WO2013054959A1 - Antibacterial composition extracted from chrysanthemum morifolium flower - Google Patents
Antibacterial composition extracted from chrysanthemum morifolium flower Download PDFInfo
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- WO2013054959A1 WO2013054959A1 PCT/KR2011/007641 KR2011007641W WO2013054959A1 WO 2013054959 A1 WO2013054959 A1 WO 2013054959A1 KR 2011007641 W KR2011007641 W KR 2011007641W WO 2013054959 A1 WO2013054959 A1 WO 2013054959A1
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- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
Definitions
- the present invention relates to an antimicrobial composition, and more particularly, to a composition containing an essential oil component extracted from chrysanthemum flowers and exhibiting antimicrobial activity against food poisoning bacteria.
- an antimicrobial composition extracted from natural materials has recently been used as a preservative to suppress the growth of food poisoning bacteria.
- these natural preservatives have a weak antibacterial effect, so they must be added in a large amount, and there are limitations on their use, such as effects only on certain food poisoning bacteria.
- Chrysanthemum morifolium a representative plant of the Compositae family, is a perennial plant and is used as drinking water or food material in gardens.
- studies on the physiological activities of chrysanthemum essential oils and extracts are insignificant.
- the present study completed the present invention by confirming that the antibacterial activity of essential oil components extracted from chrysanthemum flowers of various strains of food poisoning that occurs frequently in recent years through the antibacterial effect.
- An object of the present invention is to provide an antimicrobial composition that can be used as a food preservative or food poisoning preventive treatment and adjuvant by containing essential oil components extracted from chrysanthemum flowers as an active ingredient, showing strong antimicrobial activity against food poisoning bacteria and maintaining antimicrobial properties for a long time. It is done.
- the present invention provides an antimicrobial composition
- an essential oil component essential oil which is an extract of Chrysanthemum morifolium Flower.
- the chrysanthemum flower extract may be produced using a steam distillation method.
- the distillation fractionation method may be performed using a solvent including any one of distilled water, alcohol having 1 to 5 carbon atoms, and dimethyl sulphoxide (DMSO).
- a solvent including any one of distilled water, alcohol having 1 to 5 carbon atoms, and dimethyl sulphoxide (DMSO).
- the essential oil components include camphor, chrysanthemyl alcohol, alpha-pinene, gamma-terpinene, alpha + beta-tujone and ⁇ + ⁇ -thujone. It may include.
- the antimicrobial composition may have a minimum inhibitory concentration (MIC) of 5 ⁇ g / ml against food poisoning bacteria.
- MIC minimum inhibitory concentration
- the food poisoning bacteria include Bacillus Cereus, Aeromonas hydrophila, Salmonella choleraesuis, Salmonella enterica, Shigera sonnei, Shigella sonbrii parahaemolyticus, Vibrio vulnificus, Bacillus subtilis, Listeria monocytogenes, and Staphylococcus aureus.
- the antimicrobial composition may have a minimum bactericidal concentration (MBC) of 5 to 20 ⁇ g / ml for food poisoning bacteria.
- MBC minimum bactericidal concentration
- the food poisoning bacteria are Bacillus Cereus, Aeromonas hydrophila, Salmonella enterica, Shigerella sonnei, Vibrio parahaemolyticus, Vibrio bulnipius V. vulnificus, Bacillus subtilis, and Staphylococcus aureus.
- the present invention has the following effects.
- Antimicrobial composition according to the present invention is a camphor (chhorsanthemyl alcohol), alpha-pinene ( ⁇ -pinene), gamma-terpinene ( ⁇ -terpinene), alpha + beta-tuzone ( ⁇ + ⁇ - thujone).
- the antimicrobial composition of the present invention is Aeromonas hydrophila, Salmonella choleraesuis, Salmonella enterica, Shigera sonnei, enteritis Vibrio (Vibrio parahaemolyticus) Have strong antibacterial activity against food poisoning bacteria of Vibrio vulnificus, Bacillus subtilis, Listeria monocytogenes, and Staphylococcus aureus Can be.
- Such antimicrobial compositions can be used in food preservatives, cosmetic compositions, therapeutic agents and the like.
- Figure 1 illustrates the effect of the essential oil component extracted from the chrysanthemum flower in accordance with an embodiment of the present invention on the growth of Gram-positive food poisoning bacteria.
- Figure 2 shows the effect of the essential oil component extracted from the chrysanthemum flower in accordance with an embodiment of the present invention on the growth of Gram negative food poisoning bacteria.
- Chrysanthemum morifolium was purchased and dried in September 2009 by the Gyeongsangnam-do Herbal Agricultural Cooperative.
- Solvents for extraction and solvents for preparing samples were purchased from Wako Pure Chemical Industries, Japan.
- Food poisoning bacteria were prepared by culturing four Gram-positive bacteria and six Gram-negative bacteria, which are frequently occurring in Korea, under the conditions shown in Table 1 below.
- the four Gram-positive bacteria include Bacillus Cereus, Bacillus subtilis, Listeria monocytogenes, and Staphylococcus aureus.
- the six gram-negative bacteria are Aeromonas hydrophila, Salmonella choleraesuis, Salmonella enterica, Shigera sonnei, Enteritis vibrio, Vibrio parahaemolyticus, and Vig. Nipicus (Vibrio vulnificus).
- Each of the prepared food poisoning bacteria was stored at -70 ° C using 35% glycerol medium.
- Sterile water was mixed and adjusted to a concentration of (approximately 1 ⁇ 10 7 CFU / mL) and used.
- the medium was prepared according to the characteristics of the food poisoning bacteria and sterilized for 15 minutes at 121 °C, 1.5 atm.
- the essential oil component a chrysanthemum flower extract
- the essential oil component was prepared using a steam distillation method (G'omez NE and Witte L 2001). That is, after crushing the dry chrysanthemum flower and extracting the active ingredient from the crushed chrysanthemum flower using a ethanol-containing Clevenger-type apparatus (Hanil Labtech Ltd., Incheon, Korea), for 24 hours
- the essential oil component was prepared by drying in anhydrous sodium sulfate.
- the antimicrobial activity of essential oils, the chrysanthemum flower extract prepared as described above, was analyzed using an agar-well diffusion assay (Owen RJ and Palombo EA 2007). 100 ⁇ l of the culture solution of each food poisoning bacterium prepared as described above (OD 600 0.20.3) was evenly spread on the surface of a nutritious agar (Nutrient agar, NA, Difco, MI, USA) plate using a spreader. Wells were made on the agar surface using sterile cork-borer (5 mm diameter). 20 ⁇ l of the essential oil component and the indicator material were added to the wells, and then left at room temperature for 30 minutes to diffuse essential oil components into agar, followed by incubation at 37 ° C.
- the antimicrobial activity of the essential oil components, indicators and antibiotics against four Gram-positive food poisoning bacteria and Gram-positive food poisoning bacteria are shown in Table 2 below.
- the essential oil component did not show antimicrobial activity against Salmonella cholera suis, but showed strong antimicrobial activity against all food poisoning strains used in other experiments. It can be seen that it shows greater antimicrobial activity.
- the minimum inhibitory concentration of the essential oil component is expressed as 5 ⁇ g / ml for all food poisoning bacteria.
- the minimum inhibitory concentration of the essential oil component against food poisoning bacteria does not show a large difference between Gram-positive bacteria and Gram-negative bacteria.
- the minimum killing concentration was produced after taking 50 ml of the culture sample for each time period in the process of analyzing the minimum inhibitory concentration described above, smearing on a Nutrient agar plate, and incubating at 37 ° C. for 48 hours. When the colony number was observed, the minimum concentration at which the food poisoning bacteria were completely killed (99.9%) was set as the minimum killing concentration (Choi S et al. 2008b, Ji LL et al. 2008). All assay results were obtained through three independent experiments.
- the minimum killing concentration of the essential oil component for the food poisoning bacteria is shown in Table 4 below.
- the essential oil component was found to have no minimum killing concentration within the tested concentrations for Salmonella cholera suis and Listeria monocytogenes, but for other food poisoning bacteria, a minimum inhibitory concentration of 5 to 20 ⁇ g / ml was observed. It can be seen that it has.
- the growth inhibition assay was carried out with 100 ml of nutrient broth, 50 ml of each preparation of 2.5 ⁇ g / ml, and 50 ml of food poisoning bacteria diluted in a 96-well plate, as described above.
- the test solution prepared by sequentially adding was incubated for 24 hours while absorbance was measured at 600 nm at 4 hour intervals to examine the effect of the essential oil component on the growth of food poisoning bacteria, the results are shown in FIG.
- the essential oil component of the present invention can be seen to exhibit the effect of inhibiting the growth of all food poisoning bacteria. That is, the growth curve of the food poisoning bacteria showed a tendency to rapidly decrease the growth of the food poisoning bacteria by 8 hours and then slowly decreases by 24 hours. On the other hand, antibiotics of the control group showed a slight increase in the growth of food poisoning bacteria by 4 hours, followed by a decrease or no growth of the food poisoning bacteria.
- the essential oil component extracted from the chrysanthemum flower strongly inhibits the growth of food poisoning bacteria when it is added as a natural preservative to food, and has excellent shelf life by maintaining antibacterial property for more than 24 hours.
- the antibacterial activity, minimum inhibitory concentration, minimum killing concentration and inhibitory effect of 10 kinds of food poisoning bacteria were tested to verify the antimicrobial performance of the essential oil component of the chrysanthemum flower extract.
- the essential oil component showed excellent antimicrobial effect against both Gram-positive bacteria and Gram-negative bacteria. That is, as a result of the analysis of antimicrobial activity, the essential oil component showed stronger activity against gram positive bacteria than gram negative bacteria.
- the essential oil component showed the effect of inhibiting the growth of food poisoning bacteria at a concentration of 5 ⁇ g / ml for all strains, the minimum killing concentration analysis, Salmonella cholerasus and Listeria monocytogenes The minimum killing concentration was not observed within the experimental concentration for, but for the other strains, a minimum killing concentration of 5 to 20 ⁇ g / ml was shown.
- the essential oil component was constantly maintained antibacterial performance for 24 hours.
- the antimicrobial composition extracted from the chrysanthemum flower containing the essential oil component as an active ingredient has been demonstrated antimicrobial activity against the 10 food poisoning bacteria.
- Such an antimicrobial composition containing the essential oil component of the present invention having strong antimicrobial activity as an active ingredient may be used in food preservatives, cosmetic compositions, therapeutic agents and the like.
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Abstract
The present invention relates to an antibacterial composition which shows strong antibacterial activity toward food poisoning bacteria by containing essential oil extracted from Chrysanthemum morifolium flower as an effective component, and can be used as a food supplement, or a medicine or a supplement for treating food poisoning since an antibiotic action is maintained for a long time. An antibacterial composition of the present invention contains essential oil, which is an extract of Chrysanthemum morifolium flower, as an effective component.
Description
본 발명은 항균 조성물에 관한 것으로서, 보다 구체적으로는 국화 꽃으로부터 추출되고 식중독균에 대해 항균성을 나타내는 정유 성분을 함유하는 조성물에 관한 것이다.The present invention relates to an antimicrobial composition, and more particularly, to a composition containing an essential oil component extracted from chrysanthemum flowers and exhibiting antimicrobial activity against food poisoning bacteria.
최근, 외식 및 급식의 이용이 증가하면서 식중독이 빈번히 발생하고 있다. 이러한 식중독은 식중독균에 의한 직접적으로 발생하는 것 외에도, 식중독균과 부패미생물들이 과일, 야채, 곡물 및 가공 식품 내에 배출하는 독성대사물질에 의해 발생하기도 한다. 살모넬라균 등이 배출하는 엔테로톡신은 구토와 설사를 유발하는 원인 물질로 알려져 있다. 이러한 식중독균은 건강한 사람에게는 큰 문제를 야기하지 않지만, 면역력이 약한 어린이, 노약자 등에는 심각한 위험을 초래할 수 있다.In recent years, food poisoning has frequently occurred as the use of eating out and feeding increased. In addition to being caused directly by food poisoning bacteria, such food poisoning is also caused by toxic metabolites that food poisoning bacteria and rot microorganisms release in fruits, vegetables, grains and processed foods. Enterotoxin, released by Salmonella, is known to cause vomiting and diarrhea. These food poisoning bacteria do not cause a big problem in healthy people, but can cause serious risks for children with weak immunity, the elderly.
이에 따라, 식중독균을 생장을 억제하기 위해 다양한 화학적 합성 보존제가 사용되고 있다. 그러나, 이러한 합성 보존제는 지속적으로 사용되어 체내에 축적될 경우 돌연변이 등을 유발할 수 있다. Accordingly, various chemical synthetic preservatives have been used to suppress the growth of food poisoning bacteria. However, such synthetic preservatives can cause mutations and the like if used continuously and accumulated in the body.
이러한 문제를 해결하고자 최근 천연 재료에서 추출한 항균 조성물이 식중독균의 생장을 억제하는 보존제로 사용하고 있다. 그러나, 이러한 천연 보존제는 항균성이 약하기 때문에 다량으로 첨가해야 하며 특정 식중독균에만 효과를 나타내는 등의 사용상 한계가 있다. In order to solve this problem, an antimicrobial composition extracted from natural materials has recently been used as a preservative to suppress the growth of food poisoning bacteria. However, these natural preservatives have a weak antibacterial effect, so they must be added in a large amount, and there are limitations on their use, such as effects only on certain food poisoning bacteria.
한편, 국화과(Compositae family)의 대표적 식물인 국화(Chrysanthemum morifolium)는 다년생 식물로서, 정원의 식수나 식품 재료로 이용되고 있는데, 국화 정유 성분 및 추출물의 생리 활성에 대한 연구는 미미한 실정이다.On the other hand, Chrysanthemum morifolium, a representative plant of the Compositae family, is a perennial plant and is used as drinking water or food material in gardens. However, studies on the physiological activities of chrysanthemum essential oils and extracts are insignificant.
이에 따라, 본 연구에서는 최근 자주 발생하는 식중독의 원인 균주를 대상으로 국화 꽃으로부터 추출된 정유 성분의 항균 활성에 대한 다양한 시험을 통해 항균 효과가 있음을 확인함으로써, 본 발명을 완성하였다.Accordingly, the present study completed the present invention by confirming that the antibacterial activity of essential oil components extracted from chrysanthemum flowers of various strains of food poisoning that occurs frequently in recent years through the antibacterial effect.
본 발명은 국화 꽃으로부터 추출된 정유 성분을 유효 성분으로 함유함으로써, 식중독균에 대해 강력한 항균 활성을 나타내고 장시간 동안 항균성이 유지됨에 따라 식품 보존제 또는 식중독 예방 치료제 및 보조제로 이용 가능한 항균 조성물을 제공하는 것을 목적으로 한다.An object of the present invention is to provide an antimicrobial composition that can be used as a food preservative or food poisoning preventive treatment and adjuvant by containing essential oil components extracted from chrysanthemum flowers as an active ingredient, showing strong antimicrobial activity against food poisoning bacteria and maintaining antimicrobial properties for a long time. It is done.
상기 목적을 달성하기 위해 본 발명은, 국화 꽃(Chrysanthemum morifolium Flower)의 추출물인 정유 성분(essential oil)을 유효성분으로 함유하는 것을 특징으로 하는 항균 조성물을 제공한다.In order to achieve the above object, the present invention provides an antimicrobial composition comprising an essential oil component (essential oil) which is an extract of Chrysanthemum morifolium Flower.
상기 국화 꽃의 추출물은 증기 증류 방법(steam distillation method)을 이용하여 생산될 수 있다.The chrysanthemum flower extract may be produced using a steam distillation method.
상기 증류 분별 방법은 증류수, 탄소수가 1 내지 5인 알콜, 및 디메틸설폭사이드(dimethyl sulphoxide, DMSO) 중 어느 하나를 포함하는 용매를 이용하여 수행될 수 있다.The distillation fractionation method may be performed using a solvent including any one of distilled water, alcohol having 1 to 5 carbon atoms, and dimethyl sulphoxide (DMSO).
상기 정유 성분은 캠퍼(camphor), 크리스안서밀 알콜(chrysanthemyl alcohol), 알파-피넨(α-pinene), 감마-테르피넨(γ-terpinene), 알파+베타-튜존(α+β-thujone)을 포함할 수 있다.The essential oil components include camphor, chrysanthemyl alcohol, alpha-pinene, gamma-terpinene, alpha + beta-tujone and α + β-thujone. It may include.
상기 항균 조성물은 식중독균에 대해 5 ㎍/㎖의 최소 저해 농도(MIC, minimum inhibitory concentration)를 가질 수 있다.The antimicrobial composition may have a minimum inhibitory concentration (MIC) of 5 μg / ml against food poisoning bacteria.
상기 식중독균은 바실러스 세레우스(Bacillus Cereus), 에어로모나스 하이드로필라(Aeromonas hydrophila), 살모넬라 콜레라수이스(Salmonella choleraesuis), 살모넬라 엔터릭카(Salmonella enterica), 시게라 손네이(Shigella sonnei), 장염 비브리오(Vibrio parahaemolyticus), 비브리오 불니피쿠스(Vibrio vulnificus), 바실러스 서브틸리스(Bacillus subtilis), 리스테리아 모노사이토게네스(Listeria monocytogenes), 및 스타필로코쿠스 아루레우스(Staphylococcus aureus)를 포함할 수 있다.The food poisoning bacteria include Bacillus Cereus, Aeromonas hydrophila, Salmonella choleraesuis, Salmonella enterica, Shigera sonnei, Shigella sonbrii parahaemolyticus, Vibrio vulnificus, Bacillus subtilis, Listeria monocytogenes, and Staphylococcus aureus.
상기 항균 조성물은 식중독균에 대해 5 내지 20 ㎍/㎖의 최소 사멸 농도(MBC, minimum bactericidal concentration)를 가질 수 있다.The antimicrobial composition may have a minimum bactericidal concentration (MBC) of 5 to 20 μg / ml for food poisoning bacteria.
상기 식중독균은 바실러스 세레우스(Bacillus Cereus), 에어로모나스 하이드로필라(Aeromonas hydrophila), 살모넬라 엔터릭카(Salmonella enterica), 시게라 손네이(Shigella sonnei), 장염 비브리오(Vibrio parahaemolyticus), 비브리오 불니피쿠스(Vibrio vulnificus), 바실러스 서브틸리스(Bacillus subtilis), 및 스타필로코쿠스 아루레우스(Staphylococcus aureus)를 포함할 수 있다.The food poisoning bacteria are Bacillus Cereus, Aeromonas hydrophila, Salmonella enterica, Shigerella sonnei, Vibrio parahaemolyticus, Vibrio bulnipius V. vulnificus, Bacillus subtilis, and Staphylococcus aureus.
본 발명은 다음과 같은 효과가 있다.The present invention has the following effects.
본 발명에 따른 항균 조성물은 캠퍼(camphor), 크리스안서밀 알콜(chrysanthemyl alcohol), 알파-피넨(α-pinene), 감마-테르피넨(γ-terpinene), 알파+베타-튜존(α+β-thujone)을 포함하고 있다.Antimicrobial composition according to the present invention is a camphor (chhorsanthemyl alcohol), alpha-pinene (α-pinene), gamma-terpinene (γ-terpinene), alpha + beta-tuzone (α + β- thujone).
이에 따라, 본 발명의 항균 조성물은 에어로모나스 하이드로필라(Aeromonas hydrophila), 살모넬라 콜레라수이스(Salmonella choleraesuis), 살모넬라 엔터릭카(Salmonella enterica), 시게라 손네이(Shigella sonnei), 장염 비브리오(Vibrio parahaemolyticus), 비브리오 불니피쿠스(Vibrio vulnificus), 바실러스 서브틸리스(Bacillus subtilis), 리스테리아 모노사이토게네스(Listeria monocytogenes), 및 스타필로코쿠스 아루레우스(Staphylococcus aureus)의 식중독균에 대해 강력한 항균 활성을 가질 수 있다.Accordingly, the antimicrobial composition of the present invention is Aeromonas hydrophila, Salmonella choleraesuis, Salmonella enterica, Shigera sonnei, enteritis Vibrio (Vibrio parahaemolyticus) Have strong antibacterial activity against food poisoning bacteria of Vibrio vulnificus, Bacillus subtilis, Listeria monocytogenes, and Staphylococcus aureus Can be.
이러한, 항균 조성물은 식품 보존제, 화장품 조성물, 치료제 등에 사용될 수 있다.Such antimicrobial compositions can be used in food preservatives, cosmetic compositions, therapeutic agents and the like.
도 1은 본 발명의 일 실시예에 따른 국화 꽃으로부터 추출된 정유 성분이 그램 양성 식중독균의 생장에 미치는 효과를 도시한 것이다.Figure 1 illustrates the effect of the essential oil component extracted from the chrysanthemum flower in accordance with an embodiment of the present invention on the growth of Gram-positive food poisoning bacteria.
도 2는 본 발명의 일 실시예에 따른 국화 꽃으로부터 추출된 정유 성분이 그램 음성 식중독균의 생장에 미치는 효과를 도시한 것이다.Figure 2 shows the effect of the essential oil component extracted from the chrysanthemum flower in accordance with an embodiment of the present invention on the growth of Gram negative food poisoning bacteria.
이하, 실시예에 의해 본 발명을 상세히 설명하기로 한다. 다만, 이러한 실시예로 인해 본 발명의 권리범위가 제한되지 않는다.Hereinafter, the present invention will be described in detail by way of examples. However, the scope of the present invention is not limited by these embodiments.
재료 및 시약 준비Material and Reagent Preparation
국화(Chrysanthemum morifolium)는 경상남도 생약 농업 협동조합에서 2009년 9월에 구입 후 음건하여 준비하였다. Chrysanthemum morifolium was purchased and dried in September 2009 by the Gyeongsangnam-do Herbal Agricultural Cooperative.
국화 꽃 추출물인 정유 성분의 지표물질인, 캠퍼(camphor), 크리스안서밀 알콜(chrysanthemyl alcohol), 알파-피넨(α-pinene), 감마-테르피넨(γ-terpinene), 알파+베타-튜존(α+β-thujone) 및 상기 국화 꽃 추출물인 정유 성분의 대조군으로 사용된 항생제인, 암피실린(ampicillin), 스트렙토마이신(streptomycin)은 시그마-알드리취(Sigma-Aldrich Inc., St. Louis, MO, USA) 사로부터 구입하였다. Camphor, chrysanthemyl alcohol, alpha-pinene, gamma-terpinene, alpha + beta-tuzone Ampicillin and streptomycin, the antibiotics used as a control of the essential oil component of α + β-thujone) and the chrysanthemum flower extract, were Sigma-Aldrich Inc., St. Louis, MO. , USA).
추출용 용매, 시료를 제조하기 위한 용매들은 특급으로 와코 푸어 케미컬 인더스트리(Wako Pure Chemical Industries, Japan) 사로부터 구입하였다.Solvents for extraction and solvents for preparing samples were purchased from Wako Pure Chemical Industries, Japan.
식중독균은 대표적으로 국내에서 자주 발생하는 그램(Gram) 양성균 4종, 그램(Gram) 음성균 6종을 아래의 표 1에 나타낸 조건에서 배양시켜 준비하였다. Food poisoning bacteria were prepared by culturing four Gram-positive bacteria and six Gram-negative bacteria, which are frequently occurring in Korea, under the conditions shown in Table 1 below.
상기 그램 양성균 4종은 바실러스 세레우스(Bacillus Cereus), 바실러스 서브틸리스(Bacillus subtilis), 리스테리아 모노사이토게네스(Listeria monocytogenes), 스타필로코쿠스 아루레우스(Staphylococcus aureus)를 포함한다.The four Gram-positive bacteria include Bacillus Cereus, Bacillus subtilis, Listeria monocytogenes, and Staphylococcus aureus.
상기 그램 음성균 6종은 에어로모나스 하이드로필라(Aeromonas hydrophila), 살모넬라 콜레라수이스(Salmonella choleraesuis), 살모넬라 엔터릭카(Salmonella enterica), 시게라 손네이(Shigella sonnei), 장염 비브리오(Vibrio parahaemolyticus), 비브리오 불니피쿠스(Vibrio vulnificus)를 포함한다.The six gram-negative bacteria are Aeromonas hydrophila, Salmonella choleraesuis, Salmonella enterica, Shigera sonnei, Enteritis vibrio, Vibrio parahaemolyticus, and Vig. Nipicus (Vibrio vulnificus).
[표 1]TABLE 1
[규칙 제91조에 의한 정정 11.05.2012]
[Correction under Rule 91 11.05.2012]
상기 준비된 각 식중독균은 35% 글리세롤 미디움(glycerol medium)을 사용하여 -70 ℃에서 보관하였다. 상기 보관된 식중독균을 표 1에 명기된 각 식중독균주 배지에 접종하여 37도에서 약 48 시간 동안 배양한 식중독균액은 UV-스펙트로미터(SpectraMax M2, Molecular Decives, USA)를 이용하여 O.D.=0.2 ~ 0.3(대략 1×107 CFU/㎖) 농도로 멸균수를 혼합하여 조정한 후 사용하였다. 상기 배지는 상기 식중독균의 특성에 맞게 제조하여 121℃, 1.5기압 하에서 15분간 멸균하여 사용하였다.Each of the prepared food poisoning bacteria was stored at -70 ° C using 35% glycerol medium. The food poisoning bacteria inoculated on each food poisoning strain medium specified in Table 1 and incubated for 48 hours at 37 ° C. were OD = 0.2 to 0.3 using UV-Spectrometer (SpectraMax M2, Molecular Decives, USA). Sterile water was mixed and adjusted to a concentration of (approximately 1 × 10 7 CFU / mL) and used. The medium was prepared according to the characteristics of the food poisoning bacteria and sterilized for 15 minutes at 121 ℃, 1.5 atm.
실시예 : 정유 성분(essential oil)의 제조Example: Preparation of Essential Oils
국화 꽃 추출물인 정유 성분은 증기 증류 방법(steam distillation method, G'omez NE 와 Witte L 2001)을 이용하여 제조하였다. 즉, 음건한 국화 꽃을 분쇄하고 에탄올이 담기 클리빈저-타입 장치(Clevenger-type apparatus, Hanil Labtech Ltd., Incheon, Korea)를 사용하여 상기 분쇄된 국화 꽃으로부터 유효성분을 추출한 후, 24 시간 동안 무수황산나트륨 중에서 건조시켜 정유 성분이 제조되었다.The essential oil component, a chrysanthemum flower extract, was prepared using a steam distillation method (G'omez NE and Witte L 2001). That is, after crushing the dry chrysanthemum flower and extracting the active ingredient from the crushed chrysanthemum flower using a ethanol-containing Clevenger-type apparatus (Hanil Labtech Ltd., Incheon, Korea), for 24 hours The essential oil component was prepared by drying in anhydrous sodium sulfate.
실험예 1 : 항균 활성 측정(Agar well diffusion assay)Experimental Example 1: Determination of antimicrobial activity (Agar well diffusion assay)
상술한 바와 같이 제조된 국화 꽃 추출물인 정유성분의 항균 활성은 아가-웰 확산법(agar-well diffusion assay)를 사용하여 분석하였다(Owen RJ 와 Palombo EA 2007). 상술한 바와 같이 준비된 각 식중독균의 배양액(O.D.600=0.20.3) 100 ㎕을 스프레더(spreader)로 뉴트리언트 아가(Nutrient agar, NA, Difco, MI, USA) 플레이트의 표면에 균일하게 도말한 후, 멸균된 콜크-보러(cork-borer, 5 ㎜ diameter)를 사용하여 아가(agar) 표면에 웰(well)을 만들었다. 이러한 웰에 정유 성분과 지표물질을 각각 20 ㎕ 가한 후, 아가(agar) 내로 정유 성분 등이 확산시키기 위해 30 분간 상온에서 방치 후, 37 ℃에서 12 시간 동안 배양하였다. 모든 실험은 3회 실시하였으며, 아가 웰(agar well) 주위로 미생물의 성장이 저해되는 클리어 존(clear zone)의 크기(㎜)를 칼리퍼(caliper)로 측정하여 항균력의 크기를 계산하였다(Fazeli MR 등 2007). 또한 정유 성분을 녹이는데 사용된 용매인 디메틸설폭사이드(dimethyl sulphoxide, DMSO, St. Louis, MO, USA)를 음성 대조군으로 사용하고, 항생제인 암피실린(ampicillin), 스트렙토마이신(streptomycin)을 양성 대조군으로 사용하였다. The antimicrobial activity of essential oils, the chrysanthemum flower extract prepared as described above, was analyzed using an agar-well diffusion assay (Owen RJ and Palombo EA 2007). 100 μl of the culture solution of each food poisoning bacterium prepared as described above (OD 600 = 0.20.3) was evenly spread on the surface of a nutritious agar (Nutrient agar, NA, Difco, MI, USA) plate using a spreader. Wells were made on the agar surface using sterile cork-borer (5 mm diameter). 20 μl of the essential oil component and the indicator material were added to the wells, and then left at room temperature for 30 minutes to diffuse essential oil components into agar, followed by incubation at 37 ° C. for 12 hours. All experiments were performed three times, and the size of the antimicrobial activity was calculated by measuring the size of the clear zone (mm) in which the growth of microorganisms was inhibited around the agar well (mm) (Fazeli). MR et al. 2007). In addition, dimethyl sulphoxide (DMSO, St. Louis, MO, USA), a solvent used to dissolve essential oil components, was used as a negative control, and ampicillin and streptomycin, antibiotics, were used as a positive control. Used.
그램(Gram) 양성 식중독균 4종과 그램(Gram) 양성 식중독균에 대한 상기 정유 성분, 지표물질 및 항생제의 항균 활성 결과는 아래의 표 2에 나타내었다.The antimicrobial activity of the essential oil components, indicators and antibiotics against four Gram-positive food poisoning bacteria and Gram-positive food poisoning bacteria are shown in Table 2 below.
[표 2]TABLE 2
[규칙 제91조에 의한 정정 11.05.2012]
[Correction under Rule 91 11.05.2012]
위 표 2에서와 같이, 상기 정유 성분은 살모넬라 콜레라수이스에 대해서는 항균 활성을 나타내지 않았으나, 그 외의 실험에 사용된 모든 식중독 균주에 대하여 강한 항균 활성을 보였으며, 특히 그램 양성 식중독균에 대해서는 대조군인 항생제보다 더 큰 항균 활성을 나타냄을 알 수 있다.As shown in Table 2, the essential oil component did not show antimicrobial activity against Salmonella cholera suis, but showed strong antimicrobial activity against all food poisoning strains used in other experiments. It can be seen that it shows greater antimicrobial activity.
실험예 2 : 최소 저해 농도(Minimum inhibitory concentration, MIC) 분석Experimental Example 2: Minimum inhibitory concentration (MIC) analysis
상기 정유 성분의 상기 각 식중독균에 대한 최소 저해 농도 분석은 액체 배지 희석법(broth-dilution method)을 사용하여 수행하였다(Mann CM 와 Markham JL 1998, NCLLS 1999, Yu JQ 등 2004). 상기 정유 성분, 지표물질, 및 항생제를 디메틸설폭사이드를 이용하여 0에서 20 ㎍/㎖ 농도로 희석하여 조제액을 준비하였다. 상기 각 식중독균은 O.D.600=0.2로 농도를 조절하여 사용하였다. 96-웰 플레이트(96-well plate, Falcon, USA)에 뉴트리언트 브로스(Nutrient Broth) 100 ㎖, 각 조제액 50 ㎖, 식중독균 희석액 50 ㎖를 순차적으로 첨가한 뒤 4 시간마다 UV-스펙트로포토미터(spectrophotometer)를 이용해 흡광도 값을 측정하면서 24 시간 동안 37 ℃에서 배양하였다. 이때, 식중독균의 생육 곡선상에서 식중독균의 생장 (turbidity)이 검출되지 않는 최소 농도를 최소 저해 농도로 설정하였다. Analysis of the minimum inhibitory concentration of the essential oil component for each food poisoning bacterium was carried out using a broth-dilution method (Mann CM and Markham JL 1998, NCLLS 1999, Yu JQ et al. 2004). The essential oil component, the indicator substance, and the antibiotic were diluted using a dimethyl sulfoxide at a concentration of 0 to 20 µg / ml to prepare a preparation solution. Each food poisoning bacterium was used by adjusting the concentration to OD 600 = 0.2. To the 96-well plate (Falcon, USA), 100 ml of Nutrient Broth, 50 ml of each preparation, and 50 ml of food poisoning diluent were sequentially added, followed by a UV-spectrophotometer every 4 hours. Spectrophotometer) was incubated at 37 ℃ for 24 hours while measuring the absorbance value. At this time, the minimum concentration at which the growth of food poisoning bacteria was not detected on the growth curve of the food poisoning bacteria was set as the minimum inhibitory concentration.
상기 각 식중독균에 대한 상기 정유 성분의 최소 저해 농도에 대한 결과는 아래의 표 3에 나타내었다. The results for the minimum inhibitory concentration of the essential oil component for each of the food poisoning bacteria are shown in Table 3 below.
[표 3]TABLE 3
[규칙 제91조에 의한 정정 11.05.2012]
[Correction under Rule 91 11.05.2012]
위 표 3에서와 같이, 상기 정유 성분의 최소 저해 농도는 모든 식중독균에 대해 5 ㎍/㎖로 나타내었다. 또한, 상기 정유 성분의 식중독균에 대한 최소 저해 농도는 그램 양성균과 그램 음성균에 대해 큰 차이가 나타나지 않음을 알 수 있다. As shown in Table 3 above, the minimum inhibitory concentration of the essential oil component is expressed as 5 ㎍ / ㎖ for all food poisoning bacteria. In addition, it can be seen that the minimum inhibitory concentration of the essential oil component against food poisoning bacteria does not show a large difference between Gram-positive bacteria and Gram-negative bacteria.
실험예 3 : 최소 사멸 농도(minimum bactericidal concentrations, MBC)의 분석Experimental Example 3 Analysis of Minimum Bactericidal Concentrations (MBC)
상기 최소 사멸 농도는 상술한 최소 저해 농도의 분석 과정에서 각 시간대 별 배양 시료 50 ㎖를 취해 뉴트리언트 아가(Nutrient agar) 플레이트(plate)에 도말하여, 48 시간 동안 37 ℃에서 배양한 후, 생성되는 콜로니(colony) 수를 관찰하였을 때 상기 식중독균을 완전히 사멸(99.9%)되는 최소 농도를 최소 사멸 농도로 설정하였다(Choi S 등 2008b, Ji LL 등 2008). 모든 분석 결과는 3번의 독립적인 실험을 통해 얻었다.The minimum killing concentration was produced after taking 50 ml of the culture sample for each time period in the process of analyzing the minimum inhibitory concentration described above, smearing on a Nutrient agar plate, and incubating at 37 ° C. for 48 hours. When the colony number was observed, the minimum concentration at which the food poisoning bacteria were completely killed (99.9%) was set as the minimum killing concentration (Choi S et al. 2008b, Ji LL et al. 2008). All assay results were obtained through three independent experiments.
상기 정유 성분의 상기 식중독균에 대한 최소 사멸 농도는 아래의 표 4에 나타내었다. The minimum killing concentration of the essential oil component for the food poisoning bacteria is shown in Table 4 below.
[표 4]TABLE 4
[규칙 제91조에 의한 정정 11.05.2012]
[Correction under Rule 91 11.05.2012]
위 표 4에서와 같이, 상기 정유 성분은 살모넬라 콜레라수이스와 리스테리아 모노사이토게네스에 대해서는 실험한 농도 내에서 최소 사멸 농도가 관찰되지 않았으나 다른 식중독균에 대해서는 5 ~ 20 ㎍/㎖의 최소 저해 농도를 가지는 것을 알 수 있다.As shown in Table 4 above, the essential oil component was found to have no minimum killing concentration within the tested concentrations for Salmonella cholera suis and Listeria monocytogenes, but for other food poisoning bacteria, a minimum inhibitory concentration of 5 to 20 ㎍ / ml was observed. It can be seen that it has.
실험예 4 : 생육저해 분석(Time-killing assay)Experimental Example 4: Time-killing assay
상기 생육저해 분석은 상술한 최소 저해 농도의 분석 과정에서 상술한 바와 같이 96-웰 플레이트에 뉴트리언트 브로스(Nutrient Broth) 100 ㎖, 2.5 ㎍/㎖ 농도의 각 조제액 50 ㎖, 식중독균 희석액 50 ㎖를 순차적으로 첨가하여 제조된 시험 용액을 24 시간 동안 배양하면서 4시간 간격으로 600 ㎚에서 흡광도를 측정하여 상기 정유 성분이 식중독균의 생장에 미치는 영향을 검토하였고, 그 결과를 도 1에 나타내었다.The growth inhibition assay was carried out with 100 ml of nutrient broth, 50 ml of each preparation of 2.5 μg / ml, and 50 ml of food poisoning bacteria diluted in a 96-well plate, as described above. The test solution prepared by sequentially adding was incubated for 24 hours while absorbance was measured at 600 nm at 4 hour intervals to examine the effect of the essential oil component on the growth of food poisoning bacteria, the results are shown in FIG.
도 1에서와 같이, 본 발명의 정유 성분은 모든 식중독균의 생장을 저해하는 효과를 나타냄을 알 수 있다. 즉, 식중독균의 생장곡선은 8 시간까지 상기 식중독균의 생장이 급격히 감소하고 이어서 24 시간까지 완만히 감소하는 경향을 나타내었다. 반면, 대조군인 항생제는 4 시간까지 식중독균의 생장이 약간 증가하고 이어서 상기 식중독균의 생장이 없거나 감소하는 경향을 나타내었다. As shown in Figure 1, the essential oil component of the present invention can be seen to exhibit the effect of inhibiting the growth of all food poisoning bacteria. That is, the growth curve of the food poisoning bacteria showed a tendency to rapidly decrease the growth of the food poisoning bacteria by 8 hours and then slowly decreases by 24 hours. On the other hand, antibiotics of the control group showed a slight increase in the growth of food poisoning bacteria by 4 hours, followed by a decrease or no growth of the food poisoning bacteria.
따라서, 국화 꽃으로부터 추출된 정유 성분은 식품에 천연 보존제로서 첨가될 경우 식중독균의 생장을 강력하게 저해하고 24 시간 이상 항균성을 지속함으로써 저장성이 우수함을 알 수 있다. Therefore, it can be seen that the essential oil component extracted from the chrysanthemum flower strongly inhibits the growth of food poisoning bacteria when it is added as a natural preservative to food, and has excellent shelf life by maintaining antibacterial property for more than 24 hours.
상술한 바와 같이 국화 꽃 추출물인 정유 성분의 식중독균에 대한 항균 성능을 검증하기 위해 10종의 식중독균에 대한 항균 활성, 최소 저해 농도, 최소 사멸 농도 및 생육저해 효과 실험을 하였다. As described above, the antibacterial activity, minimum inhibitory concentration, minimum killing concentration and inhibitory effect of 10 kinds of food poisoning bacteria were tested to verify the antimicrobial performance of the essential oil component of the chrysanthemum flower extract.
상기 실험 결과 상기 정유 성분은 그램 양성균 및 그램 음성균 모두에 대해서 탁월한 항균 효과가 나타났다. 즉, 항균 활성 분석 결과, 상기 정유 성분은 그램 음성균보다 그램 양성균에 대해서 강력한 활성을 나타내었다. 또한, 최소 저해 농도 분석 결과, 상기 정유 성분은 모든 균주에 대하여 5 ㎍/㎖의 농도에서 식중독균의 생장을 저해하는 효과가 나타났고, 최소 사멸 농도 분석 결과, 살모넬라 콜레라수이스와 리스테리아 모노사이토게네스에 대해서는 실험 농도 내에서 최소 사멸 농도가 관찰되지 않았으나 다른 균주에 대해서는 5 내지 20 ㎍/㎖의 농도의 최소 사멸 농도를 나타내었다. 또한, 생육저해 효과 분석 결과, 상기 정유 성분은 24시간 동안 꾸준히 항균 성능을 유지하였다. As a result of the experiment, the essential oil component showed excellent antimicrobial effect against both Gram-positive bacteria and Gram-negative bacteria. That is, as a result of the analysis of antimicrobial activity, the essential oil component showed stronger activity against gram positive bacteria than gram negative bacteria. In addition, as a result of the minimum inhibitory concentration analysis, the essential oil component showed the effect of inhibiting the growth of food poisoning bacteria at a concentration of 5 ㎍ / ㎖ for all strains, the minimum killing concentration analysis, Salmonella cholerasus and Listeria monocytogenes The minimum killing concentration was not observed within the experimental concentration for, but for the other strains, a minimum killing concentration of 5 to 20 μg / ml was shown. In addition, as a result of the growth inhibitory effect analysis, the essential oil component was constantly maintained antibacterial performance for 24 hours.
이에 따라, 캠퍼(camphor), 크리스안서밀 알콜(chrysanthemyl alcohol), 알파-피넨(α-pinene), 감마-테르피넨(γ-terpinene), 알파+베타-튜존(α+β-thujone)을 포함하는 정유성분을 유효성분으로 함유하는 국화 꽃으로부터 추출된 항균 조성물은 상기 10종의 식중독균에 대한 항균 활성이 입증되었다.Accordingly, it includes camphor, chrysanthemyl alcohol, alpha-pinene, gamma-terpinene, alpha + beta-tujone. The antimicrobial composition extracted from the chrysanthemum flower containing the essential oil component as an active ingredient has been demonstrated antimicrobial activity against the 10 food poisoning bacteria.
이와 같은, 강력한 항균 활성을 갖는 본 발명의 정유 성분을 유효성분으로 함유하는 항균 조성물은 식품 보존제, 화장품 조성물, 치료제 등에 사용될 수 있을 것이다.Such an antimicrobial composition containing the essential oil component of the present invention having strong antimicrobial activity as an active ingredient may be used in food preservatives, cosmetic compositions, therapeutic agents and the like.
Claims (8)
- 국화 꽃(Chrysanthemum morifolium Flower)의 추출물인 정유 성분(essential oil)을 유효성분으로 함유하는 것을 특징으로 하는 항균 조성물.An antimicrobial composition comprising an essential oil ingredient (essential oil) which is an extract of Chrysanthemum morifolium Flower as an active ingredient.
- 제1항에 있어서,The method of claim 1,상기 국화 꽃의 추출물은 증기 증류 방법(steam distillation method)을 이용하여 생산된 것을 특징으로 하는 항균 조성물.The extract of the chrysanthemum flower is an antimicrobial composition, characterized in that produced using a steam distillation method (steam distillation method).
- 제2항에 있어서,The method of claim 2,상기 증류 분별 방법은 증류수, 탄소수가 1 내지 5인 알콜, 및 디메틸설폭사이드(dimethyl sulphoxide, DMSO) 중 어느 하나를 포함하는 용매를 이용하는 것을 특징으로 하는 항균 조성물.The distillation fractionation method is an antimicrobial composition, characterized in that using a solvent containing any one of distilled water, alcohol having 1 to 5 carbon atoms, and dimethyl sulphoxide (DMSO).
- 제1항에 있어서,The method of claim 1,상기 정유 성분은 캠퍼(camphor), 크리스안서밀 알콜(chrysanthemyl alcohol), 알파-피넨(α-pinene), 감마-테르피넨(γ-terpinene), 알파+베타-튜존(α+β-thujone)을 포함하는 항균 조성물.The essential oil components include camphor, chrysanthemyl alcohol, alpha-pinene, gamma-terpinene, alpha + beta-tujone and α + β-thujone. Antimicrobial composition comprising.
- 제1항에 있어서,The method of claim 1,상기 항균 조성물은 식중독균에 대해 5 내지 20 ㎍/㎖의 최소 사멸 농도(MBC, minimum bactericidal concentration)를 갖는 것을 특징으로 하는 항균 조성물.The antimicrobial composition is characterized in that it has a minimum bactericidal concentration (MBC) of 5 to 20 ㎍ / ㎖ for food poisoning bacteria.
- 제5항에 있어서,The method of claim 5,상기 식중독균은 바실러스 세레우스(Bacillus Cereus), 에어로모나스 하이드로필라(Aeromonas hydrophila), 살모넬라 엔터릭카(Salmonella enterica), 시게라 손네이(Shigella sonnei), 장염 비브리오(Vibrio parahaemolyticus), 비브리오 불니피쿠스(Vibrio vulnificus), 바실러스 서브틸리스(Bacillus subtilis), 및 스타필로코쿠스 아루레우스(Staphylococcus aureus)를 포함하는 항균 조성물.The food poisoning bacteria are Bacillus Cereus, Aeromonas hydrophila, Salmonella enterica, Shigerella sonnei, Vibrio parahaemolyticus, Vibrio bulnipius V. antimicrobial composition comprising vulnificus, Bacillus subtilis, and Staphylococcus aureus.
- 제1항에 있어서,The method of claim 1,상기 항균 조성물은 식중독균에 대해 5 ㎍/㎖의 최소 저해 농도(MIC, minimum inhibitory concentration)를 갖는 것을 특징으로 하는 항균 조성물.The antimicrobial composition is characterized in that it has a minimum inhibitory concentration (MIC, minimum inhibitory concentration) against food poisoning bacteria.
- 제7항에 있어서,The method of claim 7, wherein상기 항균 조성물은 바실러스 세레우스(Bacillus Cereus), 에어로모나스 하이드로필라(Aeromonas hydrophila), 살모넬라 콜레라수이스(Salmonella choleraesuis), 살모넬라 엔터릭카(Salmonella enterica), 시게라 손네이(Shigella sonnei), 장염 비브리오(Vibrio parahaemolyticus), 비브리오 불니피쿠스(Vibrio vulnificus), 바실러스 서브틸리스(Bacillus subtilis), 리스테리아 모노사이토게네스(Listeria monocytogenes), 및 스타필로코쿠스 아루레우스(Staphylococcus aureus)를 포함하는 항균 조성물.The antimicrobial composition may include Bacillus Cereus, Aeromonas hydrophila, Salmonella choleraesuis, Salmonella enterica, Shigera sonnei, Shigella sonnei, and enteritis vibrio. An antimicrobial composition comprising Vibrio parahaemolyticus, Vibrio vulnificus, Bacillus subtilis, Listeria monocytogenes, and Staphylococcus aureus.
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