KR102191432B1 - Lactobacillus taiwanensis strain having antibacterial activity against harmful bacteria and uses thereof - Google Patents

Abstract

The present invention relates to a Lactobacillus taiwanensis strain having antibacterial and antifungal activity and uses thereof.
The composition containing the Lactobacillus taiwanensis NIBRBA00501006 strain of the present invention or a culture solution thereof exhibits a broad antimicrobial spectrum against various bacteria, and the composition of the present invention can be applied to various bacterial infections caused by harmful bacteria.
In addition, the composition of the present invention can be usefully used for antibacterial purposes in various fields such as medicine, health functional food, or feed preservative.

Description

Lactobacillus taiwanensis strain having antibacterial activity against harmful bacteria and uses thereof {Lactobacillus taiwanensis strain having antibacterial activity against harmful bacteria and uses thereof}

The present invention relates to a Lactobacillus taiwanensis strain having an antibacterial activity against harmful bacteria and a use thereof.

Some of the substances that play an important role in the process of maintaining the homeostasis of living organisms are bioactive substances derived from various organisms. There are many studies on numerous bioactive substances so far, and among them, studies on antibacterial and antifungal substances are very important areas in the fields of life science and medicine.

All living organisms are known to produce antibacterial and antifungal substances for survival, and a lot of research has been conducted on microorganisms that produce antibacterial and antifungal substances, and microorganisms that produce antibacterial and antifungal substances or substances separated from them are used. Studies to develop antibacterial agents have been attempted for a long time.

On the other hand, lactic acid bacteria is a bacillus that produces lactic acid by fermenting sugar. It produces useful substances that help our body health, and improves immunity, antioxidants, antibacterials, anti-cholesterol, anti-obesity, carcinogenic and mutant substances. It is known to have a function of preventing the generation of.

As antimicrobial metabolites produced by lactic acid bacteria, organic acids, hydrogen peroxide, diacetyl, beta-hydroxypropionaldehyde, and bacteriocins have been reported ( Adams and Hall 1988; 1993; Plockova 1997). Among them, bacteriocin is a natural substance with antibacterial activity that is distinct from other antibiotics, and is known as a proteinaceous antibiotic that affects the growth of Gram-positive and Gram-negative bacteria (Abdel-Bar 1987, ten Brink 1994). Although many studies on the antibacterial activity of lactic acid bacteria against various pathogens and harmful microorganisms have been conducted domestically and internationally (Fank 1991, Gilliland and Speck 1977, Lewus 1991, Kim 1997), it is still strong enough to be useful industrially. There are not many lactic acid bacteria that have antibacterial activity, and it is not clear which substances among the fermentation products of lactic acid bacteria have beneficial effects on the human body (Mcfarland and Elmer 1995, Sanders 1999).

In addition, in food, lactic acid bacteria determine the taste of fermented foods and characteristics of fermented products, and contribute to the storage of food by generating substances having antibacterial activity such as organic acids and bacteriocin. Bacteriocin is an antimicrobial active protein or peptide produced from various Gram-positive bacteria, and bacteriocin derived from lactic acid is attracting attention as a substance that can be used as a natural preservative in the food industry and as a substitute for antibiotics. Klaenhammer classified bacteriocins produced by lactic acid bacteria according to molecular structure and physiological characteristics as follows.

Class I bacteriocins contain specific amino acids such as lanthionine as a result of post-translational modification. Class II bacteriocin is a bacteriocin that does not contain lanthionine and has a small molecular weight (<13 kDa) and does not form a tertiary structure due to its small molecular weight, so it is relatively stable to heat, and is generally a membrane potential helix composed of about 20 hydrophobic amino acids. It is characterized by having a (transmembrane helix) structure. Class III bacteriocin is a high molecular weight (>30 kDa), heat-labile antibacterial active protein. For example, the nisin produced by Lactococcus lactis belongs to the Class I type and has a relatively broad antimicrobial spectrum and inhibits the proliferation of most Gram-positive bacteria. Nisin is heat-stable and active even in low pH conditions, and is used as a natural food preservative. In addition, most of the bacteriocin activity is known to be strong in weak acidity and unstable in a basic environment, and it was difficult to establish an experimental method that can be widely applied to determine whether bacteriocin is produced from the culture medium of the genus Lactobacillus that produces acid. .

Under this background, the present inventors made diligent efforts to discover lactic acid bacteria strains having excellent antibacterial activity, as a result of which the new strain Lactobacillus Taiwan Nsis NIBRBA00501006 strain has the effect of secreting bacteriocin having excellent antibacterial activity, antibacterial agents, health functional foods After confirming that it can be usefully used for food preservatives and feed additives, this invention was completed.

Korean Patent Registration Publication No. 10-1772870 Korean Patent Registration Publication No. 10-1757623 Korean Patent Registration Publication No. 10-1750468 Korean Patent Registration Publication No. 10-1658377

Accordingly, the main object of the present invention is to provide a Lactobacillus taiwanensis strain having antibacterial activity.

Another object of the present invention is to provide an antibacterial composition using the Lactobacillus taiwanensis strain.

According to one aspect of the invention, the present invention provides a Lactobacillus Taiwan N-Sys (Lactobacillus taiwanensis) NIBRBA00501006 strain entrusted with Accession No. KCTC 18771P.

Terms of the present invention, "Lactobacillus Taiwan N-Sys (Lactobacillus taiwanensis) NIBRBA00501006 strain" may be used interchangeably as the novel Lactobacillus Taiwan N-Sys strains identified separating newly by the present inventors, and "NIBRBA00501006 strain" or "NIBRBA00501006".

The present inventors investigated and analyzed the antibacterial effect of lactic acid bacteria for several years for the development of a new antibacterial agent, and as a result, verified the strong antibacterial effect of Lactobacillus taiwanensis and completed the present invention (see FIGS. 1 to 8).

The present inventors compared the gene sequence of the strain of the present invention with the gene sequences of conventionally known strains, and confirmed that the strain of the present invention is a new strain belonging to Lactobacillus taiwanensis. It was named NIBRBA00501006, and it was deposited with the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center on May 10, 2019, and was given the accession number KCTC 18771P.

In addition, any one Lactobacillus Taiwan N-Sys (Lactobacillus taiwanensis) NIBRBA00501006 strain of the present invention is selected from Salmonella spp, S. Cherry teeth sp, Streptococcus sp, Bacillus sp, and the group consisting of a recess in the strain in Saccharomyces Rome It is characterized in that it has antibacterial activity against the above pathogens. Examples of the strain of the genus Salmonella include Salmonella gallinarum , and examples of the strain of the genus Escherichia include E. coli. In addition, examples of the strain of the genus Streptococcus include Streptococcus iniae , and examples of the strain of the genus Bacillus include B. cereus , and examples of the strain of the genus Saccharomyces include Saccharomyces. Rome Esces cerevisiae (S. cerevisiae) may be mentioned, but is not limited thereto.

According to another aspect of the invention there is provided Lactobacillus Taiwan N-Sys (Lactobacillus taiwanensis) NIBRBA00501006 strain (Accession No. KCTC 18771P), the culture medium of the strain, at least one selected of the culture solution concentrate and from the group consisting of a dried product of the culture broth It provides a starter for fermentation comprising the above as an active ingredient. In the present invention, the fermentation starter refers to a preparation or composition comprising any one selected from the group consisting of a microorganism including lactic acid bacteria or bacteria involved in fermentation, a culture thereof, a concentrate of the culture, and a dried product of the culture In addition, the fermentation starter is used to provide microorganisms that can grow in fermented foods or as dominant species by adding them in the production of fermented foods. In the case of manufacturing fermented food using the fermentation starter, the quality of the fermented food can be constantly controlled by microorganisms included in the fermentation starter, or the speed or stage of fermentation can be controlled, and fermentation that achieves a specific purpose You can make food. In addition, the fermentation starter may further include commonly used additives.

The Lactobacillus taiwanensis of the present invention The NIBRBA00501006 strain inhibits the growth of pathogenic bacteria, has excellent antibacterial activity against various pathogenic microorganisms, and is harmless to the human body, so it can be usefully used as a fermentation starter in the manufacture of safe fermented foods.

According to another aspect of the present invention, the present invention is Lactobacillus taiwanensis It provides a fermented food produced using the NIBRBA00501006 strain (accession number KCTC 18771P) or the fermentation starter. The fermented food refers to a food prepared by adding one or two or more microorganisms such as lactic acid bacteria or enzymes, and using the fermentation action of the microorganism, and in detail, a food prepared by adding and aging fermented food seeds to a food substrate. it means. The fermented food includes all non-sterile open fermented foods such as liquor, bread, kimchi, salted fish, miso, soy sauce, cheese, butter, and yogurt.

According to another aspect of the invention, the invention provides a fermented food manufacturing method comprising inoculation of Lactobacillus Taiwan N-Sys (Lactobacillus taiwanensis) for NIBRBA00501006 strain (Accession No. KCTC 18771P) or the fermented starter to the raw material. The fermented food manufacturing method may be a method of manufacturing kimchi, cheese, butter, yogurt, etc., and may be used in the manufacture of fermented food by inoculating the Lactobacillus Taiwan Nsis NIBRBA00501006 strain or a fermentation starter.

According to another aspect of the present invention, the present invention is entrusted with accession number KCTC 18771P, and is selected from the group consisting of Salmonella sp. strain, Escherichia sp. strain, Streptococcus sp. strain, Bacillus sp. strain, and Saccharomyces sp. It shows antimicrobial activity against any one or more pathogens, and Lactobacillus taiwanensis It provides an antimicrobial pharmaceutical composition containing as an active ingredient at least one selected from the group consisting of NIBRBA00501006 strain, a culture solution of the strain, a concentrate of the culture solution, and a dried product of the culture solution.

In the present invention, the Lactobacillus taiwanensis NIBRBA00501006 strain or a culture solution thereof is characterized in that it exhibits antibacterial activity.

In the pharmaceutical composition for preventing or treating infections of the present invention, examples of the strain of the genus Salmonella include Salmonella gallinarum , and examples of the strain of the genus Escherichia include Escherichia coli (E. coli). An example of the strain of the genus Streptococcus is Streptococcus iniae , an example of the strain of the genus Bacillus is Bacillus cereus , and an example of the strain of the genus Saccharomyces is S. cerevisiae can be mentioned, but is not limited thereto.

On the other hand, the Lactobacillus taiwanensis NIBRBA00501006 strain of the present invention or an extract of the culture solution may be prepared in the form of a pharmaceutically acceptable salt. Specifically, salts can be formed by adding an acid, for example, inorganic acids (eg, hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, etc.), organic carboxylic acids (eg, halo acids such as acetic acid and trifluoroacetic acid). Acetic acid, propionic acid, maleic acid, succinic acid, malic acid, citric acid, tartaric acid, salicylic acid), acidic sugars (glucuronic acid, galacturonic acid, gluconic acid, ascorbic acid), acidic polysaccharides (e.g. hyaluronic acid, chondroitin sulfate, arginine) Acid), an organic sulfonic acid (eg, methane, sulfonic acid, p-toluene sulfonic acid) including a sulfonic acid sugar ester such as chondroitin sulfate may be added to form a salt.

The Lactobacillus taiwanensis NIBRBA00501006 strain or its culture medium of the present invention is a kind of lactic acid bacteria isolated from edible foods and can be administered orally or parenterally during clinical administration, and can be used in the form of a general pharmaceutical formulation.

That is, the new strain NIBRBA00501006 of the present invention or its culture solution can be administered in various oral or parenteral formulations. In the case of formulation, diluents such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. It is formulated using excipients. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, liurinji, glycerogelatin, and the like may be used.

In addition, the new strain NIBRBA00501006 of the present invention or its culture medium may be used in combination with various carriers permitted as drugs such as physiological saline or organic solvent, and glucose, sucrose or dextran and Antioxidants such as carbohydrates, ascorbic acid or glutathione, chelating agents, small molecule proteins or other stabilizers can be used as pharmaceuticals.

The effective dose of the new strain NIBRBA00501006 or its culture medium of the present invention is 0.01 mg/kg to 10 mg/kg, preferably 0.1 mg/kg to 1 mg/kg, and may be administered once to three times a day.

In the pharmaceutical composition of the present invention or its culture medium, the total effective amount may be administered to a patient in a single dose by infusion or the like in a bolus form or for a relatively short period of time, and multiple doses (multiple does) can be administered by a fractionated treatment protocol that is administered over a long period of time. Since the concentration is determined by taking into account various factors such as the patient's age and health condition, as well as the route of administration and the number of treatments of the drug, the effective dosage of the patient is determined. It will be possible to determine an appropriate effective dose according to the specific use of the new strain of NIBRBA00501006 or its culture as a pharmaceutical composition.

According to another aspect of the present invention, the present invention is entrusted with accession number KCTC 18771P, and is selected from the group consisting of Salmonella sp. strain, Escherichia sp. strain, Streptococcus sp. strain, Bacillus sp. strain, and Saccharomyces sp. It shows antimicrobial activity against any one or more pathogens, and Lactobacillus taiwanensis It provides an antimicrobial health functional food composition containing as an active ingredient any one or more selected from the group consisting of NIBRBA00501006 strain, a culture solution of the strain, a concentrate of the culture solution, and a dried product of the culture solution.

In the antimicrobial health functional food composition of the present invention, examples of the Salmonella genus strain include Salmonella gallinarum , and examples of the Escherichia genus strain include Escherichia coli (E. coli), and An example of the strain of the genus Streptococcus is Streptococcus iniae , an example of the strain of the genus Bacillus is Bacillus cereus , and an example of the strain of the genus Saccharomyces is Saccharomae Ses cerevisiae (S. cerevisiae) can be mentioned, but is not limited thereto.

The health functional food of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients. The natural carbohydrates described above are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as taumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame can be used.

The ratio of the natural carbohydrate may be selected from 0.01 to 0.04 parts by weight per 100 parts by weight of the health functional food of the present invention, specifically about 0.02 to 0.03 parts by weight.

In addition to the above, the health functional food of the present invention includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, Carbonating agents used in carbonated beverages may be contained. These components may be used independently or in combination. The ratio of these additives is not very important, but may be selected from 0.01 to 0.1 parts by weight per 100 parts by weight of the health food of the present invention.

According to another aspect of the present invention, the present invention is entrusted with accession number KCTC 18771P, and is selected from the group consisting of Salmonella sp. strain, Escherichia sp. strain, Streptococcus sp. strain, Bacillus sp. strain, and Saccharomyces sp. It shows antibacterial activity against any one or more pathogens, and any one or more selected from the group consisting of the Lactobacillus taiwanensis NIBRBA00501006 strain, the culture solution of the strain, the concentrate of the culture solution, and the dried product of the culture solution as an active ingredient It provides an antibacterial food preservative composition containing.

The term "preservation" as used in the present invention means preventing food from spoiling due to the action of microorganisms, and is used as a generic term for antibiotics and antibacterials. In addition, the term "preservative" refers to a drug added to preservative for the purpose of preserving food. Therefore, there should be no harm to the human body, and the addition should not impair the quality of food.

In the antibacterial food preservative composition of the present invention, examples of the Salmonella genus strain include Salmonella gallinarum , and examples of the Escherichia genus strain include Escherichia coli (E. coli), and An example of the strain of the genus Streptococcus is Streptococcus iniae , an example of the strain of the genus Bacillus is Bacillus cereus , and an example of the strain of the genus Saccharomyces is Saccharomae Ses cerevisiae (S. cerevisiae) can be mentioned, but is not limited thereto.

According to another aspect of the present invention, the present invention is entrusted with accession number KCTC 18771P, and is selected from the group consisting of Salmonella sp. strain, Escherichia sp. strain, Streptococcus sp. strain, Bacillus sp. strain, and Saccharomyces sp. It shows antibacterial activity against any one or more pathogens, and any one or more selected from the group consisting of the Lactobacillus taiwanensis NIBRBA00501006 strain, the culture solution of the strain, the concentrate of the culture solution, and the dried product of the culture solution as an active ingredient It provides an antibacterial feed additive composition containing.

In the antibacterial feed additive composition of the present invention, examples of the Salmonella genus strain include Salmonella gallinarum , and examples of the Escherichia genus strain include Escherichia coli (E. coli). An example of the strain of the genus Streptococcus is Streptococcus iniae , an example of the strain of the genus Bacillus is Bacillus cereus , and an example of the strain of the genus Saccharomyces is Saccharomyces ces. Cerevisiae (S. cerevisiae) may be mentioned, but is not limited thereto.

In particular, the feed additive of the present invention has a strong antibacterial activity against highly pathogenic Salmonella strains collected directly from livestock farming sites, so it is possible to prevent bacterial diseases by steadily ingesting poultry, livestock, etc., and to improve bacterial diseases that have already occurred. have. Feed can be classified into several categories according to nutritional value, main ingredient, distribution, water content, blending status, and processing type. Forage, roughage, thick feed, supplemental feed, protein feed, starch feed, fat feed or fiber feed can be used. .

The feed additive may additionally contain an acceptable carrier, for example, the feed additive may be used as it is, or a known carrier or stabilizer may be added. In addition, if necessary, various nutrients such as vitamins, amino acids, minerals, antioxidants, antibiotics, antibacterial agents, and other additives may be added, and the shape may be powder, granules, pellets, suspensions, etc. . The feed additive of the present invention can be supplied alone or mixed with feed.

According to another aspect of the invention there is provided a pathogenic bacteria by administration to a subject in need of such treatment a composition containing a deposit number KCTC is entrusted to 18771P, Lactobacillus Taiwan N-Sys (Lactobacillus taiwanensis) NIBRBA00501006 strain or its culture broth It provides a method of treating a bacterial infection disease in a non-human animal comprising the step of killing.

Although the treatment method according to the present invention is a method of treating animals other than humans, it does not mean that such treatment methods are not effective in humans. In addition, in the case of humans, considering having a disease caused by a bacterial or fungal infection in which symptoms can be improved by administration of the therapeutic composition according to the present invention, it can be sufficiently used for human treatment.

In the present invention, the term "animals other than humans" refers to cattle, horses, sheep, pigs, chickens, except for humans with diseases caused by infection of bacteria or fungi that can improve symptoms by administration of the therapeutic composition according to the present invention. , Goat, camel, antelope, dog and other animals. By administering the therapeutic composition according to the present invention to animals other than humans, diseases caused by bacterial and fungal infections can be effectively prevented and treated.

In the present invention, the term "administration" means introducing a predetermined substance to an animal by any suitable method, and the route of administration of the therapeutic composition according to the present invention is oral or through any general route as long as it can reach the target tissue. It can be administered parenterally. In addition, the therapeutic composition according to the present invention may be administered using any device capable of moving the active ingredient to target cells.

A preferred dosage of the therapeutic composition according to the present invention varies depending on the condition and weight of the animal to be treated, the degree of disease, the drug form, the route of administration and the duration, but may be appropriately selected by those skilled in the art. However, for a desirable effect, the pharmaceutical composition of the present invention is 0.01 to 10 mg/kg, preferably 0.1 mg/kg to 1 mg/kg, and may be administered once to three times a day.

The features and advantages of the present invention are summarized as follows:

(I) The present invention provides an antimicrobial composition comprising a food-derived Lactobacillus Taiwan Nsis NIBRBA00501006 strain or a culture solution thereof as an active ingredient.

(Ii) The composition containing the Lactobacillus taiwanensis NIBRBA00501006 strain or its culture medium of the present invention exhibits a broad antibacterial spectrum against various bacteria, and the composition of the present invention can be applied to various bacterial infections caused by harmful bacteria. Do.

(Iii) In addition, the composition of the present invention can be usefully used for antibacterial purposes in various fields such as medicine, health functional food, or feed preservative.

1 shows Salmonella gelin arum (Gram negative, Salmonella gallinarum ), Escherichia coli (Gram negative, E. coli), Streptococcus inie (Gram positive, Streptococcus iniae ), Bacillus cereus (Gram positive, B. cereus) ), and Saccharomyces cerevisiae (yeast, S. cerevisiae) strain of the present invention Lactobacillus taiwanensis ( Lactobacillus taiwanensis) NIBRBA00501006 It is a graph measuring the antibacterial activity of the strain.
Figure 2 is a graph measuring the antimicrobial activity of the culture supernatant of the Lactobacillus taiwanensis NIBRBA00501006 strain of the present invention according to changes in the amount of pathogenic bacteria.
Figure 3 is a graph measuring the antimicrobial activity after the treatment of Proteinase K in the culture supernatant of Lactobacillus taiwanensis NIBRBA00501006 strain of the present invention.
Figure 4 is a graph measuring the antibacterial activity after treatment with EDTA in the culture supernatant of Lactobacillus taiwanensis NIBRBA00501006 strain of the present invention.
Figure 5 is a graph measuring the antimicrobial activity of the 50% methanol fraction of the culture supernatant of Lactobacillus taiwanensis NIBRBA00501006 strain culture of the present invention.
6 is a graph measuring the antimicrobial activity of the purified Sep-Pak R18 column against the methanol fraction / chloroform water layer of the Lactobacillus taiwanensis NIBRBA00501006 strain of the present invention.
7 is a graph measuring the antibacterial activity of the purified Sep-Pak R18 column against the methanol fraction/Ethyl acetate water layer of the Lactobacillus Taiwan Nsis NIBRBA00501006 strain of the present invention.
Figure 8 is a SEM photograph observed after treating the Sep-Pak R18 column purified product (FT) of the Lactobacillus taiwanensis NIBRBA00501006 strain of the present invention in Salmonella gallinarum, a pathogenic bacterium.

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. Securing strain and preparing L. taiwanesis culture supernatant

1-1. Strain

The strain used in the experiment was L. taiwanensis (L. taiwanensis) NIBRBA00501006 strain, which was isolated and stored in Korea by the National Institute of Biological Resoures (NIBR), and was used in this study.

The pathogenic microorganisms Salmonella gallinarum , pathogenic E. coli, and Streptococcus iniae are the laboratory of Prof. Jin Heo of Chungbuk National University (Iksan, Korea) and Tae- Sung Jeong of Gyeongsang National University. It was used after being sold in the professor's lab. Bacillus cereus (ATCC 1178) and S. cerevisiae (KCTC 27134) strains are sold and used at the Korea Research Institute of Bioscience and Biotechnology (KCTC, Korean Collection for Type Cultures) strains. I did.

Salmonella gelin arum, pathogenic E. coli, Streptococcus inie, and Bacillus cereus were cultured at 37°C, whereas Saccharomyces cerevise was cultured at 30°C.

1-2. L. taiwanesis Preparation of culture supernatant

Lactobacillus taiwanensis (L. taiwanensis) NIBRBA00501006 single strain was obtained by inducing a single colony. The obtained single strain was inoculated in 5mL MRS liquid medium at 1/100 and cultured at 37°C for 24 to 48 hours. After incubation, 1 mL was aliquoted and centrifuged at 13,500 RPM. After centrifugation, the supernatant was collected, filtered with a 0.2um syringe filler, and stored at -20℃ for use in experiments.

Example 2. Analysis of minimal inhibitory concentration (MIC) of L. taiwanesis strain

For the MIC measurement of the selected strains, Gram-negative bacteria Salmonella gallinarum (SG) and pathogenic E. coli JOL418 (EC) were used. All of these strains are causes of livestock disease. In particular, Salmonella gelin arum is known as the causative agent of typhus in chickens. In addition, as Gram-positive bacteria, Bacillus cereus (BC), which is the causative agent of food poisoning in humans, and Streptococcus iniae (SI), which are fish disease bacteria, were used. In addition, in order to find out the antibacterial activity against fungi, Saccharomyces cerevise, which is not a disease-causing bacterium, was used.

The five pathogens were analyzed for minimal inhibitory concentration (MIC) by DISC or microtiter plate method. For the antibacterial analysis, the strain pre-cultured overnight (O/N) was adjusted to 1×10 ⁶ CFU/ml and used in the experiment. After adding the reaction solution to a microtiter plate or dropping it on a disc, the reaction properties were observed while incubating O/N at 37°C.

As a result, MIC values of 12.2ul and 12.0ul were observed for gram-negative bacteria, Salmonella gelin arum and pathogenic E. coli, respectively, showing high antibacterial activity. This antimicrobial activity value is an antimicrobial activity superior to the MIC value of 13.4ul of the L. plantarum strain, which is a strain having strong antibacterial activity previously studied (see FIG. 1).

In addition, gram-positive bacteria Streptococcus iniae ( Streptococcus iniae ) and Bacillus cereus (B. cereus ) also showed antibacterial activity, but showed 5.3 to 5.7 times lower activity compared to Gram (-). On the other hand, it also showed activity against Saccharomyces cerevise, but showed 2.4[Gram(+)] to 13.6[Gram(-)] times lower activity than that of bacteria.

With the results shown in the culture supernatant of Lactobacillus Taiwan N-Sys (Lactobacillus taiwanensis) NIBRBA00501006 strain of the present invention exhibit antibacterial activity against a wide range of microorganisms, it was confirmed that in particular exhibits a high antibacterial activity against gram-negative bacteria.

Example 3. Analysis of antibacterial activity according to the amount of target strain

In previous studies, most of the antimicrobial peptides (AMPs) produced by microorganisms showed inversely proportional to the concentration of the target strain. Thus Bacillus of the present invention Lactobacillus bacteria Taiwan N-Sys (Lactobacillus taiwanensis) the antibacterial substances contained in the culture supernatant of NIBRBA00501006 strain is gram to verify has the same properties voice strain of Salmonella gel rinah room (Salmonella gallinarum) and gram-positive strains celebrity Mouse (B. cereus ) strain was controlled to a concentration of 10 ⁴ ~ 10 ⁸ CFU / ml to analyze the characteristics of antibacterial activity.

Gram supernatant for antimicrobial activity assays according to Bacillus cereus (B. cereus) Salmonella strain amount of gel rinah room voice strain (Salmonella gallinarum) and Gram-positive strain is Bacillus cereus (B. cereus) and Bacillus cereus (B cereus ) The MIC concentration determined at 10 ⁶ CFU/ml was used.

For analysis according to the amount of strain, Salmonella gallinarum and Bacillus cereus strains were at concentrations of 10 ⁸ , 10 ⁷ , 10 ⁶ , 10 ⁵ , 10 ⁴ , and 10 ³ CFU/ml. It was diluted and used. After adding the reaction solution to a microtiter plate or dropping it on a disc, the reaction properties were observed while incubating O/N at 37°C.

As a result, as shown in Figure 2, the Lactobacillus taiwanensis ( Lactobacillus taiwanensis) NIBRBA00501006 strain culture supernatant of the present invention is active in inverse proportion to the concentration of Salmonella gallinarum and Bacillus cereus (B. cereus ) strains. Appeared, and it was estimated to contain antimicrobial peptides (AMPs).

Example 4. Evaluation of activity according to Proteinase K treatment

The antimicrobial materials contained in the N-Sys Taiwan Lactobacillus (Lactobacillus taiwanensis) NIBRBA00501006 strain culture supernatant was treated with proteinase K to determine whether AMPs material.

The experiment was performed using a Gram-negative bacteria, Salmonella gallinarum , by a disc or microtiter plate method. The supernatant was used as the MIC result of 12.19ul, and the strain was used at a concentration of 1×10 ⁶ CFU/ml. Proteinase K was used at a concentration of 0.2mg/ml, and the culture temperature was set to 37°C for evaluation.

As a result, it was confirmed that most of the antibacterial activity was lost when proteinase K was treated as shown in FIG. 3. Therefore Lactobacillus Taiwan antibacterial substance of N-Sys (Lactobacillus taiwanensis) NIBRBA00501006 strain culture supernatant was estimated to exhibit most or all of the active substance by the AMPs.

Example 5. Evaluation of activity according to simultaneous treatment with EDTA

EDTA is a chelating agent and has a function of inhibiting enzyme activity dependent on divalent cations. Since many proteases require divalent cations for activity, reducing the soluble content of divalent cations in the solution has the effect of reducing the activity of the enzyme. Therefore, it was determined that the effect of improving the stability of the supernatant to be treated with respect to the protease can be induced.

For the measurement, analysis was performed by adding to a microtiter plate or dropping onto a disc using Salmonella gallinarum , which is a gram-negative bacterium. The supernatant was used as the MIC value of 12.19ul, and the strain was used as a concentration of 1×10 ⁸ CFU/ml. EDTA was treated at concentrations of 0, 1, 5, 10, 15, 20, 25, and 30 mM, and the culture temperature was set to 37°C for evaluation.

As a result, as shown in FIG. 4, the antimicrobial activity tended to increase compared to the control group not treated with EDTA, and the antimicrobial activity was observed to increase in proportion to the concentration of EDTA. However, at a concentration of 16 mM EDTA or higher, it was saturated and no further enhancement of activity was observed. Antimicrobial agents of N-Sys Taiwan Lactobacillus (Lactobacillus taiwanensis) NIBRBA00501006 strain culture supernatant from these results, it was confirmed that the activity shown by the AMPs material.

Example 6. Purification of culture supernatant by organic solvent fraction

The substance contained in the N-Sys Taiwan Lactobacillus (Lactobacillus taiwanensis) NIBRBA00501006 strain culture supernatant was subjected to partial purification to ensure that the AMPs material.

First, in the fractions according to the methanol concentration, the precipitates among the fractions according to the methanol concentration were all discarded because the previous study showed no activity. That is, the general characteristics of AMPs are that it is well known that having an amino acid having hydrophobicity of about 50% exhibits high activity, so it was estimated that the precipitates of the methanol fraction capable of showing higher hydrophobicity have very low or no activity, There was no activity in the actual analysis results.

The concentrated culture supernatant was subjected to organic solvent fractionation with 30%, 50%, and 70% methanol. After adjusting with each concentration of methanol, the reaction was performed at 4° C. for 1 hour, and centrifugation was performed at 2,000 rpm for 20 minutes to separate the supernatant and the precipitate, respectively. The precipitate was resuspended with the same amount of distilled water as the supernatant, and then concentrated 10 times to the original amount.

As a result of analyzing the activity with a solution resuspended in distilled water after concentration, as shown in FIG. 5, antibacterial activity was present in all methanol extracts, and in particular, it was found that the highest antibacterial activity was found in 50% methanol extract (see FIG. 5).

The 50% methanol supernatant (MS50; supernatant of 50% methanol fractionation) was partitioned using ethyl acetate and chloroform solvent, and each water layer was collected. The organic solvent layer was discarded for the same reason that the precipitate was removed from the methanol fraction above.

Ethyl acetate and chloroform aqueous layer were mixed with ice cold 0.1% trifluoroacetic acid (TFA) in the same amount. The mixed solution was centrifuged at 12,000 rpm for 10 minutes at 4°C to remove the precipitate.

The collected supernatant was loaded into Sep- ^Pak® plus C18 cartridge (Waters). After performing two washings by 10 bed volumes with 0.05% TFA, a solution composed of 10, 20, 30, 50, and 80% ACN (acetonitrile) in 0.05% TFA solution at low concentration From then, 20ml was sequentially loaded at a high concentration and elution was performed. The eluted solution was concentrated so that the final amount was concentrated 10 times relative to the initially used amount.

Sep-Pak R18 elution results by 50% methanol supernatant/chloroform aqueous layer and ethyl acetate aqueous layer are shown in FIGS. 6 and 7 respectively. The elution results of both columns showed that all fractions had antibacterial activity, but the highest activity was shown in FT (flowthrow). Accordingly, since 50% of the methanol supernatant and the highest activity in the FT appeared AMPs substance contained in the N-Sys Taiwan Lactobacillus (Lactobacillus taiwanensis) NIBRBA00501006 strain culture supernatant is estimated to be a material composed of peptides having both the hydrophilic and hydrophobic tendencies.

Example 7. SEM analysis of antimicrobial activity properties

Lactobacillus Taiwan ensis by the method of Example 6 (Lactobacillus taiwanensis) The lysis of the NIBRBA00501006 strain according to the treatment time was observed using a scanning electron microscope (SEM).

In the experiment, the most active Sep-Pak R18 eluate was treated with Salmonella gallinarum , and the culture was sampled at 0, 6, 12, and 24 hours. After the sampled culture was centrifuged at 13,500RPM/1min, only an appropriate amount of the precipitate was aliquoted on the cover-glass, water was removed with alcohol, coated with platinum, and observed using SEM.

As a result, as shown in FIG. 8, it was observed that the number of bacteria having holes (marked by arrows) in the cell membrane increased as time passed.

Taken together, Lactobacillus taiwanensis The antimicrobial activity of the NIBRBA00501006 strain culture supernatant was indicated by the AMPs substance, and was inversely proportional to the concentration of the target harmful strains, while it was found to be proportional to the concentration of the antibacterial substance. The antimicrobial peptides (AMPs) have been shown to show a partial hydrophilic tendency and cause lysis by forming a hole in the cell wall.

As described above, specific parts of the present invention have been described in detail, and it is obvious that these specific techniques are only preferred embodiments and are not intended to limit the scope of the present invention to those of ordinary skill in the art. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Korea Research Institute of Bioscience and Biotechnology KCTC18771P 20190510

Claims (13)

Accession No. KCTC the Lactobacillus Taiwan N-Sys (Lactobacillus taiwanensis) NIBRBA00501006 strain entrusted to 18771P. The method of claim 1,
The strain is characterized by having antimicrobial activity against any one or more pathogens selected from the group consisting of Salmonella sp. strain, Escherichia sp. strain, Streptococcus sp. strain, Bacillus sp. strain, and Saccharomyces sp.
The strain of the genus Salmonella is Salmonella gallinarum , the strain of the genus Escherichia is E. coli, the strain of the genus Streptococcus is Streptococcus iniae , and the The strain of the genus Bacillus is Bacillus cereus (B. cereus ), and the strain of the genus Saccharomyces is characterized in that the strain of the genus Saccharomyces is S. cerevisiae.
Lactobacillus taiwanensis NIBRBA00501006 strain. delete Claim 1 Lactobacillus Taiwan N-Sys (Lactobacillus taiwanensis) NIBRBA00501006 strain (Accession No. KCTC 18771P), fermentation containing at least one selected culture of the above strain, the culture solution concentrate and from the group consisting of a dried product of the culture broth as an active ingredient For starter. Paragraph (1) N-Sys Taiwan Lactobacillus (Lactobacillus taiwanensis) NIBRBA00501006 strain (Accession No. KCTC 18771P) or 4 of the fermentation the fermented food produced using the starter for. Paragraph (1) N-Sys Taiwan Lactobacillus (Lactobacillus taiwanensis) NIBRBA00501006 strain (Accession No. KCTC 18771P) or fermented food manufacturing method includes the step of inoculation of a starter for 4 of the fermentation raw material. Deposited with the accession number KCTC 18771P, it exhibits antimicrobial activity against any one or more pathogens selected from the group consisting of Salmonella sp. strain, Escherichia sp. strain, Streptococcus sp. strain, Bacillus sp. strain, and Saccharomyces sp. strain. ,
The strain of the genus Salmonella is Salmonella gallinarum , the strain of the genus Escherichia is E. coli, the strain of the genus Streptococcus is Streptococcus iniae , and the The strain of the genus Bacillus is Bacillus cereus (B. cereus ), and the strain of the genus Saccharomyces is characterized in that the strain of the genus Saccharomyces is S. cerevisiae.
Lactobacillus taiwanensis ( Lactobacillus taiwanensis ) NIBRBA00501006 Antibacterial pharmaceutical composition containing the strain as an active ingredient. delete Deposited with the accession number KCTC 18771P, it exhibits antimicrobial activity against any one or more pathogens selected from the group consisting of Salmonella sp. strain, Escherichia sp. strain, Streptococcus sp. strain, Bacillus sp. strain, and Saccharomyces sp. strain. ,
The strain of the genus Salmonella is Salmonella gallinarum , the strain of the genus Escherichia is E. coli, the strain of the genus Streptococcus is Streptococcus iniae , and the The strain of the genus Bacillus is Bacillus cereus (B. cereus ), and the strain of the genus Saccharomyces is characterized in that the strain of the genus Saccharomyces is S. cerevisiae.
Antibacterial health functional food composition containing the strain of Lactobacillus taiwanensis NIBRBA00501006 as an active ingredient. delete Deposited with the accession number KCTC 18771P, it exhibits antimicrobial activity against any one or more pathogens selected from the group consisting of Salmonella sp. strain, Escherichia sp. strain, Streptococcus sp. strain, Bacillus sp. strain, and Saccharomyces sp. strain. ,
The strain of the genus Salmonella is Salmonella gallinarum , the strain of the genus Escherichia is E. coli, the strain of the genus Streptococcus is Streptococcus iniae , and the The strain of the genus Bacillus is Bacillus cereus (B. cereus ), and the strain of the genus Saccharomyces is characterized in that the strain of the genus Saccharomyces is S. cerevisiae.
Lactobacillus taiwanensis (Lactobacillus taiwanensis) NIBRBA00501006 Antibacterial food preservative composition containing the strain as an active ingredient. delete Deposited with the accession number KCTC 18771P, it exhibits antimicrobial activity against any one or more pathogens selected from the group consisting of Salmonella sp. strain, Escherichia sp. strain, Streptococcus sp. strain, Bacillus sp. strain, and Saccharomyces sp. strain. ,
The strain of the genus Salmonella is Salmonella gallinarum , the strain of the genus Escherichia is E. coli, the strain of the genus Streptococcus is Streptococcus iniae , and the The strain of the genus Bacillus is Bacillus cereus (B. cereus ), and the strain of the genus Saccharomyces is characterized in that the strain of the genus Saccharomyces is S. cerevisiae.
Lactobacillus taiwanensis (Lactobacillus taiwanensis) NIBRBA00501006 Antibacterial feed additive composition containing the strain as an active ingredient.

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