KR20050083098A - Natural anti-biotics containing the culture broth of leuconostoc kimchii - Google Patents

Abstract

The present invention relates to the natural antibacterial effect of the culture of the new lactic acid bacteria Leukonostok Kimchi I found in Korea Kimchi, more specifically, the preservation of the product by having an antiseptic and moisturizing function in food, medicine, cosmetics, household goods, etc. The present invention relates to a novel use of leukonostock kimchi eye culture medium as a natural antimicrobial agent that can increase stability and is safe and does not cause resistance in mammals including humans.

Description

Natural Anti-biotics Containing the Culture broth of Leuconostoc kimchii}

The present invention while having the antioxidant and skin moisturizing and anti-aging activity as antibacterial activity is related to the excellent natural antimicrobial agent, more specifically, current Pocono stock kimchi child main culture medium, concentrated liquid or culture solution of the dried product of the culture of (Leuconostoc kimchii) The present invention relates to a multifunctional natural antimicrobial agent having an active ingredient, having an antioxidant function and an anti-aging activity, having high antibacterial activity, and having a broad antibacterial spectrum.

In order to maintain the quality of food, medicine, cosmetics, etc. for a long time, preservatives that prevent decay by microorganisms are essential, but in the case of cosmetics with a long shelf life and a lot of microbial nutrients due to the characteristics of the product.

However, even parabens preservatives, which are the safest as preservatives and are widely used in cosmetics and medicines, are allergic to skin (Andrea Counti et al., Contact Dermatitis, 1997,37; 35-36) and their potential as environmental hormones (Edwin et al. , Tocxicology and Applied Pharmacology, 1998,153; 12-19) and resistant bacteria. In addition, preservatives for foods are distrusted in the use of acceptable standards, and new problems such as acute and chronic toxicity and mutagenesis due to continuous accumulation in the body are emerging (Xinhua, Food Science and Industry, 1990, 23 (4) 68). -72).

 Due to these problems, research on natural preservatives with excellent safety and economical efficiency of products is ongoing, and as a result, extracts of animal and plant origin such as spices, essential oils, herbal medicines, and natural antimicrobial substances such as bacteriocin have been reported. .

 Alkaloids (alkaloids), flavonoids (phytonoids), phytoalexin (antibacterial peptides), antimicrobial peptides and the like are known as natural antimicrobial substances, and antimicrobial agents such as organic acids and fatty acids are also known. Most of them are estimated to be the main mechanism of acid pH and chelate effects (El-shenawa, MA et al., J. Food Protec. 1989,52 (11): 771-778) (Bizri, JN). J Food Science, 1994, 59 (1), 130-135).

However, most of the reported natural antimicrobial substances have not been commercialized due to color, deterioration of stability, narrow antimicrobial spectrum, and problems in formulation.They are a white extract, hinokitiol, magnolia extract, megnonol and grapefruit. Only a few, such as seed extract DF-100, are commercially available.

In the case of DF-100, which is the most developed product among them, the antibacterial activity is known to be due to the organic acid and synthetic preservative Benzethonium chloride contained in DF-100. Due to the limitation of the scope of use, such as natural antibacterial agent that is capable of advanced and commercialized development is urgent.

 Meanwhile, antimicrobial agents currently used in general products are not intended to maintain product quality, but synthetic antibiotics are widely used to prevent and treat diseases of humans and livestock caused by microorganisms, but various resistant strains have appeared due to misuse and abuse. Esau forms a vicious loop that requires the use of high-level antibiotics to treat infections, and the public health issues that cause susceptibility of causative organisms to the livestock and that antibiotics remain.

Methicillin-resistant Staphylococcus aureus (Methicillin Resistant Staphylococcus aureus; MRSA) : use of two or more antibiotics to combat (Voss, such as A, Int J. Antimicrob.Agents, 1995,5 101-106) or a combination of vancomycin (Vancomycin ) Is used. However, resistant strains such as vancomycin-resistant enterococci (Billot-klein D. Antimicrob. Agents, Chemother, 1992, 36: 1487-1490) resistant to vancomycin have emerged.

 Accordingly, in recent years, it has been reported that natural products were used with flavonoids to increase the sensitivity of antibiotics (IAIN X, Liu et al. J. Pharm. Pharmacol, 2000, 52: 361-366), and antimicrobial peptides (Giacometti A Et al., J. Antimicrob. Chemother 2000 Nov; 46 (5): 807-811), although many studies have been conducted as alternatives for the combined effect, antimicrobial peptides have only limited antimicrobial properties and problems in economic mass production methods. Lee JH et al., Protein.Expr.Purif. 1998 Feb; 12 (1), 53-60).

Nisin is a peptide that has antimicrobial activity against some bacteria that rot food or cosmetics. It is a fermentation product of Streptococcus lactics , an edible bacterium, and has excellent safety and efficacy as a food preservative. It is widely used in milk and cheese around the world. Nisin is included in the category of antimicrobial material, Lantibiotics, containing an amino acid called lanthionine, and has been approved by the FDA as a food preservative and as a cosmetic ingredient by CTFA.

However, nisin also has a narrow range of antibacterial activity. Nisin does not show antibacterial activity, for example, to Bacillus, a gram-positive bacterium having endogenous spores, to Yeast, a fungi, and the like, of eukaryotic microorganisms.

The present invention solves the human hazards of conventional chemical preservatives, which are safe from humans (harmless to humans), easy to learn, economical, and also have a narrow range of antimicrobial spectrum of conventional natural preservatives, more specifically Gram. Unlike antimicrobial peptides such as nisin, which only act as antibacterial agents, the antimicrobial spectrum is broad enough to have an antimicrobial effect against almost all microorganisms, more specifically viruses, Gram-negatives, benign bacteria, yeasts, and fungi. In addition, the object of the present invention is to provide a new natural antimicrobial material that is stable in physical properties and can be applied to various areas.

It is another object of the present invention to provide a food, medicine and cosmetics containing the natural antimicrobial substance.

The present invention for achieving the above object relates to a natural antimicrobial agent and a natural antiviral agent comprising a culture solution of Leuconostoc kimchii , concentrated solution of the culture solution or dried product of the culture as an active ingredient.

Kimchi, a traditional Korean fermented food, is a fermented fermentation of cabbage, radish, carrots, peppers, etc. Korean adults consume an average of 50-200g every day. Taxonomically diverse groups such as Leuconostoc mesenteroides , Leuconostoc pseudomesenteroides and Leuconostoc lactis , as well as Leuconostoc brevis and lactose Lactic acid bacteria such as Lactobacillus plantarum , including Lactobacillus plantarum , are found in the fermentation process of kimchi. Recently, Professor Kim Jung-ho discovered a new lactic acid bacterium in Kimchi and reported it to academia (Int'l J. of Systematic & Evolutionary Microbiology (2000), 50,1915-1919). I have published a genome map for Korea.

The present invention has been completed by finding the fact that the culture medium of the microorganism shows a strong antibacterial effect. Leuconostoc kimchii IMSNU11154 was distributed by the Microbial Resources Center of the Seoul National University Microbiological Research Institute.

The present invention also relates to foods, pharmaceuticals and cosmetics containing a natural antimicrobial agent or a natural antiviral agent as an active ingredient of the culture medium, the concentrated solution of the culture medium or the dried product of the culture medium.

As confirmed in the following examples and applications, the natural antimicrobial agent according to the present invention has an excellent antimicrobial spectrum which is far superior to that of parabens, which are widely known and widely used.

In the following Examples and Applications, the experiment was not specifically applied to the natural antibacterial or natural antiviral agent according to the present invention to food or pharmaceuticals. However, the strain of leukonostock kimchi eye was found in kimchi, and the kimchi was proved to be safe as it was ingested in humans for a long time. Therefore, the strain is harmless to the human body (safety), and thus, the strain is naturally in the culture medium of the strain. It has been confirmed that there is antibacterial or natural antiviral activity. And when the culture was applied to cosmetics of various formulations it was confirmed that the product maintains antiseptic power. Although practical experiments have not been carried out, it is possible for foods, beverages, and medicines having a formulation similar to the certified cosmetics to impart antimicrobial or antiviral power by injecting a natural antimicrobial agent or a natural antiviral agent according to the present invention. Will be taken for granted.

When the natural antimicrobial agent or the natural antiviral agent according to the present invention is applied to food, medicine or cosmetics, it may be contained in an amount of 0.1 to 30% by volume depending on the characteristics of the application target, the manufacture, distribution, storage and use of the application target.

Hereinafter, the present invention will be described in detail with reference to Examples and Application Examples. The following examples and the like are merely illustrative of the present invention, and thus the scope of the technical spirit of the present invention is not changed or reduced. It will also be apparent to those skilled in the art that various embodiments or applications are possible in addition to the following examples or applications.

EXAMPLES Preparation of Culture Media of Leukonostock Kimchi

For analysis, the strain Leuconostoc kimchii IMSNU11154 was distributed at the Microbial Resource Center of the Seoul National University Microbiological Research Institute.

100 ml of MRS (Difco) broth was autoclaved at 121 ° C. for 15 minutes, inoculated with Leuconostoc kimchii and incubated at 30 ° C. for 16 hours. The culture solution was centrifuged at 5000 X g for 3 minutes, and then filtered using a membrane filter having a pore size of 0.22 μm to maintain bacteria-free state.

In case of concentration of the culture solution, the culture solution obtained above was filtered through 10,000 Da membrane, and then concentrated to 1,000 Da membrane. The concentrated culture solution was filtered using a membrane filter (pore size 0.22 μm) to maintain bacteria-free state.

The final pH of the filtrate was adjusted to 7 at all times.

Application Example 1: Check the antimicrobial activity of the culture medium of the leukonostock kimchi eye 1

The antimicrobial activity of the culture solution of Leuconostoc kimchii bacteria according to the present invention obtained by the above-described example was measured.

(1) Preparation of contaminating bacteria

In order to measure the antimicrobial activity of the culture medium of Leuconostoc kimchii bacteria according to the present invention, the target microorganisms having various taxonomic distributions were selected as shown in Table 1 below.

Bacteria and yeast were inoculated in 50 ml (250 ml flask) of the medium described in Table 1 one day before the antimicrobial activity measurement experiment was incubated overnight. Mold was pre-incubated for 1-2 weeks in potato dextrose agar plate or slant. At this time, antibiotics chloramphenicol were added to the fungal medium to prevent bacteria from growing.

First, E. coli , S. aureus , and P. aerruginosa belonging to group 1 overnight incubation, and then mixed 1: 1: 1 to prepare a group 1 inoculum. In the mold belonging to group 4, 10 ml of saline solution was added to the culture medium for 1-2 weeks, and the surface was gently scraped to harvest spores, and then diluted 1/10 to 1/100 ( A.niger is 1/10 to 1/100, Penicillium The dilution is at least 1/100 to be able to count), and the inoculation solution was prepared by counting directly with a hemocytometer and measuring cell #. Finally, the inoculum was diluted to 10 ⁸ cells / ml for Bacteria and 10 ⁷ cells / ml for yeast and mold.

(2) Inoculation of contaminating bacteria and analysis of antibacterial activity

Fourteen conical tubes containing 50 ml of the culture medium of Leuconostoc kimchii according to the present invention obtained in Example 1 were prepared, and 0.5 ml of Group 1 inoculation solution (final bacteria concentration was about 10 ^{7 in} each tube). cells / ml), 0.4 to 0.45 ml of group 2 microorganism overnight culture medium, 0.5 ml of group 3 overnight culture medium (final cell concentration is about 10 ⁵ cells / ml) and mold cultures of group 4 (final cell concentration of each mold is Inoculate to a final concentration of 5 × 10 ⁵ cells / ml (four experiments each).

Samples inoculated with group 1 and group 2 were incubated at 30-35 ° C., samples inoculated with group 3 and group 4 were incubated at 25-30 ° C. with 0, 1, 2, 7, 14, At 21 and 28 days, a portion of the sample (0.1ml or 0.1g) was taken and spread on the prepared plate and incubated in an incubator. After 2 days of culture (for group 1) and 1 week after culture (for groups 2 to 4), the plates were taken out, and the number of viable bacteria was measured and shown in Table 2 below. The numerical values shown in Table 2 are average values of four experimental samples.

Application Example 2: Confirmation of antimicrobial activity of the culture medium of the leukonostock kimchi eye

In order to confirm whether the antimicrobial activity of the culture solution of the leukonostock kimchi eye according to the present invention also works in cosmetics, by applying the culture solution according to the present invention to a skin lotion which is one of the well-known basic cosmetics, the antibacterial activity of the paraben is most commonly used. And the comparative analysis (Test Composition 1, 2, 3 in Table 3). However, the present invention is not limited to skin lotion as an example of the applied cosmetic composition.

Experimental method was inoculated in the same manner as in Example 1 by inoculating the microbial group of Groups 1 to 4 in the same manner as in Example 1, using the test compositions Examples 1, 2, and 3 as samples, respectively, in the same manner as in the Kimchi lactic acid bacteria culture medium in Example 1. After culturing at a temperature of 14 days finally confirmed the state of the antimicrobial activity (see Figures 1 to 4). 1, 2, 3, and 4 are photographs of the experiment results of Group 1, Group 2, Group 3, and Group 4, respectively. In Figures 1, 2, and 3 are the test compositions 1, 2, 3 is shown.

As can be seen in the figure, as a whole, the growth of a large number of bacteria in the test composition 1 of all subject groups was confirmed (except in FIG. 4, too many microbial colonies in the test composition 1 as a whole microbial layer on the medium. As a result, it appears as if there are no microbial colonies in the photograph.) However, in group 4 (fungus), the most mold growth was found in the test composition 2 containing parabens. On the other hand, almost no growth was found in the test composition 3 of all groups, that is, the composition containing the natural antibiotic according to the present invention.

Accordingly, it was confirmed that the natural antibiotic according to the present invention has an excellent antibiotic effect while having a broad spectrum.

Application Example 3: Identification of antiviral activity of leukonostock kimchi eye

In order to confirm whether the culture medium of the leukonostock kimchi eye according to the present invention has antiviral activity, an experiment was performed in which the influenza virus was treated with the culture medium according to the present invention.

Madin-Darby Canine Kidney cell (MDCK) ATCC CCL 34 and Influenza virus A1H3N2 were distributed from the National Institutes of Health. MDCK cells (80% confluent in 6 well plates) were washed three times with Phosphate-Buffered Saline (PBS, manufactured by GIBCO) at pH 7.4. Influenza virus was serially diluted with Dulbecco's Modified Eagle Medium (MEM medium, manufactured by GIBCO) without Fetal Bovine Serum.

After influenza virus was inoculated into MDCK cells at a concentration of 0.1-0.2 ml / 6 well plates, the dilution (control) or the culture solution of Leuconostoc kimchii (treatment group) were added to each well to 10% and at 33-34 ° C. Incubated for 1 hour. At this time, the plate was shaken every 15 minutes. After removing the virus and washing the cells with PBS, the cells were covered with medium containing 2 times concentrated MEM, 2 times concentrated agarose (2%), and trypsin (1.2 g / ml) and maintained at 42 ° C. I let you. The medium was solidified at room temperature and then incubated at 33-34 ° C. for 3-4 days until clear zone was observed. When clear zones appeared, 100-200 μl of 5% glutaraldehyde was added to each well and left at 33-34 ° C. for 2 hours to fix. Plates were washed with water to remove the covered medium and stained with 0.1% neutral red. At this time, dead cells were white and living cells were stained red (FIG. 5). The left plate in the control and treatment groups in FIG. Is a sample without virus treatment.

As can be seen in the virus-treated control group (right plate of the control picture), the virus actively attacked the host cell, resulting in a lot of white virus plaques. On the other hand, in the treatment group in which the virus was treated in the state containing the antiviral agent according to the present invention (right plate of the treatment group photo), a significantly smaller number of virus flushes were formed than in the control group.

Therefore, the antiviral power of the antiviral agent by this invention was confirmed.

As discussed and proved above, the culture solution of the leukonostock kimchi eye, the concentrate of the culture solution, or the dried product of the culture medium of the present invention exhibit a wide and excellent antibacterial and antiviral activity. Therefore, when utilizing this, it is possible to give antimicrobial or antiviral power to food, medicine, cosmetics safely and effectively.

1 to 4 is a photograph of the experimental results showing the antimicrobial activity of leukonostock kimchi eye culture solution according to the present invention.

Figure 5 is a photograph of the experimental results showing the antiviral power of the leukonostock kimchi eye culture medium according to the present invention.

Claims (14)

A natural antimicrobial agent comprising a culture solution of Leuconostoc kimchii , a concentrate of the culture solution or a dried product of the culture solution as an active ingredient. Food containing the natural antimicrobial agent of Claim 1. The method of claim 2, Food, characterized in that containing 0.1 to 30% by volume of the natural antibacterial agent. Pharmaceuticals containing the natural antibacterial agent according to claim 1. The method of claim 4, wherein Medicines, characterized in that containing 0.1 to 30% by volume of the natural antibacterial agent. Cosmetics containing the natural antibacterial agent according to claim 1. The method of claim 6, Cosmetics, characterized in that containing 0.1 to 30% by volume of the natural antibacterial agent. A natural antiviral agent comprising a culture solution of Leuconostoc kimchii , a concentrate of the culture solution or a dried product of the culture solution as an active ingredient. Food containing the natural antiviral agent of Claim 8. The method of claim 9, Food characterized in that the natural antiviral agent is contained 0.1 to 30% by volume. A pharmaceutical product containing the natural antiviral agent according to claim 8. The method of claim 11, Medicines, characterized in that containing 0.1 to 30% by volume of the natural antiviral agent. Cosmetics containing the natural antiviral agent of Claim 8. The method of claim 13, Cosmetics, characterized in that containing 0.1 to 30% by volume of the natural antiviral agent.