KR20180069593A - Antimicrobial composition comprising Cyclic dipeptide purified from fermented culture medium of Lactobacillus paraplantarum KNUC25 and manufacturing method thereof - Google Patents

Abstract

The present invention relates to an antimicrobial composition comprising, as an active component, a cyclic dipeptide compound purified from a fermented culture medium of lactobacillus paraplantarum KNUC 25, a method for manufacturing the compound and a purpose thereof. The cyclic dipeptide compound according to the present invention, purified from the fermented culture medium of the lactobacillus paraplantarum KNUC 25, shows excellent antibacterial activity against pathogenic bacteria including gram negative bacteria and gram positive bacteria, and against pathogenic fungi, thereby being usefully utilized as an antibacterial additive composition when foods, medicines, cosmetics, etc. are manufactured. In addition, the method according to the present invention stably separates and purifies the cyclic dipeptide from the culture medium, thereby making the cyclic dipeptide stable and enhancing industrial usage values thereof as compared with a compound existing in microorganism itself.

Description

[0001] The present invention relates to an antimicrobial composition comprising a cyclic diepeptide compound isolated from a fermentation broth of Lactobacillus paraflutarum strain KNUC25, and a method for producing the antimicrobial composition comprising the cyclic dipeptide compound from Lactobacillus paraplantarum KNUC25,

The present invention relates to a composition having an antimicrobial activity, which comprises a cyclic di-peptide compound isolated from a fermentation broth of Lactobacillus paraparentaum KNUC25 strain, a method for producing the compound, and a use thereof.

Since the discovery of penicillin, a number of antibiotics have been developed and contributed greatly to the health of humankind. However, the emergence of resistant strains to antibiotics has made it difficult to easily disinfect existing antibiotics, and it is constantly demanding the development of antibiotics that exhibit activity as a new mechanism. Efforts to develop new antibiotics were not enough to keep pace with the rate of resistance to antibiotics, and resistance to antibiotics is now becoming a more serious social problem as more pharmaceutical companies abandon the study of new antibiotic development.

Recently, the possibility of influx of foreign pathogenic microorganisms is increasing more and more in the economic and cultural exchange in Korea. As the import of livestock products increases, it is expected that the spread of new bacteria will accelerate. In order to cope with rapidly increasing bacterial drug resistance and effectively treat various bacteria, development of new drugs (molecular structure, efficacy of drugs, etc.) different from conventional antibiotics is urgently required. Recently, the development of new antibiotics is urgently required due to the emergence of super bacteria resistant to antibiotics. In addition, the conditions for these new drugs should be safe drugs that have no adverse effects on human and animal cells as well as sterilization effects.

Meanwhile, kimchi is a traditional fermented food in Korea. In order to maintain the preservability and aging of the product, various fermented condiments (red pepper powder, garlic, ginger, Food. Lactic acid bacteria found in kimchi include Ryukono stock, Lactobacillus, and Bacillus. Lactic acid bacteria are used for the production of foods such as dairy products, kimchi, and brewed foods by preventing the growth of pathogenic bacteria and harmful bacteria by lactic acid produced by lactic acid fermentation. In addition, it is reported that the possibility of prevention of cardiovascular diseases by inhibiting the absorption of cholesterol in the intestines by immune function of the host, which is an important bacteria used as a formulant, by preventing abnormal fermentation by bacteria inhabiting in the mammalian intestines has been reported Among the lactic acid bacteria isolated from kimchi, strains having antiviral activity against avian influenza virus were also found. Based on these results, the lactic acid bacteria found in kimchi have attracted attention as probiotic because of their ease of ingestion and ease of application of food additives.

The cyclic dipeptide (CDP) is an eco-friendly substance derived from microorganisms, and it has not only the effects of anticancer, antivirus (Sinha, S. et al. Nucleosides, Nucleotides Nucleic Acids 2004, 23, 1815) It has been reported that it has a plant immunoenhancement effect such as growth promoting effect and anti-cold effect. Therefore, if such a cyclic di-peptide can be separated and purified from microorganisms, it will be more stable and more industrially useful.

Thus, the present inventors fermented the Lactobacillus paraplora room strain isolated from kimchi, isolated the cyclic di-peptide from the fermentation broth, and confirmed its excellent antibacterial activity.

The present inventors obtained a fermentation broth of a Lactobacillus paraplora room strain to isolate a cyclic di-peptide therefrom, and confirmed that the compound is a pathogenic bacterium including fungi (Gram-) and gram-positive bacteria (Gram +) and fungi (Fungi) and the like. Based on this finding, the present invention has been completed.

Accordingly, it is an object of the present invention to provide a composition for antibiosis against a pathogenic microorganism, which comprises a cyclic di-peptide compound as an active ingredient.

Another object of the present invention is to provide a food additive composition, a pharmaceutical composition, and an antibacterial cosmetic additive composition comprising the cyclic dipeptide compound as an active ingredient.

It is still another object of the present invention to provide a method for producing a cyclic dipeptide compound having an antibacterial activity.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention, the present invention provides a composition for antibacterial activity against a pathogenic microorganism, which comprises a cyclic dipeptide compound represented by the following general formula (1) as an active ingredient.

[Chemical Formula 1]

The present invention also provides a food additive composition for antimicrobial use comprising the cyclic dipeptide compound as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for antimicrobial use comprising the cyclic dipeptide compound as an active ingredient.

The present invention also provides a cosmetic additive composition for antimicrobial use comprising the cyclic dipeptide compound as an active ingredient.

In one embodiment of the invention, the cyclic dipeptide compound may be isolated from a fermentation broth of Lactobacillus paraplantarum KNUC25 strain (Accession No. KCTC 10958BP).

In another embodiment of the present invention, the pathogenic microorganism may be a pathogenic bacterium or fungus.

In another embodiment of the present invention, the pathogenic bacterium is selected from the group consisting of Escherichia coli , Salmonella enterica subsp . enterica ), Streptococcus mutans , Enterococcus faecalis or Bacillus subtilis .

In another embodiment of the present invention, the pathogenic fungus may be Candida albicans .

In addition, the present invention provides a method for producing a cyclic dipeptide compound having antimicrobial activity, comprising the steps of:

(a) inoculating and culturing a strain of Lactobacillus paraplantarum strain in a medium to obtain a fermented product, and then separating only the fermentation supernatant;

(b) treating the fermentation supernatant with ammonium sulfate and allowing it to settle, and then separating the ammonium sulfate by centrifugation;

(c) dialyzing the separated fermentation supernatant for 12 to 36 hours after filtration;

(d) centrifuging the dialysis solution to separate only the substance having a size of 3 kDa or more; And

(e) lyophilizing the separated material to obtain a cyclic dipeptide compound.

In one embodiment of the present invention, the strain may be a Lactobacillus paraplantarum KNUC25 strain (Accession No. KCTC 10958BP).

In another embodiment of the present invention, in step (a), the strain may be cultured at 25 to 30 DEG C for 1 to 5 days.

The cyclic dipeptide compound according to the present invention is isolated from a fermentation broth of a Lactobacillus paraplantarum strain and exhibits excellent antimicrobial activity against pathogenic bacteria and pathogenic fungi including Gram-negative bacteria and Gram-positive bacteria, Can be usefully used as an antibiotic additive composition in the production of foods, medicines, cosmetics and the like.

In addition, according to the production method of the present invention, it is expected that the cyclic di-peptide can be stably isolated and purified from the strain, so that it is more stable and more industrially useful than the compound existing in the microorganism itself.

1 is a diagram illustrating a process for preparing a cyclic di-peptide according to the present invention and a chemical structural formula thereof.
FIG. 2 and FIG. 3 are the results of GC-MS analysis of a substance having a size of 3 kDa or more separated from the fermentation broth of Lactobacillus paraparentaum KNUC25 strain.
Figure 4 is a graph lt; RTI ID = 0.0 > coli < / RTI >
FIG. 5 is a schematic diagram of an Enterococcus faecalis ) of the cyclic di-peptide.
Figure 6 is a graph The results of the measurement of the inhibitory activity of the cyclic di-peptide on the growth over time for E. coli .
FIG. 7 shows the results of measuring the inhibitory activity of the cyclic di-peptide against Bacillus subtilis over time.
Figure 8 is a graphical representation of Salmonella enterica subsp . enterica ) of the cyclic di-peptide.
FIG. 9 shows the results of measuring the inhibitory activity of the cyclic di-peptide against Streptococcus mutans over time.
10 shows the results of measuring the inhibitory activity of cyclic di-peptide against Candida albicans over time.

The present inventors have stably isolated a cyclic diepeptide compound from a fermentation broth of Lactobacillus paraplantarum KNUC25 strain, which is a microorganism, and confirmed that the compound exhibits excellent antimicrobial activity against various pathogenic microorganisms, Completed.

Accordingly, the present invention provides an antimicrobial composition for a pathogenic microorganism, which comprises a cyclic dipeptide compound represented by the following general formula (1) as an active ingredient.

[Chemical Formula 1]

The cyclic dipeptide compound may be isolated from a fermentation broth of Lactobacillus paraplantarum strain, more preferably Lactobacillus paraplantarum KNUC25 strain (Accession No. KCTC 10958BP).

Further, the cyclic diepeptide compound is preferably a compound wherein IUPAC name is 2,3,6,7,8,8a-hexahydropyrrolo [1,2-a] pyrazine-1,4-dione, more preferably (8aR) -2 , 3,6,7,8,8a-hexahydropyrrolo [1,2-a] pyrazine-1,4-dione.

The pathogen may be a pathogenic bacteria and pathogenic fungi, including Gram-positive bacteria and gram negative bacteria, wherein the pathogenic bacteria is Enterococcus faecalis (Enterococcus faecalis), Escherichia coli (Escherichia E. coli , Bacillus subtilis , Salmonella enterica subsp . enterica , Streptococcus mutans , and the pathogenic fungus may be, but is not limited to, Candida albicans .

In the present invention, the antimicrobial activity of the cyclic dipeptide compound was evaluated against various pathogenic microorganisms.

In one embodiment of the present invention, the cyclic dipeptide compound is treated at a different concentration (1 M to 31.25 mM) in E. coli and the growth curve of the microorganism is measured to determine the minimum inhibitory concentration MIC). As a result, it was confirmed that the initial growth of the microorganism was remarkably inhibited when the concentration of about 1 mol was treated (see Example 2-2).

In addition, when the cyclic diepeptide was treated with various pathogenic microorganisms at the minimum inhibitory concentration determined through the above-mentioned Examples, excellent antimicrobial activity was confirmed for each strain (see Example 2-3).

Based on the above results, the present invention provides an antibacterial food additive composition comprising the cyclic di-peptide as an active ingredient.

The food additive may be used in the form of a food preservative, and the food preservative may be prepared by further adding a pharmaceutically acceptable solvent or diluent to the cyclic di-peptide according to the present invention for the purpose of enhancing the shelf- can do. The food preservative of the present invention may be formulated together with the raw material in the process of producing food or may be added as a separate aqueous suspension. In addition, the food preservative according to the present invention can be used to immerse the target food or spray the food preservative of the present invention to the target food.

The present invention also provides a pharmaceutical composition for antibacterial use which comprises a cyclic di-peptide compound as an active ingredient.

The present invention also provides an antibacterial cosmetic additive composition comprising a cyclic dipeptide compound as an active ingredient.

More specifically, the cyclic diepeptide compound according to the present invention can be used as a disinfectant, a sterilizing detergent, and the like.

The disinfecting agent may be prepared by using the cyclic diep peptide of the present invention as it is or by diluting it with a diluent or a solvent which is dermatologically and pharmacologically acceptable so as to have a suitable concentration. The disinfectant may be used on the surface of the organism, preferably on the skin of mammals, most preferably on human skin. In addition, the disinfectant may be used on surfaces of non-living organisms such as wood, metal, glass, ceramic, plastic, paper and cloth. The disinfecting agent may be applied to the surface of the organism or organism using methods including immersion, swab, spray and brushing. Preferably, the disinfectant may be used in hospitals and general households for use on wound surfaces, pre-operative skin disinfection, surgical instrument disinfection, and catheter disinfection.

In the case of the germicidal detergent, the cyclic diepeptide according to the present invention can be easily prepared according to a known method in the art, including one or more excipients and additives generally used in the field of manufacturing detergents. The sterilizing detergent may be used for a kitchen or a food. In addition to the cyclic diepeptide processed in the sterilizing detergent for the kitchen, an anionic surfactant, a nonionic surfactant, an amphoteric surfactant, and the like may be further included. Examples of the anionic surfactant include alkylbenzene sulfonate, alpha olefin sulfate, sodium or ammonium lauryl ether sulfate, and examples of the nonionic surfactant include fatty acid amide, alkylpolyglucoside, ethoxylated fatty alcohol type, And the like. Examples of amphoteric surfactants include betaine surfactants, amine oxides, and the like. In addition to the surfactant, a diluent such as fragrance, pigment and water may be optionally added. In the case of a food sterilizing detergent, it can be prepared by additionally adding a pharmaceutically acceptable solvent or diluent to the cyclic di-peptide according to the present invention and diluting it to an appropriate concentration. The food sterilizing detergent can be used for sterilizing and washing fruits, fish and meat, for example.

The present invention also provides a method for producing a cyclic di peptide having the above-mentioned antimicrobial activity.

(a) inoculating and culturing a strain of Lactobacillus paraplantarum strain in a medium to obtain a fermented product, and then separating only the fermentation supernatant;

(b) treating the fermentation supernatant with ammonium sulfate and allowing it to settle, and then separating the ammonium sulfate by centrifugation;

(c) dialyzing the separated fermentation supernatant for 12 to 36 hours after filtration;

(d) centrifuging the dialysis solution to separate only the substance having a size of 3 kDa or more; And

(e) lyophilizing the separated material to obtain a cyclic dipeptide compound.

In said step (a), the Lactobacillus paraplora room strain may preferably be Lactobacillus paraplora room KNUC25.

The fermented product is inoculated into the MRS (de Man, Rogosa & Sharpe broth) medium of the Lactobacillus paraparentaum KNUC25 strain and then incubated at 25 ° C to 30 ° C for 1 day to 5 days, more preferably at 30 ° C for about 2 For 3 days.

In the step (b), the filtration can be performed using a membrane filter, but a method commonly used by a person skilled in the art can be appropriately selected and used.

The dialyzed dialyzed supernatant can be dialyzed and dialyzed against 50 mM citrate phosphate buffer (pH 5.0) for 12 to 36 hours, preferably about 24 hours.

In the step (c), the step of separating only the substance having a size of 3 kDa or more may be performed using Centricon, and the separation of substances according to the size may be repeated several times.

A cyclic di-peptide compound having a size of 3 kDa or more according to the present invention can be obtained by the above-described method.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[ Example ]

Example  1. Ring type Dipeptide  Compound manufacturing

1-1. Isolation of microbial fermentation broth-derived extract

Lactobacillus paraplantarum strain KNUC25 was inoculated in 2 L of MRS (de Man, Rogosa & Sharpe broth) medium and cultured for 2 days in an incubator maintained at 30 캜. To obtain a fermentation supernatant of the microorganism strain from the culture, the culture was centrifuged at 8,000 rpm for 20 minutes at 4 ° C to separate the bacterial and supernatant. The supernatant was treated with 70% ammonium sulfate and incubated at 4 ° C Precipitated. Then, centrifugation was carried out at 4 ° C and 20,000 rpm for 1 hour to separate ammonium sulfate, and the separated fermentation supernatant was filtered using a 0.22 μm filter (Millipore, USA) to obtain a sterile supernatant. The brown supernatant was then dialyzed against 50 mM citrate phosphate buffer (pH 5.0) for 24 h. The extracts were centrifuged at 8,000 rpm at 4 ° C using Centricop (Centriprep / Ultracel YM-3, Merck Millipore) to separate the material into 3 kDa size. The Centricon-based material separation can be repeated several times, and the extracts thus obtained were collected and classified into a substance size of 3 kDa or more and a substance size of 3 kDa or less. As a result, 160 ㎖ of extract was obtained from 2 L of total fermentation supernatant.

The above process and the chemical structure of the separated cyclic di-peptide are shown in Fig. 1.

1-2. GC Compound analysis via -MS

The extract obtained from the fermentation broth of Lactobacillus paraparentaum KNUC25 obtained according to the method of Example 1-1 was dissolved in DMSO and the substance was analyzed by GC-MS. The material analysis conditions using the GC-MS are shown in Table 1 below.

GC Material analysis conditions using -MS GC-Mass model name Agilent 7890-5975C GC / MSD Column HP-INNOWax 30m * 0.25mm (inner diameter) Injection temperature 240 ℃ Column flow 1.0 ml / min solvent delay 4 min Oven temperature Raising 5 ° C per minute at 130 ° C to 260 ° C Hold 30min

Samples of size cut-off at 3 kDa or more at the last stage of centrifugation were analyzed according to the above method, and as a result, it was confirmed that they were cyclic di-peptides (Cyclo (Pro-Gly)) as shown in Figs.

Example  2. Ring type Dipeptide  Evaluation of antimicrobial activity of compounds

2-1. Antimicrobial activity test method

The antimicrobial activity of the cyclic di-peptide isolated from the fermentation broth of the Lactobacillus paraplora-room KNUC25 strain was evaluated through the method of Example 1. For this purpose, changes in growth curve and minimum inhibitory concentration (MIC) over time were analyzed. Target strains for the antimicrobial activity experiments were Enterococcus faecalis KCTC3206, Escherichia coli coli) K12, Bacillus subtilis (Bacillus subtilis) KCTC3135, Salmonella Entebbe Rica (Salmonella enterica subsp. enterica) ATCC13076 , Streptococcus mutans (Streptococcus mutans) KCTC3065, Candida albicans (Candida albicans ) KCTC17712 were used. The E. faecalis KCTC3206 was prepared by adding BHI agar (Brain Heart Infusion, 8.0 g of Brain Heart, Infusion from Solids, 5.0 g of peptic digest of animal tissue, 16.0 g of pancreatic digest of casein, 5.0 g of sodium chloride, 2.0 g of glucose , disodium hydrogen phosphate 2.5g) medium, E. coli K12 medium was used TSA (Tryptic Soy Agar, 15.0g of BactoTM tryptone per liter, 5.0g of bactosoyone, 5.0g of sodium chloride) and B. subtilis KCTC3135 TSA badges, S. e. subsp . enterica ATCC13076 is BHI culture medium, S. mutans is a BHI medium, C. albicans is to keep each and cultured in YM (Yeast Mold, 1 yeast extract 3.0 g, malt extract 3.0 g, peptone 5.0 g, dextrose 10.0 g per L) medium Respectively.

For the antimicrobial activity assay, each strain to be tested was inoculated into 10 ml of the medium, cultured at 30 ° C, and the whole culture was inoculated into a new medium. When the OD600 value was 0.15-0.2 at 30 ° C, 180 [mu] L of each well was added to each well of a microtiter plate, and 20 [mu] L of the cyclic dipeptide compound was treated. After incubation at 30 ° C, absorbance was measured at 595 nm (ELISA reader, Thermo Miltiskan EX) at 0, 1, 2, and 4 hours intervals. In all experiments, cultures in which the material was not treated were used as a negative control and 0.5% or 1% lactic acid as a positive control.

2-2. E. coli ( Escherichia coli ) Ring type Dipeptide  Minimal inhibition concentration analysis

In order to determine the minimum inhibitory concentration of the cyclic di-peptide required to inhibit microbial growth, the cyclic di-peptide compound was treated at different concentrations (1 M to 32.25 mM) in E. coli according to the method of 2-1 (1, 2, 4, 8 hours (h)), and the absorbance was measured at OD595 to determine the growth curve of E. coli .

As a result, as shown in FIG. 4, it was confirmed that the initial growth of the microorganism was inhibited at 1 mol, and the antimicrobial activity of the cyclic di-peptide was superior to that of the positive control, 0.1% lactic acid.

2-3. Ring Dipeptide  Antimicrobial activity range analysis

As a result of Example 2-2, it was confirmed that the cyclic diep peptide isolated from Lactobacillus paraparenta KNUC25 fermentation broth exhibited antimicrobial activity, and furthermore, the range of the antimicrobial activity of various strains was determined I would like to see. To this end, the Gram-positive bacteria, Gram-negative bacteria and fungi described in 2-1 above were treated with 1 M, 500 mM of cyclic dipeptide compound, which was the concentration for inhibiting the growth of microorganisms identified in Example 2-2, The growth curves were observed over time. As a result, as shown in Figs. 5 to 10, E. coli , S. e. subsp. Enterica , S. mutans , E. faecalis , B. subtilis, and C. albicans were found to have excellent antimicrobial activity and overall inhibition of bacterial growth was observed.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. There will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (11)

1. An antimicrobial composition for a pathogenic microorganism comprising, as an active ingredient, a cyclic dipeptide compound represented by the following formula (1).
[Chemical Formula 1]

The method according to claim 1,
Wherein said cyclic dipeptide compound is isolated from a fermentation broth of Lactobacillus paraplantarum KNUC25 strain (Accession No. KCTC 10958BP).
The method according to claim 1,
Wherein said pathogenic microorganism is a pathogenic bacterium or fungus.
The method of claim 3,
The pathogenic bacterium is Escherichia E. coli , Salmonella enterica subsp . enterica ), Streptococcus mutans , Enterococcus faecalis , or Bacillus subtilis .
The method of claim 3,
Wherein said pathogenic fungus is Candida albicans .
A food additive composition for antimicrobial use comprising the cyclic di-peptide compound of claim 1 as an active ingredient.
A pharmaceutical composition for antimicrobial use, which comprises the cyclic di-peptide compound of claim 1 as an active ingredient.
An antimicrobial cosmetic additive composition comprising the cyclic diepeptide compound of claim 1 as an active ingredient.
A method for producing a cyclic dipeptide compound having antimicrobial activity, comprising the steps of:
(a) inoculating and culturing a strain of Lactobacillus paraplantarum strain in a medium to obtain a fermented product, and then separating only the fermentation supernatant;
(b) treating the fermentation supernatant with ammonium sulfate and allowing it to settle, and then separating the ammonium sulfate by centrifugation;
(c) dialyzing the separated fermentation supernatant for 12 to 36 hours after filtration;
(d) centrifuging the dialysis solution to separate only the substance having a size of 3 kDa or more; And
(e) lyophilizing the separated material to obtain a cyclic dipeptide compound.
10. The method of claim 9,
Wherein said strain is Lactobacillus paraplantarum KNUC25 strain (Accession No. KCTC 10958BP).
10. The method of claim 9,
Wherein in step (a), the strain is cultured at 25 占 폚 to 30 占 폚 for 1 to 5 days.

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