WO2006009394A1

WO2006009394A1 - Natural antibiotic and composition comprising thereof

Info

Publication number: WO2006009394A1
Application number: PCT/KR2005/002346
Authority: WO
Inventors: Dong-Il Jang; Jung-Hwa Lee
Original assignee: Cotde, Ltd.
Priority date: 2004-07-20
Filing date: 2005-07-20
Publication date: 2006-01-26
Also published as: KR100583836B1; KR20050087741A

Abstract

Disclosed herein are a natural antibiotic having high inhibitory activity against a broad spectrum of microbes, obtainable using lactic acid bacteria isolated from Kimchi, and a composition comprising the natural antibiotic. The natural antibiotic is prepared from a culture solution of the lactic acid bacteria isolated from Kimchi during the fermentation of Kimchi, a concentrate of the culture solution, a dehydrate of the culture solution, or combinations thereof. The natural antibiotic can be applied to compositions, such as fodder, beverages, food, medicines, and cosmetics, imparting antimicrobial activity thereto, without harmful effects on the body.

Description

NATURAL ANTIBIOTIC AND COMPOSITION COMPRISING

THEREOF

Technical Field

[1] The present invention relates to a natural antibiotic having high inhibitory activity against a broad spectrum of microbes, obtainable using lactic acid bacteria isolated from Kimchi, and the application of the natural antibiotic to compositions, such as beverages, fodder, medicines, cosmetics, food, etc. Background Art

[2] Most feedstuffs, beverages, foods, medicines and cosmetics resort exclusively to preservatives against microbial putrefaction in order to maintain their quality for a long period of time. This is particularly true of almost all cosmetics because they have a relatively long distribution period and are rich in nutrients suitable for microbes.

[3] Even parabens, which are known to be the most safe preservatives and are widely used in cosmetics and medicines, are found to have the problem of causing dermal allergies (Andrea Counti et al., Contact Dermatitis, 1997, 37;35-36), likely act as endocrine disruptors (Edwin et al., Toxicology and Applied Pharmacology,1998,153;12-19) and cause the advent of resistant bacteria. Besides, preservatives for foods are mistrusted even when they are used in an amount within approved standards and are viewed with suspicion if they cause new problems, such as acute/chronic toxicity and mutations, when accumulated within the body (Shin Dong- Hwa, Food Science and Industry, 1990, 23(4) 68-72).

[4] In order to overcome these problems, research continues to be conducted into natural preservatives realizing good safety and economical benefits. As a result, extracts from animals or plants, such as spices, essential oils, herbal medicines, and materials originating from microorganisms, such as bacteriocin, are reported as natural antibiotics.

[5] For example, alkaloids, flavonoids, Phytoalexin, and some peptides are known as natural antibiotics. Some organic acids and fatty acids also show antimicrobial activity. The antibiotic mechanism of these materials is believed to be, for the most part, based on a pH effect or chelating effect (El-shenawa, MA et al., J. Food Protec. 1989, 52(ll):771-778; Bizri, JN et al., J food Science,1994, 59(l),130-135).

[6] Most of the materials that have been reported, thus far, to have antibacterial activity have not been commercialized because of their colors and odors, poor stability, narrow antimicrobial spectra, formulation problems, etc. Out of the materials reported, a few have been commercialized, such as Hinokitiol, extracted from Chamaecyparis obtusa, Megnonol, extracted from magnolia, and DF- 100, extracted from grapefruit seeds.

[7] DF- 100, which has the longest history of commercialization, owes its antimicrobial activity to organic acids and benzethonium chloride, a synthetic preservative. Other natural antimicrobial agents suffer from the disadvantages of being economically un¬ favorable, having a narrow spectrum of microbial targets, and showing poor physical properties. Therefore, there is an imperative need for natural antibiotics that have improved properties and can be included in commercial products.

[8] Meanwhile, synthetic antibiotics are widely used in various products to prevent and treat bacterial diseases in humans and animals rather than to maintain qualities of the products. However, the misuse and overuse of synthetic antibiotics give rise to the advent of various mutants resistant thereto, which are killed only with antibiotics of higher potency. Thus, a vicious circle of emerging resistant mutants and the use of an¬ tibiotics of higher potency occurs. As a result, synthetic antibiotics become less inhibitive against pathogens in livestock animals. Besides, synthetic antibiotics ad¬ ministered to livestock do not completely degrade, but remain therein, threatening public health.

[9] For instance, methicillin resistant Staphylococcus aureus (MRSA) (Voss, A et al.,

Int J. Antimicrob. Agents, 1995, 5:101-106) is now treated with two or more an¬ tibiotics in combination, or with vancomycin. However, strains resistant to vancomycin, such as vancomycin-resistant enterococci (Billot-klein D. Antimicrob. Agents, Chemother, 1992, 36:1487-1490), have emerged.

[10] Recently, it has been reported that the antimicrobial activity of synthetic antibiotics can be improved when they are used in combination with natural materials such as flavonoids (IAIN X, Liu et al., J. Pharm. Pharmacol, 2000, 52:361-366). Antibacterial peptides (Giacometti A et al., J. Antimicrob. Chemother 2000 Nov; 46(5):807-811) have been also extensively studied as alternatives for synergic antibacterial effects. However, antibacterial peptides show limited inhibitory activity and are difficult to produce at a profit (Lee J. H. et al., Protein. Expr. Purif. 1998 Feb;12(l), 53-60).

[11] Nisin is a peptide that acts as a bacteriocin, inhibiting the germination and growth of some bacteria capable of putrefying foods or cosmetics. Expressed in from the food bacteria Streptococcus lactis, this peptide has been found to have good safety and efficacy as a food preservative and is widely used in diary products, such as milk and cheese, all over the world, including the U.S. A. Containing the amino acid lantionin, nisin falls within the category of lantibiotics, and has been FDA approved for use as a food preservative and CTFA approved for use in cosmetics.

[12] However, nisin acts against a limited spectrum of bacteria. For example, nisin cannot inhibit the growth of gram-positive bacteria with endospores, such as Bacillus spp., and progressive microorganisms, such as yeasts and fungi. Disclosure of Invention

Technical Problem

[13] Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and an object of the present invention is to provide a novel natural antibiotic, which is safe to the body without the irritation of con¬ ventional synthetic preservatives, is readily obtainable and economically favorable in its preparation, and shows inhibitory activity against a wide spectrum of bacteria.

[14] In addition, it is another object of the present invention to provide a composition containing the natural antibiotic as an active ingredient, which can be included in various products. Technical Solution

[15] In accordance with an aspect of the present invention, the objects of the present invention could be accomplished by the provision of a natural antibiotic, prepared from one selected from a group consisting of a culture solution of the lactic acid bacteria isolated from Kimchi during the fermentation of Kimchi, a concentrate of the culture solution, a dehydrate of the culture solution, and combinations thereof.

[16] In the natural antibiotic, the lactic acid bacteria may be selected from a group consisting of Leuconostoc spp., Lactobacillus spp., Weissella spp., and combinations thereof.

[17] Preferably, the Leuconostoc spp. useful in the natural antibiotic of the present invention include Leuconostoc citreum, Leuconostoc lactis, Leuconostoc mesenteroides subsp. dextranicum, Leuconostoc mesenteroides subsp. Mesenteroides, Leuconostoc argentinum, Leuconostoc carnosum, Leuconostoc gellidum, Leuconostoc kimchii, Leuconostoc inhae, Leuconostoc gasicomitatum and combinations thereof.

[18] Preferably, the Lactobacillus spp. useful in the natural antibiotic of the present invention include Lactobacillus brevis, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus plantarum, Lactobacillus kimchii, Lactobacillus para- plantarum, Lactobacillus curvatus subsp. curvatus, Lactobacillus sakei subsp. sakei and combinations thereof.

[19] Preferably, Weissella spp. useful in the natural antibiotic of the present invention include Weissella koreens, Weissella hanii, Weissella kimchii, Weissella soli, Weissella confusa and combinations thereof.

[20] In accordance with another aspect of the present invention, the object of the present invention could be accomplished by the provision of a composition that comprises a natural antibiotic prepared from one selected from a group consisting of a culture solution of the lactic acid bacteria isolated from Kimchi during the fermentation of Kimchi, a concentrate of the culture solution, a dehydrate of the culture solution, and combinations thereof, as an active ingredient.

[21] In this regard, the composition is preferably in the form of one of a beverage, fodder, food, a medicine or a cosmetic. Advantageous Effects

[22] Culture solutions of Kimichi lactic acid bacteria, or their concentrates or dried materials have excellent antimicrobial activity against a wide spectrum of microbes and can endow foods, fodders, beverages, medicines and cosmetics with antimicrobial activity with safety and efficiency. Best Mode for Carrying Out the Invention

[23] Leading to the present invention, intensive and thorough research, conducted by the present inventors, into the preservation of foods and cosmetics, resulted in the finding that a culture of lactic acid bacteria isolated from Kimchi (hereinafter referred to as Kimchi lactic acid bacteria) can act as a multifunctional natural antibiotic that has potent antimicrobial activity against a broad spectrum of bacteria. As well known, Kimchi lactic acid bacteria emerge during the fermentation of Kimchi, which is generally prepared by pickling radish, cabbage and cucumber with salt, mixing the pickles with a condiment composition including powdered pepper, crushed garlic, chopped green onions, salted seafood, etc. and fermenting the pickled vegetables. Mode for the Invention

[24] A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limit of the present invention.

[25]

[26] EXAMPLES

[27]

[28] - Preparation of Culture Solution of Kimchi Lactic Acid Bacteria-

[29]

[30] As seen in Table 1, a culture was prepared using Kimchi lactic acid bacteria as follows.

[31] First, 100 ml of MRS (Difco) broth was autoclaved at 121°C for 15 min, and

Kimchi lactic acid bacteria was inoculated in the broth and cultured at 30°C for 16 hours with agitation.

[32] After the centrifugation of the resulting culture at 5000xg for 3 min, the supernatant was filtered through a membrane filter having a pore size of 0.22 D to afford a bacteria- free solution.

[33] Subsequently, this bacteria-free solution was passed through a membrane having a cut-off size of 10,000 Da. The resulting filtrate was concentrated using the membrane having a cut-off size of 1,000 Da. The concentrate thus obtained was again filtered through a membrane filter having a pore size of 0.22 D so as to maintain the bacteria- free state. The final culture solution was adjusted to a pH of 7 in order to exclude the pH-dependent effect of antimicrobial activity.

[34] Table 1

[35] [36] -Assay of Culture Solution of Kimchi Lactic Acid bacteria for Antimicrobial Activity -

[37] [38] (1) Preparation of Contaminants [39] In order to assay the antimicrobial activity of the Leuconostoc spp. culture solution according to the present invention, a wide taxonomical range of target microorganisms were selected and are given in Table 2, below.

[40] Table 2

[41] [42] Bacteria and yeasts were inoculated into 50 ml of each of the media listed in Table 1 (in 250 ml flasks) one day before the assays for antimicrobial activity, and cultured overnight. Molds were pre-cultured on potato dextrose agar plates or slants for about 1-2 weeks prior to being assayed. In this regard, chloramphenicol was added to the culture medium for fungi so as to inhibit the growth of bacteria therein.

[43] First, E. coli, S. aureus, and P. aeruginosa of Group 1 were cultured overnight and the respective culture media were mixed in a volume ratio of 1 : 1 : 1 to yield an in¬ oculation solution of Group 1. 10 ml of saline was poured onto the agar on which molds of Group 4 had been cultured for 1-2 weeks, and spores were harvested by carefully scraping off the agar, followed by dilution with saline by a factor of 10¹ - 10² (cell counting was impossible before 10- to 100-fold dilution for A. niger and at least 100-fold dilution for Penicillium). Using a hemocytometer, living cells were counted.

[44] Finally, appropriate dilutions of the culture solutions were made in sterilized biological saline to yield inoculation solutions that contained bacteria at a population

8 7 7 of 10 cells/ml, yeast at a population of 10 cells/ml, and mold at a population of 10 cells/ml, respectively.

[45] [46] (2) Inoculation of contaminants and Assays for antimicrobial activity [47] Culture solution samples 1 to 12 were added in an amount of 50 ml into respective conical tubes. Into each tube was added 0.5 ml of the inoculation solution of Group 1, (final cell density about 10 cells/ml), 0.4~0.45ml of the overnight cultured solution of Group 2, and 0.5ml of the overnight cultured solution of Group 3 (final cell density about 10 cells/ml) or the culture solution of Group 4 (each mold had a final cell density of 5x10⁵ cells/ml) (in quadruple).

[48] While being incubated at 30~35°C, for Groups 1 and 2, and 25~30°C, for Groups 3 and 4, each of the samples was withdrawn in an amount of 0.1 ml or 0.1 g at 0, 1, 2, 7, 14, 21, and 28 days after inoculation, and spread over plates which were then incubated in an incubator. Two days (Group 1) and one week (Groups 2 to 4) after the incubation, the plates were taken out of the incubator and measured for numbers of surviving cells. The results are given in Table 3 for sample 1, Table 4 for sample 2, Table 5 for sample 3, Table 6 for sample 4, Table 7 for sample 5, Table 8 for sample 6, Table 9 for sample 7, Table 10 for sample 8, Table 11 for sample 9, Table 12 for sample 10, Table 13 for sample 11, and Table 14 for sample 12. Numerals listed in the following tables are averages of the values obtained in quadruple experiments.

[49] Table 3

[50]

[51]

[52] Table 6 Day O Day 1 Day 2 Day 7 Day 14 Day 21 Day 28

Group 1 1.0* 10⁷ 1.0* 10⁶ 8.6* 10⁴ O O O O

Group 2 1.0* 10⁵ 1.0* 10⁵ 1.0* 10⁵ 9*10 3*10 O O

Group 3 1.0* 10⁵ 1.0* 10⁵ 3.0* 10⁴ 2.5*10² 3.8*10 O O

Group 4 1.0* 10⁵ 1.0* 10⁵ 1.0* 10⁵ 8.0* 10⁴ 6.0* 10⁴ O O

[53] Table 7

Day O Day 1 Day 2 Day 7 Day 14 Day 21 Day 28

Group 1 1.0* 10⁷ 1.0* 10⁶ 9.0* 10⁴ O O O O

Group 2 1.0* 10⁵ 1.0* 10⁵ 1.0* 10⁵ 9*10⁴ 6*10 O O

Group 3 1.0* 10⁵ 1.0* 10⁵ 3.1*10⁴ 2.7* 10² 3.8*10 O O

Group 4 1.0* 10⁵ 1.0* 10⁵ 1.0* 10⁵ 1.0*10⁵ 1.0*10⁵ O O

[54] Table 8

Day O Day 1 Day 2 Day 7 Day 14 Day 21 Day 28

Group 1 1.0* 10⁷ 1.2*10⁶ 8.6* 10⁴ O O O O

Group 2 1.0* 10⁵ 1.0* 10⁵ 1.0* 10⁴ 8*10³ 4*10³ O O

Group 3 1.0* 10⁵ 1.0* 10⁵ 3.1*10⁴ 2.1*10² 2.8*10 O O

Group 4 1.0* 10⁵ 1.0* 10⁵ 1.0* 10⁵ 1.0*10⁵ 1.0*10⁵ O O

[55] Table 9

Day O Day 1 Day 2 Day 7 Day 14 Day 21 Day 28

Group 1 1.0* 10⁷ l.l*10⁶ 9.6* 10⁴ O O O O

Group 2 1.0* 10⁵ 1.0* 10⁵ 1.0* 10⁵ 9*10⁴ 6*10³ O O

Group 3 1.0* 10⁵ 1.0* 10⁵ 3.1*10⁴ 2.7* 10² 3.8*10 O O

Group 4 1.0* 10⁵ 1.0* 10⁵ 1.0* 10⁵ 1.0*10⁵ 1.0*10⁴ O O

[56] Table 10

Day O Day 1 Day 2 Day 7 Day 14 Day 21 Day 28

Group 1 1.0* 10⁷ l.l*10⁶ 7.6* 10⁴ O O O 0

Group 2 1.0* 10⁵ 1.0* 10⁵ 1.0* 10⁵ 7*10⁴ 6*10³ O 0

Group 3 1.0* 10⁵ 1.0* 10⁵ 3.1*10⁴ 2.6* 10² 3.0*10 O 0

Group 4 1.0* 10⁵ 1.0* 10⁵ 1.0*10⁵ 1.0*10⁵ 1.0*10⁴ O 0 [57] Table 11

Day O Day 1 Day 2 Day 7 Day 14 Day 21 Day 28

Group 1 1.0* 10⁷ 1.0* 10⁶ 5.7*10⁴ 0 0 O 0

Group 2 1.0* 10⁵ 1.0* 10⁵ 1.0* 10⁵ 9*10⁴ 5*10³ O 0

Group 3 1.0* 10⁵ 1.0* 10⁵ 3.1*10⁴ 2.0* 10² 3.0*10 O 0

Group 4 1.0* 10⁵ 1.0* 10⁵ 1.0* 10⁵ 9*10⁴ 9*10⁴ O 0

[58] Table 12

Day O Day 1 Day 2 Day 7 Day 14 Day 21 Day 28

Group 1 1.0* 10⁷ 1.2*10⁶ 9.0* 10⁴ 0 0 O 0

Group 2 1.0* 10⁵ 1.0* 10⁵ 1.0* 10⁵ 9*10⁴ 6*10³ O 0

Group 3 1.0* 10⁵ 1.0* 10⁵ 3.1*10⁴ 2.7* 10² 3.8*10 O 0

Group 4 1.0* 10⁵ 1.0* 10⁵ 1.0* 10⁵ 1.0*10⁵ 1.0* 10⁵ O 0

[59] Table 13

Day O Day 1 Day 2 Day 7 Day 14 Day 21 Day 28

Group 1 1.0* 10⁷ 1.2*10⁶ 9.4* 10⁴ O 0 0 0

Group 2 1.0* 10⁵ 1.0* 10⁵ 1.0* 10⁵ 8*10⁴ 6*10³ 0 0

Group 3 1.0* 10⁵ 1.0* 10⁵ 3.1*10⁴ 2.5*10² 3.3*10 0 0

Group 4 1.0* 10⁵ 1.0* 10⁵ 1.0* 10⁵ 1.0*10⁵ 1.0* 10⁴ 0 0

[60] Table 14

Day O Day 1 Day 2 Day 7 Day 14 Day 21 Day 28

Group 1 1.0* 10⁷ 1.2*10⁶ 9.4* 10⁴ 1.0*10 O 0 0

Group 2 1.0* 10⁵ 1.0* 10⁵ 1.0* 10⁵ 6*10⁴ 6*10³ 0 0

Group 3 1.0* 10⁵ 1.0* 10⁵ 3.1*10⁴ 1.7*10² 3.8*10 0 0

Group 4 1.0* 10⁵ 1.0* 10⁵ 1.0* 10⁵ 1.0*10⁵ 1.0*10⁴ 0 0

[61] As is apparent from the data in the tables, the natural antibiotic of the present invention inhibits the growth of Gram-negative and positive bacteria, yeasts, and fungi, thereby showing action against a wide antimicrobial spectrum.

[62] [63] -Comparison of Antimicrobial Activity between Culture Solution of Kimchi Lactic Acid Bacteria and Paraben-

[64] [65] To compare the antimicrobial activity of the culture solution of Kimchi lactic acid bacteria with that of paraben, a skin lotion composition was combined with culture solution samples 1-4 of Leuconostoc spp., culture solution samples 5-10 of Lac¬ tobacillus spp., culture samples 11-12 of Weissella spp., or paraben, to prepare test solutions as in Table 15.

[66] Table 15

[67] In the same manner as in the assay for antimicrobial activity, each of the test com¬ positions 1-14 was inoculated with microbes of Groups 1 to 4, and then incubated at the same temperatures. 14 days after the incubation, living cells were counted and the results are given in Table 16, below.

[68] [69] Table 16

[70] [71] The test composition 1, as seen in Table 16, allowed microbes of all groups to grow actively. The test composition 2 containing the conventional synthetic preservative was found to be susceptible particularly to the bacterial mixture while inhibiting the growth of yeasts and fungi to some extent. In contrast, almost no growth of microbes of all groups was found in the test compositions 3 to 14, each containing the natural antibiotic according to the present invention.

[72] As described hereinbefore, the natural antibiotic according to the present invention shows more potent antimicrobial activity and has a wider antimicrobial spectrum than does paraben, a conventional popular synthetic antibiotic.

[73] As in cosmetic compositions, the antimicrobial activity of the culture solution of the present invention can be realized in beverages, food, fodder, and medicines. When the culture solution of the present invention is applied to cosmetics, beverages, food, fodder, medicines, etc., those skilled in the art will appreciate that various modi¬ fications, additions and substitutions in the culture solution are possible according to properties of the subjects and embodiments of preparation, distribution, storage and use of the subjects, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

[74] Various experiments conducted by the present inventor proved that a composition such as beverage, fodder, food, medicine, cosmetic, etc., preferably contains the natural antibiotic of the present invention in an amount from 0.1 to 30 % by volume based on the total volume of the composition.

[75] In addition, since the natural antibiotic of the present invention is prepared from a culture solution of Kimchi lactic acid bacteria that emerges during the fermentation of the Korean traditional food Kimchi, there is no doubt about the safety of the natural antibiotic to the body even if additional safety tests are not conducted. Industrial Applicability

[76] Taken together, the data obtained in Examples demonstrate that culture solutions of

Kimichi lactic acid bacteria, or their concentrates or dried materials have excellent an¬ timicrobial activity against a wide spectrum of microbes and can endow foods, fodders, beverages, medicines and cosmetics with antimicrobial activity with safety and efficiency.

[77]

Claims

[1] A natural antibiotic, prepared from one selected from a group consisting of a culture solution of the lactic acid bacteria isolated from Kimchi during the fer¬ mentation of Kimchi, a concentrate of the culture solution, a dehydrate of the culture solution, and combinations thereof.

[2] The natural antibiotic as set forth in claim 1, wherein the lactic acid bacteria is selected from among Leuconostoc spp., Lactobacillus spp., Weissella spp., and combinations thereof.

[3] The natural antibiotic as set forth in claim 2, wherein the Leuconostoc spp. includes Leuconostoc citreum, Leuconostoc lactis, Leuconostoc mesenteroides subsp. dextranicum, Leuconostoc mesenteroides subsp. Mesenteroides, Leuconostoc argentinum, Leuconostoc carnosum, Leuconostoc gellidum, Leuconostoc kimchii, Leuconostoc inhae, Leuconostoc gasicomitatum, and com¬ binations thereof.

[4] The natural antibiotic as set forth in claim 2, wherein the Lactobacillus spp. include Lactobacillus brevis, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus plantarum, Lactobacillus kimchii, Lactobacillus para- plantarum, Lactobacillus curvatus subsp. curvatus, Lactobacillus sakei subsp. sakei and combinations thereof.

[5] The natural antibiotic as set forth in claim 2, wherein Weissella spp. include

Weissella koreensi, Weissella hanii, Weissella kimchii, Weissella soli, Weissella confusa and combinations thereof.

[6] A composition, comprising a natural antibiotic prepared from one selected from a group consisting of a culture solution of the lactic acid bacteria isolated from Kimchi during the fermentation of Kimchi, a concentrate of the culture solution, a dehydrate of the culture solution, and combinations thereof, as an active ingredient.

[7] The composition as set forth in claim 6, wherein the lactic acid bacteria is selected from among Leuconostoc spp., Lactobacillus spp., Weissella spp., and combinations thereof.

[8] The composition as set forth in claim 7, wherein the Leuconostoc spp. include

Leuconostoc citreum, Leuconostoc lactis, Leuconostoc mesenteroides subsp. dextranicum, Leuconostoc mesenteroides subsp. Mesenteroides, Leuconostoc argentinum, Leuconostoc carnosum, Leuconostoc gellidum, Leuconostoc kimchii, Leuconostoc inhae, Leuconostoc gasicomitatum and combinations thereof.

[9] The composition as set forth in claim 7, wherein the Lactobacillus spp. include Lactobacillus brevis, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lac¬ tobacillus plantarum, Lactobacillus kimchii, Lactobacillus paraplantarum, Lac¬ tobacillus curvatus subsp. curvatus, Lactobacillus sakei subsp. sakei and com¬ binations thereof.

[10] The composition as set forth in claim 7, wherein the Weissella spp. include

[11] The composition as set forth in any one of claims 6 to 10, being selected from a group consisting of beverages, fodders, foods, medicines and cosmetics.