KR100583836B1 - Natural antibiotic and Composition comprising thereof - Google Patents

Abstract

The present invention relates to a multifunctional natural antimicrobial agent having a high antimicrobial activity and a broad antimicrobial spectrum prepared by using kimchi lactic acid bacteria appearing during the ripening process of kimchi, and a composition such as a beverage, feed, medicine, cosmetics, food, etc. Kimchi lactobacillus culture according to the invention, the concentrated solution of the culture solution or the dried product of the culture medium shows a wide and excellent antimicrobial activity, when used, it can be safely and effectively impart antimicrobial activity to feed, beverages, food, medicine, cosmetics.

Description

Natural antibiotic and composition comprising same

The present invention relates to a multifunctional natural antimicrobial agent having a high antibacterial activity and a broad antimicrobial spectrum produced by using kimchi lactic acid bacteria appearing during the ripening process of kimchi and a composition such as beverages, feeds, pharmaceuticals, cosmetics, foods and the like.

In order to maintain the quality of feed, beverages, food, medicines, cosmetics, etc. for a long time, preservatives that prevent decay by microorganisms are essential.

However, even parabens preservatives, which are the safest as conventional preservatives and are widely used in cosmetics and medicines, are also known as skin allergies (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, food preservatives are distrusted in the use of accepted standards, and new problems such as acute, chronic toxicity, and mutagenesis due to persistent body accumulation are emerging (Xinhua, Food Science and Industry, 1990, 23 (4) 68). -72).

Due to these problems, studies on natural preservatives with excellent safety and economical efficiency of products continue, and as a result, natural antibacterial substances of microbial origin such as extracts of copper and plant origin such as spice, essential oil, herbal medicine, and bacteriocin are 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, formulation problems, and the like. Henokinitiol, a cypress extract, Magnonol, a magnolia extract, and grapefruit Only a few, such as seed extract DF-100, are commercially available.

In the case of DF-100, the most advanced product development, the antimicrobial activity is known to be due to the organic acid and synthetic preservative Benzethonium chloride (Benzethonium chloride) contained in the DF-100, and other natural antimicrobial agents are economical There is a need for a natural antimicrobial agent that can be developed and commercialized due to problems such as narrow antimicrobial spectrum or limited use range due to physical properties.

Meanwhile, antimicrobials currently used in general products are not used to maintain the quality of products, but synthetic antibiotics are widely used to prevent and treat diseases of humans and livestock caused by microorganisms, but various resistant strains appear due to misuse and abuse. Esau forms a vicious cycle that requires the use of high-level antibiotics to treat infectious diseases, and the susceptibility of causative organisms to animals is low, and important public health problems 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 are 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 has many limitations in the range of antibacterial activity, for example, Bacillus, an endogenous spore, Bacillus , Yeast, Fungi, etc. have.

Therefore, the technical problem to be achieved by the present invention is a natural origin that can solve the disadvantages of the conventional chemical preservatives have a stimulus to the human body is safe, easy to learn and economical, and also has a narrow range of existing natural preservatives Overcoming the antimicrobial spectrum to provide a new natural antimicrobial material having a broad antimicrobial spectrum.

In addition, another technical problem to be achieved by the present invention is to improve the composition that can be applied to various products including the natural antimicrobial substance as described above as an active ingredient.

In order to achieve the above technical problem, the present invention

It provides any one natural antimicrobial agent selected from the group consisting of a culture of kimchi lactic acid bacteria, a concentrate of the culture, dried products of the culture and mixtures thereof appearing during the ripening of kimchi.

In the natural antimicrobial agent according to the present invention as described above, the lactic acid bacteria of the kimchi lactic acid bacteria of the genus Leuconostoc , Kimchi lactic acid bacteria of the genus Lactobacillus ( Lactobacillus ), Kimchi lactic acid bacteria of the genus Weissella ( Weissella ) and mixed lactic acid bacteria thereof It is preferably any one selected.

In the natural antimicrobial agent according to the present invention as described above, the lactic acid bacteria of the genus Leuconostoc is Leuconostoc citreum , Leuconostoc lactis , Leuconostoc lactis , Leukonosstock metheteroid sub Spigot. Leuconostoc mesenteroides subsp.dextranicum , Leukonostok mesentroid subspi. Leuconostoc mesenteroides subsp.Mesenteroides, Leuconostoc argentinum , Leuconostoc carnosum , Leuconostoc gellidum , Leuconoststock kimchi child ( Leuconostoc kimchi) , Leuconostoc inhae ( Leuconostoc inhae ), Leuconostoc gasicomitatum ( Leuconostoc gasicomitatum ) is preferably any one selected from the group consisting of mixed lactic acid bacteria thereof.

In the natural antimicrobial agent according to the present invention as described above, the Lactobacillus ( Lactobacillus ) kimchi lactic acid bacteria are Lactobacillus brevis ( Lactobacillus brevis ), Lactobacillus acidophilus ( Lactobacillus acidophilus ), Lactobacillus vulgaricus (Lactobacillus bulgaricus) , Lactobacillus plantarum , Lactobacillus kimchii , Lactobacillus paraplantarum , Lactobacillus curbatus subsiphy. Lactobacillus curvatus subsp.curvatus , Lactobacillus sakei subcipi . Lactobacillus sakei subsp. Sakei and is preferably any one selected from the group consisting of mixed lactic acid bacteria.

In the natural antimicrobial agent according to the present invention as described above, the weissella genus kimchi lactic acid bacteria Weissella koreensi ( Weissella koreensi ), Weissella hanii ( Weissella hanii ), Weissella kimchii ( Weissella kimchii ), It is preferably one selected from the group consisting of Weissella soli , Weissella confusa and mixed lactic acid bacteria thereof.

In order to achieve another technical problem, the present invention includes any one of the natural antimicrobial agent selected from the group consisting of a culture solution of kimchi lactic acid bacteria, a concentrate of the culture solution, dried products of the culture solution and mixtures thereof appearing during the ripening process of kimchi as an active ingredient It provides a composition.

The composition according to the present invention as described above is preferably any one selected from the group consisting of beverages, feed, food, medicines and cosmetics.

 In the present invention, pickled radish, cabbage, cucumber, etc., or salted red pepper, garlic, green onion, ginger, salted fish, and so on, are fermented at low temperature and fermented at low temperature. The culture of kimchi lactic acid bacteria is high antimicrobial activity, antimicrobial spectrum was found to be able to play a role of a wide range of multifunctional natural antimicrobial agent will be described in detail with reference to the present invention. The following examples are merely illustrative of the present invention, and thus the scope of the technical spirit of the present invention is not changed or reduced.

<Example>

-Kimchi Lactobacillus Culture Solution Preparation-

To prepare a culture using kimchi lactic acid bacteria as shown in Table 1 Looking at the process as follows.

First, 100 ml of MRS (Difco) broth was autoclaved at 121 ° C. for 15 minutes, and then inoculated with kimchi lactic acid bacteria and shaken at 30 ° C. for 16 hours.

The culture solution was centrifuged at 5000 X g for 3 minutes to obtain a supernatant, and the supernatant was filtered using a membrane filter having a pore size of 0.22 µm to obtain a bacteria-free culture solution.

Subsequently, the culture solution was passed through a 10,000 Da membrane to obtain a filtrate, and then concentrated to 1,000 Da membrane, and filtered using a membrane filter having a pore size of 0.22 μm to maintain the concentrated culture solution in a bacteria-free state. Final experimental cultures were obtained. The final pH of this culture was always adjusted to 7 to exclude the effect of pH.

TABLE 1

-Antibacterial Activity Confirmation Test of Kimchi Lactic Acid Bacteria Culture-

(1) Preparation of contaminating bacteria

In order to measure the antimicrobial activity of the culture solution of lactic acid bacteria of the genus Leukonostalk according to the present invention, the target microorganisms having various taxonomic distributions were selected as shown in Table 2 below.

TABLE 2

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 (overnight). The molds were preincubated for one to two weeks in a potato dextrose agar plate or a slant. At this time, antibiotics chloramphenicol were added to the fungal medium to prevent bacteria from growing.

First, E. coli, S. aureus, and P. aeruginosa belonging to group 1 were cultured overnight (overnight), 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). Should be diluted to at least 1/100 to be countable (counting)) and counted directly with a hemocytometer to measure the cell number (cell #) to prepare the inoculum.

Finally, the number of microorganisms in the inoculum was diluted using sterile saline solution 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

14 conical tubes containing 50 ml of the culture solution samples obtained in Example 1 were prepared for each sample, and 0.5 ml of Group 1 inoculation solution (final cell concentration was about 10 ⁷ cells / ml) and Group 2 microorganism overnight were prepared in each tube. 0.4 to 0.45 ml of overnight culture, 0.5 ml of Group 3 overnight culture (final cell concentration is approximately 10 ⁵ cells / ml) and mold cultures in Group 4 (final cell concentration of each mold is the final Inoculate to a 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, 0.1 ml or 0.1 g was taken and spread on the prepared plates 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 removed and the number of viable cells was measured. For Sample 3, Table 5, For Sample 4, Table 6, For Sample 5, Table 7, For Sample 6, Table 8, For Sample 8, Table 10, For Sample 9, Table 11, Sample 10 In Table 12, Table 11 for Sample 11 and Table 14 for Sample 12 are shown. The numerical values shown in the table below are the average values of four experiments.

TABLE 3

TABLE 4

TABLE 5

TABLE 6

TABLE 7

TABLE 8

TABLE 9

TABLE 10

TABLE 11

TABLE 12

TABLE 13

TABLE 14

As described in the above table, it can be seen that the natural antimicrobial agent according to the present invention showed a very broad antimicrobial spectrum having antimicrobial effect on Gram-negative, positive bacteria, yeasts, and fungi.

-Antibacterial activity comparison test of Kimchi lactic acid bacteria culture medium and paraben-

In order to compare the antibacterial activity of the kimchi lactic acid bacteria culture medium according to the present invention and the antibacterial activity of parabens, the skin lotion, which is one of the basic cosmetics, samples 1-4 of the genus Leuconostoc , and 5-10 samples of the genus Lactobacillus. Weissella genus 11 ~ 12 and the parabens were applied to prepare a test composition as shown in Table 15 to compare the antimicrobial activity.

TABLE 15

As shown in the above antimicrobial activity test, the test compositions were inoculated to the microorganisms of Groups 1 to 4 in each of Examples 1 to 14, cultured at the same temperature, and finally, after 14 days, the number of viable bacteria was measured. Indicated.

TABLE 16

As can be seen from Table 16, overall, growth of many bacteria was confirmed in Test Composition 1 of all subject groups. In the case of the test composition using the conventional chemical preservatives 2 showed a particularly weak side in the bacterial mixture, the fungal microorganisms such as yeast or fungi showed some degree of inhibiting the growth. On the other hand, almost no growth was found in the test compositions 3 to 14 of all groups, that is, the composition containing the natural antibiotic according to the present invention.

As described above, it was confirmed that 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 addition to the cosmetic composition tested for antimicrobial activity as described above, even if included in the composition, such as beverages, feed, food, medicines, etc. is expected to show antimicrobial activity, if applied to the composition, such as beverages, feed, food, medicines, etc. Appropriately adjusting the content of the culture medium according to the present invention according to the characteristics, the preparation, distribution, storage and use of the application targets, such as those of ordinary skill in the art to which the present invention belongs It will be possible enough.

According to various experiments of the present inventors, it was found that it is appropriate to include 0.1 to 30% by volume with respect to the whole composition such as beverages, feeds, foods, medicines, cosmetics, and the like.

In addition, since the natural antimicrobial agent according to the present invention is a culture of kimchi lactic acid bacteria appearing in the ripening process of kimchi which has been ingested by humans for a long time, there is no doubt about its stability even without a separate stability test.

As discussed and proved above, the culture of kimchi lactic acid bacteria, the concentrate of the culture solution or the dried product of the culture medium of the present invention show a wide and excellent antibacterial activity. Therefore, when utilizing this, it is possible to safely and effectively give antimicrobial activity to feed, beverages, food, medicine, cosmetics.

Claims (11)

It consists of Weissella koreensi , Weissella hanii , Weissella kimchii , Weissella soli , Weissella confusa and their mixed lactic acid bacteria Any one natural antimicrobial agent selected from the group consisting of any kimchi lactic acid bacteria culture medium selected from the group, the concentrated solution of the culture solution, the dried product of the culture solution and mixtures thereof. delete delete delete delete It consists of Weissella koreensi , Weissella hanii , Weissella kimchii , Weissella soli , Weissella confusa and their mixed lactic acid bacteria A composition comprising any one natural antimicrobial agent selected from the group consisting of any kimchi lactic acid bacteria culture medium selected from the group, the concentrated solution of the culture solution, the dried product of the culture solution and mixtures thereof as an active ingredient. delete delete delete delete The composition of claim 6, wherein the composition is any one selected from the group consisting of beverage, feed, food, medicine and cosmetics.