KR20220037034A - Antibacterial peptide derived from Haliotis discus discus - Google Patents
Antibacterial peptide derived from Haliotis discus discus Download PDFInfo
- Publication number
- KR20220037034A KR20220037034A KR1020200119256A KR20200119256A KR20220037034A KR 20220037034 A KR20220037034 A KR 20220037034A KR 1020200119256 A KR1020200119256 A KR 1020200119256A KR 20200119256 A KR20200119256 A KR 20200119256A KR 20220037034 A KR20220037034 A KR 20220037034A
- Authority
- KR
- South Korea
- Prior art keywords
- antibacterial
- peptide
- discus
- antibacterial peptide
- present
- Prior art date
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Classifications
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Abstract
Description
본 발명은 둥근전복(Haliotis discus discus)으로부터 유래한 서열번호1의 아미노산 서열로 이루어진 항균 펩타이드 및 이를 포함하는 항균용 조성물에 관한 것이다.The present invention relates to an antibacterial peptide comprising the amino acid sequence of SEQ ID NO: 1 derived from Haliotis discus discus and an antibacterial composition comprising the same.
우리나라에 서식하는 대표적인 전복은 둥근전복(까막전복, Haliotis discus discus), 북방전복(참전복, H. discus hannai), 왕전복(H. madaka), 말전복(H. gigantea)이며 종에 따라 겨울철 12℃ 등온선을 경계로 제주도 근해부터 전 연안에 서식한다. 이 중 북방전복과 둥근전복은 우리나라에서 주로 양식하는 종으로 2019년 기준 천해양식 생산량 18,436 M/T, 생산금액 591,741,551천원이며 양식규모가 매년 상승세에 있다. 전복양식 생산량의 98%가 전라남도에서 생산되며 그 외 경상남도, 충청남도, 제주도, 울산광역시 등에서 나머지 2%를 생산한다.Typical abalone inhabiting Korea are round abalone ( Haliotis discus discus ), northern abalone ( H. discus hannai ), king abalone ( H. madaka ), and horse abalone ( H. gigantea ). It inhabits all coasts from the sea near Jeju Island to the 12 ℃ isothermal line. Among them, northern abalone and round abalone are mainly cultivated in Korea. As of 2019, shallow sea production was 18,436 M/T, production amount was 591,741,551 thousand won, and the scale of aquaculture is increasing every year. 98% of abalone production is produced in Jeollanam-do, and the remaining 2% is produced in Gyeongsangnam-do, Chungcheongnam-do, Jeju-do, and Ulsan Metropolitan City.
페니실린 이후 수많은 종류의 항생제가 개발되어 사용되어 왔으나, 근래에 들어서 항생제 내성을 가지는 균주들이 증가하고 있다. 미국 질병통제센터(CDC)의 AMR(antimicrobial resistance) 병원 항생제 보고서에 따르면, 항생제의 오남용으로 인해 2006년 한해 동안 약 10만명이 메티실린 내성 황색포도상구균(Methicillin-resistant Staphylococcus aureus, MRSA)에 감염되었고 그 중 사망자는 약 2만명이었다. 2010년에는 국내에서도 슈퍼박테리아라 일컬어지는 NDM-1(New Delhi metalo-beta-lactamase) 유전자를 함유한 카바페넴 내성 장내균(Carbapenem-resistant Enterobacteriaceae, CRE)이 발견되는 등 내성균에 의한 위험성이 증가하고 있다. 현재 전 세계적으로 이루어지는 감염성 질환에 대한 연구의 대부분은 병원균의 대사나 생합성 경로를 선택적으로 억제·제어하는 항생 물질의 개발과 약독화 병원균의 배양에 의한 백신 개발에 초점이 맞추어져 있는데 기존의 항생 물질의 경우 숙주의 대사 및 생합성 경로를 조절하는 효소도 동시에 억제하여 부작용과 독성을 나타내는 경우가 많고 내성 문제로 사용이 제한되고 있으며, 백신 개발만으로는 신종 병원균에 신속하게 대처할 수 없는 문제점이 있다. 따라서 감염성 질환을 초기에 효과적으로 제어할 수 있는 새로운 패러다임의 감염 치료제의 개발이 시급한 실정이며, 최근 들어 국내외 연구진들은 기존의 문제점을 해결할 수 있는 방안으로 항균 펩타이드(antimicrobial peptide, AMP)에 주목하고 있다.Numerous types of antibiotics have been developed and used since penicillin, but recently, strains with antibiotic resistance are increasing. According to the US Centers for Disease Control and Prevention's (CDC) antimicrobial resistance (AMR) hospital antibiotic report, in 2006, about 100,000 people became infected with methicillin-resistant Staphylococcus aureus (MRSA) due to misuse of antibiotics. Of these, about 20,000 died. In 2010, carbapenem-resistant Enterobacteriaceae (CRE) containing the New Delhi metalo-beta-lactamase (NDM-1) gene, which is also called a super bacterium in Korea, was discovered, and the risk of resistant bacteria is increasing. . Most of the current research on infectious diseases around the world is focused on the development of antibiotics that selectively inhibit and control the metabolic or biosynthetic pathways of pathogens and the development of vaccines by culturing attenuated pathogens. In the case of enzymes that control the host's metabolic and biosynthetic pathways, side effects and toxicity are often inhibited at the same time, and their use is limited due to resistance problems, and there is a problem in that it is not possible to quickly cope with new pathogens only by developing a vaccine. Therefore, there is an urgent need to develop a new paradigm of an infection treatment agent that can effectively control infectious diseases at an early stage.
항균 펩타이드(antimicrobial peptide, AMP)란 생물체가 발현시키는 작은 펩타이드 단백질로 체내에 들어온 병원균이나 곰팡이, 기생충, 바이러스 등에 대항하는 면역작용이나 항염 작용을 담당한다고 알려져 있다. 그 길이가 10~50개 아미노산으로 매우 짧고 주로 양전하(+2 내지 +9)를 띠며 30% 이상의 소수성 아미노산 잔기를 포함하는 것이 특징이다. AMP는 음전하를 띠는 세균의 세포막에 접촉하여 알파 헬릭스(α-helix) 또는 베타 시트(β-sheet)를 형성하면서 ‘배럴-스테이브(barrel-stave)’, ‘카펫(carpet)’, 또는 ‘환상명의 포어(toroidal-pore)’ 기작으로 세포막의 구멍을 내어 세균을 죽이는 것으로 알려져 있다. AMP는 생물체에서 유래한 단백질이기 때문에 효과적인 항균 활성을 가지면서 부작용이나 독성이 없다고 알려져 의약품이나 향장품 원료로 활용되고 있다Antimicrobial peptide (AMP) is a small peptide protein expressed by an organism and is known to be responsible for immune or anti-inflammatory action against pathogens, fungi, parasites, and viruses that enter the body. It is very short with a length of 10 to 50 amino acids, mainly positively charged (+2 to +9), and characterized in that it contains more than 30% of hydrophobic amino acid residues. AMP is in contact with the negatively charged bacterial cell membrane to form an alpha-helix or beta-sheet, forming a 'barrel-stave', 'carpet', or It is known to kill bacteria by puncturing the cell membrane with a 'toroidal-pore' mechanism. Since AMP is a protein derived from living organisms, it is known that it has effective antibacterial activity and no side effects or toxicity, and is used as a raw material for pharmaceuticals and cosmetics.
본 발명에서는 전복의 유전자원을 기반으로 전복 유래 활성물질을 탐색하고자 둥근전복의 전사체 데이터를 기반으로 항균 펩타이드(AMP) 후보를 탐색하였으며 이들의 항균 활성을 확인하였다.In the present invention, in order to search for active substances derived from abalone based on the genetic resources of abalone, antibacterial peptide (AMP) candidates were searched based on transcriptome data of round abalone, and their antibacterial activity was confirmed.
본 발명의 목적은 둥근전복(Haliotis discus discus)으로부터 유래한 서열번호1의 아미노산 서열로 이루어진 항균 펩타이드 및 이를 포함하는 항균용 약학 조성물, 항균용 화장료 조성물, 항균용 사료첨가제, 위생용품 및 식중독 예방 또는 치료용 약학 조성물을 제공하는 것이다. An object of the present invention is an antibacterial peptide consisting of the amino acid sequence of SEQ ID NO: 1 derived from round abalone ( Haliotis discus discus ) and an antibacterial pharmaceutical composition, antibacterial cosmetic composition, antibacterial feed additive, hygiene product and food poisoning prevention or To provide a pharmaceutical composition for treatment.
상기와 같은 본 발명의 목적을 달성하기 위하여, 하나의 양태로 둥근전복(Haliotis discus discus)으로부터 유래한 서열번호1의 아미노산 서열로 이루어진 항균 펩타이드를 제공한다.In order to achieve the object of the present invention as described above, in one aspect, it provides an antibacterial peptide consisting of the amino acid sequence of SEQ ID NO: 1 derived from abalone ( Haliotis discus discus ).
상시 항균 펩타이드는 고초균(Bacillus subtilis), 대장균(Escherichia coli), 녹농균(Pseudomonas aeruginosa) 및 장염비브리오균(Vibrio parahaemolyticus)으로 이루어진 군에서 선택되는 어느 1종 이상에 대해 항균 활성을 가질 수 있다.The permanent antibacterial peptide may have antibacterial activity against any one or more selected from the group consisting of Bacillus subtilis , Escherichia coli , Pseudomonas aeruginosa , and Vibrio parahaemolyticus .
또한 상기 항균 펩타이드를 유효성분으로 하는 항균용 약학 조성물을 제공한다. In addition, there is provided an antibacterial pharmaceutical composition comprising the antibacterial peptide as an active ingredient.
또한 상기 항균 펩타이드를 유효성분으로 하는 항균용 식품 조성물을 제공한다.In addition, there is provided an antibacterial food composition comprising the antimicrobial peptide as an active ingredient.
또한 상기 항균 펩타이드를 유효성분으로 하는 항균용 화장료 조성물을 제공한다. In addition, there is provided an antibacterial cosmetic composition comprising the antibacterial peptide as an active ingredient.
또한 상기 항균 펩타이드를 유효성분으로 하는 항균용 사료첨가제를 제공한다. It also provides an antibacterial feed additive containing the antibacterial peptide as an active ingredient.
또한, 상기 항균 펩타이드를 유효성분으로 하는 위생용품을 제공한다. In addition, there is provided a hygiene product containing the antibacterial peptide as an active ingredient.
또한, 상기 항균 펩타이드를 유효성분으로 하는 식중독 예방 또는 치료용 약학 조성물을 제공한다.In addition, there is provided a pharmaceutical composition for preventing or treating food poisoning using the antibacterial peptide as an active ingredient.
본 발명 따른 둥근전복에서 유래된 항균 펩타이드는 양성 및 음성 균주에 대해 항균활성을 지니기 때문에 항균용 약학조성물, 식품 조성물, 화장품 조성물, 위생용품, 사료 조성물 등에 활용가능하다.Since the antibacterial peptide derived from round abalone according to the present invention has antibacterial activity against positive and negative strains, it can be used in antibacterial pharmaceutical compositions, food compositions, cosmetic compositions, hygiene products, feed compositions, and the like.
도 1은 AMP 후보 탐색과정(workflow)을 도식화 한 것이다.
도 2는 동근전복(Haliotis discus discus) 유래 펩타이드(HD22925_1)의 시료농도에 따른 항균력 검사 결과이다(1~10; 시료 농도, mg/㎖). 1 is a schematic diagram of an AMP candidate search process (workflow).
2 is an antimicrobial activity test result according to the sample concentration of the abalone ( Haliotis discus discus )-derived peptide (HD22925_1) (1-10; sample concentration, mg/ml).
본 발명은 상기의 과제를 해결하기 위해 본 발명은 둥근전복(Haliotis discus discus)으로부터 유래한 서열번호1의 아미노산 서열로 이루어진 항균 펩타이드를 제공한다.The present invention provides an antibacterial peptide comprising the amino acid sequence of SEQ ID NO: 1 derived from abalone ( Haliotis discus discus ) in order to solve the above problems.
본 발명에서 항균 펩타이드는 서열번호1의 아미노산 서열로 이루어진다. In the present invention, the antibacterial peptide consists of the amino acid sequence of SEQ ID NO: 1.
본 발명의 실시예에서 서열번호1의 펩타이드가 3303.94 Da, 등전점(pI) 11.247, 및 전하 +7임을 확인하였는바, 본 발명의 펩타이드는 3303.94 Da, 등전점(pI) 11.247, 및 전하 +7인 것을 특징으로 할 수 있다.In the example of the present invention, it was confirmed that the peptide of SEQ ID NO: 1 has 3303.94 Da, isoelectric point (pI) 11.247, and charge +7, so that the peptide of the present invention has 3303.94 Da, isoelectric point (pI) 11.247, and charge +7 can be characterized.
본 발명의 실시예에서 둥근전복(Haliotis discus discus)에서 RNA를 추출하고, 라이브러리를 제작하여 전사체를 확보하였으며, 둥근전복 전사체 서열을 단백질 서열로 변환하여 항균 펩타이드 후보물질을 탐색하였다. 구체적으로 80 아미노산(amino acid) 이하이면서, 등전점(isoelectronic point, pI) 범위가 8~12 사이, 알파 헬릭스 경향(alpha helix propensity) 범위가 0 내지 25, 베타 시트 경향(beta sheet propensity) 범위가 25 내지 100, 생체 외 응집(in vitro aggregation, AGG)이 500 이하, 생체내 응집(in vivo aggregation, Na4vSS) 범위가 -40 내지 60인 서열들 중 항균 펩타이드를 선별하였다. 이중 신규한 항균 펩타이드 33개를 선정하였다. 선정된 항균 펩타이드 중 서열번호1(HD22925_1)의 항균 펩타이드가 그람 양성균인 고초균(Bacillus subtilis)과 그람 음성균인 대장균(Escherichia coli), 녹농균(Pseudomonas aeruginosa) 및 장염비브리오균(Vibrio parahaemolyticus)에 대해 항균 효과를 가짐을 확인하였다. In an example of the present invention, RNA was extracted from Haliotis discus discus , a library was prepared to secure transcripts, and antibacterial peptide candidates were searched for by converting the round abalone transcript sequence into a protein sequence. Specifically, 80 amino acids or less, the isoelectronic point (pI) range is between 8 and 12, the alpha helix propensity range is 0 to 25, and the beta sheet propensity range is 25 Antibacterial peptides were selected from sequences having an in vitro aggregation (AGG) of 500 or less, and an in vivo aggregation (Na4vSS) range of -40 to 60. Among them, 33 novel antibacterial peptides were selected. Among the selected antibacterial peptides, the antibacterial peptide of SEQ ID NO: 1 (HD22925_1) has an antibacterial effect on Gram-positive bacteria Bacillus subtilis and Gram-negative bacteria Escherichia coli , Pseudomonas aeruginosa and Vibrio parahaemolyticus was confirmed to have
따라서 본 발명의 서열번호1로 이루어진 펩타이드는 항균활성을 가진다. Therefore, the peptide consisting of SEQ ID NO: 1 of the present invention has antibacterial activity.
본 발명에서 “항균”이란, 유해성을 가지는 모든 미생물의 생장 또는 성장을 억제하는 것을 말하며, 본 발명에서 상기 미생물은 그람 양성균, 그람 음성균일 수 있고, 상기 미생물은 구체적으로 고초균(Bacillus subtilis), 대장균(Escherichia coli), 녹농균(Pseudomonas aeruginosa) 및 장염비브리오균(Vibrio parahaemolyticus)으로 이루어진 군에서 선택되는 어느 1종 이상일 수 있다. In the present invention, "antibacterial" refers to inhibiting the growth or growth of all microorganisms having a harmful effect, and in the present invention, the microorganism may be a gram-positive bacteria or a gram-negative bacteria, and the microorganisms are specifically Bacillus subtilis , Escherichia coli ( Escherichia coli ), Pseudomonas aeruginosa ) and Vibrio enteritis ( Vibrio parahaemolyticus ) It may be any one or more selected from the group consisting of.
또한 본 발명의 실시예에서 서열번호1의 아미노산 서열의 펩타이드를 둥근 전복에서 유래하였는바, 본 발명의 항균 펩타이드는 둥근전복(Haliotis discus discus)에서 분리된 것일 수 있다.In addition, in an embodiment of the present invention, the peptide of the amino acid sequence of SEQ ID NO: 1 was derived from abalone, and the antibacterial peptide of the present invention may be isolated from abalone ( Haliotis discus discus ).
본 발명의 항균용 펩타이드는 항균용 조성물로 이용될 수 있다. The antibacterial peptide of the present invention can be used as an antibacterial composition.
상기 항균용 조성물은 항미생물제를 총칭하는 의미인 항생제와 같은 의미일 수 있고, 항균제, 살균제, 방부제, 보존제 또는 제균제와 같은 의미일 수 있다.The antimicrobial composition may have the same meaning as an antibiotic, which is a generic term for antimicrobial agents, and may have the same meaning as an antibacterial agent, a bactericide, an antiseptic, a preservative, or a bactericide.
상기 항균용 조성물은 항균을 목적으로 다양한 용도로 사용될 수 있는데, 이의 예로는 약학 조성물, 식품 조성물, 화장료 조성물, 사료 첨가제, 위생용품, 의약외품 등을 들 수 있다.The antibacterial composition can be used for various purposes for the purpose of antibacterial, and examples thereof include pharmaceutical compositions, food compositions, cosmetic compositions, feed additives, hygiene products, quasi-drugs, and the like.
또한, 상기 항균 펩타이드는 항균 활성을 보이는 고초균(Bacillus subtilis), 대장균(Escherichia coli), 녹농균(Pseudomonas aeruginosa) 및 장염비브리오균(Vibrio parahaemolyticus)으로부터 대표적으로 유발되는 질병인 식중독 등의 질병을 예방 또는 치료하는 것이 가능하기 때문에 이러한 항균 펩타이드를 유효성분으로 함유하여 식중독 예방 또는 치료용 약학 조성물로 활용될 수 있다.In addition, the antibacterial peptide prevents or treats diseases such as food poisoning, a disease typically caused by Bacillus subtilis , Escherichia coli , Pseudomonas aeruginosa , and Vibrio parahaemolyticus showing antibacterial activity Because it is possible to contain such an antibacterial peptide as an active ingredient, it can be used as a pharmaceutical composition for preventing or treating food poisoning.
본 발명의 실시예에서 서열번호1의 항균 펩타이드의 고초균에 대해 최소억제농도(MIC)는 0.5mg/㎖, 최소살균농도(MBC)는 1mg/㎖를 나타내며, 녹농균에 대해서 최소억제농도(MIC)는 2mg/㎖, 최소살균농도(MBC)는 3mg/㎖, 장염비브리오균에 대해 최소억제농도(MIC)는 3mg/㎖, 최소살균농도(MBC)는 6mg/㎖, 대장균에 대해 최소억제농도(MIC)는 7mg/㎖, 최소살균농도(MBC)는 10mg/㎖ 초과로 나타났다. In an embodiment of the present invention, the minimum inhibitory concentration (MIC) of the antibacterial peptide of SEQ ID NO: 1 against Bacillus subtilis is 0.5 mg/ml, the minimum bactericidal concentration (MBC) is 1 mg/ml, and the minimum inhibitory concentration (MIC) for Pseudomonas aeruginosa is 2 mg/ml, minimum bactericidal concentration (MBC) is 3 mg/ml, minimum inhibitory concentration (MIC) for enteritis vibrio bacteria is 3 mg/ml, minimum bactericidal concentration (MBC) is 6 mg/ml, minimum inhibitory concentration for E. coli ( MIC) was 7 mg/ml, and the minimum bactericidal concentration (MBC) was more than 10 mg/ml.
따라서 본 발명의 항균 조성물은 서열번호1의 펩타이드를 고초균에 대해 최소억제 농도로 확인된 0.5mg/㎖ 이상으로 포함하는 것일 수 있으며, 고초균(Bacillus subtilis), 대장균(Escherichia coli), 녹농균(Pseudomonas aeruginosa) 및 장염비브리오균(Vibrio parahaemolyticus)에 동시에 항균 효과를 나타내기 위해 항균 펩타이드를 7mg/㎖의 농도로 포함하는 것일 수 있다. Therefore, the antibacterial composition of the present invention may contain the peptide of SEQ ID NO: 1 at a minimum inhibitory concentration of 0.5 mg/ml or more, Bacillus subtilis , Escherichia coli , Pseudomonas aeruginosa ) and enteritis Vibrio ( Vibrio parahaemolyticus ) It may be to include an antibacterial peptide at a concentration of 7 mg/ml to exhibit an antibacterial effect at the same time.
또한 본 발명은 상기 항균 펩타이드를 유효성분으로 하는 항균용 약학 조성물을 제공한다.The present invention also provides an antibacterial pharmaceutical composition comprising the antibacterial peptide as an active ingredient.
본 발명에서 항균 펩타이드에 관한 설명은 전술한 바와 같다.The description of the antibacterial peptide in the present invention is the same as described above.
본 발명에서 항균용 약학 조성물은 미생물 감염에 의해 발생하는 질환을 예방 또는 치료하는 것으로, 구체적으로 그람 양성균 또는 그람 음성균에 의해 유발되는 감염성 질환을 치료하는 것을 목적으로 할 수 있다. In the present invention, the antibacterial pharmaceutical composition is to prevent or treat diseases caused by microbial infection, specifically, it may aim to treat infectious diseases caused by gram-positive bacteria or gram-negative bacteria.
본 발명에서 상기 감염성 질환은 고초균(Bacillus subtilis), 대장균(Escherichia coli), 녹농균(Pseudomonas aeruginosa) 및 장염비브리오균(Vibrio parahaemolyticus)로 이루어지 군에서 선택되는 1종의 미생물에 의해 유발되는 것일 수 있다. In the present invention, the infectious disease may be caused by one type of microorganism selected from the group consisting of Bacillus subtilis , Escherichia coli , Pseudomonas aeruginosa , and Vibrio parahaemolyticus . .
상기 약학 조성물은 약학 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 또는 희석제를 더 포함할 수 있다.The pharmaceutical composition may further include an appropriate carrier, excipient or diluent commonly used in the preparation of the pharmaceutical composition.
본 발명에서 사용 가능한 담체, 부형제 또는 희석제로는, 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 또는 광물유 등을 들 수 있다.Carriers, excipients or diluents usable in the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil.
상기 약학 조성물은 약학 조성물은 정맥내, 동맥내, 복강내, 근육내, 동맥내, 복강내, 흉골내, 경피, 비측내, 흡입, 국소, 직장, 경구, 안구내 또는 피내 경로를 통해 통상적인 방식으로 대상체로 투여할 수 있다.The pharmaceutical composition may be administered via a conventional intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal route. manner can be administered to the subject.
상기 항균 펩타이드의 바람직한 투여량은 대상체의 상태 및 체중, 질환의 종류 및 정도, 약물 형태, 투여경로 및 기간에 따라 달라질 수 있으며 당업자에 의해 적절하게 선택될 수 있다. 본 발명의 일실시예에 따르면 이에 제한되는 것은 아니지만 1일 투여량이 0.01 내지 200 mg/kg, 구체적으로는 0.1 내지 200 mg/kg, 보다 구체적으로는 0.1내지 100 mg/kg 일 수 있다. 투여는 하루에 한 번 투여할 수도 있고 수회로 나누어 투여할 수도 있으며, 이에 의해 본 발명의 범위가 제한되는 것은 아니다.The preferred dosage of the antimicrobial peptide may vary depending on the condition and weight of the subject, the type and extent of the disease, the drug form, the route and duration of administration, and may be appropriately selected by those skilled in the art. According to an embodiment of the present invention, although not limited thereto, the daily dose may be 0.01 to 200 mg/kg, specifically 0.1 to 200 mg/kg, and more specifically 0.1 to 100 mg/kg. Administration may be administered once a day or may be administered in several divided doses, thereby not limiting the scope of the present invention.
본 발명에 있어서, 상기 '대상체'는 인간을 포함하는 포유동물일 수 있으나, 이들 예에 한정되는 것은 아니다.In the present invention, the 'subject' may be a mammal including a human, but is not limited to these examples.
또한 본 발명은 상기 항균 펩타이드를 유효성분으로 하는 항균용 식품 조성물을 제공한다.The present invention also provides an antibacterial food composition comprising the antimicrobial peptide as an active ingredient.
본 발명에서 항균 펩타이드에 관한 설명은 전술한 바와 같다.The description of the antibacterial peptide in the present invention is the same as described above.
본 발명에 따른 식품 조성물은 항균효과가 있다고 알려진 공지의 활성 성분과 함께 혼합하여 조성물 형태로 제조 할 수 있으며 기능성 식품(functional food), 영양 보조제(nutritional supplement), 건강식품(health food) 및 식품 첨가제(food additives) 등의 모든 형태를 포함한다. 상기 유형의 식품 조성물은 당 업계에 공지된 통상적인 방법에 따라 다양한 형태로 제조할 수 있다. The food composition according to the present invention can be prepared in the form of a composition by mixing with a known active ingredient known to have an antibacterial effect, and functional food, nutritional supplement, health food, and food additive. (food additives) and the like. Food compositions of this type can be prepared in various forms according to conventional methods known in the art.
또한 본 발명은 상기 항균 펩타이드를 유효성분으로 하는 항균용 사료 첨가제를 제공한다.In addition, the present invention provides an antibacterial feed additive comprising the antibacterial peptide as an active ingredient.
본 발명의 항균 펩타이드는 항균 효과를 가져 사료에 첨가되는 항생제 대체 물질로 사용 가능하다. The antibacterial peptide of the present invention has an antibacterial effect and can be used as a substitute for antibiotics added to feed.
상기 사료첨가제는 닭, 오리, 거위, 꿩, 돼지, 소, 염소, 개 및 고양이 중 어느 하나 이상의 동물용 배합사료 내 첨가 혼합되는 사료첨가제일 수 있다.The feed additive may be a feed additive that is added and mixed in a compound feed for any one or more of chickens, ducks, geese, pheasants, pigs, cattle, goats, dogs and cats.
또한, 상기 사료첨가제는 서열번호1의 아미노산 서열로 이루어진 펩타이드 외의 추가적으로 항생물질을 더 포함할 수 있다. In addition, the feed additive may further include an antibiotic in addition to the peptide consisting of the amino acid sequence of SEQ ID NO: 1.
또한 본 발명은 상기 항균 펩타이드를 유효성분으로 하는 위생용품을 제공한다.The present invention also provides a hygiene product containing the antibacterial peptide as an active ingredient.
본 발명에서 항균 펩타이드에 관한 설명은 전술한 바와 같다. The description of the antibacterial peptide in the present invention is the same as described above.
본 발명에서 위생용품은 비누, 세정제, 물티슈, 손 소독제, 구강청정제, 구강소독제를 포함한다. In the present invention, hygiene products include soap, detergent, wet tissue, hand sanitizer, mouthwash, and mouth sanitizer.
또한 본 발명은 상기 항균 펩타이드를 유효성분으로 하는 식중독 예방 또는 치료용 약학 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating food poisoning comprising the antibacterial peptide as an active ingredient.
본 발명에서 항균 펩타이드에 관한 설명은 전술한 바와 같다. The description of the antibacterial peptide in the present invention is the same as described above.
본 발명에서 식중독은 식품의 섭취에 연관된 인체에 유해한 미생물 또는 유독 물질에 의해 발생했거나 발생한 것으로 판단되는 감염성 또는 독소형 질환을 의미한다. In the present invention, food poisoning refers to an infectious or toxin-type disease caused or judged to be caused by microorganisms or toxic substances harmful to the human body related to ingestion of food.
본 발명에서 상기 식중독은 고초균(Bacillus subtilis), 대장균(Escherichia coli), 녹농균(Pseudomonas aeruginosa) 및 장염비브리오균(Vibrio parahaemolyticus)으로 이루어진 군에서 선택되는 1종 이상에 의해 발병된 것일 수 있다. In the present invention, the food poisoning may be caused by one or more selected from the group consisting of Bacillus subtilis , Escherichia coli , Pseudomonas aeruginosa , and Vibrio parahaemolyticus .
이하, 본 발명을 실시예에 의해 보다 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기의 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail by way of Examples. However, the following examples are only illustrative of the present invention, and the content of the present invention is not limited to the following examples.
1. 둥근전복 유래 항균 펩타이드 후보 탐색 및 합성1. Search and synthesis of antibacterial peptide candidates derived from round abalone
둥근전복(Haliotis discus discus)은 제주특별자치도 해양수산연구원에서 제공받았으며 전복의 외전근(abduct muscle), 소화관, 눈, 족부, 아가미, 생식소, 혈구(hemocyte), 간췌장, 맨틀(mantle), 촉수(tentacle) 등의 조직을 적출하여 액체질소로 급속 냉동하였다. 냉동된 조직 50 내지 100 mg으로부터 RNAiso plus(Total RNA extraction reagent, TaKaRa) 방법에 따라 total RNA를 추출하였다.Round abalone ( Haliotis discus discus ) was provided by the Jeju Special Self-Governing Province Research Institute of Oceans and Fisheries, and the abduct muscle, digestive tract, eyes, feet, gills, gonads, hemocytes, hepatopancreas, mantle, and tentacles Tissues such as tentacles were removed and rapidly frozen with liquid nitrogen. Total RNA was extracted from 50 to 100 mg of frozen tissue according to the RNAiso plus (Total RNA extraction reagent, TaKaRa) method.
각 조직으로부터 추출한 total RNA를 동량으로 혼합하고 ㈜랩지노믹스에 의뢰하여 약 150 bp 크기의 라이브러리 어댑터(library adapter)를 포함하는 RNA 라이브러리(library)를 제작하였다. 제작된 RNA library 중에서 어댑터를 제외한 길이가 500 bp 이상의 염기서열들을 대상으로 ㈜인실리코젠에서 제공하는 Miseq platform으로 시퀀싱하였고 De novo assembly 과정을 통해 각 전복에 대한 전사체(transcriptome)를 확보하였다.Total RNA extracted from each tissue was mixed in the same amount, and an RNA library including a library adapter of about 150 bp in size was prepared by requesting Lab Genomics. Among the prepared RNA libraries, nucleotide sequences over 500 bp in length excluding adapters were sequenced with the Miseq platform provided by Insilicogen, and transcriptomes for each abalone were secured through the de novo assembly process.
각 전복 전사체로부터 전복에서 특이적으로 발현되는 AMP 후보를 ㈜인실리코젠에 의뢰하여 탐색하였다. AMP candidates specifically expressed in abalone from each abalone transcript were searched for by Insilicogen Co., Ltd.
전복 전사체 서열을 단백질 서열로 변환하여 PEPSTATS, TANGO, AGGRESCAN 등의 분석 방법으로 AMP 후보 물질을 탐색하였다. AMP 후보는 크기가 80 아미노산(amino acid) 이하이면서, 등전점(isoelectronic point, pI) 범위가 8~12 사이, 알파 헬릭스 경향(alpha helix propensity) 범위가 0 내지 25, 베타 시트 경향(beta sheet propensity) 범위가 25 내지 100, 생체 외 응집(in vitro aggregation, AGG)이 500 이하, 생체내 응집(in vivo aggregation, Na4vSS) 범위가 -40 내지 60인 서열들 중에서 아직까지 발표되지 않은 신규 펩타이드만을 선별하였다(도 1).By converting the abalone transcript sequence into a protein sequence, AMP candidates were searched for by analysis methods such as PEPSTATS, TANGO, and AGGRESCAN. AMP candidates have a size of 80 amino acids or less, and an isoelectronic point (pI) range of 8 to 12, an alpha helix propensity range of 0 to 25, and a beta sheet propensity. Among sequences with a range of 25 to 100, an in vitro aggregation (AGG) of 500 or less, and an in vivo aggregation (Na4vSS) range of -40 to 60, only novel peptides that have not yet been published were selected. (Fig. 1).
그 결과 둥근 전복에서 33개의 신규한 AMP 후보를 선정하였다. 표 1은 둥근 전복의 AMP 분석결과이다. 선정된 AMP 후보 중에서 일부를 선택하여 애니젠(주)에 의뢰하여 합성하였다(표 2).As a result, 33 novel AMP candidates were selected from round abalone. Table 1 shows the results of AMP analysis of round abalone. Some of the selected AMP candidates were selected and synthesized by Anygen Co., Ltd. (Table 2).
(mer)(mer)
M.W.M.W.
(Da)(Da)
(H. discus discus)round abalone
( H. discus discus )
(서열번호1)KERSKGKFKIFFKKKTDFQLRDIWTK
(SEQ ID NO: 1)
2. 둥근 전복 유래 펩타이드의 항균 효과 분석2. Analysis of antibacterial effect of round abalone-derived peptides
항균활성 실험에는 그람 양성균인 고초균(Bacillus subtilis)과 그람 음성균인 대장균(Escherichia coli), 녹농균(Pseudomonas aeruginosa), 장염비브리오균(Vibrio parahaemolyticus)를 한국미생물보존센터(KCCM, Korea Culture Center of Microorganisms)에서 분양 받아 사용하였다(표 3).In the antibacterial activity experiment, Gram-positive bacteria Bacillus subtilis , Gram-negative bacteria Escherichia coli , Pseudomonas aeruginosa , and Vibrio parahaemolyticus were used at the Korea Culture Center of Microorganisms (KCCM). It was received and used (Table 3).
항균활성 실험은 CLSI(Clinical and Laboratory Standards Institute)의 디스크 확산법을 변형하여 실시하였다. 순수 배양된 집락을 LB(Luria Bertani) 액체배지에 McFarland 0.5가 되도록 현탁 하였다. 면봉으로 균액을 묻여 LB 고체배지의 표면에 골고루 바른 다음 무균상태의 실온에서 10~15분간 방치하였다. 검사하고자 하는 AMP를 1 드롭(drop)한 후 30℃에서 18시간 배양한 후 억제환 생성 유무를 확인하여 항균력을 판정하였다.The antibacterial activity test was carried out by modifying the disc diffusion method of CLSI (Clinical and Laboratory Standards Institute). The pure cultured colonies were suspended in LB (Luria Bertani) liquid medium so as to become McFarland 0.5. The bacterium solution was applied with a cotton swab and applied evenly to the surface of the LB solid medium, and then left at room temperature under aseptic conditions for 10 to 15 minutes. After 1 drop of the AMP to be tested was incubated at 30° C. for 18 hours, the antibacterial activity was determined by checking the presence or absence of inhibitory rings.
AMP는 증류수에 완전히 녹여 사용했으며 시료의 농도를 10mg/㎖에서 1mg/㎖까지 점차적으로 희석하여 항균 활성을 확인하였다. 검사를 통하여 대략적인 항균활성 농도를 확인하였으며, 이를 바탕으로 CLSI 기준에 따라 최소억제농도 (MIC, Minimal Inhibitory Concentration)와 최소살균농도(MBC, Minimal Bactericidal Concentration)를 측정하였다.AMP was completely dissolved in distilled water and the concentration of the sample was gradually diluted from 10 mg/ml to 1 mg/ml to confirm the antibacterial activity. The approximate antibacterial activity concentration was confirmed through the test, and based on this, the minimum inhibitory concentration (MIC, Minimal Inhibitory Concentration) and the minimum bactericidal concentration (MBC, Minimal Bactericidal Concentration) were measured according to the CLSI standard.
LB 액체배지에 균을 접종하여 30℃에서 배양한 후, McFarland 0.5(1~5×106 CFU/㎖)로 농도를 조절하여 균 현탁액을 준비하였다. AMP를 0.125 내지 10 ㎎/㎖까지 단계별로 희석한 후, 균 현탁액을 각각 접종하고 18시간 배양 후 MIC 값을 판정하였다. MIC 판정을 위해 처리된 각 배양액을 LB 고체배지에 도말하여 18시간 배양한 후 최초 접종 균수의 99.9%를 사멸시키는 최대희석배수의 농도를 MBC로 판정하였다.After inoculating the bacteria in LB broth and culturing at 30°C, the concentration was adjusted with McFarland 0.5 (1-5×10 6 CFU/ml) to prepare a bacterial suspension. After stepwise dilution of AMP to 0.125 to 10 mg/ml, the bacterial suspension was inoculated, respectively, and the MIC value was determined after incubation for 18 hours. Each culture treated for MIC determination was smeared on LB solid medium and cultured for 18 hours. Then, the concentration of the maximum dilution factor that kills 99.9% of the first inoculated bacteria was determined by MBC.
분석결과, 고초균에 대해서는 1 내지 10 mg/㎖ 농도에서 모두 항균 활성이 나타났으며, 대장균은 7 mg/㎖ 이상, 녹농균 및 장염비브리오균은 3 mg/㎖ 이상의 농도에서 항균활성이 나타남을 확인하였다(도 2, 표 4).As a result of the analysis, it was confirmed that all of Bacillus subtilis exhibited antibacterial activity at concentrations of 1 to 10 mg/ml, E. coli showed antibacterial activity at concentrations of 7 mg/ml or higher, and Pseudomonas aeruginosa and enteritis vibrio were 3 mg/ml or higher. (Fig. 2, Table 4).
표 5는 둥근전복 유래 AMP의 각 균주에 대한 최소억제농도(MIC)와 최소살균농도(MBC)를 분석한 결과이다. 표 5를 참조하면 고초균에 대해 최소억제농도(MIC)는 0.5mg/㎖, 최소살균농도(MBC)는 1mg/㎖를 나타내며, 녹농균에 대해서 최소억제농도(MIC)는 2mg/㎖, 최소살균농도(MBC)는 3mg/㎖, 장염비브리오균에 대해 최소억제농도(MIC)는 3mg/㎖, 최소살균농도(MBC)는 6mg/㎖, 대장균에 대해 최소억제농도(MIC)는 7mg/㎖, 최소살균농도(MBC)는 10mg/㎖ 초과로 나타났다(표 5). Table 5 shows the results of analyzing the minimum inhibitory concentration (MIC) and minimum sterilization concentration (MBC) for each strain of AMP derived from round abalone. Referring to Table 5, the minimum inhibitory concentration (MIC) for Bacillus subtilis is 0.5 mg/mL, and the minimum bactericidal concentration (MBC) is 1 mg/mL, and the minimum inhibitory concentration (MIC) for P. aeruginosa is 2 mg/mL, minimum bactericidal concentration (MBC) is 3mg/ml, the minimum inhibitory concentration (MIC) for enteritis vibrio is 3mg/ml, the minimum bactericidal concentration (MBC) is 6mg/ml, the minimum inhibitory concentration (MIC) for Escherichia coli is 7mg/ml, the minimum The bactericidal concentration (MBC) was found to be greater than 10 mg/ml (Table 5).
<110> Jeju National University Industry-Academic Cooperation Foundation
<120> Antibacterial peptide derived from Haliotis discus discus
<130> DP20200170
<160> 1
<170> KoPatentIn 3.0
<210> 1
<211> 26
<212> PRT
<213> Artificial Sequence
<220>
<223> HD22925_1
<400> 1
Lys Glu Arg Ser Lys Gly Lys Phe Lys Ile Phe Phe Lys Lys Lys Thr
1 5 10 15
Asp Phe Gln Leu Arg Asp Ile Trp Thr Lys
20 25
<110> Jeju National University Industry-Academic Cooperation Foundation
<120> Antibacterial peptide derived from Haliotis discus discus
<130> DP20200170
<160> 1
<170> KoPatentIn 3.0
<210> 1
<211> 26
<212> PRT
<213> Artificial Sequence
<220>
<223> HD22925_1
<400> 1
Lys Glu Arg Ser Lys Gly Lys Phe Lys Ile Phe Phe Lys
Claims (8)
상기 항균 펩타이드는 고초균(Bacillus subtilis), 대장균(Escherichia coli), 녹농균(Pseudomonas aeruginosa) 및 장염비브리오균(Vibrio parahaemolyticus)으로 이루어진 군에서 선택되는 1종 이상에 대해 항균 효과를 가지는 것인, 펩타이드.The method of claim 1,
The antibacterial peptide is Bacillus subtilis ), Escherichia coli , Pseudomonas aeruginosa ) and Vibrio parahaemolyticus ) It will have an antibacterial effect against at least one selected from the group consisting of, the peptide.
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KR20130109544A (en) * | 2012-03-27 | 2013-10-08 | 부경대학교 산학협력단 | Novel antimicrobial peptide from the abalone, haliotis discus discus, and uses thereof |
KR101668229B1 (en) | 2015-08-14 | 2016-10-21 | 군산대학교 산학협력단 | Antibacterial peptide derived from Crassostrea gigas and antibacterial pharmaceutical composition containing the same |
KR20180053867A (en) * | 2016-11-14 | 2018-05-24 | 대한민국(관리부서:국립수산과학원) | Antimicrobial Peptides derived from abalone bactericidal??permeability-increasing protein and uses thereof |
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KR101668229B1 (en) | 2015-08-14 | 2016-10-21 | 군산대학교 산학협력단 | Antibacterial peptide derived from Crassostrea gigas and antibacterial pharmaceutical composition containing the same |
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