KR101766145B1 - Antimicrobial composition comprising citraconic anhydride purified from fermented culture medium of Lactobacillus paraplantarum KNUC25 and isolating method thereof - Google Patents

Abstract

The present invention relates to a composition, which includes citraconic anhydride isolated from a fermentation broth of Lactobacillus paraplantarum KNUC25 strains as an active ingredient while having an antibacterial activity, a compound separation method, and a use thereof. According to the present invention, the citraconic anhydride is isolated from the fermentation broth of Lactobacillus paraplantarum strains, has an antibacterial activity superior to the fermentation supernatant of the strains, shows an advanced antibacterial activity against pathogenic bacteria and pathogenic fungi including gram-negative bacteria and gram-positive bacteria, and is useful as an antibacterial additive composition in the production of food, medicine, and cosmetic products. The separation method is used to stably separate and refine the citraconic anhydride from the strains to be more reliable and industrially valuable than a microbial culture solution with contents not clearly defined in advance or compounds present in the microorganisms itself.

Description

[0001] The present invention relates to an antimicrobial composition comprising citraconic anhydride separated from a fermentation broth of Lactobacillus paraplaterum strain KNUC25, and a method for isolating it from a fermentation broth of Lactobacillus paraplantarum KNUC25. The antimicrobial composition comprises citraconic anhydride purified from fermented culture medium of Lactobacillus paraplantarum KNUC25 and isolating method

The present invention relates to an antimicrobial composition comprising citraconic anhydride separated from Lactobacillus paraplorum KNUC25 fermentation broth as an active ingredient, a method for separating 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.

Citraconic anhydride is used as a maleimide, pyrrolidine, and terpolymer synthesis and as an N-terminal protecting reagent for amino acids. It selectively reacts with histidine-containing peptides and is used for the acylation of amino acid residues. . Since the antimicrobial activity of citraconic anhydride produced by microorganisms has not been reported so far, the present inventors have isolated the citraconic anhydride from the fermentation broth of Lactobacillus paraplora room isolated from kimchi, The antimicrobial activity was confirmed.

The present inventors obtained a fermentation broth of the Lactobacillus paraplora room strain to isolate citraconic anhydride therefrom. It was confirmed that the compound exhibited excellent antimicrobial activity against pathogenic bacteria and fungi of Gram-negative bacteria and Gram-positive bacteria, On this basis, the present invention has been completed.

Accordingly, it is an object of the present invention to provide a method for separating citraconic anhydride having an antibacterial activity.

It is another object of the present invention to provide an antimicrobial composition for a pathogenic microorganism, which comprises citraconic anhydride as an active ingredient.

It is still another object of the present invention to provide a food additive composition, a pharmaceutical composition, or an antimicrobial cosmetic additive composition comprising the citraconic anhydride as an active ingredient.

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 method for separating citraconic anhydride having antibacterial activity, which comprises the following steps.

(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 substances having a size of 3 kDa or less; And

(e) lyophilizing the separated material to obtain citraconic anhydride.

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 ° C to 30 ° C for 1 to 5 days.

The present invention also provides an antimicrobial composition for pathogenic microorganisms comprising citraconic anhydride represented by the following formula (1) as an active ingredient.

[Chemical Formula 1]

The present invention also provides a food additive composition for antimicrobial use comprising the citraconic anhydride as an active ingredient.

The present invention also provides a pharmaceutical composition for antimicrobial use comprising the citraconic anhydride as an active ingredient.

The present invention also provides a cosmetic additive composition for antimicrobial use comprising the citraconic anhydride as an active ingredient.

In one embodiment of the present invention, the citraconic anhydride may be isolated from the 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 a pathogenic fungus.

In another embodiment, the pathogenic bacteria are Bacillus subtilis (Bacillus subtilis), E. coli (Escherichia coli), Enterococcus faecalis (Enterococcus faecalis), Streptococcus mutans (Streptococcus mutans), Streptococcus small debris Taunus (Streptococcus sobrinus , or Lactobacillus sakei .

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

The citraconic anhydride according to the present invention is isolated from a fermentation broth of a Lactobacillus paraplantarum strain and has an antimicrobial activity superior to that of the fermentation supernatant of the above strain and is a pathogenic bacterium including Gram-negative bacteria and Gram- Exhibits a wide range of antimicrobial activity against pathogenic fungi and can be usefully used as an antimicrobial additive composition in the production of foods, medicines, cosmetics and the like.

In addition, according to the separation method of the present invention, citraconic anhydride can be stably isolated and purified from the strain, thereby making it more stable and industrially useful as compared with a microorganism culture solution in which a compound or component present in the microorganism itself is not clearly identified It is expected to be high.

1 is a diagram illustrating a process of separating citraconic anhydride according to the present invention and a chemical structural formula thereof.
FIGS. 2 and 3 are the results of GC-MS analysis of a substance having a size of 3 kDa or less isolated from Lactobacillus paraparentaum KNUC25 fermentation broth.
Fig. 4 shows the results of comparing the antimicrobial activities of the fermentation supernatant of Lactobacillus paraparentaum KNUC25 strain and the citraconic anhydride separated therefrom.
Figure 5 shows the result of measuring the minimal inhibitory concentration of citraconic anhydride against Enterococcus faecalis .
6 shows the results of measuring the inhibitory activity of citraconic anhydride over time against Bacillus subtilis .
FIG. 7 shows the results of measuring the inhibitory activity of citraconic anhydride over time against Escherichia coli .
8 shows the results of measuring the growth inhibitory activity of citraconic anhydride over time against Streptococcus mutans .
FIG. 9 shows the results of measuring the growth inhibitory activity of citraconic anhydride over time against Streptococcus sobrinus .
Figure 10 shows the results of measuring the inhibitory activity of citraconic anhydride over time against Candida albicans .
FIG. 11 shows the results of measuring the growth inhibitory activity of citraconic anhydride with respect to Lactobacillus sakei over time.

The present inventors have stably isolated citraconic anhydride 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, thereby completing the present invention Respectively.

Accordingly, the present invention provides a method for separating citraconic anhydride having antimicrobial activity including the following steps.

(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 substances having a size of 3 kDa or less; And

(e) lyophilizing the separated material to obtain citraconic anhydride.

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

The citraconic anhydride having a size of 3 kDa or less according to the present invention can be obtained by the above-mentioned method.

In the present invention, it was confirmed that citraconic anhydride isolated from the fermentation broth of Lactobacillus paraplatanum strain exhibited excellent antimicrobial activity by the above method.

In one embodiment of the present invention, the antimicrobial activity of the fermentation supernatant of the strain and the citraconic anhydride according to the present invention against C. albicans was compared, showing that the antimicrobial activity of citraconic anhydride was superior to that of the fermentation supernatant (See Example 2-2).

In another embodiment of the present invention, citraconic acid anhydride is treated at different concentrations (0.1-0.0031 mol) in E. faecalis 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 0.1 ~ 0.05 M was applied (see Example 2-3).

In another embodiment of the present invention, citraconic acid anhydrides were treated with various pathogenic microorganisms at the minimum inhibitory concentration determined by the above examples, and as a result, excellent antimicrobial activity against each strain was confirmed (see Example 2-4).

Based on the above results, the present invention provides an antimicrobial composition for pathogenic microorganisms, comprising citraconic anhydride represented by the following general formula (1) as an active ingredient.

[Chemical Formula 1]

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

The pathogenic microorganism may be pathogenic bacteria and pathogenic fungi including gram negative bacteria and gram positive bacteria and the pathogenic bacteria may be Bacillus subtilis , Escherichia coli , Enterococcus faecalis , Streptococcus mutans, Streptococcus mutans , Streptococcus sobrinus or Lactobacillus sakei , and the pathogenic fungus may be Candida albicans , but is not limited thereto.

The present invention also provides a food additive composition for antimicrobial use comprising the above citraconic anhydride as an active ingredient.

The food additive may be used in the form of a food preservative. The food preservative may be prepared by further adding a pharmaceutically acceptable solvent or diluent to the citraconic anhydride of the present invention for the purpose of enhancing the preservability of the food. 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 antimicrobial use comprising citraconic anhydride as an active ingredient.

The present invention also provides an antimicrobial cosmetic additive composition comprising citraconic anhydride as an active ingredient.

More specifically, the citraconic anhydride 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 citraconic anhydride 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, citric acid anhydrides according to the present invention can be easily prepared by known methods in the art, including one or more excipients and additives commonly used in the field of manufacturing detergents. The sterilizing detergent may be used for a kitchen or a food. In addition to the processed citraconic anhydride, the sterilizing detergent for the kitchen may further include an anionic surfactant, a nonionic surfactant, and an amphoteric surfactant. Examples of the anionic surfactant include alkylbenzenesulfonate, 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 further adding a pharmaceutically acceptable solvent or diluent to the citraconic anhydride 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.

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. Preparation of citraconic anhydride

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

Lactobacillus paraplantarum KNUC25 strain 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 bacteria 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 is shown in FIG.

1-2. Compound analysis via GC-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.

Material analysis conditions using GC-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

As a result of analyzing a sample cut at a size cut-off of 3 kDa or less at the final stage of centrifugation according to the above method, it was confirmed that citraconic anhydride was separated as shown in FIGS. 2 and 3.

Example 2. Evaluation of antimicrobial activity of citraconic anhydride

2-1. Antimicrobial activity test method

The antimicrobial activity of citraconic anhydride isolated from Lactobacillus paraplatanum KNUC25 fermentation broth was evaluated by the method of Example 1. For this purpose, changes in growth curve and minimum inhibitory concentration (MIC) over time were analyzed. Bacillus subtilis 168, Escherichia coli K12, Enterococcus faecalis KCTC 3206, Streptococcus mutans KCTC 3065, Streptococcus sobrinus Streptococcus sobrinus KCTC 3308, Candida albicans KCTC 7712, and Lactobacillus sakei ATCC 15521 were used. The B. subtilis and E. coli K12 used TSA (Tryptic Soy Agar, 15.0 g of Bacto ^™ tryptone, 5.0 g of Bactosoytone, and 5.0 g of sodium chloride per liter), S. mutans and S. sobrinus were BHI C. albicans was cultured in YM (Yeast Mold, 1 L), medium containing 10% fetal bovine serum, 10 g of protease peptone, 2.0 g of dextrose, 5.0 g of sodium chloride and 2.5 g of disodium phosphate. Yeast extract 3.0 g, malt extract 3.0 g, peptone 5.0 g, dextrose 10.0 g), and L. sakei were stored and cultured in MRS medium.

For the antimicrobial activity assay, each strain to be tested was inoculated in 10 ml of the medium, cultured at 30 ° C, and the whole culture was inoculated into a fresh medium. When the OD ₆₀₀ value was 0.15-0.2, 180 [mu] l of each well was added to each well and 20 [mu] l of citraconic anhydride was added to each well. 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.

In order to compare the antimicrobial effect of the fermentation supernatant of Lactobacillus paraparentaum KNUC25 strain and citraconic anhydride separated therefrom, the fermentation supernatant was prepared according to the following method.

In order to ferment the microorganism Lactobacillus paraplantarum KNUC25, the microorganism strain was inoculated in 2 L of MRS (de Man, Rogosa & Sharpe broth) medium, and then incubated for 2 days in an aseptic incubator maintained at 30 ° C. To obtain the fermentation supernatant of the microorganism strain, the microorganism and the supernatant were separated by centrifugation at 8,000 rpm for 20 minutes at 4 ° C. The separated fermentation supernatant was filtered using a pore size 0.22 μm filter (Millipore, USA) The supernatant was obtained. Afterwards, each 25 ml of fermentation supernatant was lyophilized in a container, and the sample lyophilized in the form of solid powder was concentrated 30 times in the third distilled water immediately before the experiment.

2-2. Comparative analysis of antimicrobial effect of fermentation supernatant and citraconic anhydride of Lactobacillus paraplantaum KNUC25 strain

The culture broth of the Lactobacillus paraplora Room KNUC25 strain was analyzed for its antimicrobial activity by comparing citracanic acid anhydrides isolated therefrom without clearly identifying the constituents of the composition. To this end, samples were prepared according to the method of Example 2-1, and the KNUC25 fermentation supernatant, which was concentrated 30-fold and 15-fold, respectively, and C. albicans and 0.05% or 0.005% citraconic anhydride (CA) The cells were cultured for 3 hours, and the viable cell counts were determined by diluting and smearing the bacterial solution.

As a result, as shown in Fig. 4, it was confirmed that a small amount of citraconic anhydride exhibited antimicrobial activity similar to or slightly superior to the culture supernatant (KNUC25) of the strain.

2-3. E. faecalis Analysis of the Minimum Inhibitory Concentration of Citraconic Anhydride for

To investigate the minimum inhibitory concentration of citraconic anhydride required to inhibit microbial growth, citraconic anhydride was treated at different concentrations (0.1-0.0031 mol) in E. faecalis according to the method of 2-1, The growth curve of E. faecalis was measured by measuring the absorbance at OD ₅₉₅ in the medium (0, 2, 4 hours (h)).

As a result, as shown in FIG. 5, it was confirmed that the initial growth of microorganisms was inhibited when citraconic anhydride was treated at a concentration of 0.1 to 0.05 M. When the concentration of 0.0125 M was treated, 50% of bacterial growth inhibition was observed.

2-4. Analysis of antimicrobial activity range of citraconic anhydride

The results of Examples 2-2 and 2-3 show that citraconic anhydride isolated from Lactobacillus paraparentaum KNUC25 fermentation broth exhibits antimicrobial activity. Further, it has been confirmed that antimicrobial activity against various strains I wanted to know the range. To this end, the various gram-positive bacteria, gram-negative bacteria, and fungi described in 2-1 above were treated with a citric acid anhydride compound having a minimum inhibitory concentration of microorganisms of 0.05 M as identified in Example 2-3, Growth curve. As a result, as shown in Figs. 6 to 11, it was confirmed that they had excellent antibacterial activity against B. subtilis , E. coli , S. mutans , S. sobrinus , C. albicans , and L. sakei strains, Overall, it was observed to inhibit the initial growth of the fungus.

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)

A method for separating citraconic anhydride 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 substances having a size of 3 kDa or less; And
(e) lyophilizing the separated material to obtain citraconic anhydride.
The method according to claim 1,
Wherein the strain is Lactobacillus paraplantarum KNUC25 strain (Accession No. KCTC 10958BP).
The method according to claim 1,
Wherein in step (a), the strain is cultured for 1 to 5 days at 25 ° C to 30 ° C.
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