KR20240011581A - Lactobacillus reuteri sy308, a probiotics, and feed additive comprising thereof - Google Patents

Abstract

The present invention relates to Lactobacillus reuteri SY308 strain, which is a probiotic strain, and its use.
The Lactobacillus reuteri SY308 strain of the present invention has excellent acid resistance, bile resistance, and intestinal adhesion ability, so it can be used as a probiotic agent, and has excellent heat stability, so it has good processing suitability and shows excellent performance as a feed additive. In addition, it has excellent antibacterial performance against other harmful bacteria and can be used as an antibacterial composition.

Description

Lactobacillus reuteri SY308 strain, a probiotic strain, and feed additive containing the same {LACTOBACILLUS REUTERI SY308, A PROBIOTICS, AND FEED ADDITIVE COMPRISING THEREOF}

The present invention relates to a probiotic strain, Lactobacillus reuteri SY308 strain, and a feed additive containing the same.

Probiotics are defined as microorganisms and ingredients that have a beneficial effect on the host by improving the balance of intestinal microorganisms in humans or animals. Microorganisms used as probiotics include lactic acid bacteria, Bacillus subtilis, Bacillus, and yeast. The conditions that a probiotic must have in order to help the host are safety to the host, the ability to survive in the intestine by possessing acid resistance and bile acid resistance, the ability to settle in the intestine, and the ability to produce antibacterial substances to kill pathogenic bacteria. Inhibition ability, immune activation effect, easy mass production during the manufacturing process and survivability within the distribution period, etc. Additionally, in the case of livestock probiotics, there is improvement in growth rate and feed efficiency, ease of nutrient absorption due to improved digestibility, and There is a demand for the effect of improving odor in livestock environments.

Antibacterial substances (bacterioncins) are protein or peptide-based antibacterial substances secreted extracellularly and are known to kill or inhibit the growth of microorganisms closely related to the producing strain. Currently, nisin, the only antibacterial substance that is commercially available, is used as a food preservative in processed cheese, canned vegetables and fruits, and fermented milk products in about 50 countries. Since these antibacterial substances are decomposed by various proteolytic enzymes in the digestive system, they are harmless to the human body and have the advantage of non-residual properties. Accordingly, various studies have been actively conducted recently to utilize them as natural antibiotics or food preservatives. It's going on.

Accordingly, the present inventors completed the present invention by confirming the functionality of Lactobacillus reuteri SY308 strain, identified from commercially available kimchi, as a probiotic strain and antibacterial substance that can be used as a feed additive or food probiotic.

To solve the above problems

One embodiment of the present invention is

Provides the Lactobacillus reuteri SY308 strain deposited with accession number KCCM13205P.

To solve the above problems

Another embodiment of the present invention is

Lactobacillus reuteri SY308 strain deposited with accession number KCCM13205P;

Culture medium or culture of the above strain; and

Antibacterial substances isolated from the culture fluid or culture thereof; Provided is an antibacterial composition comprising any one or more selected from among the active ingredients.

One embodiment of the present invention to achieve the above object is

It is a Lactobacillus reuteri SY308 strain deposited with the accession number KCCM13205P.

Hereinafter, the present invention will be described in detail.

The present invention provides Lactobacillus reuteri SY308 (Lactobacillus reuteri SY308) strain deposited with deposit number KCCM13205P.

The present inventors identified a strain isolated from kimchi, a naturally fermented food, and identified Lactobacillus reuteri SY308 (Lactobacillus reuteri SY308) as a strain with acid resistance, bile resistance, and immune-enhancing activity, and sent it to the Korea Microorganism Conservation Center. It was deposited on June 13, 2022 and was given deposit number KCCM13205P.

In the present invention, “acid resistance” and “biliary resistance” refers to the ability to survive by reaching the intestines without being killed by gastric acid present in the stomach or by digestive juices containing digestive enzymes such as bile. And strains with bile tolerance have a high digestive tract survival rate. In the present invention, acid resistance may be acid resistance at pH 3 to 6, and bile resistance may be bile resistance at 0.1 to 0.3%.

Additionally, the present invention provides a probiotic preparation containing Lactobacillus reuteri SY308 or its culture medium as an active ingredient.

The probiotic preparation can be manufactured and administered in various formulations and methods according to methods known in the art. For example, the strain of the present invention or its culture medium is mixed with a carrier commonly used in the pharmaceutical field to produce powders, liquids and solutions, tablets, capsules, and syrups. , it can be manufactured and administered in the form of a suspension or granule. The carrier may include, for example, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a coloring agent, and a flavoring agent, but is not limited thereto. In addition, the administered dose can be appropriately selected depending on the absorption of the active ingredient in the body, the inactivation rate, the excretion rate, the age, gender, species, condition, and severity of the disease of the recipient.

The method of culturing the strain of the present invention can be cultured according to a method commonly used in the art, and is not limited to a special method.

When using the strain or its culture obtained in the step of cultivating the strain of the present invention as an additive, the strain or its culture can be added as is or used together with other additives, and can be used appropriately according to a conventional method. . The mixing amount of active ingredients can be appropriately determined depending on the purpose of use.

In addition, the present invention provides a starter composition for producing fermented foods containing Lactobacillus reuteri SY308 or its culture medium as an active ingredient.

The “starter for producing fermented food” refers to an agent or composition containing microorganisms involved in fermentation for producing fermented food. By adding it when manufacturing fermented foods, it is used to provide microorganisms that can grow in fermented foods or microorganisms that can grow as a dominant species. When manufacturing a food using the food fermentation starter, the quality of the food is controlled to a certain level by the microorganisms contained in the food fermentation starter, or for a specific purpose, for example, not to generate an off-flavor in the food, or The purpose of reducing can be achieved. In the present invention, fermented foods with acid resistance, bile resistance, and immune-boosting activity can be produced.

Additionally, the present invention provides a feed additive containing Lactobacillus reuteri SY308 or its culture medium as an active ingredient.

The feed additive using Lactobacillus reuteri SY308 of the present invention has excellent thermal stability even in a high temperature environment, making it easy to store, easy to apply to the manufacturing method, and has excellent acid resistance, bile resistance, and tenon adhesion ability. This helps the digestion of the subject consuming the food.

Another embodiment of the present invention for achieving the above object is Lactobacillus reuteri SY308 strain deposited with accession number KCCM13205P;

Culture medium or culture of the above strain; and

Antibacterial substances isolated from the culture fluid or culture thereof; It is an antibacterial composition containing one or more selected from among the active ingredients.

The antibacterial composition includes Clostridium difficile , Salmonella enteritidis, Vibrio vulnificus , Actinomyces viscosus , and Escherichia coli. coli ), Streptococcus sobrinu s, Salmonella typhimuriu m, Shigella sonnei , Vibrio parahaemolyticus , Salmonella enterica ( Salmonella enterica ), Fusobacterium nucleatum, ( Streptococcus mutan s), and Staphylococcus aureus ( Staphylococcus aureus ) Antibacterial against any one or more selected from the group consisting of It may be characterized as having activity.

The antibacterial composition may further include glycerol.

When the antibacterial composition further contains glycerol, the antibacterial activity against harmful bacteria is superior due to the synergistic effect of Lactobacillus reuteri SY308 strain and glycerol. At this time, the antibacterial composition of the present invention is Clostridium difficile , Staphylococcus epidermidis , Salmonella enteritidis , Vibrio vulnificus , and Actinomyces viscosus , Escherichia coli, Streptococcus sobrinu s, Salmonella typhimuriu m, Shigella sonnei , Vibrio parahaemolyticus , Salmonella enterica , Fusobacterium nucleatum , Streptococcus mutan s, Streptococcus mitis mitis ) and Staphylococcus aureus.

In addition, the antibacterial composition of the present invention can be used in pharmaceutical compositions, food compositions, food additive compositions, fertilizer compositions, fertilizer additive compositions, cleaning compositions, cleaning additive compositions, spray compositions, spray additive compositions, feed compositions, feed additive compositions, It may be an antibacterial composition that is a cosmetic composition or a quasi-drug composition.

The method of producing an antibacterial substance from the strain of the present invention further includes the step of treating the culture obtained in the step of culturing the strain of the present invention by one or more of the following processes: filtration process, concentration process, extraction process, and drying process. More may be included. For example, it may further include centrifuging the culture, filtering the supernatant obtained above, and concentrating the filtrate obtained above. The filtration process, concentration process, extraction process, and drying process can all be applied to conventional methods that a person skilled in the art can use for microbial cultures. Therefore, commonly used filter filtration, membrane filtration, ultrafiltration, centrifugation, heat concentration, freeze-drying, crushing, grinding, solvent extraction, and hot water extraction can be used alone or in combination.

As used in the present invention, the term "culture" refers to the result obtained by inoculating a medium and culturing it for a certain period of time. The culture medium itself is a crude culture medium obtained by culturing the strain according to the present invention in a suitable liquid medium, and the culture medium is filtered or centrifuged to isolate the strain. The filtrate or centrifuged supernatant from which the It includes, but is not limited to, concentrates obtained by concentrating extracts, etc. and their dried products.

The term “concentrate” used in the present invention refers to concentrated culture medium. “Extract” means extracted from the culture medium or its concentrate, and is not limited thereto, as long as it is an extract that can exhibit the antibacterial effect of the present invention, and is not limited to extract, diluted or concentrated liquid of the extract, dried product obtained by drying the extract, or these. It may include all crude products, purified products, and fractions thereof.

The term “dried material” used in the present invention refers to dried culture medium, its concentrate, and its extract. Drying methods include ventilation drying, natural drying, spray drying, and freeze drying, but are not limited thereto.

Therefore, the probiotic or antibacterial composition according to the present invention refers to the dried culture solution, its concentrate, and its extract. Drying methods include ventilation drying, natural drying, spray drying, and freeze drying, but are not limited thereto.

As used herein, the term “antibacterial” refers to the ability to reduce, prevent, inhibit, or eliminate the growth or survival of microorganisms at a specific concentration. Antibacterial composition may have the same meaning as antibiotic, which is a general term for antimicrobial agents, and may have the same meaning as antibacterial agent, bactericide, preservative, preservative or sterilizing agent, and is preferably gram-positive bacteria, gram-negative bacteria and fungi (yeast and mold). ) refers to a substance that can inhibit or inhibit the growth and life functions of one or more types of microorganisms selected from the group consisting of, that is, a substance with antibacterial and antifungal effects.

The antibacterial composition according to the present invention can be widely used in pesticides, medicines, cosmetics, food, food additives, feed, feed additives, household goods, etc. to achieve purposes such as antibacterial, sterilization, disinfection, and preservative purposes. Specifically, in agriculture, for antibacterial, sterilization, and disinfection purposes, in medicine, for purposes such as antibiotics or anti-pollution agents, in food, for preservative or antibacterial purposes, and in cosmetics and household goods, for anti-dandruff, athlete's foot, and armpit use. It can be used in products directly related to microorganisms, such as for anti-acne collection or for anti-acne, or for preservative, antibacterial, or sterilizing purposes such as cleaning detergents or dishwashing detergents, but is not limited to these purposes.

In this case, the strain of the present invention, its culture, and any one or more of the antibacterial substances produced by the strain are included as active ingredients.

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrating the present invention in more detail, and the scope of the present invention is not limited to these examples.

The Lactobacillus reuteri SY308 strain of the present invention has excellent acid resistance, bile resistance, and intestinal adhesion ability, so it can be used as a probiotic agent, and has excellent heat stability, so it has good processing suitability and shows excellent performance as a feed additive. In addition, it has excellent antibacterial performance against other harmful bacteria and can be used as an antibacterial composition.

Meanwhile, the effects that can be obtained from the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below. You will be able to.

Figure 1 shows the results of DNA sequence analysis of Lactobacillus reuteri SY308 strain.
Figure 2 shows the results of identification of Lactobacillus reuteri SY308 strain.
Figure 3 shows the results of hemolytic and bile decomposition characteristics of Lactobacillus reuteri SY308 strain.
Figure 4 shows the antibacterial activity evaluation results of Lactobacillus rhamnosus GG strain and Lactobacillus reuteri SY308 strain.
Figure 5 shows the inhibitory effect of E. coli ATCC 43896 strain according to the concentration of glycerol added with Lactobacillus reuteri SY308 strain.
Figure 6 shows the acid resistance and bile resistance evaluation results of Lactobacillus reuteri SY308 strain.
Figure 7 shows the intestinal adhesion (blue) and pathogen adhesion inhibition ability (red) of the Lactobacillus reuteri SY308 strain.
Figure 8 shows the heat stability evaluation results of Lactobacillus reuteri SY308 strain.

Hereinafter, embodiments of the present invention will be described in more detail. Embodiments of the present invention may be modified in various forms, and the scope of the present invention should not be construed as being limited to the following embodiments. This example is provided to more completely explain the present invention to those skilled in the art.

Example 1. Results of DNA sequence analysis and identification of strains

The lactobacillus used in this experiment was Lactobacillus reuteri SY308 (hereinafter also referred to as L. Reuteri SY308) strain derived from commercially available kimchi as a sample. Lactobacillus reuteri SY308 used in the present invention was obtained by diluting commercially available kimchi with sterilized saline solution and then smearing the diluted solution on selective medium to recover the detected colonies.

The full-length DNA sequence of the strain was analyzed using Pacbio sequence data. The full-length DNA sequence was amplified using a DNA polymerase complex (Sequel Binding kit 3.0), v4 sequencing primer, and annealed to the SMRTbell template to amplify the gene of the strain. To increase the sequencing accuracy of the amplified gene, excess primers and polymerase were removed using AMPure Purryption, then sequencing was performed using SMRT Cell 1M v2 and Sequencing Kit v3.0, and information from EZbioCloud, Swissprot, KEGG, and SEED databases. Based on this, the full-length DNA sequence was analyzed using the UBLAST program, and the results are shown in Figures 1 and 2. As a result of full-length DNA sequence analysis and identification, it was confirmed that it was a Lactobacillus reuteri strain, as shown in Figures 1 and 2.

Example 2. Hemolytic and bile degradative evaluation experiment

To evaluate the safety of this strain as a feed additive or food probiotic, hemolytic and bile degradative properties were evaluated.

To investigate whether the strain is hemolytic, the selection strain was inoculated into a medium prepared by adding 5% sheep blood from which fiber was removed to blood agar, and then cultured at 37°C for 48 hours to determine whether a transparent ring formed around the bacterial cells was formed. Hemolysis was determined.

Meanwhile, bile salt hydrolase (BSH) activity was tested by inoculating MRS-bile salt agar medium containing 0.5% taurodeoxycholic acid (TDCA) and culturing under anaerobic conditions at 37°C for 48 hours. . After incubation, the activity of bile salt decomposing enzyme was judged by whether bile salt was hydrolyzed and a white ring was formed around the colony, which is formed due to precipitation of bile acid.

The above experimental results are shown in Figure 3. As a result of the experiment, E. coli generally exhibits red blood cell hemolysis, but the L. reuteri SY308 strain does not have this characteristic and bile decomposition characteristics that reduce digestion power were not observed, making it safe as a feed additive or food probiotic. confirmed

Example 3 . L. reuteri Antibacterial activity evaluation experiment of SY308 strain and culture

To analyze the antibacterial activity of the L. reuteri SY308 strain, a total of 19 types of pathogens and 4 types of lactic acid bacteria were tested for antibacterial activity (including some periodontal disease bacteria), and Lactobacillus rhamnosus GG (hereinafter L. rhamnosus GG) was used as a control. ) strains were used for comparison. For the antibacterial experiment, 0.7% soft agar medium was prepared and analyzed using the paper disk method. In the case of L. reuteri SY308 strain, the supernatant was removed by centrifugation, 10 mL of 300 mM glycerol aqueous solution was added, and cultured for 2 hours. Afterwards, the culture was centrifuged and the supernatant was sterilized using a 0.45μm syringe filter. After dispensing 10 mL of 0.7% soft agar medium into a Petri dish, the strains were plated. 1 mL of the culture medium of the strain was taken and a sufficiently absorbed paper disk was placed on the medium. The culture was incubated at 37°C for 24 hours, and the size of the inhibitory ring formed around the well was measured using a caliper to analyze the antibacterial activity.

Meanwhile , L. reuteri SY308 was cultured in a medium supplemented with glycerol concentrations of 0, 20, 40, 60, 80, 100, 200, and 300 mM, then centrifuged to recover the supernatant and adjust the pH to 6.5. Sterilization was performed using a 0.45 ㎛ syringe filter. After mixing 5 mL of supernatant and 45 mL of TSB, E. coli ATCC 43896 was inoculated and cultured at 37°C. The growth potential of the bacteria was measured at OD 600 using a spectrophotometer in the culture medium at 0, 2, 4, 6, 8, 10, and 12 hours.

The results of antibacterial tests conducted on L. rhamnosus GG strain and L. reuteri SY308 strain are shown in Table 1 and Figure 4 below.

When glycerol was added to the growth medium, the L. reuteri SY308 strain had a better antibacterial range and antibacterial activity against pathogens than the L. rhamnosus GG strain, and showed excellent antibacterial activity against Salmonella strains, which are a problem in livestock industries such as poultry farming, especially Clos. It was confirmed that the antibacterial activity against Tridium strains was very excellent. (Table 1 and Figure 4)

In addition, after growing the L. reuteri SY308 strain in a medium containing glycerol, the growth inhibition effect on E. coli ATCC 43896 in the culture was examined, and the results showed that the culture showed antibacterial activity when more than 100 mM of glycerol was added to the medium. (Figure 5)

Example 4. L. reuteri Experiment to evaluate acid resistance, bile resistance and intestinal adhesion of strain SY308

To confirm its potential as a probiotic, acid resistance, bile resistance, and intestinal adhesion of L. reuteri SY308 strain were evaluated.

Resistance to artificial gastric acid is determined by preparing artificial gastric acid using 1 mg/mL pepsin (Sigma-Aldrich) solution and HCl to adjust the pH to 2, inoculating each lactic acid bacteria, and then incubating at 37°C for 0 hours, 1 hour, and 3 hours. The number of viable bacteria was measured. To evaluate resistance to artificial bile acids, each lactic acid bacteria was inoculated into an artificial bile acid solution containing pancreatin (Sigma-Aldrich) and bile, adjusted to pH 7.0 using HCl and NaOH, and incubated at 37°C for 0 hours, 12 days. The number of viable bacteria was measured at 24 hours. The number of viable bacteria in artificial gastric juice and artificial bile acid was measured by inoculating MRS agar medium and culturing at 37°C for 48 hours.

CaCo-2 cells were grown using minimum essential (MEM, Gibco) medium supplemented with 10% FBS (fatal bovine serum), 1% streptomycin/penicillin (10,00 IU/mL), and 25 mM HEPS, at 37°C. Cultured under 5% CO ₂ conditions. In order to confirm the adhesion of lactic acid bacteria and the ability to inhibit attachment to pathogens, the strains were Lactobacillus rhamnosus GG ( L. rhamnosus GG), Lactobacillus acidophilus ( L. acidophilus) ATCC 4356 , and Lactobacillus reuteri SY308 (L. reuteri). SY308) was used, and E. coli ATCC 43896 was used as the pathogen. Lactic acid bacteria were cultured in MRS medium and the cells were recovered by centrifugation (4000 RPM, 5 min). The cells were washed three times with PBS and then suspended at 1x108 CFU/mL in serum free MEM. CaCo-2 cells were inoculated into a 24-well plate at a concentration of 106 cells/well, cultured until the cells formed a monolayer, and then the strain was dispensed at 0.5 ml/well. The 24-well plate was incubated for 2 hours at 37°C and 5% CO ₂ and then washed with PBS to remove bacteria that failed to adhere. Pathogens were suspended in serum-free MEM in the same manner as lactic acid bacteria and dispensed at 0.5 mL/well. After culturing in a 24-well plate at 37°C and 5% CO2 conditions for 2 hours, the cells were washed with PBS. To measure the number of viable cells, 1 mL of 0.05% trition X-100 was added to each well and left for 10 minutes. Cells attached with PBS were recovered, serially diluted with sterilized 0.85% NaCl, plated on MRS agar medium, and cultured at 37°C for 48 hours. The viable cell count of E. coli was measured using MacConkey agar medium. The attachment rate (%) was determined as the ratio of the number of attached bacteria to the initial number of bacteria introduced into CaCo-2 cells. All experiments were repeated three times and the results were calculated as the average value.

The acid resistance and bile resistance of the L. reuteri SY308 strain were similar to those of the L. rhamnosus GG strain, which has proven efficacy as a conventional probiotic strain, and was therefore confirmed to be of sufficient value as a probiotic strain. (Figure 6)

In addition, as a result of examining the intestinal adhesion of the strain itself using the Caco-2 cell line, the intestinal adhesion was significantly higher than that of the L. rhamnosus GG strain and the L. acidophilus ATCC 4356 strain used as controls , and E. coli The ATCC 43896 adhesion inhibitory effect was lower than that of the L. rhamnosus GG strain, but showed a similar inhibitory effect to that of the L. acidophilus ATCC 4356 strain (Figure 7)

Example 5. L. reuteri Thermal stability evaluation experiment of SY308 strain

The thermal stability of L. reuteri SY308 strain was evaluated to confirm its processing suitability as a feed additive or food probiotic.

Thermal stability was measured by inoculating 100 uL of the subcultured culture into 10 mL of MRS broth and culturing at 60°C and 65°C for 5 and 15 minutes. The medium cultured at each time was step-by-step diluted and inoculated onto MRS plate medium, then cultured at 37°C for 48 hours, and the number of viable cells was measured to calculate the survival rate.

As a result of the experiment, as shown in Figure 8, all three strains tested ( L. rhamnosus GG, L. reuteri SY308, L. acidophilus ATCC 4356) showed high survival rates when treated at 60°C for 15 minutes, especially the L. acidophilus ATCC 4356 strain. Thermal stability was the highest, and the L. reuteri SY308 strain showed similar thermal stability to the L. rhamnosus GG strain.

In addition, even when treated at 65°C for 5 minutes, the survival rate of all strains tended to remain above 95%, and in particular , the L. reuteri SY 308 strain and the L. acidophilus ATCC 4356 strain showed significant differences compared to the L. rhamnosus GG strain. It showed high thermal stability. When treated at 65℃ for 15 minutes, the survival rate of all strains tended to decrease. The thermal stability of the L. reuteri SY308 strain was lower than that of the L. acidophilus ATCC 4356 strain, but the thermal stability was significantly higher than that of the L. rhamnosus GG strain. .

The above detailed description is illustrative of the present invention. Additionally, the foregoing is intended to illustrate preferred embodiments of the present invention, and the present invention can be used in various other combinations, modifications, and environments. That is, changes or modifications can be made within the scope of the inventive concept disclosed in this specification, a scope equivalent to the written disclosure, and/or within the scope of technology or knowledge in the art. The written examples illustrate the best state for implementing the technical idea of the present invention, and various changes required for specific application fields and uses of the present invention are also possible. Accordingly, the detailed description of the invention above is not intended to limit the invention to the disclosed embodiments. Additionally, the appended claims should be construed to include other embodiments as well.

Korea Center for Microbial Conservation (KCCM) KCCM13205P 20220613

Claims (10)

Lactobacillus reuteri SY308 (Lactobacillus reuteri SY308) strain deposited with accession number KCCM13205P. The strain according to claim 1, wherein the strain has acid resistance and bile resistance. A probiotic preparation comprising the strain of claim 1 or its culture medium as an active ingredient. A starter composition for producing fermented food comprising the strain of claim 1 or its culture medium as an active ingredient. A feed additive containing the strain of claim 1 or its culture solution as an active ingredient. Lactobacillus reuteri SY308 strain deposited with accession number KCCM13205P;
Culture medium or culture of the above strain; and
Antibacterial substances isolated from the culture medium or culture thereof; An antibacterial composition comprising one or more selected from among the active ingredients. The method of claim 6, wherein the antibacterial composition is Clostridium difficile , Salmonella enteritidis, Vibrio vulnificus , Actinomyces viscosus , Escherichia coli , Streptococcus sobrinu s, Salmonella typhimuriu m , Shigella sonnei , Vibrio parahaemolyticus , Salmonella enterica , Fusobacterium nucleatum , Streptococcus mutans , and Staphylococcus aureus . An antibacterial composition, characterized in that it has antibacterial activity against one or more substances. According to clause 6,
The antibacterial composition is a food composition, food additive composition, fertilizer composition, fertilizer additive composition, cleaning composition, cleaning additive composition, spray composition, spray additive composition, feed composition, feed additive composition, cosmetic composition, or quasi-drug composition. An antibacterial composition. According to clause 6,
An antibacterial composition further comprising glycerol. The method of claim 9, wherein the antibacterial composition is Clostridium difficile , Staphylococcus epidermidis , Salmonella enteritidis , Vibrio vulnificus , Actinomyces viscosus , Escherichia coli, Streptococcus sobrinu s, Salmonella typhimuriu m, Shigella sonnei , Vibrio parahaemolyticus , Salmonella enterica , Fusobacterium nucleatum , Streptococcus mutan s, Streptococcus mitis ( An antibacterial composition, characterized in that it has antibacterial activity against at least one selected from the group consisting of Streptococcus mitis ) and Staphylococcus aureus .