KR102001990B1 - Pediococcus acidilactici SRCM102615 strain having immunity activity, antiviral activity and probiotics properties and uses thereof - Google Patents
Pediococcus acidilactici SRCM102615 strain having immunity activity, antiviral activity and probiotics properties and uses thereof Download PDFInfo
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- KR102001990B1 KR102001990B1 KR1020180070651A KR20180070651A KR102001990B1 KR 102001990 B1 KR102001990 B1 KR 102001990B1 KR 1020180070651 A KR1020180070651 A KR 1020180070651A KR 20180070651 A KR20180070651 A KR 20180070651A KR 102001990 B1 KR102001990 B1 KR 102001990B1
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- srcm102615
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Abstract
Description
본 발명은 면역 활성, 항바이러스 활성 및 프로바이오틱스 특성을 갖는 페디오코커스 애시디락티시 SRCM102615 균주 및 이의 용도에 관한 것이다.The present invention relates to the Pediococcus aceyltactii SRCM102615 strain having immunological activity, antiviral activity and probiotics characteristics and uses thereof.
프로바이오틱스 (Probiotics)라는 말은 'for life'라는 뜻으로 그리스어에서 유래되었다. 프로바이오틱스는 1965년에 '미생물이 분비하는 물질로 다른 미생물의 성장을 자극하는 물질'이라고 정의되었고, 1974년에는 '숙주동물에게 유익한 효과를 주는 생물 또는 물질'이라고 정의되었다. 그 후 1989년에 풀러 (Fuller)가 프로바이오틱스를 다시 정의하기를 '장내 균총을 개선시킴으로서 숙주에게 유익한 영향을 주는 생균제제'라고 하여 살아있는 미생물에 대한 개념이 도입되었다. 이러한 프로바이오틱스에는 호기성균군, 혐기성균군, 유산균, 효모균들이 사용되고 있으나 주로 유산균들이 주요 균주로 이용되고 있다.Probiotics is derived from the Greek word for life. Probiotics was defined in 1965 as "a substance secreted by a microorganism that stimulates the growth of other microorganisms." In 1974, it was defined as "a creature or substance that has a beneficial effect on the host animal." Then in 1989, Fuller redefined probiotics as "a probiotic agent that beneficially influenced the host by improving the intestinal microflora" and introduced the concept of living microorganisms. Aerobic bacteria, anaerobic bacteria, lactic acid bacteria, and yeast bacteria are used in these probiotics, but lactic acid bacteria are mainly used as a major strain.
유산균은 자연계에 널리 존재하며 탄수화물을 혐기적으로 이용하여 유산을 생산한다. 유산균이 발견되는 자연환경은 다양한데, 사람이나 동물의 장내에 존재할 뿐 아니라, 다양한 채소와 과일에서도 발견되며 우리나라의 김치나 요구르트, 독일의 사우어크라우트 (sauerkraut)와 같은 발효식품에서 그 발효과정에 중요한 역할을 담당한다. 이들 유산균은 장내로 유입된 후 장내 상피세포에 착생하게 되어 유해미생물의 장 정착을 방지하고 항균물질을 분비함으로써 유해미생물의 생육을 억제하고 설사와 변비를 개선할 뿐만 아니라, 면역활성 증진, 항암 작용, 혈청 콜레스테롤 저하 등의 작용을 수행한다. 또한, 일반적으로 이들 유산균은 직접 혹은 간접적으로 식품에 첨가되어 이들의 대사산물인 유산에 의해 식품의 저장성을 향상시키며 식품의 향미와 조직을 개선한다고 알려져 있다.Lactic acid bacteria are widely found in nature and produce carbohydrates by anaerobic use of carbohydrates. The natural environment in which lactic acid bacteria are found is not only present in human or animal intestines but also in various vegetables and fruits and plays an important role in the fermentation process in fermented food such as kimchi, yogurt and German sauerkraut in Korea . These lactic acid bacteria enter the intestines and multiply in the intestinal epithelium, thereby preventing the colonization of harmful microorganisms and secreting antimicrobial substances, thereby suppressing the growth of harmful microorganisms, improving diarrhea and constipation, , Serum cholesterol lowering and the like. In addition, it is generally known that these lactic acid bacteria are directly or indirectly added to foods to improve the storage stability of foods by lactic acid, which is a metabolite thereof, and to improve the flavor and texture of foods.
한편, 한국등록특허 제1635997호에는 '페디오코쿠스 에시디락티시(Pediococcus acidilactici)를 유효성분으로 포함하는 Th1-매개 면역 질환 또는 Th2-매개 면역 질환의 예방, 개선 또는 치료용 조성물'이 개시되어 있고, 한국공개특허 제2018-0059372호에는 '신규한 페디오코커스 악시딜락티시를 이용한 돼지 설사증 예방 또는 치료용 조성물'이 개시되어 있으나, 본 발명에서와 같이, '면역 활성, 항바이러스 활성 및 프로바이오틱스 특성을 갖는 페디오코커스 애시디락티시 SRCM102615 균주 및 이의 용도'에 대해서는 밝혀진 바가 전혀 없다.Korean Patent No. 1635997 discloses a composition for preventing, ameliorating or treating a Th1-mediated disease or a Th2-mediated immune disease comprising Pediococcus acidilactici as an active ingredient Korean Patent Laid-Open Publication No. 2018-0059372 discloses a composition for preventing or treating porcine diarrhea using a novel pediococcus axicilyllacticia. However, as in the present invention, 'immunological activity, antiviral activity, The Pediococcus aceyltactii SRCM102615 strain having probiotic properties and its use have never been disclosed.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명에서는 장아찌로부터 항바이러스 활성, 면역활성, 항산화 활성, 유단백질 응고성, 내산성, 내담즙성, 항생제 내성, β-글루코시다제(β-glucosidase) 분비능, γ-용혈 활성, 유해미생물에 대한 항균활성이 있고, 바이오제닉 아민, 유해물질 및 유해효소를 생성하지 않는 페디오코커스 애시디락티시(Pediococcus acidilactici) SRCM102615 균주(KCCM12241P)를 분리하였다.SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned needs, and it is an object of the present invention to provide an anticancer agent, an antioxidant activity, an antioxidant activity, a milk protein coagulant, an acid tolerance, a bile acid resistance, an antibiotic resistance, a β-glucosidase ) secretion, γ- hemolytic activity, and antimicrobial activity of the microorganisms, were isolated biogenic amines, harmful substances and does not produce the toxic enzyme Phedi O Lactococcus her CD rakti when (Pediococcus acidilactici) SRCM102615 strain (KCCM12241P).
본 발명의 페디오코커스 애시디락티시 SRCM102615 균주는 상기와 같은 프로바이틱스의 특징을 모두 가지므로, 정장제로 유용하게 사용될 수 있으며, 또한 품질 좋은 발효제품 제조를 위한 스타터 균주 및 바실러스 세레우스, 바실러스 스패리쿠스, 엔테로코커스 패시움, 리스테리아 이바노비, 리스테리아 모노시토게네스, 스타필로코커스 아우레우스, 엔테로코쿠스 에로게네스, 에스케리키아 콜라이, 쉬겔라 플렉스네리 또는 쉬겔라 손네이인 유해미생물에 대한 항균용 제제로도 사용이 가능함을 확인함으로써, 본 발명을 완성하였다.Since the strain Pediococcus agilactii SRCM102615 of the present invention has all the characteristics of the above-mentioned provitics, it can be used effectively as a formulating agent, and can also be used as a starter strain for producing a fermented product of good quality, a Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, Enterococcus erogenes, Escherichia coli, Shigella flexneri, or Shigella sonnaiin harmful microorganisms, such as Escherichia, The present invention has been accomplished by confirming that it can be used as an anti-bacterial agent.
상기 과제를 해결하기 위해, 본 발명은 항바이러스 활성, 면역활성, 항산화 활성, 유단백질 응고성, 내산성, 내담즙성, 항생제 내성, β-글루코시다제(β-glucosidase) 분비능, γ-용혈 활성, 유해미생물에 대한 항균활성이 있고, 바이오제닉 아민, 유해물질 및 유해효소를 생성하지 않는 페디오코커스 애시디락티시(Pediococcus acidilactici) SRCM102615 균주(KCCM12241P)를 제공한다.In order to solve the above-mentioned problems, the present invention provides a pharmaceutical composition comprising an antiviral activity, an immunological activity, an antioxidative activity, a milk protein coagulant, an acid tolerance, a bile acid tolerance, an antibiotic resistance, a β-glucosidase secretion, Pediococcus acidilactici SRCM102615 strain (KCCM12241P), which has antimicrobial activity against harmful microorganisms and does not produce biogenic amines, harmful substances and harmful enzymes, is provided.
또한, 본 발명은 상기 균주, 이의 배양액, 상기 배양액의 농축액 또는 그의 건조물을 유효성분으로 포함하는 프로바이오틱스 제제를 제공한다.The present invention also provides a probiotic preparation comprising the strain, a culture thereof, a concentrate of the culture broth, or a dried product thereof as an active ingredient.
또한, 본 발명은 상기 균주, 이의 배양액, 상기 배양액의 농축액 또는 그의 건조물을 유효성분으로 함유하는 바실러스 세레우스, 바실러스 스패리쿠스, 엔테로코커스 패시움, 리스테리아 이바노비, 리스테리아 모노시토게네스, 스타필로코커스 아우레우스, 엔테로코쿠스 에로게네스, 에스케리키아 콜라이, 쉬겔라 플렉스네리 또는 쉬겔라 손네이에 대한 항균용 조성물을 제공한다.The present invention also relates to a method for producing a microorganism, which comprises culturing the above strain, a culture thereof, a concentrate of the culture broth or a dried product thereof as an active ingredient, such as Bacillus cereus, Bacillus sparkicus, Enterococcus sp., Listeria ivanovi, Listeria monocytogenes, There is provided a composition for antimicrobial use against Cercus aureus, Enterococcus erogenes, Escherichia coli, Shigella flexneri, or Shigella sinnai.
또한, 본 발명은 상기 균주, 이의 배양액, 상기 배양액의 농축액 또는 그의 건조물을 유효성분으로 함유하는 발효식품 제조용 스타터(starter) 조성물을 제공한다.The present invention also provides a starter composition for the production of a fermented food, which contains the strain, a culture solution thereof, a concentrate of the culture solution or a dried product thereof as an active ingredient.
본 발명의 페디오코커스 애시디락티시(Pediococcus acidilactici) SRCM102615 균주(KCCM12241P)는 항바이러스 활성, 면역활성, 항산화 활성, 유단백질 응고성, 내산성, 내담즙성, 항생제 내성, β-글루코시다제(β-glucosidase) 분비능, γ-용혈 활성, 유해미생물에 대한 항균활성이 있고, 바이오제닉 아민, 유해물질 및 유해효소를 생성하지 않는 점을 확인하였다.The Pediococcus acidilactici strain, SRCM102615 strain (KCCM12241P) of the present invention is useful as an antiviral activity, an immunological activity, an antioxidative activity, a milk protein solidification property, an acid resistance, a bile acid resistance, an antibiotic resistance,? -Glucosidase -glucosidase) secretion, γ-hemolytic activity, and antimicrobial activity against harmful microorganisms, and did not produce biogenic amines, harmful substances and harmful enzymes.
따라서, 본 발명의 페디오코커스 애시디락티시 SRCM102615 균주는 정장제, 발효식품 제조를 위한 스타터 균주 및 항균용 미생물 제제 등 관련 산업에 다양하게 사용될 수 있는 유용한 균주로 판단된다.Therefore, the strain Pediococcus epidermal growth factor SRCM102615 of the present invention is considered to be a useful strain that can be used in a variety of industries, such as a starter, a starter strain for producing a fermented food, and an antibacterial microorganism preparation.
도 1은 본 발명에서 분리한 페디오코커스 애시디락티시(Pediococcus acidilactici) SRCM102615 균주의 FACS를 이용한 인플루엔자 A 바이러스(Influenza A viruses) 감염억제 효과를 나타낸다.
도 2는 본 발명에서 분리한 페디오코커스 애시디락티시(Pediococcus acidilactici) SRCM102615 균주의 IFN-β의 분비능 측정 결과를 나타낸다.
도 3은 본 발명에서 분리한 페디오코커스 애시디락티시(Pediococcus acidilactici) SRCM102615 균주의 면역 활성을 나타낸다.
도 4는 본 발명에서 분리한 페디오코커스 애시디락티시(Pediococcus acidilactici) SRCM102615 균주와 아스코르브산(Ascorbic acid)의 항산화 활성 비교 결과이다.
도 5는 본 발명에서 분리한 페디오코커스 애시디락티시(Pediococcus acidilactici) SRCM102615 균주의 계통도를 나타낸다.1 shows the effect of inhibiting Influenza A virus infection with FACS of Pediococcus acidilactici strain SRCM102615 isolated in the present invention.
FIG. 2 shows the measurement result of the secretion ability of IFN-.beta. Of Pediococcus acidilactici strain SRCM102615 isolated in the present invention.
Figure 3 shows the immunological activity of Pediococcus acidilactici strain SRCM102615 isolated in the present invention.
FIG. 4 shows the antioxidative activity of Pediococcus acidilactici SRCM102615 isolated from the present invention and ascorbic acid.
FIG. 5 shows a flow diagram of the Pediococcus acidilactici strain SRCM102615 isolated in the present invention. FIG.
본 발명의 목적을 달성하기 위하여, 본 발명은 항바이러스 활성, 면역활성, 항산화 활성, 유단백질 응고성, 내산성, 내담즙성, 항생제 내성, β-글루코시다제(β-glucosidase) 분비능, γ-용혈 활성, 유해미생물에 대한 항균활성이 있고, 바이오제닉 아민, 유해물질 및 유해효소를 생성하지 않는 페디오코커스 애시디락티시(Pediococcus acidilactici) SRCM102615 균주(KCCM12241P)를 제공한다.In order to accomplish the object of the present invention, the present invention provides a pharmaceutical composition comprising an antiviral activity, an immunological activity, an antioxidative activity, a milk protein coagulant, an acid tolerance, a bile resistance, an antibiotic resistance, a β- activity, the antimicrobial activity of the microorganisms, and provides a biogenic amine, harmful substances and Phedi O Lactococcus Ke does not produce the harmful enzymes CD rakti when (Pediococcus acidilactici) SRCM102615 strain (KCCM12241P).
본 발명에서 장아찌로부터 균주를 분리하였고, 그 중 항바이러스 활성, 면역활성, 항산화 활성, 유단백질 응고성, 내산성, 내담즙성, 항생제 내성, β-글루코시다제(β-glucosidase) 분비능, γ-용혈 활성, 유해미생물에 대한 항균활성이 있고, 바이오제닉 아민, 유해물질 및 유해효소를 생성하지 않는 페디오코커스 애시디락티시(Pediococcus acidilactici) SRCM102615 균주를 확인하였으며, 이를 페디오코커스 애시디락티시(Pediococcus acidilactici) SRCM102615 균주로 동정하여 2018년 03월 28일 한국미생물보존센터(KCCM)에 기탁하여 기탁번호 KCCM12241P를 부여받았다.In the present invention, strains were isolated from pickles, and antibiotic activity, immunological activity, antioxidative activity, lipid-lowering activity, acid-fast bacilli, antibiotic resistance, β-glucosidase secretion ability, Pediococcus acidilactici SRCM102615 strain, which has antimicrobial activity against active and harmful microorganisms and does not produce biogenic amines, harmful substances and harmful enzymes, was identified, and it was confirmed that Pediococcus acidiflactii ( Pediococcus acidilactici Pediococcus acidilactici ) SRCM102615, deposited on March 28, 2018 at the Korean Society for Microbiological Conservation (KCCM) and received the deposit number KCCM12241P.
본 발명의 일 구현 예에 따른 균주에서, 상기 내산성은 pH3~5에서 내산성일 수 있으며, 내담즙성은 0.1~1.0% 담즙에서 내담즙성일 수 있다.In a strain according to an embodiment of the present invention, the acid resistance may be acidic at pH 3-5, and the bile resistance may be intra-bile in 0.1-1.0% bile.
본 발명의 일 구현 예에 따른 균주에서, 상기 바이러스는 인플루엔자 A 바이러스(Influenza A viruses)이고, 상기 항생제는 아미카신(Amikacin), 아목시실린(Amoxicillin)+클라불란산(Clavulanic acid), 세프타지딤(Ceftazidime), 세파렉신(Cephalexin), 시프로플록사신(Ciprofloxacin), 푸시딘산(Fusidic acid), 젠타마이신(Gentamicin), 카나마이신(Kanamycin), 네틸미신(Netilmicin), 메티실린(Methicillin), 날리딕스산(Nalidixic acid), 네오마이신(Neomycin), 노르플록사신(Norfloxacin), 오플록사신(Ofloxacin), 피페미드산(Pipemidic acid), 스트렙토마이신(Streptomycin), 테이코플라닌(Teicoplanin), 트리메소프림(Trimethoprim), 트리메소프림-설파메톡사졸(Trimethoprim-Sulfamethoxazole) 및 반코마이신(Vancomycin) 계열의 항생제이며, 상기 유해미생물은 바실러스 세레우스(Bacillus cereus), 바실러스 스패리쿠스(Bacillus sphaericus), 엔테로코커스 패시움(Enterococcus faecium), 리스테리아 이바노비(Listeria ivanovii), 리스테리아 모노시토게네스(Listeria monocytogenes), 스타필로코커스 아우레우스(Staphylococcus aureus), 엔테로코쿠스 에로게네스(Enterococcus aerogenes), 에스케리키아 콜라이(Escherichia coli), 쉬겔라 플렉스네리(Shigella flexneri) 및 쉬겔라 손네이(Shigella sonnei)이며, 상기 바이오제닉 아민은 히스타민 및 티라민이며, 상기 유해물질은 인돌 및 페닐피루브산이며, 상기 유해효소는 β-글루쿠로니다아제 및 우레아제일 수 있으나, 이에 제한되지 않는다.In a strain according to an embodiment of the present invention, the virus is Influenza A viruses and the antibiotic is selected from the group consisting of Amikacin, Amoxicillin + Clavulanic acid, A drug such as ceftazidime, cephalexin, ciprofloxacin, fusidic acid, gentamicin, kanamycin, netilmicin, methicillin, nalidixic, acid, neomycin, Norfloxacin, Ofloxacin, Pipemidic acid, Streptomycin, Teicoplanin, Trimethoprim, Trimethoprim, Trimethoprim-Sulfamethoxazole and Vancomycin, and the harmful microorganisms are selected from the group consisting of Bacillus cereus , Bacillus sphaericus , Enterococcus faecium , Listeria ivanovii , Listeria monocytogenes , Staphylococcus aureus , Enterococcus aerogenes , Escherichia, Escherichia coli , Shigella flexneri , and Shigella sonnei . The biogenic amine is histamine and tyramine. The harmful substance is indole and phenylpyruvic acid. The harmful enzyme is β - glucuronidase and urease, but are not limited thereto.
상기 페디오코커스 애시디락티시(Pediococcus acidilactici) SRCM102615 균주는 β-글루코시다제를 분비하며, 추가적으로 류신 아릴아미다제(leucine arylamidase), 발린 아릴아미다제(valine arylamidase), 시스틴 아릴아미다제(cystine arylamidase), 산성 포스파타제(acid phosphatase), α-갈락토시다제(α-galactosidase), β-갈락토시다제(β-galactosidase) 및 N-아세틸-β-글루코사미니다제(N-acetyl-β-glucosaminidase)를 분비할 수 있으나, 이에 제한되지 않는다.The Pediococcus acidilactici strain SRCM102615 secretes beta -glucosidase and additionally contains a leucine arylamidase, a valine arylamidase, a cystine arylamidase ), Acid phosphatase,? -Galactosidase,? -Galactosidase and N-acetyl-β-galactosidase. glucosaminidase, but is not limited thereto.
또한, 본 발명은 상기 균주, 이의 배양액, 상기 배양액의 농축액 또는 그의 건조물을 유효성분으로 포함하는 프로바이오틱스 제제를 제공한다.The present invention also provides a probiotic preparation comprising the strain, a culture thereof, a concentrate of the culture broth, or a dried product thereof as an active ingredient.
상기 프로바이오틱스 제제는 당업계에 공지된 방법에 따라 다양한 제형과 방법으로 제조 및 투여될 수 있다. 예를 들어, 본 발명의 페디오코커스 애시디락티시(Pediococcus acidilactici) SRCM102615 균주, 이의 배양액, 상기 배양액의 농축액 또는 그의 건조물은 약제학적 분야에서 통상적으로 사용되는 담체와 혼합하여 산제(powder), 액제(liquids and solutions), 정제(tablet), 캡슐(capsule), 시럽(syrup), 현탁제(suspension) 또는 과립제(granule) 등의 형태로 제조되어 투여될 수 있다. 상기 담체로는 예를 들어, 결합제, 활탁제, 붕해제, 부형제, 가용화제, 분산제, 안정화제, 현탁화제, 색소 및 향료 등일 수 있으나, 이에 제한되지 않는다. 또한, 투여 용량은 체내에서의 활성성분의 흡수도, 불활성률, 배설속도, 피투여자의 연령, 성별, 축종, 상태 및 질병의 중증 정도 등에 따라 적절히 선택할 수 있다.The probiotic agent can be prepared and administered in various formulations and methods according to methods known in the art. For example, the Pediococcus acidilactici strain SRCM102615 of the present invention, the culture solution thereof, the concentrated solution of the culture solution or the dried product thereof may be mixed with a carrier commonly used in the pharmaceutical field to prepare a powder, for example, in the form of liquids and solutions, tablets, capsules, syrups, suspensions or granules, and the like. Examples of the carrier include, but are not limited to, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring matters and fragrances. The administration dose can be appropriately selected depending on the degree of absorption of the active ingredient in the body, the inactivation rate, the excretion rate, the age, sex, strain, condition and severity of the disease.
본 발명의 균주를 배양하는 방법은 당업계에 통상적으로 이용되는 방법에 따라 배양할 수 있으며, 특별한 방법에 한정되는 것은 아니다.The method for culturing the strain of the present invention can be carried out according to a method commonly used in the art, and is not limited to a specific method.
본 발명의 균주를 배양하는 단계에서 얻어지는 상기 균주 또는 상기 균주의 배양물 또는 상기 균주의 배양액의 농축액을 첨가제로 사용할 경우, 상기 균주 또는 상기 균주의 배양물 또는 상기 균주의 배양액의 농축액을 그대로 첨가하거나 다른 첨가제를 함께 사용할 수 있으며, 통상적인 방법에 따라 적절하게 사용될 수 있다 유효 성분의 혼합양은 그의 사용 목적에 따라 적합하게 결정될 수 있다.When the strain obtained in the step of culturing the strain of the present invention or the culture of the strain or the concentrated liquid of the culture medium of the strain is used as an additive, the culture of the strain or the strain or the concentrated liquid of the culture of the strain is directly added Other additives can be used together and can be suitably used according to conventional methods. The amount of the active ingredient to be mixed can be suitably determined according to the purpose of use thereof.
또한, 본 발명은 상기 프로바이오틱스 제제를 포함하는 식품을 제공한다.The present invention also provides a food comprising the probiotic agent.
본 발명의 상기 프로바이오틱스 제제는 항바이러스 활성, 면역활성, 항산화 활성, 유단백질 응고성, 내산성, 내담즙성, 항생제 내성, β-글루코시다제(β-glucosidase) 분비능, γ-용혈 활성, 유해미생물에 대한 항균활성이 있고, 바이오제닉 아민, 유해물질 및 유해효소를 생성하지 않는 페디오코커스 애시디락티시(Pediococcus acidilactici) SRCM102615 균주, 이의 배양액, 상기 배양액의 농축액 또는 그의 건조물을 유효성분으로 포함한다.The probiotic agent of the present invention can be used as an antibiotic agent for the prevention and treatment of various diseases such as antiviral activity, immunological activity, antioxidant activity, milk protein solidity, acid resistance, biliary cholesterol, antibiotic resistance,? -Glucosidase secretion ability, And a Pediococcus acidilactici SRCM102615 strain which does not produce harmful substances and harmful enzymes, a culture thereof, a concentrate of the culture solution or a dried product thereof as an active ingredient.
본 발명의 상기 프로바이오틱스를 식품첨가물로 사용하는 경우, 상기 프로바이오틱스를 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효 성분의 혼합양은 그의 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품 또는 음료의 제조시에 본 발명의 프로바이오틱스는 원료에 대하여 15 중량부 이하, 바람직하게는 10 중량부 이하의 양으로 첨가된다. 그러나, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 혼합양은 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 혼합양은 상기 범위 이상의 양으로도 사용될 수 있다.When the probiotics of the present invention are used as a food additive, the probiotics can be directly added or used together with other food or food ingredients, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to its intended use (prevention, health or therapeutic treatment). Generally, the probiotics of the present invention are added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight, based on the raw material, in the production of foods or beverages. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of controlling health, the mixing amount may be less than the above range, and since there is no problem in terms of safety, the mixing amount may be used in an amount exceeding the above range.
상기 식품의 종류에는 특별한 제한은 없다. 상기 프로바이오틱스를 첨가할 수 있는 식품의 예로는 빵, 캔디류, 스낵류, 과자류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강식품을 모두 포함한다.There is no particular limitation on the kind of the food. Examples of foods to which the probiotics can be added include breads, candies, snacks, confectionery, gums, dairy products including ice cream, various soups, drinks, tea, drinks, and vitamin complexes. .
또한, 본 발명은 상기 균주, 이의 배양액, 상기 배양액의 농축액 또는 그의 건조물을 유효성분으로 함유하는 바실러스 세레우스, 바실러스 스패리쿠스, 엔테로코커스 패시움, 리스테리아 이바노비, 리스테리아 모노시토게네스, 스타필로코커스 아우레우스, 엔테로코쿠스 에로게네스, 에스케리키아 콜라이, 쉬겔라 플렉스네리 또는 쉬겔라 손네이에 대한 항균용 조성물을 제공한다.The present invention also relates to a method for producing a microorganism, which comprises culturing the above strain, a culture thereof, a concentrate of the culture broth or a dried product thereof as an active ingredient, such as Bacillus cereus, Bacillus sparkicus, Enterococcus sp., Listeria ivanovi, Listeria monocytogenes, There is provided a composition for antimicrobial use against Cercus aureus, Enterococcus erogenes, Escherichia coli, Shigella flexneri, or Shigella sinnai.
본 발명의 항균 조성물이란 항미생물제를 총칭하는 의미인 항생제와 같은 의미일 수 있고, 항진균제, 살균제, 방부제, 보존제 또는 제균제와 같은 의미일 수 있으며, 바람직하게는 바실러스 세레우스, 바실러스 스패리쿠스, 엔테로코커스 패시움, 리스테리아 이바노비, 리스테리아 모노시토게네스, 스타필로코커스 아우레우스, 엔테로코쿠스 에로게네스, 에스케리키아 콜라이, 쉬겔라 플렉스네리 또는 쉬겔라 손네이 등을 포함하는 병원성 미생물의 발육과 생활 기능을 저지 또는 억제할 수 있는 물질을 의미할 수 있으나, 이에 제한되지 않는다.The antimicrobial composition of the present invention may have the same meaning as antibiotics, which is generically referred to as an antimicrobial agent, and may have the same meaning as an antifungal agent, a bactericide, an antiseptic, a preservative or a bactericidal agent, and preferably Bacillus cereus, Bacillus sp. Development of pathogenic microorganisms including Enterococcus lasserium, Listeria monocytogenes, Staphylococcus aureus, Enterococcus erogenes, Escherichia coli, Shigella flexneri, and Shigella sinnai. And a substance capable of inhibiting or inhibiting a living function, but the present invention is not limited thereto.
본 발명의 항균 조성물은 페디오코커스 애시디락티시(Pediococcus acidilactici) SRCM102615 균주, 이의 배양액, 상기 배양액의 농축액 또는 그의 건조물 외에 당질, 단백질, 지질, 비타민류 및 미네랄류를 포함할 수 있다. 상기 당질, 단백질, 지질, 비타민류 또는 미네랄류는 그 사용 목적 및 용도에 따라 적의 선택될 수 있으며, 일 예로 상기 당질은 벌꿀, 덱스트린, 수크로오스, 팔라티노스, 포도당, 과당, 물엿, 당알콜(sugar alcohol), 소르비톨, 크실리톨, 말티톨일 수 있고 상기 단백질은 카제인(casein), 유청 단백질(whey protein) 등의 우유 유래 단백질, 대두 단백질, 이들 단백질의 트립신, 펩신 등의 동물 유래 효소 및 뉴트라아제(neutrase), 알칼라아제(alkilase)에 의한 가수분해물일 수 있으며, 상기 지질은 제1가 포화지방산, 다가 불포화지방산을 포함하는 해바라기유, 채종유(rapeseed oil), 올리브유, 홍화유(safflower oil), 옥수수유, 대두유, 팜유(palm oil), 야자유 등의 각종 식물 유래 유지, 중쇄 지방산(middle-chain fatty acid), EPA, DHA, 대두유래 인지질, 우유 유래 인지질일 수 있고, 상기 미네랄류는 인산칼륨, 탄산칼륨, 염화칼륨, 염화나트륨, 유산칼슘, 글루콘산칼슘, 판토텐산칼슘, 카제인칼슘, 염화마그네슘, 황산제1철, 탄산수소나트륨일 수 있으나, 각각의 예에 의해 특별히 제한되는 것은 아니다.The antimicrobial composition of the present invention may contain saccharides, proteins, lipids, vitamins and minerals in addition to the Pediococcus acidilactici SRCM102615 strain, the culture thereof, the concentrate of the culture or the dried product thereof. The saccharide may be selected from the group consisting of honey, dextrin, sucrose, palatinose, glucose, fructose, starch syrup, sugar alcohol and the like. The saccharide, protein, lipid, vitamins or minerals, , Sorbitol, xylitol and maltitol. The protein may be milk-derived proteins such as casein, whey protein, soybean protein, animal-derived enzymes such as trypsin and pepsin of these proteins, and neutrase The lipid may be a first saturated fatty acid, a sunflower oil containing a polyunsaturated fatty acid, a rapeseed oil, an olive oil, a safflower oil, a corn oil, Derived fat such as soybean oil, palm oil and palm oil, middle-chain fatty acid, EPA, DHA, soybean-derived phospholipid, milk-derived phospholipid, The RAL fluids may be potassium phosphate, potassium carbonate, potassium chloride, sodium chloride, calcium lactate, calcium gluconate, calcium pantothenate, casein calcium, magnesium chloride, ferrous sulfate, sodium bicarbonate, but are not particularly limited by each example .
또한, 본 발명은 상기 균주, 이의 배양액, 상기 배양액의 농축액 또는 그의 건조물을 유효성분으로 함유하는 발효식품 제조용 스타터(starter) 조성물을 제공한다.The present invention also provides a starter composition for the production of a fermented food, which contains the strain, a culture solution thereof, a concentrate of the culture solution or a dried product thereof as an active ingredient.
본 발명에 있어서, 발효식품 제조용 스타터(starter)란 발효식품 제조를 위해 발효에 관여하는 미생물을 포함하는 제제 또는 조성물을 의미한다. 발효식품 제조 시에 첨가함으로써 발효된 식품에서 생장할 수 있는 미생물 또는 우점종으로 생장할 수 있는 미생물을 제공하기 위하여 사용된다. 상기 식품 발효용 스타터를 사용하여 식품을 제조하는 경우, 상기 식품 발효용 스타터에 포함된 미생물에 의하여, 식품의 품질을 일정하게 조절하거나, 특정한 목적, 일 예로 식품에서 이취를 발생시키지 않거나, 감소시키는 목적을 달성할 수 있다. 본 발명에서는 페디오코커스 애시디락티시(Pediococcus acidilactici) SRCM102615 균주 또는 이의 배양액을 스타터 균주로 이용함으로써, 항바이러스 활성, 면역활성, 항산화 활성, 유단백질 응고성, 내산성, 내담즙성, 항생제 내성, β-글루코시다제(β-glucosidase) 분비능, γ-용혈 활성, 유해미생물에 대한 항균활성이 있고, 바이오제닉 아민, 유해물질 및 유해효소를 생성하지 않는 발효 식품을 제조할 수 있다. In the present invention, a starter for fermented food preparation means a preparation or a composition containing microorganisms involved in fermentation for producing fermented food. And is used to provide a microorganism capable of growing in a fermented food or a microorganism capable of growing as a dominant species by adding at the time of producing fermented food. When the foodstuff is produced using the foodstuff fermenting starter, the microorganism contained in the foodstuff fermentation starter may be used to control the quality of the foodstuffs constantly or to control the quality of the foodstuffs for a specific purpose, Purpose can be achieved. In the present invention, Pediococcus acidilactici SRCM102615 strain or a culture thereof is used as a starter strain for antiviral activity, immunological activity, antioxidative activity, milk protein solidity, acid resistance, bile resistance, antibiotic resistance, A fermented food which has a β-glucosidase releasing activity, a γ-hemolytic activity, an antimicrobial activity against harmful microorganisms, and does not produce a biogenic amine, a harmful substance and a harmful enzyme can be produced.
본 발명의 일 구현 예에 따른 발효식품 제조용 스타터 조성물에서, 상기 발효식품은 김치, 장류 또는 발효유 등일 수 있으나, 이에 제한되지 않는다.In the starter composition for manufacturing fermented food according to an embodiment of the present invention, the fermented food may be, but not limited to, kimchi, soybean paste or fermented milk.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.
재료 및 방법Materials and methods
고추장아찌로부터 유산균의 분리Isolation of lactic acid bacteria from pepper pickles
유산균 분리를 위하여 냉장 보관한 고추장아찌 시료 1g을 취하여 멸균된 0.85% NaCl 용액 9 ㎖에 각 단계별로 희석하였다. 희석액 100 ㎕을 락토바실러스 MRS 아가 (DifcoTM,MI, USA) 배지를 함유하는 플레이트 상에 도말하여 37℃에서 48시간 배양한 후 유산균과 유사한 형태적 특징을 갖는 균주를 1차 선별하였다. 순수 분리한 유산균은 MRS 고체 배지에 배양하여 10% 스킴 밀크(BD Difco, Sparks, MD, USA) 용액에 현탁하고 -80℃에 보관하여 사용하였다.For the isolation of lactic acid bacteria, 1 g of red pepper jellyfish sample was chilled and stored in 9 ml of sterilized 0.85% NaCl solution. 100 μl of the diluted solution was plated on a plate containing Lactobacillus MRS agar (Difco ™, MI, USA) medium and cultured at 37 ° C for 48 hours, and strains having morphological characteristics similar to those of lactic acid bacteria were firstly screened. The purely isolated lactic acid bacteria were cultured on MRS solid medium, suspended in 10% skim milk (BD Difco, Sparks, MD, USA) and stored at -80 ° C.
면역 및 항바이러스 활성 측정을 위한 유산균 배양액 제조 및 세포 배양Preparation of Lactic Acid Bacteria Culture and Cell Culture for Measurement of Immune and Antiviral Activity
선별 유산균의 면역 및 항바이러스 활성 측정을 위한 배양액을 제조하기 위해 50 ㎖ MRS 브로스에 접종하고 30℃에서 150rpm으로 24시간 배양하였다. 이 유산균 배양액은 3000rpm으로 30분간 원심분리하여 균체를 제거한 후 회수된 상층액은 중성 pH로 적정한 후, 0.22㎛ 필터로 여과하여 본 실험에 사용하였다. 선별 유산균의 항바이러스 활성과 면역 증강 효과를 확인하기 위하여 마크로파지 세포 라인(macrophage cell line)인 RAW264.7(TIB-71)을 사용하였다. RAW264.7(TIB-71)은 10% FBS (Fetal Bovine Serum, Gibco, #16000-044 500mililiter)와 1% 항세균 및 항진균 물질(Anti-Anti 100X, Gibco, #15240-062)이 함유된 DMEM (Dulbecco's Modified Eagles Medium, High Glucose, HyClone #SH30243.01) 배지를 사용하여 37℃, 5% CO2 배양기에서 하루에 한번 계대를 진행하며 배양하였다.In order to prepare a culture medium for measuring immunity and antiviral activity of the lactic acid bacteria, 50 ml of MRS broth was inoculated and cultured at 30 DEG C and 150 rpm for 24 hours. The culture broth of the lactic acid bacteria was centrifuged at 3000 rpm for 30 minutes to remove the cells, and the recovered supernatant was titrated with neutral pH and filtered with a 0.22 μm filter. RAW264.7 (TIB-71), a macrophage cell line, was used to confirm the antiviral activity and immunostimulating effect of the selective lactobacillus. RAW264.7 (TIB-71) was cultured in DMEM containing 10% FBS (Fetal Bovine Serum, Gibco, # 16000-044 500 mililiter) and 1% antibacterial and antifungal substances (Anti-Anti 100X, Gibco, # 15240-062) (Dulbecco's Modified Eagles Medium, High Glucose, HyClone # SH30243.01) medium at 37 ° C in a 5% CO 2 incubator once a day.
항바이러스 활성Antiviral activity
분리 균주의 항바이러스 활성을 측정하기 위해 마우스 마크로파지(Mouse macrophage)에서 A/PR(Puerto Rico)/8/34-GFP 감염억제 효과와 사이토카인(cytokine)의 일종으로 바이러스 감염에 대한 생체의 방어기구인자로 알려진 인터페론-β(IFN-β)의 분비능을 측정하였다.In order to measure the antiviral activity of the isolated strain, a mouse macrophage inhibited the A / PR (Puerto Rico) / 8/34-GFP infection and a cytokine, a vital defense agent against viral infection (IFN-?), Which is known to be an inhibitor of interferon-beta.
먼저 마우스 마크로파지에서 A/PR(Puerto Rico)/8/34-GFP 감염억제 효과를 확인하기 위해 RAW264.7(TIB-71) 세포는 10% FBS와 1% 항세균 및 항진균 물질이 함유된 DMEM 배지를 이용하여 24-웰 배지 플레이트(CORNING)에 2×105 세포/웰의 농도로 분주하여 37℃, 5% CO2 배양기에서 약 16시간 배양한 후, FBS를 제거하기 위해 500㎕ PBS 버퍼를 각 웰에 첨가하여 세포를 세척하였다. 분리 균주 배양상등액은 1% FBS와 1% 항생물질이 함유된 DMEM 배지를 사용하여 최종농도가 4%가 되도록 희석하여 세포에 처리하고 37℃, 5% CO2 배양기에서 8시간 배양한 후, 배양 상등액을 제거하고 PBS 완충액을 사용하여 세척하였다. 인플루엔자 A 바이러스 (Green Fluorescent Protein (GFP)-tagged A/PR/8/34, A/PR/8/34-GFP, H1N1-GFP-PR8)는 0.1MOI가 되도록 FBS(-) DMEM (무혈청) 배지를 사용하여 희석한 후 500 ㎕/웰씩 세포에 넣어준 뒤 1시간 감염 과정을 수행하였다. PBS 완충용액을 사용하여 세척한 후, 1% FBS와 1% 항생물질 함유된 DMEM 배지를 500 ㎕/웰씩 넣어주고 37℃, 5% CO2 배양기에서 12시간 배양하였다. Raw cell 264.7에 인플루엔자 A 바이러스의 감염억제 효과의 확인은 Fluorescence-activated cell sorting (FACS, Guava® easyCyte Flow Cytometers)을 이용하여 측정하였다. 인플루엔자 A 바이러스의 감염억제 효과의 확인을 위한 양성대조구 물질로는 LPS(lipopolysaccharide)와 항바이러스 활성 보유 특허균주인 류코노스톡 메센테로이데스(Leuconostoc mesenteroides) BLS35를 사용하였다.RAW264.7 (TIB-71) cells were cultured in DMEM medium containing 10% FBS and 1% anti-bacterial and antifungal substances to confirm the inhibitory effect of A / PR (Puerto Rico) / 8 / Well plate (CORNING) at a concentration of 2 × 10 5 cells / well, and cultured in a 5% CO 2 incubator at 37 ° C. for about 16 hours. Then, 500 μl of PBS buffer was added to remove FBS Were added to each well to wash the cells. The isolated culture supernatant was diluted to a final concentration of 4% using DMEM medium containing 1% FBS and 1% antibiotic, and cultured for 8 hours at 37 ° C in a 5% CO 2 incubator. The supernatant was removed and washed with PBS buffer. (-) DMEM (serum-free) to be 0.1 MOI for influenza A virus (Green Fluorescent Protein (GFP) -tagged A / PR / 8/34, A / PR / 8/34-GFP, H1N1- After dilution with the medium, the cells were added to the cells at 500 μl / well, followed by infection for 1 hour. After washing with PBS buffer, DMEM medium containing 1% FBS and 1% antibiotic was added at a rate of 500 μl / well and cultured at 37 ° C in a 5% CO 2 incubator for 12 hours. Raw cell 264.7 was tested for inhibition of influenza A virus infection using fluorescence-activated cell sorting (FACS, Guava ® easyCyte Flow Cytometers). LPS (lipopolysaccharide) and Leuconostoc mesenteroides BLS35, a patented antiviral activity strain, were used as positive control substances to confirm infection inhibition effect of influenza A virus.
IFN-β의 분비능을 측정하기 위해 RAW264.7(TIB-71) 세포는 10% FBS와 1% 항세균 및 항진균 물질이 함유된 DMEM 배지를 이용하여 24-웰 배양 플레이트 (CORNING)에 2×105 세포/웰의 농도로 분주하여 37℃, 5% CO2 배양기에서 약 16시간 배양한 후, FBS를 제거하기 위해 500㎕ PBS 버퍼를 각 웰에 첨가하여 세포를 세척하였다. 분리 균주 배양상등액은 1% FBS와 1% 항생물질이 함유된 DMEM 배지를 사용하여 최종농도가 4%가 되도록 희석하여 세포에 처리하고 37℃, 5% CO2 배양기에서 24시간 배양한 후, 상등액으로 분비된 IFN-β의 양을 VerkineTM Interferon beta ELISA kit (PBL Assay Science, NJ, USA)을 이용하여 측정하였다. 회수한 상등액 시료는 희석 버퍼로 10배 희석하여 항체가 코팅된 96-웰 플레이트에 10㎕씩 넣어준 뒤 1시간 상온에서 방치하였다. 세척 버퍼를 300 ㎕/웰씩 넣어 3회 세척 후, 항체 용액을 농축 용액으로 희석하여 제조한 후 100㎕씩 플레이트에 넣어준 뒤 1시간 상온에서 방치하였다. 세척 버퍼를 300 ㎕/웰씩 넣어 3회 세척 후, HRP 콘쥬게이트 농축액을 농축 용액으로 희석하여 이를 100 ㎕/웰씩 넣어준 후 1시간 상온에서 방치하였다. 세척 버퍼를 300 ㎕/웰씩 넣어 3회 세척 후, TMB 기질을 100㎕/웰씩 넣은 후 암소에서 15분 방치하였다. 정지 용액(Stop solution)을 웰당 100 ㎕씩 넣어 반응을 중지시키고, SpectraMax 190 마이크로플레이트 리더 (Molecular Devices, US)를 이용하여 450 nm에서 흡광도를 측정하였다. 450 nm에서 흡광도를 측정하여 얻어진 O.D값을 표준물질을 이용해 작성한 표준곡선과 비교하여 IFN-β의 양을 측정하였다. 표준물질로는 마우스 IFN-β 스탠다드를 사용하였으며 농도는 15.6 pg/㎖에서 1000 pg/㎖까지 희석버퍼로 2배씩 희석하여 얻은 표준곡선과 비교하여 계산하였다. IFN-β 분비능의 양성대조구 물질로는 LPS(lipopolysaccharide)와 항바이러스 활성 보유 특허균주인 류코노스톡 메센테로이데스(Leuconostoc mesenteroides) BLS35를 사용하였다. RAW264.7 (TIB-71) cells were cultured in a 24-well culture plate (CORNING) using DMEM medium containing 10% FBS and 1% anti-bacterial and antifungal substances at a concentration of 2 x 10 < 5 cells / well, and cultured at 37 ° C in a 5% CO 2 incubator for about 16 hours. To remove FBS, 500 μl PBS buffer was added to each well to wash the cells. The isolated culture supernatant was diluted to a final concentration of 4% using DMEM medium containing 1% FBS and 1% antibiotic, and cultured for 24 hours at 37 ° C in a 5% CO 2 incubator. Then, Was measured using a Verkine Interferon beta ELISA kit (PBL Assay Science, NJ, USA). The recovered supernatant samples were diluted 10-fold with dilution buffer and added to the 96-well plate coated with antibody in an amount of 10 μl, and then left at room temperature for 1 hour. The washing solution was washed three times with 300 μl / well of the washing buffer, and the antibody solution was diluted with the concentrated solution. Then, 100 μl of the antibody solution was added to the plate, followed by standing at room temperature for 1 hour. Washing buffer was added 300 μl / well and washed three times. HRP conjugate concentrate was diluted with concentrated solution, and 100 μl / well was added thereto. Then, the mixture was left at room temperature for 1 hour. Wash buffer was added at a rate of 300 μl / well. After washing three times, the TMB substrate was added at a rate of 100 μl / well and left in a dark place for 15 minutes. The reaction was stopped by adding 100 μl of stop solution per well and the absorbance was measured at 450 nm using a SpectraMax 190 microplate reader (Molecular Devices, US). The OD value obtained by measuring the absorbance at 450 nm was compared with a standard curve prepared using a standard substance to measure the amount of IFN- ?. Mouse IFN-β standard was used as a standard, and the concentration was calculated by comparing with the standard curve obtained by diluting 2 times with dilution buffer from 15.6 pg / ㎖ to 1000 pg / ㎖. Lipopolysaccharide (LPS) and Leuconostoc mesenteroides BLS35, a patented antiviral activity strain, were used as positive control substances for IFN-β secretion.
면역활성 보유 미생물 평가 Evaluation of immunologically active microorganisms
분리 균주의 면역 증강 효과를 확인하기 위해 사이토카인인 TNF-α(tumor necrosis factor-α)의 분비능을 측정하였다. TNF-α의 분비능을 측정하기 위해 RAW264.7(TIB-71) 세포는 10% FBS와 1% 항세균 및 항진균 물질이 함유된 DMEM 배지를 이용하여 24-웰 배양 플레이트(CORNING)에 2×105 세포/웰의 농도로 분주하여 37℃, 5% CO2 배양기에서 약 16시간 배양한 후, FBS를 제거하기 위해 500 ㎕ PBS 완충용액을 각 웰에 첨가하여 세포를 세척하였다. 분리 균주 배양상등액은 1% FBS와 1% 항생제가 함유된 DMEM 배지를 사용하여 최종농도가 4%가 되도록 희석하여 세포에 처리하고 37℃, 5% CO2 배양기에서 24시간 배양한 후, 상등액으로 분비된 TNF-α의 양을 OptEIATM ELISA kit (BD Biosciences, SanDiego, CA, USA)을 이용하여 측정하였다. anti-cytokine capture antibody를 코팅 버퍼 (0.2 M 소듐 포스페이트, pH 6.5)에 희석하여 96 웰 플레이트(NUNC Co., Roskilde, Denmark)에 100 ㎕/웰씩 넣은 후, 4℃에서 하룻밤 방치하여 코팅(coating)하였다. Tween 20 (Sigma)을 PBS(phosphate buffered saline)에 0.05% (v/v)가 되도록 가한 PBS/Tween 용액으로 플레이트를 3회 세척한다. Assay diluent (PBS with 10% FBS)를 플레이트에 200 ㎕/웰씩 넣은 후 상온에서 1시간 방치하였다. PBS/Tween으로 3회 세척 후, 배양 상등액을 각 웰에 100 ㎕씩 넣어준 뒤 2시간 상온에서 방치하였다. PBS/Tween으로 5회 세척 후, 검출 항체인 비오틴화 항체와 효소 용액인 스트렙트아비딘-호스라디쉬 퍼록시다제 컨쥬게이트(streptavidin-horseradish peroxidase conjugate)를 분석 희석제(10% FBS를 포함하는 PBS)에 희석하여 플레이트에 100 ㎕/웰씩 넣은 후 상온에서 1시간 방치하였다. PBS/Tween으로 7회 세척 후, 기질 용액(TMB substrate reagent Set, BD Biosciences)를 100 ㎕/웰씩 넣은 후 암소에서 30분 방치하였다. 정지 용액인 2 N 황산용액을 웰당 50 ㎕ 씩 넣어 반응을 중지시키고, SpectraMax 190 Microplate Reader (Molecular Devices, US)를 이용하여 450 nm에서 흡광도를 측정하였다. 450 nm에서 흡광도를 측정하여 얻어진 O.D값을 표준물질을 이용해 작성한 표준곡선과 비교하여 TNF-α의 양을 측정하였다. 표준물질로는 재조합 마우스 TNF를 사용하였으며 농도는 15.6 pg/㎖에서 1000 pg/㎖까지 assay diluent (PBS with 10% FBS)로 2배씩 희석하여 얻은 표준곡선과 비교하여 계산하였다. TNF-α 분비능의 양성대조구 물질로는 LPS(lipopolysaccharide)와 항바이러스 활성 보유 특허균주인 류코노스톡 메센테로이데스(Leuconostoc mesenteroides) BLS35를 사용하였다. In order to confirm the immunostimulating effect of the isolated strain, the secretion ability of the cytokine TNF-α (tumor necrosis factor-α) was measured. RAW264.7 (TIB-71) cells were cultured in a 24-well culture plate (CORNING) using DMEM medium containing 10% FBS, 1% anti-bacterial and antifungal substances at a concentration of 2 x 10 < 5 cells / well, and cultured at 37 ° C in a 5% CO 2 incubator for about 16 hours. To remove FBS, 500 μl PBS buffer solution was added to each well to wash the cells. The isolated culture supernatant was diluted to a final concentration of 4% using DMEM medium containing 1% FBS and 1% antibiotic, and cultured in a 37 ° C, 5% CO 2 incubator for 24 hours. The amount of secreted TNF-α was measured using OptEIATM ELISA kit (BD Biosciences, San Diego, Calif., USA). anti-cytokine capture antibody was diluted in a coating buffer (0.2 M sodium phosphate, pH 6.5) and added to a 96-well plate (NUNC Co., Roskilde, Denmark) at a rate of 100 μl / well. Respectively. Tween 20 (Sigma) is washed three times with PBS / Tween solution added to PBS (phosphate buffered saline) to 0.05% (v / v). Assay diluent (PBS with 10% FBS) was added to the plate at 200 μl / well and left at room temperature for 1 hour. After washing three times with PBS / Tween, 100 μl of the culture supernatant was added to each well, followed by standing at room temperature for 2 hours. After washing 5 times with PBS / Tween, a biotinylated antibody as a detection antibody and streptavidin-horseradish peroxidase conjugate (an enzyme solution, streptavidin-horseradish peroxidase conjugate) were diluted in PBS containing 10% FBS, And 100 쨉 l / well was added to the plate, followed by allowing to stand at room temperature for 1 hour. After washing 7 times with PBS / Tween, 100 μl / well of a substrate solution (TMB substrate reagent set, BD Biosciences) was added and left in a dark place for 30 minutes. The reaction was stopped by adding 50 μl of 2 N sulfuric acid solution as a stop solution, and the absorbance was measured at 450 nm using a SpectraMax 190 Microplate Reader (Molecular Devices, US). The OD value obtained by measuring the absorbance at 450 nm was compared with a standard curve prepared using a standard substance to measure the amount of TNF- ?. Recombinant mouse TNF was used as the reference material and the concentration was calculated by comparing the standard curve obtained by diluting 2 times with the assay diluent (PBS with 10% FBS) from 15.6 pg / ㎖ to 1000 pg / ㎖. Positive control substances of TNF-a secretion ability include LPS (lipopolysaccharide) and the patented antiviral activity strain Leuconostoc mesenteroides BLS35 was used.
바이오제닉 아민 생성 확인Identification of biogenic amine production
바이오제닉 아민의 생성 여부 확인을 위한 정성실험 방법에 의하여 확인하였다. 분리 균주의 바이오제닉 아민 생성 확인 고체배지는 0.25% 글리세롤, 0.006% 브로모크레졸 퍼플, 0.1% 히스티딘 또는 티로신이 포함된 MRS 아가(pH 5.3) 배지를 제조하여 사용하였다. 분리 균주를 각 배지에 획선 도말 후 37℃에서 24시간 배양 후 대조구인 일반 MRS 배지와 발색 정도를 비교함에 따라 바이오제닉 아민의 생성 여부를 확인하였다.It was confirmed by the qualitative test method for confirming the generation of biogenic amine. Identification of biosynthetic amine production of isolated strains The solid medium was prepared by using MRS agar (pH 5.3) medium containing 0.25% glycerol, 0.006% bromocresol purple, 0.1% histidine or tyrosine. The isolated strains were streaked on each medium and cultured at 37 ° C for 24 hours. Then, the generation of the biogenic amines was confirmed by comparing the degree of color development with the control MRS medium.
항산화 활성 측정Antioxidant activity measurement
선별 균주의 항산화 활성을 측정하기 위해 DPPH(2,2-diphenyl-1-picryl-hydrazyl, Sigma-aldrich, MO, USA)의 방법에 따라 실험하였다. 분리 균주를 MRS 아가 배지에 획선 도말한 후 37℃ 배양기에서 48시간 배양하였으며 단일 콜로니를 5 ㎖ MRS 브로스에 접종하여 37℃, 150rpm 조건에서 24시간 배양하였다. 배양액 1 ㎖을 1.7 ㎖ 튜브에 취한 후 13,000rpm에서 10분간 원심분리하여 상등액을 회수하였다. 100 μM DPPH 메탄올 용액 180 ㎕에 20 ㎕의 상등액을 첨가한 후 반응 산물은 30분 동안 실온에서 반응시킨 후 마이크로플레이트 리더로 517nm에서 흡광도를 측정하여 아래 식에 따라 항산화 활성을 축정하였다. DPPH 라디컬 소거 활성의 비교는 천연 항산화제인 아스코르브산을 표준물질로 하여 측정하였다. DPPH (2,2-diphenyl-1-picryl-hydrazyl, Sigma-aldrich, MO, USA) was used to measure the antioxidant activity of the strain. The isolates were streaked on MRS agar medium and cultured in a 37 ° C incubator for 48 hours. Single colonies were inoculated into 5 ml MRS broth and cultured at 37 ° C and 150 rpm for 24 hours. 1 ml of the culture was taken in a 1.7 ml tube and centrifuged at 13,000 rpm for 10 minutes to recover the supernatant. 20 μl of supernatant was added to 180 μl of 100 μM DPPH methanol solution. The reaction product was reacted at room temperature for 30 minutes, and the absorbance at 517 nm was measured with a microplate reader. The DPPH radical scavenging activity was compared using ascorbic acid, a natural antioxidant, as a standard.
항산화활성 (%) = [1-(샘플의 Abs517nm)/(샘플의 Abs517nm)]Х100Antioxidant activity (%) = [1- (Abs 517nm of sample) / (Abs 517nm of sample)] X100
Abs : 517㎚에서 샘플의 DPPH 용액의 흡광도Abs: Absorbance of the DPPH solution of the sample at 517 nm
Abc : 517㎚에서 샘플이 없는 DPPH 용액의 흡광도Abc: Absorbance of sample-free DPPH solution at 517 nm
식품유해 미생물에 대한 항균활성 측정Measurement of antimicrobial activity against food harmful microorganisms
항균력을 조사하기 위해 분리 유산균은 MRS 액체배지에 접종하였고 37℃에서 24시간 배양 후, 13,000 rpm에서 10분간 원심분리하여 상등액을 회수하였다. 회수된 상등액은 0.45㎛ 시린지 필터 (Sartorius, Frankfurt, Germany)로 제균 후, 0.8% 소프트 아가 플레이트를 이용한 웰 확산법에 준하여 분리 균주의 상등액을 6mm의 웰에 100㎕씩 분주하였고, 30℃에서 24시간동안 배양하여 형성된 억제환의 크기를 측정하였다. 12종의 지시균주는 미생물자원센터(Korean Collection for Type Cultures, KCTC)와 한국미생물보존센터(Korean Culture Center of Microorganisms, KCCM)에서 분양 받고(표 1), 20% 글리세롤 스톡하여 -80℃에서 보관하여 사용하였다. 유해세균들은 각각 해당 배지에 알맞게 배양하여 균일한 농도(O.D.600, 0.4)로 조절한 후 0.8% 소프트 아가 플레이트에 접종하여 항균활성 측정을 위한 배지를 제조하였다.To investigate the antibacterial activity, isolated lactic acid bacteria were inoculated into MRS liquid culture medium, cultured at 37 ° C for 24 hours, and centrifuged at 13,000 rpm for 10 minutes to recover the supernatant. The recovered supernatant was sterilized with a 0.45 μm syringe filter (Sartorius, Frankfurt, Germany), and 100 μl of the supernatant of the isolated strain was dispensed into wells of 6 mm in accordance with the well diffusion method using a 0.8% soft agar plate. And the size of the inhibitory ring formed was measured. Twelve indicative strains were purchased from the Korean Collection for Type Cultures (KCTC) and the Korean Culture Center of Microorganisms (KCCM) (Table 1), stored at -80 ° C in 20% glycerol stock Respectively. The harmful bacteria were cultured to the appropriate medium, adjusted to a uniform concentration (OD 600 , 0.4), and then inoculated on a 0.8% soft agar plate to prepare a medium for antimicrobial activity measurement.
용혈성 및 유해대사산물과 유해효소 생성 분석Analysis of hemolytic and noxious metabolites and harmful enzyme production
선별된 균주의 용혈성 확인을 위해 혈액 아가 베이스 (BD, Difco)에 5% 양 혈액(MBcell, Korea)을 첨가하여 혈액 아가 배지를 제조하였다. 선별균주는 획선 도말하여 37℃에서 24시간 배양 후 균체 주위에 생기는 환의 형태로 α, β 및 γ 용혈성을 확인하였다. 그리고 암 유발 관련 유해대사산물인 인돌 및 페닐피루브산의 생성여부를 확인하기 위해 선별된 균주를 MRS (BD, Difco) 액체배지에 접종한 후 37℃에서 24시간 동안 진탕배양하고 인돌 용액 드로퍼 (BBL, BD Difco)를 첨가하여 배지 표면의 색 변화로 인돌 생성 여부를 확인하였다. 페닐피루브산 생성 여부는 페닐알라닌 아가(fluka, Buchs, switzerland) 배지에 균주를 접종하여 37℃에서 24, 48시간 배양 후 10% 염화 제2철 (MBcell, Korea)을 3-4 방울 첨가하여 균주 주위의 색 변화로 페닐피루브산 생성 여부를 확인하였다. 그리고 우레아제(Urease) 및 β-글루쿠로니다제(β-glucuronidase)와 같은 대장암 유발 유해효소의 생성여부를 확인하였다. 우레아제 생성 여부는 우레아 신속 테스트 키트(MB 세포)에 선별 균주를 접종하였고 바셀린 오일(MB 세포, 한국)을 표면에 첨가하여 혐기상태로 37℃에서 4 ~ 24시간 배양 후 색변화를 통해 확인하였다. β-글루쿠로니다제는 트립톤 담즙 X-글루쿠로니드 (TBX, Oxoid) 아가 배지에 선별 균주를 접종하여 37℃에서 24시간 배양 후 균주의 색 변화를 관찰하여 β-글루쿠로니다제 활성 여부를 확인하였다. Blood agar medium was prepared by adding 5% sheep blood (MBcell, Korea) to a blood agar base (BD, Difco) to identify the hemolytic activity of the selected strains. The selected strains were cultured for 24 hours at 37 ℃ after streaking, and α, β and γ hemolytic activity were confirmed in the form of rings around the cells. In order to confirm the production of indole and phenylpyruvic acid, which are harmful metabolites related to cancer, MRS (BD, Difco) liquid culture medium was inoculated with the selected strains, followed by shake culture at 37 ° C for 24 hours, and the indole solution dropper (BBL, BD Difco) was added to determine whether indole formation was caused by color change of the medium surface. The production of phenylpyruvic acid was determined by inoculating a strain in a phenylalanine agar (fluka, Buchs, switzerland) medium, culturing at 37 ° C for 24 hours and 48 hours, adding 3-4 drops of 10% ferric chloride (MBcell, Korea) And the presence of phenylpyruvic acid was confirmed by color change. And the production of a colon cancer-inducing enzyme such as urease and? -Glucuronidase was confirmed. Urease production was assessed by color change after 4-24 h incubation at 37 ° C in anaerobic condition with Vaseline oil (MB cells, Korea) added to the urea rapid test kit (MB cells). β-Glucuronidase was inoculated into the tryptone bile X-glucuronide (TBX, Oxoid) agar medium and incubated at 37 ° C. for 24 hours. The color change of the strain was observed to determine β- And the activity thereof was confirmed.
선별 유산균의 유단백질 응고성Coagulability of milk protein in selected lactic acid bacteria
유단백질의 응고성은 선별된 유산균을 MRS 브로스에서 37℃로 24시간 동안 배양한 다음 멸균된 10% 스킴 밀크(difco, USA)에 1%씩 접종하였다. 균주가 접종된 스킴 밀크는 37℃에서 5일 동안 정치배양 후 커드의 형성정도를 관찰하였고, 응고 정도에 따라 발효유 및 발효식품 제조용 스타터로서의 사용 가능성을 판단하였다. The coagulability of milk proteins was determined by incubating selected lactic acid bacteria in MRS broth at 37 ° C for 24 hours and then inoculating 1% in sterilized 10% skim milk (difco, USA). The skim milk inoculated with the strains was incubated for 5 days at 37 ° C and the degree of curd formation was observed. The degree of curd formation was determined according to the degree of coagulation as a starter for fermented milk and fermented food.
β-글루코시다제(β-glucosidase) 활성을 갖는 유산균의 선별Selection of lactic acid bacteria having? -glucosidase activity
β-글루코시다제 활성을 측정하기 위해 MRS 브로스에 에스쿨린 철 아가(Esculin Iron agar)를 혼합하여 MRS-EI 배지를 제조하였다. 선별 유산균 균주를 MRS-EI 배지에 접종하여 37℃에서 48시간 배양한 후 콜로니 주변의 색의 변화를 조사하여 갈색 또는 검정색 존(zone)을 형성하는 균주를 선별하였다.MRS-EI medium was prepared by mixing Esculin Iron agar with MRS broth to measure β-glucosidase activity. The strains were inoculated on MRS-EI medium and incubated at 37 ° C for 48 hours. The strains forming the brown or black zone were selected by examining the color change around the colonies.
내산성 및 내담즙성 확인Determination of acid resistance and bile resistance
위액과 담즙에 대한 내성을 갖는 균주를 선별하기 위해 내산성, 내담즙성 실험을 수행하였다. 내산성 분석을 위해 선별균주를 MRS 액체배지에 108 CFU/㎖가 되도록 접종하였다. 그리고 pH2.0, pH3.0, pH4.0 및 pH5.0으로 조절한 MRS 액체배지에 각각 선별균주를 1%씩 접종하여 37℃에서 3시간 정치배양 후 생균수를 측정하였다. pH를 조절 전 생균수를 대조구로 사용하였다. 또한 담즙산에 대한 내성을 분석하기 위해 선별된 균주는 MRS 액체배지에 초기 균수가 108 CFU/㎖가 되도록 접종하였고 옥스-바일 (Neogen, USA)의 농도가 0.1%, 0.3%, 0.5% 및 1.0%로 조절된 MRS 액체배지에 각각 1%씩 접종하여 37℃에서 6시간 배양 후 생균수를 측정하였다. 옥스갈(Oxgall) 첨가 전 생균수를 대조구로 사용하였으며, 생존율을 계산하여 내담즙성 정도를 확인하였다.In order to select strains resistant to gastric juice and bile, acid tolerance and bile test were performed. For acid resistance analysis, the strain was inoculated into the MRS liquid medium at a concentration of 10 8 CFU / ml. Then, 1% of each strain was inoculated into the MRS liquid medium adjusted to pH 2.0, pH 3.0, pH 4.0 and pH 5.0, and the number of viable cells was measured after incubation at 37 ° C for 3 hours. The number of viable cells before the pH control was used as a control. In order to analyze the tolerance to bile acids, the selected strains were inoculated into the MRS broth at an initial bacterial count of 10 8 CFU / ml, and the concentration of OX-Vyl (Neogen, USA) was 0.1%, 0.3%, 0.5% and 1.0 %, Respectively. After incubation at 37 ° C for 6 hours, the number of viable cells was measured. The number of viable cells before addition of Oxgall was used as a control, and the survival rate was calculated to confirm the degree of biliary bite.
항생제 감수성 확인Confirm antibiotic susceptibility
선별된 균주의 항생제 감수성 확인을 위하여 디스크 확산법(diffusion method)으로 실시하였다. 사용한 항생제 디스크(BBL Sensi-disc, Becton Dickinson Co, USA)는 아목시실린(Amoxicillin, 25㎍, AML), 아미카신(Amikacin, 30㎍, AK), 아목시실린 + 클라불란산(Clavulanic acid, 3㎍, AUG), 앰피실린(Ampicillin, 10㎍, AMP), 아지트로마이신(Azithromycin, 15㎍, AZM), 세파클러(Cefaclor, 30㎍, CEC), 세포탁심(Cefotaxime, 30㎍, CTX), 세프타지딤(Ceftazidime, 30㎍, CAZ), 세프록심(Cefuroxime, 30㎍, CXM), 세파렉신(Cephalexin, 30㎍, CL), 시프로플록사신(Ciprofloxacin, 5㎍, CIP), 클린다마이신(Clindamycin, 10㎍, CD), 에리트로마이신(Erythromycin, 15㎍, E), 푸시딘산(Fusidic acid, 30㎍, FC), 젠타마이신(Gentamicin, 30㎍, CN), 이미페넴(Imipenem, 10㎍, IMI), 카나마이신(Kanamycin, 30㎍, K), 린코마이신(Lincomycin, 15㎍, MY), 네틸미신(Netilmicin, 30㎍, NET), 메티실린(Methicillin, 5㎍, MET), 날리딕스산(Nalidixic acid, 30㎍, NA), 네오마이신(Neomycin, 30㎍, N), 니트로푸란토인(Nitrofurantoin, 300㎍, F), 노르플록사신(Norfloxacin, 10㎍, NOR), 오플록사신(Ofloxacin, 5㎍, OFX), 페니실린 G(Penicillin G, 1IU, P), 페니실린 G(10IU, P), 페니실린 G(2IU, P), 피페미드산(Pipemidic acid, 20㎍, PI), 피페라실린(Piperacillin, 100㎍, PRL), 리팜피신(Rifampicin, 5㎍, RD), 록시스로마이신(Roxithromycin, 15㎍, RXT), 스트렙토마이신(Streptomycin, 10㎍, S), 테이코플라닌(Teicoplanin, 30㎍, TEC), 테트라사이클린(Tetracycline, 30㎍, TE), 트리메소프림(Trimethoprim, 5㎍, TM), 트리메소프림-설파메톡사졸(Trimethoprim-Sulfamethoxazole, 25㎍, SXT), 반코마이신(Vancomycin, 30㎍, VA)으로 총 38종을 사용하였다. 감수성 시험방법은 최종균주를 이용하여 MRS 고체배지를 만든 후 디스크 디스펜서 8 (Liofilchem, Italy)에 항생제를 꽂아 순서대로 디스크를 올려놓고 37℃에서 24시간 배양하였다. 각 항생제에 대한 억제환 크기를 측정하고 CLSI(Clinical Laboratory Standards Institute)에 의해 감수성과 내성을 확인하였다. The antibiotic susceptibility of selected strains was examined by diffusion method. Amikacillin (25 μg, AML), Amikacin (30 μg, AK), amoxicillin + clavulanic acid (3 μg, AFP), Ampicillin (10 μg, AMP), Azithromycin (15 μg, AZM), Cefaclor (30 μg, CEC), Cefotaxime Cefuroxime (30 μg, CZM), cephalexin (30 μg, CL), ciprofloxacin (5 μg, CIP), clindamycin CD, Erythromycin, 15 μg, E, Fusidic acid, 30 μg, FC, Gentamicin, 30 μg, CN, Imipenem, 10 μg, IMI, Kanamycin , 30 μg of K), lincomycin (15 μg of MY), netilmicin (30 μg of NET), methicillin (5 μg of MET), nalidixic acid NA), neomycin (30 mu g, N), nitrofuran (Nifroxacin, 10 μg, NOR), Ofloxacin (5 μg, OFX), Penicillin G (1 IU, P), Penicillin G (10 IU, Pipemidic acid (20 μg, PI), Piperacillin (100 μg, PRL), Rifampicin (5 μg, RD), Roxithromycin Rexithromycin, 15 μg, RXT), streptomycin (10 μg, S), Teicoplanin, 30 μg, TEC, Tetracycline, 30 μg TE, Trimethoprim (Vectomycin, 30 μg, VA) were used in total of 38 species. For the susceptibility test, MRS solid medium was prepared using the final strain, and antibiotics were inserted into disk dispenser 8 (Liofilchem, Italy), and the disks were placed in order and cultured at 37 ° C for 24 hours. The inhibitory ring size for each antibiotic was measured and the sensitivity and tolerance were confirmed by CLSI (Clinical Laboratory Standards Institute).
당 이용성 확인Confirmation of availability
선별 균주의 당 이용성을 확인하기 위하여 총 49종의 탄수화물 이용성을 기초로 제작된 API 50 CHL kit (BioMerieux, France)를 사용하였다. 선별 균주를 5 ㎖의 MRS 액체배지에 계대배양하여 30℃에서 24시간 배양한 후 배양액 1 ㎖을 13,000 rpm에서 20분간 원심 분리하여 상층액을 걷어내고 세포 펠렛만 회수하여 PBS (phosphate buffered saline, 0.1M, pH 7.0)로 2회 세척한 뒤 실험에 사용하였다. PBS와 McFarland 스탠다드를 이용하여 2와 같은 탁도로 맞추어 현탁액을 제조하였고, API 50 CHL 배지에 현탁한 균주 배양액 1 ㎖을 첨가 후 혼합하여, API 50 CH 스트립에 120㎕씩 분주하고, 미네랄 오일(mineral oil)로 튜브 당 2~3방울씩 첨가하여 37℃에서 24시간 및 48시간 동안 배양한 후 각각의 당 발효패턴을 비교하였다. 당 발효 패턴은 튜브의 색 변화를 확인하여 양성 및 음성으로 판독하였다.
효소 생성능 확인 Confirm enzyme production ability
페디오코커스 애시디락티시 SRCM102615 균주의 효소 생성 여부를 조사하기 위해 API ZYM kit(bioMeriux Co., France)를 사용하여 알칼리성 포스파타제 외 19가지 효소활성을 검사하였다. 페디오코커스 애시디락티시 SRCM102615를 MRS 고체 배지에서 배양한 후 균체를 회수하여 0.85% NaCl 용액에 현탁해 5~6 Macfarland 스탠다드로 조정하였다. API ZYM kit의 각 각두(cupule)에 현탁액을 분주하고 37℃에서 4시간 배양한 후에 ZYM-A 및 ZYM-B 시약을 한 방울씩 떨어뜨려 색깔의 변화를 관찰함으로써, 효소 활성 정도를 조사하였다.In order to investigate the enzyme production of Pediococcus acidiflucis SRCM102615, 19 enzymatic activities such as alkaline phosphatase were examined using API ZYM kit (bioMeriux Co., France). After culturing Pediococcus acidiflucice SRCM102615 in MRS solid medium, the cells were collected, suspended in 0.85% NaCl solution and adjusted to 5-6 Macfarland standard. The suspension was added to each cupule of the API ZYM kit and incubated at 37 ° C for 4 hours. After that, the ZYM-A and ZYM-B reagents were dropped one by one to observe the change of color, and the degree of enzyme activity was examined.
선별 균주의 동정 및 계통수 작성Identification of strain and selection of strain
선별된 균주의 분자학적 동정 및 계통수 작성을 위해 균주를 TSB 브로스에 접종하여 37℃에서 24시간 배양한 후 원심분리하여 균체를 회수하고 ZR Fungal/Bacterial DNA Miniprep kit (Zymo Research Corp., CA., USA)를 이용하여 DNA를 추출하였다. 추출한 DNA는 유니버셜 프라이머인 785F(5'-GGATTAGATACCCTGGTA-3' : 서열번호 2)와 907R(5'-CCGTCAATTCMTTTRAGTTT-3' : 서열번호 3) 프라이머로 합성하여 16S rRNA 유전자 단편을 증폭시켰고, 증폭된 PCR 산물은 QIAquick PCR 정제 키트 (QIAGEN)를 사용하여 정제한 후 ㈜마크로젠에 의뢰하여 염기서열을 분석하였다. 분석결과를 NCBI(National Center for Biotechnology Information, Bethesda, MD, USA)의 뉴클레오티드 BLAST를 사용하여 GeneBank에 등록된 염기서열과 상동성을 비교하였다. 염기서열은 ExTaxone-e 서버(http://www.eztaxon.org)를 통해 표준균주의 염기서열을 확보하여 MEGA 7.0 프로그램을 사용하였으며, 염기서열간 상호비교 후 계통도를 작성하였다. 계통도는 Neighbor-joining 알고리즘을 사용하였으며, 1,000회 반복을 통해 부트스트랩핑하여 작성한 계통도의 견고성을 확인하였다.The strain was inoculated on TSB broth and incubated at 37 ° C for 24 hours. The cells were recovered by centrifugation and the ZR Fungal / Bacterial DNA Miniprep kit (Zymo Research Corp., CA., USA). The extracted DNA was synthesized with primers 785F (5'-GGATTAGATACCCTGGTA-3 ': SEQ ID NO: 2) and 907R (5'-CCGTCAATTCMTTTRAGTTT-3': SEQ ID NO: 3) to amplify 16S rRNA gene fragments. The product was purified using a QIAquick PCR purification kit (QIAGEN), and then submitted to Macrogen Co., Ltd. for analysis of the base sequence. The nucleotide BLAST of the NCBI (National Center for Biotechnology Information, Bethesda, MD, USA) was used to compare the homology with the nucleotide sequence registered in GeneBank. The nucleotide sequence of the standard strain was obtained through the ExTaxone-e server (http://www.eztaxon.org) and the MEGA 7.0 program was used. Neighbor-joining algorithm was used for the system diagram, and the robustness of the system was confirmed by bootstrapping over 1,000 iterations.
실시예 1. 선별 균주의 동정Example 1. Identification of a selection strain
SRCM102615의 16S rRNA 유전자 염기서열 분석 결과를 바탕으로 BLAST 검색한 결과 페디오코커스 애시디락티시(Pediococcus acidilactici)로 판명되었으며, Eztaxon server에서 표준 균주와 상동성을 비교 분석하였다. 서열 정렬 분석에 사용한 파라미터 및 옵션 값은 단백질 무게 매트릭스(protein weight matrix)로 고넷 시리즈(Gonnet series)를 사용하였고, 갭 오프닝 15, 갭 익스텐션 6.66, 딜레이 트란지션 웨이트 0.5로 설정하였으며, 갭 패널티는 5, K-터플 사이즈는 2, 탑 디아고날스(top diagonals)는 4, 윈도우 사이즈는 4로 설정하였다. 그 결과 SRCM102615(1,515 bp, 서열번호 1)는 페디오코커스 애시디락티시(DSM20284)와 99%의 상동성을 나타내었다. 염기서열을 이용하여 계통수를 작성하기 위해 분류학적 거리의 추론은 맥시멈 컴포지트 라이크후드(maximum composite Likehood) 방법을 모델로 사용하였다. 최종적으로 선별된 균주를 페디오코커스 애시디락티시(Pediococcus acidilactici) SRCM102615로 명명하였고, 2018년 3월 28일 한국미생물보존센터(KCCM)에 기탁하여 기탁번호 KCCM12241P를 부여받았다. Based on the results of 16S rRNA gene sequencing analysis of SRCM102615, BLAST was found to be Pediococcus acidilactici and the homology with standard strains was compared and analyzed on Eztaxon server. The parameters and option values used for the sequence alignment analysis were the Gonnet series as the protein weight matrix, the gap opening 15, the gap extension 6.66 and the delay transition weight 0.5, and the gap penalty was 5 , A K-tuple size of 2, top diagonals of 4, and a window size of 4. As a result, SRCM102615 (1,515 bp, SEQ ID NO: 1) showed 99% homology with Pediococcus acidiflucici (DSM20284). In order to construct the phylogenetic tree using the nucleotide sequence, the taxonomic distance inference was modeled by the maximum composite Likehood method. The finally selected strain was named Pediococcus acidilactici SRCM102615 and deposited on March 28, 2018 with the Korean Society for Microbiological Conservation (KCCM) to receive the deposit number KCCM12241P.
실시예 2. 항바이러스 활성Example 2. Antiviral activity
SRCM102615의 항바이러스 활성 확인을 위해 마우스 대식세포주인 Raw cell 264.7에 유산균의 배양여액을 처리한 후, GFP가 테그된 바이러스(A/PR(Puerto Rico)/8/34-GFP)에 감염시켜 대조군에 비해 바이러스에 감염 억제 정도를 확인하기 위해 FACS를 이용하여 측정하였다(도 1). 인플루엔자 A 바이러스 감염 억제 효과를 살펴본 결과, 페디오코커스 애시디락티시 SRCM102615는 75%, 대조구인 류코노스톡 메센테로이데스(Leuconostoc mesenteroides) BLS35는 65%로 확인되었다. 대조구보다 페디오코커스 애시디락티시 SRCM102615 균주가 인플루엔자 A 바이러스 감염 억제 효과가 우수함을 확인하였다.In order to confirm the antiviral activity of SRCM102615, GFP was infected with the tagged virus (A / PR (Puerto Rico) / 8/34-GFP) after treatment of lactic acid bacteria culture filtrate with mouse macrophage cell line Raw cell 264.7 (FACS) in order to confirm the degree of inhibition of virus infection (FIG. 1). Influenza A virus infection inhibitory effect was examined. As a result, Pediococcus acycloviridae SRCM102615 contained 75%, control group Leuconostoc mesenteroides , BLS35 was found to be 65%. It was confirmed that the strain Pediococcus asiadultiti SRCM102615 was superior to the control in that it inhibited influenza A virus infection.
SRCM102615의 IFN-β의 분비능은 항바이러스 특허 보유 균주인 류코노스톡 메센테로이데스 BLS35를 대조구로 하여 측정 결과를 도 2와 같이 나타내었다. IFN-β는 바이러스, 암세포 등 외부물질과 반응하여 분비되는 사이토카인으로 세포내 항암 및 항바이러스 작용을 일으켜 면역반응을 유도하는 물질이다. BLS35 균주의 IFN-β의 분비능을 측정한 결과 0.15±0 ng/㎖이 나왔으며 페디오코커스 애시디락티시 SRCM102615 균주는 0.24±0.01 ng/㎖으로 측정되었다. 대조구인 류코노스톡 메센테로이데스 BLS35 균주보다 페디오코커스 애시디락티시 SRCM102615 균주가 160% 활성이 우수한 것을 확인하였다.The secretion potency of IFN-? Of SRCM102615 was measured using Ryukono Stokeshenceteroids BLS35, which is an antiviral patented strain, as a control, and the measurement results are shown in Fig. IFN-β is a cytokine secreted by reacting with external substances such as viruses and cancer cells, and induces an immune response by causing an anti-cancer and anti-viral action. The secretion of IFN-? Of the BLS35 strain was measured to be 0.15 ± 0 ng / ml, and that of Pediococcus aceyltracis SRCM102615 was measured to be 0.24 ± 0.01 ng / ml. Control group Ryukono Stokeshen terroidis It was confirmed that the Pediococcus aceyltracis SRCM102615 strain was more excellent in the 160% activity than the BLS35 strain.
실시예 3. 면역 활성Example 3. Immunological activity
SRCM102615의 면역 활성은 항바이러스 특허 보유 균주인 류코노스톡 메센테로이데스 BLS35를 대조구로 하여 면역 활성 결과를 도 3과 같이 나타내었다. BLS35 균주의 TNF-α는 65.7±3.23 ng/㎖이 나왔으며 페디오코커스 애시디락티시 SRCM102615은 206.26±1.40 ng/㎖로 측정되었다. 대조구인 류코노스톡 메센테로이데스 BLS35 균주보다 페디오코커스 애시디락티시 SRCM102615 균주가 313.9% 면역 활성이 우수한 것을 확인하였다.The immunological activity of SRCM102615 was shown in FIG. 3, using Ryukono Stokeshenceteroids BLS35, which is an antiviral patented strain, as a control. TNF-α of the BLS35 strain was 65.7 ± 3.23 ng / ml, and that of Pediococcus acycloviricus SRCM102615 was 206.26 ± 1.40 ng / ml. Control group Ryukono Stokeshen terroidis It was confirmed that the Pediococcus aceyltracis SRCM102615 strain was superior to the BLS35 strain by 313.9%.
실시예 4. 바이오제닉 아민 생성 확인Example 4. Confirmation of production of biogenic amine
바이오제닉 아민은 주로 단백질이 함유되어 있는 발효식품에서 높은 농도로 검출되며, 식품의 발효 및 숙성과 저장과정에서 아미노산이나 탈탄산효소(decarboxylase) 생산 미생물에 의해 생성된다. 바이오제닉 아민 중 히스타민은 사람이 섭취 시 식중독과 알레르기를 유발하기도 하며, 티라민의 경우 혈관수축인자들을 분비해 고혈압을 일으킬 수 있다. 따라서 바이오제닉 아민 정성 실험 결과 전구체 아미노산인 히스티딘과 티로신을 첨가한 배지에서 SRCM102615 균주가 이를 이용하지 않은 것으로 나타나 바이오제닉 아민을 생성하지 않는 것으로 확인되었다.Biogenic amines are mainly detected in fermented foods containing protein, and are produced by amino acids or decarboxylase-producing microorganisms during the fermentation, aging and storage of foods. Among biogenic amines, histamine can cause food poisoning and allergies when consumed, and tyramine can secrete vasoconstrictors to cause hypertension. Therefore, as a result of biosgenic amine qualitative experiment, it was confirmed that SRCM102615 strain did not use the precursor amino acid histidine and tyrosine in the medium, so that it did not produce biogenic amine.
실시예 5. 항산화 활성 측정Example 5. Antioxidant activity measurement
DPPH는 항산화 활성을 측정하기 위한 대표적인 기질로 페놀성 물질에 대한 항산화 작용의 지표중 하나이며, 자유 라디칼에 전자를 공여하여 식품 내 지방질 산화를 억제하고, 인체 내 활성산소에 의한 노화를 억제하여 질병 및 노화를 예방하는데 중요한 역할을 한다. 따라서 DPPH를 이용하여 SRCM102615 균주의 항산화 활성을 측정한 결과, 아스코르브산(Ascorbic acid) 30㎍/㎖은 22.93%이었을 때, SRCM102615 균주의 항산화 활성은 38.48%로 높은 활성을 보였다.DPPH is one of the indicators of antioxidant activity against phenolic substances as a representative substrate for measuring antioxidant activity. It inhibits lipid oxidation in foods by donating electrons to free radicals, inhibits aging by reactive oxygen species in the body, And to prevent aging. Therefore, the antioxidative activity of SRCM102615 strain was measured using DPPH. As a result, the antioxidative activity of SRCM102615 strain was as high as 38.48% when 30 ㎍ / ㎖ of ascorbic acid was 22.93%.
실시예 6. 식품유해 미생물에 대한 항균활성 측정Example 6. Measurement of antimicrobial activity against food harmful microorganisms
총 12종의 병원성 미생물에 대하여 항균 활성을 측정한 결과, 다양한 지시균주에 대하여 항균활성을 갖고 있었으며, 저해 효과 또한 우수하였다. 특히, SRCM102615는 바실러스 스패리쿠스 KCTC1184와 에스케리키아 콜라이 KCCM11234에서 높은 항균 효과를 나타내었다. 전통 장류 발효 및 식품 제조에 다양한 항균 스펙트럼을 갖는 미생물의 사용은 식품 제조업체에서의 병원성 미생물에 의한 오염의 문제 해결에 유용할 것으로 보이며, 천연식품 보존제나 사료보존제로서 뿐만 아니라 새로운 항생제 대체 의약소재로의 응용이 가능함을 보여주었다. The antimicrobial activity of the 12 pathogenic microorganisms was measured and the antimicrobial activity against various indicator strains was found to be excellent. In particular, SRCM102615 discloses that Bacillus sp. KCTC1184 and Escherichia coli KCCM11234 showed high antimicrobial activity. The use of microorganisms with a variety of antimicrobial spectra in traditional soybean fermentation and food manufacturing is expected to be useful in addressing the problem of contamination by pathogenic microorganisms in food manufacturers, as well as natural preservatives and feed preservatives, as well as new antibiotic substitutes Application.
실시예 7. 용혈성 및 유해대사산물과 유해효소 생성 분석Example 7. Analysis of hemolytic and noxious metabolites and harmful enzyme production
SRCM102615 균주를 대상으로 헤몰리신(hemolysin)을 유리하여 적혈구를 용해하거나 혈액을 변색시키는 용혈성을 보유하고 있는지 확인한 결과 선별 균주는 γ-용혈현상을 나타내는 것으로 확인되었다. 선별 균주는 β-글루쿠로니다아제(β-Glucuronidase) 및 우레아제(Urease)와 같은 암 형성에 관여할 수 있는 효소를 생성하지 않았고, 유해대사산물로 알려진 인돌(indole)과 페닐피루브산(phenylpyruvic acid)를 생성하지 않았다. 따라서 용혈현상, 유해효소 및 유해대사산물을 가지고 있지 않아 프로바이오틱스 균주로써 안전성이 있을 것으로 확인 되었다. The SRCM102615 strains were tested for hemolysin by dissolving red blood cells or hemolysis that discolored the blood. As a result, the strain was found to exhibit the gamma-hemolysis phenomenon. The selective strains did not produce enzymes that could be involved in cancer formation such as β-glucuronidase and urease, and the indole and phenylpyruvic acid, known as the noxious metabolites, ). Therefore, it has been confirmed that it is safe as a probiotic strain because it does not have hemolysis, harmful enzymes and harmful metabolites.
실시예 8. 선별 유산균의 단백질 응고성Example 8. Protein coagulation of selected lactic acid bacteria
SRCM102615 균주의 유단백질 응고성을 확인하기 위하여 환원탈지유에서의 커드형성 특성을 조사하였다. SRCM102615 균주는 배양 48시간에 커드를 형성하여 유단백질 발효 활성이 우수한 것으로 확인하였다. 우수한 커드형성 능력은 발효유용 스타터로서 적합하게 이용될 수 있을 것으로 생각된다.Curd formation characteristics in reduced skim milk were investigated to confirm the coagulability of SRCM102615 strain. The strain SRCM102615 was found to have an excellent fermentation activity of milk protein by forming a curd at 48 hours of incubation. It is believed that excellent curd forming ability can be suitably used as a fermentation starter.
실시예 9. β-글루코시다제(β-glucosidase) 활성을 갖는 유산균의 선별Example 9. Selection of lactic acid bacteria having? -Glucosidase activity
에스쿨린 철 아가를 이용하여 SRCM102615 균주의 β-글루코시다제 정성적 평가 결과는 표 6과 같다. 대두 발효식품에는 인체에 유익한 성분인 대두단백, 펩티드, 올리고당, 인지질, 이소플라본, 사포닌, 무기질 및 비타민이 포함되어 있다. 이러한 당 성분이 혼합되어 있는 아글리콘 형태의 배당체는 이소플라본을 예로 들 수 있다. 배당체로 존재하는 이소플라본은 유산균이 생산하는 β-글루코시다제에 의하여 비배당화 형태의 다이제인(daizein)과 제니스틴(genistein)으로 전환되어 체내에 흡수된다. 제니스틴은 항암작용이 있는 것으로 알려져 있으며 이러한 효소를 생산하는 미생물을 발효에 이용하였을 때, 발효종균으로 사용할 수 있어 SRCM102615는 프로바이오틱스 식품의 접목 가능성을 보여주었다.The qualitative evaluation results of β-glucosidase of SRCM102615 strain using esculin iron agar are shown in Table 6. Soybean fermented foods contain soybean protein, peptides, oligosaccharides, phospholipids, isoflavones, saponins, minerals and vitamins, which are beneficial to the human body. Examples of the aglycone-type glycosides in which such sugar components are mixed include isoflavones. Isoflavones present as glycosides are converted into non-glycosylated forms of daisyin and genistein by β-glucosidase produced by lactic acid bacteria and then absorbed into the body. Genistein is known to have anticancer activity, and SRCM102615 showed the possibility of grafting of probiotic food as it could be used as a fermentation seed when the enzyme producing microorganism was used for fermentation.
실시예 10. 내산성 및 내담즙성 확인Example 10. Identification of acid resistance and bile resistance
프로바이오틱스 특성 중 낮은 위액의 pH와 미생물의 세포막에 영향을 주어 사멸하게 하는 담즙산에 대한 내성을 확인하였다. 내산성은 pH 조절 전 생균수 대비 pH 5.0, pH 4.0, pH 3.0, pH 2.0 조건에서 생존율을 확인하였다. 그 결과, pH 2.0을 제외한 나머지 pH 3.0, pH4.0, pH 5.0 조건에서 90%가 넘는 생존율을 보여주었다. 내담즙성의 경우 모든 농도에서 90%가 넘는 생존율을 보여주어 담즙산에 내성이 강한 균주로 확인되었다. 따라서 선별균주는 내산성, 내담즙성이 우수하여 이 균주를 이용할 때 낮은 위액의 pH와 담즙산 미생물 세포막 분해를 견디고 장까지 이동이 가능할 것으로 판단된다.Among the probiotics characteristics, the pH of the lower gastric juice and the tolerance to the bile acid causing the microbial cell membrane to be affected were confirmed. The survival rate of acid tolerance was determined at pH 5.0, pH 4.0, pH 3.0 and pH 2.0. As a result, the survival rate was over 90% at pH 3.0, pH 4.0 and pH 5.0 except pH 2.0. In the case of bile, the survival rate was more than 90% at all concentrations, indicating that the strain was resistant to bile acid. Therefore, the selective strains are excellent in acid resistance and bile resistance, and it is considered that they can tolerate low gastric juice pH and bile acid microbial cell membrane decomposition and move to the intestines when using this strain.
102615SRCM
102615
항생제 감수성 확인Confirm antibiotic susceptibility
SRCM102615의 항생제 감수성을 조사한 결과 이미페넴(Imipenem)에 대하여 생육이 억제됨을 확인하였고, 아미카신(Amikacin), 아목시실린(Amoxicillin)+클라불란산(Clavulanic acid), 세프타지딤(Ceftazidime), 세파렉신(Cephalexin), 시프로플록사신(Ciprofloxacin), 푸시딘산(Fusidic acid), 젠타마이신(Gentamicin), 카나마이신(Kanamycin), 네틸미신(Netilmicin), 메티실린(Methicillin), 날리딕스산(Nalidixic acid), 네오마이신(Neomycin), 노르플록사신(Norfloxacin), 오플록사신(Ofloxacin), 피페미드산(Pipemidic acid), 스트렙토마이신(Streptomycin), 테이코플라닌(Teicoplanin), 트리메소프림(Trimethoprim), 트리메소프림-설파메톡사졸(Trimethoprim-Sulfamethoxazole), 반코마이신(Vancomycin) 계열의 항생제에 대하여 내성을 지니고 있음을 확인하였다.The antimicrobial susceptibility of SRCM102615 was confirmed to be inhibited against Imipenem and the growth was inhibited by Amikacin, Amoxicillin + Clavulanic acid, Ceftazidime, Sepharaxine (For example, cephalexin), ciprofloxacin, fusidic acid, gentamicin, kanamycin, netilmicin, methicillin, nalidixic acid, neomycin ), Norfloxacin, Ofloxacin, Pipemidic acid, Streptomycin, Teicoplanin, Trimethoprim, Trimethoprim-sulfa It has been confirmed that it is resistant to antibiotics based on methoxazole (Trimethoprim-Sulfamethoxazole) and vancomycin (Vancomycin).
당 이용성 확인Confirmation of availability
당 이용성을 확인하기 위하여 페디오코커스 에시디락티시 SRCM102615 균주를 API 50 CHL Kit(바이오메리오사)를 사용하여 확인하였다. 그 결과, 당 이용성은 L-아라비노스, D-리보스, D-자일로스, D-갈락토스, D-글루코스, D-프럭토스, D-만노스, N-아세틸-D-글루코사민, 에스쿨린, 살리신, D-셀로비오스, D-락토스, D-멜리비오스, D-라피노스, D-타가토스를 탄소원으로 이용할 수 있음을 확인하였다.To confirm the glycosyltransferase activity, the strain Sicilactii SRCM102615 was identified in Pediococcus using an
효소 생성능 확인Confirm enzyme production ability
페디오코커스 애시디락티시 SRCM102615 균주의 효소 생성 여부를 확인하기 위하여 API ZYM Kit(바이오메리오사)를 이용해 19개 효소에 대한 생산성을 확인하였다. 그 결과는 표 10에 나타내었으며 선별균주는 류신 아릴아미다제(leucine arylamidase), 발린 아릴아미다제(valine arylamidase), 시스틴 아릴아미다제(cystine arylamidase), 산성 포스파타제(acid phosphatase), α-갈락토시다제(α-galactosidase), β-갈락토시다제(β-galactosidase), β-글루코시다제(β-glucosidase), N-아세틸-β-글루코사미니다제(N-acetyl-β-glucosaminidase)의 활성을 가지고 있음을 확인하였다.Productivity of 19 enzymes was confirmed by using API ZYM Kit (BioMerosha) to confirm the enzyme production of Pediococcus acidiflucis SRCM102615. The results are shown in Table 10. The selected strains were leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase,? -Galactoside Glucosidase,? -Galactosidase,? -Glucosidase, N-acetyl-β-glucosaminidase, and the like. Respectively.
<110> Microbial Institute for Fermentation Industry <120> Pediococcus acidilactici SRCM102615 strain having immunity activity, antiviral activity and probiotics properties and uses thereof <130> PN18201 <160> 3 <170> KopatentIn 2.0 <210> 1 <211> 1515 <212> DNA <213> Pediococcus acidilactici SRCM102615 <400> 1 tcaggatgaa cgctggccgg cgtgccttaa tacatgcaag tcggaacgaa cttcccgtta 60 attgatttat acggtgcttg cactgaatga gattttaaca cgaagtgagt ggcggacggg 120 tgagtaacac gtgggtaacc tgcccagaag caggggataa cacctggaaa cagatgctaa 180 taccgtataa cagagaaaac cgcctggttt tcttttaaaa gatggctctg ctatcacttc 240 tggatggacc cgcggcgcat tagctagttg gtgaggtaac ggctcaccaa ggcgatgatg 300 cgtagccgac ctgagagggt aatcggccac attgggactg agacacggcc cagactccta 360 cgggaggcag cagtagggaa tcttccacaa tggacgcaag tctgatggag caacgccgcg 420 tgagtgaaga agggtttcgg ctcgtaaagc tctgttgtta aagaagaacg tgggtgagag 480 taactgttca cccagtgacg gtatttaacc agaaagccac ggctaactac gtgccagcag 540 ccgcggtaat acgtaggtgg caagcgttat ccggatttat tgggcgtaaa gcgagcgcag 600 gcggtctttt aagtctaatg tgaaagcctt cggctcaacc gaagaagtgc attggaaact 660 gggagacttg agtgcagaag aggacagtgg aactccatgt gtagcggtga aatgcgtaga 720 tatatggaag aacaccagtg gcgaaggcgg ctgtctggtc tgtaactgac gctgaggctc 780 gaaagcatgg gtagcgaaca ggattagata ccctggtagt ccatgccgta aacgatgatt 840 actaagtgtt ggagggtttc cgcccttcag tgctgcagct aacgcattaa gtaatccgcc 900 tggggagtac gaccgcaagg ttgaaactca aaagaattga cgggggcccg cacaagcggt 960 ggagcatgtg gtttaattcg aagctacgcg aagaacctta ccaggtcttg acatcttctg 1020 ccaacctaag agattaggcg ttcccttcgg ggacagaatg acaggtggtg catggttgtc 1080 gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttattacta 1140 gttgccagca ttcagttggg cactctagtg agactgccgg tgacaaaccg gaggaaggtg 1200 gggacgacgt caaatcatca tgccccttat gacctgggct acacacgtgc tacaatggat 1260 ggtacaacga gttgcgaaac cgcgaggttt agctaatctc ttaaaaccat tctcagttcg 1320 gactgtaggc tgcaactcgc ctacacgaag tcggaatcgc tagtaatcgc ggatcagcat 1380 gccgcggtga atacgttccc gggccttgta cacaccgccc gtcacaccat gagagtttgt 1440 aacacccaaa gccggtgggg taacctttta ggagctagcc gtctaaggtg ggacagatga 1500 ttagggtgaa gtcgt 1515 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ggattagata ccctggta 18 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ccgtcaattc mtttragttt 20 <110> Microbial Institute for Fermentation Industry <120> Pediococcus acidilactici SRCM102615 strain having immunity activity, antiviral activity and probiotics properties and uses the <130> PN18201 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 1515 <212> DNA <213> Pediococcus acidilactici SRCM102615 <400> 1 tcaggatgaa cgctggccgg cgtgccttaa tacatgcaag tcggaacgaa cttcccgtta 60 attgatttat acggtgcttg cactgaatga gattttaaca cgaagtgagt ggcggacggg 120 tgagtaacac gtgggtaacc tgcccagaag caggggataa cacctggaaa cagatgctaa 180 taccgtataa cagagaaaac cgcctggttt tcttttaaaa gatggctctg ctatcacttc 240 tggatggacc cgcggcgcat tagctagttg gtgaggtaac ggctcaccaa ggcgatgatg 300 cgtagccgac ctgagagggt aatcggccac attgggactg agacacggcc cagactccta 360 cgggaggcag cagtagggaa tcttccacaa tggacgcaag tctgatggag caacgccgcg 420 tgagtgaaga agggtttcgg ctcgtaaagc tctgttgtta aagaagaacg tgggtgagag 480 taactgttca cccagtgacg gtatttaacc agaaagccac ggctaactac gtgccagcag 540 ccgcggtaat acgtaggtgg caagcgttat ccggatttat tgggcgtaaa gcgagcgcag 600 gcggtctttt aagtctaatg tgaaagcctt cggctcaacc gaagaagtgc attggaaact 660 gggagacttg agtgcagaag aggacagtgg aactccatgt gtagcggtga aatgcgtaga 720 tatatggaag aacaccagtg gcgaaggcgg ctgtctggtc tgtaactgac gctgaggctc 780 gaaagcatgg gtagcgaaca ggattagata ccctggtagt ccatgccgta aacgatgatt 840 actaagtgtt ggagggtttc cgcccttcag tgctgcagct aacgcattaa gtaatccgcc 900 tggggagtac gaccgcaagg ttgaaactca aaagaattga cgggggcccg cacaagcggt 960 ggagcatgtg gtttaattcg aagctacgcg aagaacctta ccaggtcttg acatcttctg 1020 ccaacctaag agattaggcg ttcccttcgg ggacagaatg acaggtggtg catggttgtc 1080 gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttattacta 1140 gttgccagca ttcagttggg cactctagtg agactgccgg tgacaaaccg gaggaaggtg 1200 gggacgacgt caaatcatca tgccccttat gacctgggct acacacgtgc tacaatggat 1260 ggtacaacga gttgcgaaac cgcgaggttt agctaatctc ttaaaaccat tctcagttcg 1320 gactgtaggc tgcaactcgc ctacacgaag tcggaatcgc tagtaatcgc ggatcagcat 1380 gccgcggtga atacgttccc gggccttgta cacaccgccc gtcacaccat gagagtttgt 1440 aacacccaaa gccggtgggg taacctttta ggagctagcc gtctaaggtg ggacagatga 1500 ttagggtgaa gtcgt 1515 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ggattagata ccctggta 18 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ccgtcaattc mtttragttt 20
Claims (5)
상기 바이러스는 인플루엔자 A 바이러스(Influenza A viruses)이고, 상기 유해미생물은 바실러스 세레우스(Bacillus cereus), 바실러스 스패리쿠스(Bacillus sphaericus), 엔테로코커스 패시움(Enterococcus faecium), 리스테리아 이바노비(Listeria ivanovii), 리스테리아 모노시토게네스(Listeria monocytogenes), 스타필로코커스 아우레우스(Staphylococcus aureus), 엔테로코쿠스 에로게네스(Enterococcus aerogenes), 에스케리키아 콜라이(Escherichia coli), 쉬겔라 플렉스네리(Shigella flexneri) 및 쉬겔라 손네이(Shigella sonnei)이며, 상기 바이오제닉 아민은 히스타민 및 티라민이며, 상기 유해물질은 인돌 및 페닐피루브산이며, 상기 유해효소는 β-글루쿠로니다아제 및 우레아제인 것을 특징으로 하는 균주.Antibiotic activity, β-glucosidase secretion activity, γ-hemolytic activity, antimicrobial activity against harmful microorganisms, and bioavailability, bioavailability, antioxidant activity, antioxidant activity, Pediococcus acidilactici SRCM102615 strain (KCCM12241P) which does not produce genic amines, toxic substances and harmful enzymes,
Wherein the virus is an influenza A virus (Influenza A viruses), and wherein the microorganisms are Bacillus cereus (Bacillus cereus), Bacillus sphaericus (Bacillus sphaericus), Enterococcus passive help (Enterococcus faecium), L. Ivanova ratio (Listeria ivanovii) Listeria monocytogenes , Staphylococcus aureus , Enterococcus aerogenes , Escherichia coli , Shigella flexneri , and the like. sh Gela hand Nei (Shigella sonnei), and the biogenic amine is histamine and tyramine, the harmful substances are indole and phenylpyruvic acid, the harmful enzymes are strains, characterized in that is a β- glucuronidase dehydratase and urease.
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