liquid medium again, mixed with a cryopreservative, and stored at minus 70 ° C. Table 1 shows the isolated lactic acid bacteria. Table 1 shows the lactic acid bacteria isolated from the sample.

sample Lactobacillus Miso Pediococcus acidilactici (Pediococcua acidilactici) Kimchi Lactobacillus pentosus Kimchi Lactobacillus sakei Miso Bacillus Coagulans

1.3 Preparation of four lactic acid bacteria mixtures

The lactic acid bacteria type 4 mixture is a powdered beneficial bacteria mixture (or mixture) and is prepared for blended feedstock and real care. Pediococcus acidilactici , Lactobacillus pentosus , Lactobacillus sakei , Bacillus coagulans isolated from kimchi and miso are 1x10 ⁸ CFU / More than g was used as a raw material, and fructooligosaccharide was mixed as an excipient to prepare a supplementary feed. The prepared lactic acid bacteria 4-type mixture was diluted well with 20 g of 1,000 sugar in 1.5 liter lukewarm water and mixed with negative water to feed the chickens.

Example 2: In vitro S.Enteritidis Antibacterial activity test

2.1 Salmonella preparation

In this experiment, a strain having chloramphenicol resistance among Salmonella enteritica serovar Enteritidis strains pre-sold from the Salmonella Genetic Stock Center (SGSC) in Canada was used.

2.2 In vitro S.Enteritidis Antibacterial activity test

After dissolving kimchi and miso separated lactic acid bacteria 4 mixture products (Kav Lactobacillus prototype, KCAV) and 1 minute of the positive control PoultryStar ^{® in} 30 mL of MRS liquid medium, cultured at 37 ° C. for 24 hours. The culture medium of lactic acid bacteria cultured in MRS liquid medium was centrifuged at 3000 rpm for 20 minutes, and the supernatant was prepared by filtering (0.22 μm). Thereafter, S. Enteritidis standard strains were cultured in Nutrient Broth 20 ml at 37 ° C for 24 hours. 100 µl of standard strain S. Enteritidis cultured in Mueller Hinton agar was evenly dispensed. After making wells (6 mm) in Muller Hinton agar coated with S. Enteritidis standard strain, 75 µl of each filtered lactic acid bacteria supernatant was dispensed. 75 µl of MRS broth, which was not cultured as a control, was dispensed into one well. After incubation at 37 ° C. for 24 hours, the length of the inhibition section (diameter) of each well was measured (Table 2). Table 2 shows the inhibition period and antibacterial activity of S. Enteritidis of the lactic acid bacteria mixture culture and the positive control PoultryStar. As shown in Table 2, it was confirmed that the supernatant in the culture mixture of lactic acid bacteria showed an inhibitory effect on the growth of S. Enteritidis and a significant antibacterial effect.

division Type of lactic acid bacteria Antibacterial activity of lactic acid bacteria culture supernatant against S. Enteritidis Suppression section ^A Antibacterial power ^B One Lactobacillus mixture - +++ 2 PoultryStar® - +++

^A S. Enteritidis inhibition section (diameter) length of the wells with filtered lactic acid bacteria supernatant

^B +: 6 <= inhibition interval <= 9, ++: 10 <= inhibition interval <= 13, +++: 14 <= inhibition interval <= 16

Example 3: In vitro laboratory anti-oxidative and anti-biliary testing of lactic acid bacteria mixture

Artificial bile was prepared by adding 0.3% Bile salt (Fisher science) to the MRS liquid medium. After adjusting the pH to 2.5 by adding 1N HCL in sterile PBS, artificial gastric acid was prepared by adding 3 mg / ml (1000 unit / ml) of Pepsin (Acros Organics, Fair Lawn, NJ). After washing the mixture of four lactic acid bacteria products twice with PBS, the turbidity was adjusted to an OD value of 0.4 at 600 nm. After that, lactic acid bacteria were added to artificial bile juice and artificial gastric juice at a content of 1%, respectively. Artificial gastric juice was measured for changes in the number of bacteria after the reaction for 0 hours at room temperature and 3 hours after adding lactic acid bacteria. Artificial bile juice was measured for changes in the number of bacteria after incubation at 0 hours and 37 ° C. for 24 hours after the addition of lactic acid bacteria. As a control drug, the same test was performed for Biomin's PoultryStar® and compared.

division Lactic acid bacteria Initial number of bacteria (0 hour)
(log cfu / ml) Antioxidant
(log cfu / ml) Anti-biliary
(log cfu / ml) 3 hours after the reaction 24 hours after reaction One Lactobacillus mixture 2.7x10 ⁴ CFU / ml 2.2x10 ⁷ CFU / ml 2.4x10 ⁹ CFU / ml 2 PoultryStar® 4.2x10 ⁴ CFU / ml <10 ³ CFU / ml 4.6x10 ⁶ CFU / ml

Example 4: In vitro antibiotic susceptibility test for lactic acid bacteria mixture

Lactic acid bacteria mixture is pre-cultured in LSM agar at 37 ° C for 24 hours (Preculture). The lactic acid bacteria mixture cultured in LSM agar was dissolved in sterile physiological saline (0.85% NaCl) to fit 1 McFarland standard turbidity (3x10 ⁸ CFU / ml) (OD600 = 0.123). A dilution of the lactic acid bacteria mixture was added to the LSM Broth at a ratio of 1: 500 (= 6x10 ⁵ CFU / ml). Each antibiotic listed in Table 4 was 2-fold-serial dilution in a 96-well plate with LSM broth (50 μl volume). Table 4 shows the types of antibiotics and their concentrations.

Ampicillin (AMP) 0.5 ~ 64 μg / ml Vancomycin (VAN) 1 to 128 μg / ml Gentamicin (GEN) 2 to 256 μg / ml Kanamycin (KAN) 8 ~ 1024 μg / ml Streptomycin (STR) 2 to 256 μg / ml Erythromycin (ERY) 0.12 ~ 16 μg / ml Tetracycline (TCL) 1 to 128 μg / ml Chloramphenicol (CHL) 1 to 128 μg / ml

In a well diluted with EFSA-designated antibiotic (Guidance on the assessment of bacterial antimicrobial susceptibility), 50 µl of the lactic acid bacteria mixture dilution is added and reacted 1: 1. Dispense 100 µL of LSM broth to the negative control well and 100 µL of the lactic acid bacteria mixture to the positive control well. The result is read after a reaction at 37 ° C for 48 hours. If the MIC dilution factor was higher than the break point, it was judged to be resistant.

Table 5 is a result showing the resistance of the lactic acid bacteria mixture dilution to antibiotics. As shown in Table 5, the lactic acid bacteria mixture diluent shows antibiotic resistance against VAN, KAN, STR, ERY, CHL, and CLD.

division AMP VAN GEN KAN STR ERY TCL CHL CLD Lactobacillus mixture N / A o N / A o o o N / A o o

Example 5: Broiler Salmonella enteritidis inhibition efficacy evaluation

45 1-day-old broilers were divided into 3 groups of 15 animals and kept in quarantine. Group 1 was orally administered for 3 days after injecting 1 minute of the mixed preparation of 4 lactic acid bacteria. Group 2 was orally administered Poultrystar® sol from Biomin, which is known as a control drug, for 3 days. Group 3 was placed as an untreated positive control without medication. After administration of the test drug for 3 days, 100 μl (1.0x10 ⁶ cfu / ml) of broiler-derived Salmonella enteritidis strains were orally inoculated into all test groups to confirm the effect of the administered drug. Quantitative analysis of Salmonella enteritidis was performed by randomly selecting 5 animals from each test group 2 days after the 4th day of challenge and collecting the appendix by autopsy after euthanasia.

5.1 Quantitative analysis of Salmonella enteritidis

Quantitative analysis of Salmonella enteritidis was challenged for each chicken undergoing autopsy after euthanasia. After exposing the cecum through autopsy, feces in the intestine were collected, weighed, and diluted in 1x Phosphate buffer saline of 10 times the corresponding capacity. Subsequently, PBS containing the feces was serially diluted in 10 µl portions in XLD agar, a medium for selectively detecting Salmonella species, and then evenly spread using beads. XLD medium was prepared by adding 1% Chloramphenicol antibiotic to suppress E. coli in the intestine, and the challenge strain was set as Chloramphenicol resistant Salmonella enteritidis. Thereafter, the plated XLD agar was cultured in a 37 ° C incubator for 24 hours to determine the re-separation titer of Salmonella enteritidis using a dilution factor of a medium in which 10 colonies or less were observed.

5.2 Results of Salmonella enteritidis inhibition efficacy evaluation according to lactobacillus administration

The test group administered with a mixture of four lactic acid bacteria showed reduced Salmonella discharge re-separation in the appendix compared to the positive control group. Salmonella enteritidis On the second day after the challenge inoculation, Salmonella enteritidis was not detected as a result of re-separation in the group treated with the four lactic acid bacteria mixture and Poultrystar® sol, and in the positive control group, Salmonella enteritidis of 10 ^4.86 cfu / ml was detected (Table) 6). Table 6 shows the results of re-separation of Salmonella enteritidis in the appendix after 2 days of challenge, according to the administration of lactic acid bacteria.

group Lactic acid bacteria administration Average Mean log cfu / ml ^A G1 Lactobacillus mixture 0.00 G2 PoultryStar® 0.00 G3 - 4.86

^A The average value of Salmonella enteritidis quantitative values in 5 appendices (Average Mean log cfu / ml)

Four days after the inoculation also attack was not detected in the 10 ^5.31 cfu / ml, but a detection of S.ENTERITIDIS, 4 kinds of lactic acid bacteria preparations test group and control group Poultrystar® sol (Table 7). Table 7 shows the results of re-separation of S. Enteritidis in appendix stool 4 days after the challenge, according to lactic acid bacteria administration.

group Lactic acid bacteria administration Average Mean log cfu / ml ^A G1 Lactobacillus mixture 0.00 G2 PoultryStar® 0.00 G3 - 5.31

^A Mean of mean S. Enteritidis quantitative value in 5 autopsies (Average Mean log cfu / ml)

In the re-separation 7 days after challenge inoculation, Salmonella enteritidis of 10 ^6.71 cfu / ml in the positive control group and 10 ^4.40 cfu / ml in the Poultrystar® sol group were detected, but S.ENTERITIDIS was not separated in the case of the 4 lactic acid bacteria mixture ( Table 8). Table 8 shows the results of re-separation of Salmonella enteritidis in the appendix after 7 days of challenge, according to the administration of lactic acid bacteria.

group Lactic acid bacteria administration Average Mean log cfu / ml ^A G1 Lactobacillus mixture 0.00 G2 PoultryStar® 4.40 G3 - 6.71

^A The average value of Salmonella enteritidis quantitative values in 5 appendices (Average Mean log cfu / ml)

As a result of analyzing the average value of Salmonella enteritidis re-separation results in appendix stool on the 2nd, 4th, and 7th day after administration of the drug (see Table 9), the number of Salmonella enteritidis in the Poultrystar® sol group and the mixture of 4 lactic acid bacteria was statistically It was confirmed that it decreased significantly (P <0.001).

In addition, in case of lactic acid bacteria type 4 mixture, Salmonella enteritidis re-separation result was significantly lower after the challenge compared to Poultrystar® sol group used as a control drug. Table 9 shows the average Salmonella enteritidis re-separation in the appendix after administration of lactic acid bacteria.

group Lactic acid bacteria administration Average Mean log cfu / ml ^A G1 Lactobacillus mixture 0.00 G2 PoultryStar® 1.47 G3 - 5.63

^A Average of the mean value of Salmonella enteritidis in the appendix of the 5-day, 7-day, and 10-day necropsy system (Average Mean log cfu / ml)

Korea Research Institute of Bioscience and Biotechnology KCTC18709P 20180823

Claims (12)

Pediococcus acidilactici , Lactobacillus pentosus , Lactobacillus sakei , and one or more strains selected from the group consisting of Bacillus coagulans or a culture thereof It has an antibacterial activity against Salmonella spp. Containing as an active ingredient, a composition for antibacterial. The method according to claim 1, wherein the antimicrobial composition is Pediococcus acidilactici , Lactobacillus pentosus , Lactobacillus sakei , and Bacillus coagulans Antibacterial composition comprising a combination. The method according to claim 1, The antimicrobial composition is an antimicrobial composition that is anti-acid and anti-biliary. The method according to claim 1, The antimicrobial composition is an antimicrobial composition that is resistant to one or more antibiotics selected from the group consisting of vancomycin, kanamycin, streptomycin, erythromycin, clofamfeticol, and clindamycin. The method according to claim 1, The antimicrobial composition is a composition for antimicrobial to Salmonella Enteritidis ( Salmonella Enteritidis ). The method according to claim 1, The antimicrobial composition is an antimicrobial composition for the prevention or treatment of Salmonella infection disease. The method according to claim 1, The antimicrobial composition is an antimicrobial composition that is administered to poultry. The method according to claim 1, The antimicrobial composition is for oral administration is an antimicrobial composition. One or more selected from the group consisting of Pediococcus acidilactici , Lactobacillus pentosus , Lactobacillus sakei , and Bacillus coagulans as active ingredients A composition having a protective effect against Salmonella enteritidis containing. One or more selected from the group consisting of Pediococcus acidilactici , Lactobacillus pentosus , Lactobacillus sakei , and Bacillus coagulans as active ingredients Composition for preventing and treating Salmonella enteritidis containing. The composition according to claim 10, wherein the composition is for the prevention and treatment of Salmonella enteritidis for poultry. An additive for poultry feed comprising the antimicrobial composition of claim 1.

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