KR102641020B1 - Pediococcus pentosaceus strain having antibacterial activity against spoilage moulds and uses thereof - Google Patents
Pediococcus pentosaceus strain having antibacterial activity against spoilage moulds and uses thereof Download PDFInfo
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- KR102641020B1 KR102641020B1 KR1020210111880A KR20210111880A KR102641020B1 KR 102641020 B1 KR102641020 B1 KR 102641020B1 KR 1020210111880 A KR1020210111880 A KR 1020210111880A KR 20210111880 A KR20210111880 A KR 20210111880A KR 102641020 B1 KR102641020 B1 KR 102641020B1
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- present
- cheese
- strain
- mold
- spoilage
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
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- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Food Preservation Except Freezing, Refrigeration, And Drying (AREA)
Abstract
본 발명은 부패곰팡이에 항균 활성을 가지는 균주, 이를 유효성분으로 포함하는 생균제, 항균용 조성물 및 이의 용도에 관한 것으로서, 본 발명의 균주는 치즈 부패 곰팡이의 생장 및 활성을 현저하게 억제할 수 있으므로, 치즈를 비롯한 식품의 부패 방지에 유용하게 활용될 수 있다.The present invention relates to a strain having antibacterial activity against spoilage mold, a probiotic containing the same as an active ingredient, an antibacterial composition, and a use thereof. The strain of the present invention can significantly inhibit the growth and activity of cheese spoilage mold, It can be useful in preventing spoilage of food, including cheese.
Description
본 발명은 부패곰팡이에 항균 활성을 가지는 페디오코쿠스 펜토사세우스(Pediococcus pentosaceus) 균주, 이를 유효성분으로 포함하는 생균제, 항균용 조성물 및 이의 용도에 관한 것이다.The present invention relates to a Pediococcus pentosaceus strain having antibacterial activity against putrefaction mold, a probiotic containing the same as an active ingredient, an antibacterial composition, and its use.
2011년 이후 EU, 미국, 호주, 뉴질랜드 등 낙농선진국과의 FTA 체결로 수입치즈의 양이 큰 폭으로 증가하고 있으며, 국내 1인당 치즈 소비량은 2005년 1.5 kg에서 2015년 2.6 kg으로 약 73% 증가하였다. 이에 발맞추어 국내 자연치즈의 소비량 및 국내 치즈 자급율도 증가하고 있는데, '자연치즈'란 우유에 효소를 넣어 응고 및 숙성시킨 발효식품이며, 그 중에서도 자연숙성치즈는 제조된 후에도 제품의 숙성과 풍미를 확보하기 위해 저온에서 계속 발효시키는 살아있는 식품으로서, 짧게는 24시간부터 길게는 수년간의 숙성기간을 거치게 된다. 미생물은 유제품의 제조와 숙성기간 동안에 맛, 향기, 질감 및 외형에 중요한 역할을 하는데, 유제품의 미생물학적 다양성과 개체량은 우유의 품질, 취급 및 열처리, 제조, 응결 처리 조건, 숙성기간의 온도 및 습도, 염분의 양, 제조 중 또는 제조 후 외인성 미생물에 노출된 정도에 따라 변화한다. 곰팡이는 일반적으로 산성조건 및 30 ℃에서 잘 증식하나 온도가 적당하면 건조식품에서도 증식한다. 효모 또한 증식할 수 있는 pH 범위가 넓고(pH 2.0~8.2) 곰팡이와 마찬가지로 약산성에서 잘자란다. 이들은 치즈 변질의 주요 원인이며, 여러 유형의 치즈에서 천이 과정에 중대한 영향을 미치는 것으로 알려져 있다Since 2011, the amount of imported cheese has increased significantly due to the conclusion of FTAs with advanced dairy countries such as the EU, the US, Australia, and New Zealand, and domestic cheese consumption per capita has increased by approximately 73% from 1.5 kg in 2005 to 2.6 kg in 2015. did. In line with this, the consumption of domestic natural cheese and the domestic cheese self-sufficiency rate are also increasing. 'Natural cheese' is a fermented food made by coagulating and maturing milk with enzymes. Among them, naturally aged cheese is a product that retains the product's ripeness and flavor even after it is manufactured. It is a living food that is continuously fermented at low temperatures to ensure that it undergoes a maturation period ranging from as short as 24 hours to as long as several years. Microorganisms play an important role in the taste, aroma, texture and appearance of dairy products during the production and maturation period. Microbiological diversity and population of dairy products are influenced by milk quality, handling and heat treatment, manufacturing and curing conditions, temperature and maturation period. It varies depending on humidity, amount of salt, and exposure to exogenous microorganisms during or after manufacturing. Molds generally grow well under acidic conditions and at 30°C, but they also grow on dried foods if the temperature is appropriate. Yeast also has a wide pH range in which it can proliferate (pH 2.0 to 8.2), and like mold, it grows well in mildly acidic environments. They are the main causes of cheese spoilage and are known to have a significant impact on the transition process in several types of cheese.
곰팡이는 종종 특정 품종의 치즈에 첨가되어 특이적 외형, 품질의 일관성 및 풍미를 제공하고, 유통 기한을 연장시키는 것으로 알려져 있으나, 이중 몇몇 곰팡이는 자연치즈 등과 같이 장기간의 숙성을 필요로 하는 유제품 등에서 부패를 유발하는 것으로 알려져 있다. 이러한 부패 유발 곰팡이의 독소는 간과 신장의 손상, 중추신경계의 이상구토 및 설사 등이 있으며, 장기적으로 노출되었을 경우에는 만성질환, 발암, 돌연변이, 태아 기형등을 유발할 수 있다고 보고되어 있다(Food Control 22(3-4):360-368). 따라서 이러한 치즈의 부패를 방지하기 위한 조성물의 연구 개발이 시급한 실정이다.Molds are often added to certain types of cheese to provide a specific appearance, quality consistency, and flavor, and are known to extend shelf life. However, some of these molds cause spoilage in dairy products that require long-term maturation, such as natural cheese. It is known to cause . It has been reported that the toxins of these decay-causing molds cause damage to the liver and kidneys, abnormal vomiting and diarrhea in the central nervous system, and that long-term exposure can cause chronic diseases, carcinogenesis, mutations, and fetal deformities (Food Control 22). (3-4):360-368). Therefore, there is an urgent need for research and development of compositions to prevent spoilage of such cheese.
상기와 같은 배경하에, 본 발명자들은 치즈에서 주로 분리되는 유해 곰팡이에 항균효과가 있는 균주를 발굴해내기 위하여 연구 노력한 결과, 본 발명의 미생물을 처리하는 경우 치즈 유해 곰팡이 균에 대한 현저한 항균효과를 가지면서, 우수한 염분내성, 저온내성, 건조내성 및 바이오필름 형성 효과를 가지고 있음을 확인함으로써 본 발명을 완성하였다. 이에, 본 발명은 페디오코쿠스 펜토사세우스(Pediococcus pentosaceus) M132-2 균주(기탁번호: KACC 92316P)를 제공하는 것을 목적으로 한다.Against the above background, the present inventors made research efforts to discover strains that have an antibacterial effect on harmful molds mainly isolated from cheese. As a result, when treated with the microorganism of the present invention, it has a significant antibacterial effect on harmful molds in cheese. The present invention was completed by confirming that it has excellent salt tolerance, low temperature tolerance, drying resistance, and biofilm formation effect. Accordingly, the purpose of the present invention is to provide Pediococcus pentosaceus M132-2 strain (accession number: KACC 92316P).
또한, 본 발명은 상기 균주를 유효성분으로 포함하는, 생균제를 제공하는 것을 다른 목적으로 한다.Another object of the present invention is to provide a probiotic containing the above strain as an active ingredient.
또한, 본 발명은 페디오코쿠스 펜토사세우스(Pediococcus pentosaceus) M132-2 균주(기탁번호: KACC 92316P)를 유효성분으로 포함하는, 항균용 조성물을 제공하는 것을 또 다른 목적으로 한다.Another object of the present invention is to provide an antibacterial composition containing Pediococcus pentosaceus M132-2 strain (accession number: KACC 92316P) as an active ingredient.
또한, 본 발명은 페디오코쿠스 펜토사세우스(Pediococcus pentosaceus) M132-2 균주(기탁번호: KACC 92316P)를 처리하는 단계를 포함하는, 유해균 제거 또는 활성 억제 방법을 제공하는 것을 또 다른 목적으로 한다.Another object of the present invention is to provide a method for removing or inhibiting the activity of harmful bacteria, including the step of treating Pediococcus pentosaceus M132-2 strain (Accession number: KACC 92316P). .
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.
상기 상술한 목적을 달성하기 위하여, 본 발명은 페디오코쿠스 펜토사세우스(Pediococcus pentosaceus) M132-2 균주(기탁번호: KACC 92316P)를 제공한다.In order to achieve the above-described object, the present invention provides Pediococcus pentosaceus M132-2 strain (Accession Number: KACC 92316P).
본 발명의 일실시예에 있어서, 상기 균주는 서열번호 1로 표시되는 16S rRNA 염기서열을 갖는 것일 수 있다.In one embodiment of the present invention, the strain may have a 16S rRNA base sequence represented by SEQ ID NO: 1.
본 발명의 다른 실시예에 있어서, 상기 균주는 염분내성, 건조내성 및 저온내성으로 이루어진 군에서 선택되는 특성 하나 이상을 가지는 것일 수 있다.In another embodiment of the present invention, the strain may have one or more characteristics selected from the group consisting of salt tolerance, desiccation tolerance, and low temperature tolerance.
본 발명의 또 다른 실시예에 있어서, 상기 균주는 바이오필름(biofilm) 형성능을 가지는 것일 수 있다.In another embodiment of the present invention, the strain may have the ability to form biofilm.
또한, 본 발명은 상기 균주를 유효성분으로 포함하는, 생균제를 제공한다.Additionally, the present invention provides a probiotic containing the above strain as an active ingredient.
본 발명의 일실시예에 있어서, 상기 생균제는 치즈 부패곰팡이에 대해서 항균 활성을 가지는 것일 수 있다.In one embodiment of the present invention, the probiotic may have antibacterial activity against cheese spoilage mold.
본 발명의 다른 실시예에 있어서, 상기 치즈 부패곰팡이는 아스퍼질러스 나이거(Aspergillus niger), 클라도스포리움 클라도스포리오이데스(Cladosporium cladosporioides) 및 페니실린 코뮨(Penicillium commune)으로 이루어진 군에서 선택되는 하나 이상인 것일 수 있다.In another embodiment of the present invention, the cheese rot mold is selected from the group consisting of Aspergillus niger , Cladosporium cladosporioides , and Penicillium commune . There may be more than one.
또한, 본 발명은 페디오코쿠스 펜토사세우스(Pediococcus pentosaceus) M132-2 균주(기탁번호: KACC 92316P)를 유효성분으로 포함하는, 항균용 조성물을 제공한다.In addition, the present invention provides an antibacterial composition containing Pediococcus pentosaceus M132-2 strain (Accession Number: KACC 92316P) as an active ingredient.
본 발명의 일실시예에 있어서, 상기 항균용 조성물은 치즈 부패곰팡이에 항균 효과를 가지는 것일 수 있다.In one embodiment of the present invention, the antibacterial composition may have an antibacterial effect on cheese rot mold.
본 발명의 다른 실시예에 있어서, 상기 치즈 부패곰팡이는 아스퍼질러스 나이거(Aspergillus niger), 클라도스포리움 클라도스포리오이데스(Cladosporium cladosporioides) 및 페니실린 코뮨(Penicillium commune)으로 이루어진 군에서 선택되는 하나 이상인 것일 수 있다.In another embodiment of the present invention, the cheese rot mold is selected from the group consisting of Aspergillus niger , Cladosporium cladosporioides , and Penicillium commune . There may be more than one.
또한, 본 발명은 페디오코쿠스 펜토사세우스(Pediococcus pentosaceus) M132-2 균주(기탁번호: KACC 92316P)를 처리하는 단계를 포함하는, 유해균 제거 또는 활성 억제 방법을 제공한다.In addition, the present invention provides a method of removing or inhibiting the activity of harmful bacteria, comprising the step of treating Pediococcus pentosaceus M132-2 strain (Accession number: KACC 92316P).
본 발명의 일실시예에 있어서, 상기 단계는 상기 균주를 3일에 한번씩 15일 동안 처리하는 것일 수 있다.In one embodiment of the present invention, the step may be treating the strain once every 3 days for 15 days.
본 발명의 다른 실시예에 있어서, 상기 유해균은 치즈 부패곰팡이일 수 있다.In another embodiment of the present invention, the harmful bacteria may be cheese rot mold.
본 발명의 또 다른 실시예에 있어서, 상기 치즈 부패곰팡이는 아스퍼질러스 나이거(Aspergillus niger), 클라도스포리움 클라도스포리오이데스(Cladosporium cladosporioides) 및 페니실린 코뮨(Penicillium commune)으로 이루어진 군에서 선택되는 하나 이상인 것일 수 있다.In another embodiment of the present invention, the cheese spoilage mold is selected from the group consisting of Aspergillus niger , Cladosporium cladosporioides , and Penicillium commune . There may be more than one thing.
본 발명은 부패곰팡이에 항균 활성을 가지는 균주, 이를 유효성분으로 포함하는 생균제, 항균용 조성물 및 이의 용도에 관한 것으로서, 본 발명에 따른 특정 균주를 이용하여 치즈 부패곰팡이의 생장 및 활성을 억제할 수 있으므로, 치즈를 비롯한 식품의 부패 방지를 위해 유용하게 활용될 수 있다.The present invention relates to a strain having antibacterial activity against spoilage mold, a probiotic containing the same as an active ingredient, an antibacterial composition, and its use. The specific strain according to the present invention can be used to inhibit the growth and activity of cheese spoilage mold. Therefore, it can be usefully used to prevent spoilage of food, including cheese.
단, 본 발명의 효과는 상기 효과로 한정되는 것은 아니며, 본 발명의 상세한 설명 또는 청구범위에 기재된 발명의 구성으로부터 추론 가능한 모든 효과를 포함하는 것으로 이해되어야 한다.However, the effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.
도 1은 본 발명 미생물의 계통도를 나타낸 것이다.
도 2는 본 발명 미생물의 항균활성을 확인한 것으로서, 도 2의 A는 아스퍼질러스 나이거(Aspergillus niger), 도 2의 B는 클라도스포리움 클라도스포리오이데스(Cladosporium cladosporioides), 도 2의 C는 페니실린 코뮨(Penicillium commune)에 대한 항균활성을 확인한 결과이다.
도 3은 본 발명 미생물을 처리한 치즈에서 부패를 확인한 것으로서, 도 3의 A는 제조한지 1일된 치즈에서 부패를 관찰한 결과이고, 도 3의 B는 제조한지 3개월된 치즈에서 부패를 관찰한 결과이다.
도 4는 본 발명 미생물의 내염도를 NaCl 농도를 달리하면서 확인한 결과이다.
도 5는 본 발명 미생물의 건조내성을 확인한 결과이다.
도 6은 본 발명 미생물의 저온내성을 온도를 달리하면서 확인한 결과이다.
도 7은 본 발명 미생물의 바이오필름 형성능을 확인한 것으로서, 도 7a는 본 발명 미생물의 바이오필름 형성능을 확인한 것이고, 도 7b는 바이오필름이 형성된 본 발명 미생물의 생존능을 확인한 것이다.Figure 1 shows a systematic diagram of the microorganism of the present invention.
Figure 2 confirms the antibacterial activity of the microorganism of the present invention, where A in Figure 2 is Aspergillus niger , B in Figure 2 is Cladosporium cladosporioides , and Figure 2 C is the result of confirming antibacterial activity against penicillium commune .
Figure 3 shows the confirmation of spoilage in cheese treated with the microorganism of the present invention, where A in Figure 3 is the result of observing spoilage in cheese that was 1 day old, and B in Figure 3 is the result of observing spoilage in cheese that was 3 months old. It is a result.
Figure 4 shows the results of confirming the salt tolerance of the microorganism of the present invention while varying the NaCl concentration.
Figure 5 shows the results of confirming the drying tolerance of the microorganism of the present invention.
Figure 6 shows the results of confirming the low temperature tolerance of the microorganism of the present invention at different temperatures.
Figure 7 confirms the biofilm-forming ability of the microorganism of the present invention, Figure 7a confirms the biofilm-forming ability of the microorganism of the present invention, and Figure 7b confirms the viability of the microorganism of the present invention with a biofilm formed.
본 발명자들은 치즈에서 주로 분리되는 유해 곰팡이에 항균효과가 있는 균주를 발굴해내기 위하여 연구 노력한 결과, 본 발명의 미생물을 처리하는 경우 치즈 유해 곰팡이 균에 대한 현저한 항균효과를 가지면서, 우수한 염분내성, 저온내성, 건조내성 및 바이오필름 형성 효과를 가지고 있음을 확인하였는바, 이로써 본 발명을 완성하게 되었다.The present inventors have made research efforts to discover strains that have an antibacterial effect on harmful molds mainly isolated from cheese, and as a result, when treated with the microorganism of the present invention, it has a significant antibacterial effect against harmful molds in cheese, excellent salt tolerance, It was confirmed that it has low temperature resistance, drying resistance, and biofilm formation effects, thereby completing the present invention.
이에, 본 발명은 페디오코쿠스 펜토사세우스(Pediococcus pentosaceus) M132-2 균주(기탁번호: KACC 92316P)를 제공한다.Accordingly, the present invention provides Pediococcus pentosaceus M132-2 strain (Accession number: KACC 92316P).
상기 페디오코쿠스 펜토사세우스 M132-2 균주는 서열번호 1로 나타나는 염기서열의 16S rDNA를 가질 수 있으며, 미생물 기탁번호 KACC 92316P(미생물 명칭: Pediococcus pentosaceus M132-2)로 기탁된 균주일 수 있다.The Pediococcus pentosaceus M132-2 strain may have 16S rDNA of the base sequence shown in SEQ ID NO: 1, and may be a strain deposited under the microorganism deposit number KACC 92316P (microorganism name: Pediococcus pentosaceus M132-2). .
본 발명의 다른 양태로서, 본 발명은 상기 균주를 유효성분으로 포함하는, 생균제를 제공한다.In another aspect of the present invention, the present invention provides a probiotic containing the above strain as an active ingredient.
본 발명의 또 다른 양태로서, 본 발명은 상기 균주를 유효성분으로 포함하는, 항균용 조성물을 제공한다.In another aspect of the present invention, the present invention provides an antibacterial composition containing the above strain as an active ingredient.
본 발명에서 "항균"의 의미는 특정 농도에서 미생물의 성장 또는 생존을 감소, 방지, 억제, 또는 제거하는 능력을 의미한다. 항균용 조성물이란 항미생물제를 총칭하는 의미인 항생제와 같은 의미일 수 있고, 항균제, 살균제, 방부제, 보존제 또는 제균제와 같은 의미일 수 있으며, 바람직하게는 그람양성균, 그람음성균, 진균(효모 및 곰팡이)으로 이루어진 군에서 선택된 1 종 이상의 미생물의 발육과 생활 기능을 저지 또는 억제할 수 있는 물질 즉, 항세균 및 항진균 효력이 있는 물질을 의미한다.As used herein, the meaning of “antibacterial” refers to the ability to reduce, prevent, inhibit, or eliminate the growth or survival of microorganisms at a specific concentration. Antibacterial composition may have the same meaning as antibiotic, which is a general term for antimicrobial agents, and may have the same meaning as antibacterial agent, bactericide, preservative, preservative, or sterilizing agent, and is preferably gram-positive bacteria, gram-negative bacteria, and fungi (yeast and mold). ) refers to a substance that can inhibit or inhibit the growth and life functions of one or more types of microorganisms selected from the group consisting of, that is, a substance with antibacterial and antifungal effects.
본 발명에 있어서, 상기 생균제 및 항균용 조성물은 이에 제한되는 것은 아니지만, 치즈 부패곰팡이에 대하여 항균 활성을 가지는 것일 수 있으며, 상기 치즈 부패곰팡이는 아스퍼질러스 나이거(Aspergillus niger), 클라도스포리움 클라도스포리오이데스(Cladosporium cladosporioides) 또는 페니실린 코뮨(Penicillium commune)일 수 있으나, 이에 제한되지 않는다.In the present invention, the probiotic and antibacterial composition are not limited thereto, but may have antibacterial activity against cheese rot mold, and the cheese spoilage mold is Aspergillus niger and Cladosporium. It may be Cladosporium cladosporioides or Penicillium commune , but is not limited thereto.
본 발명에 따른 생균제 또는 항균용 조성물은 상술한 균주 그 자체 또는 그의 배양물을 함유할 수 있고, 나아가 적합한 부형체 또는 담체를 추가로 포함할 수도 있다.The probiotic or antibacterial composition according to the present invention may contain the above-described strain itself or its culture, and may further include a suitable excipient or carrier.
본 명세서에서 용어 "생균제"는 인체나 동물의 건강을 증진시키기 위하여 고안된 살아있는 미생물 제제이고, 이는 독자적으로 섭취 또는 투여되거나 의약품, 식품 및 사료 또는 식이 첨가제 등에 포함되어 사용될 수 있다.As used herein, the term “probiotic” refers to a live microbial preparation designed to improve the health of humans or animals, and can be ingested or administered independently or used as part of medicine, food, feed, or dietary additives.
본 명세서에서 "배양물"은 균주를 배지에 접종하고 일정시간 배양하여 얻은 결과물로서, 본 발명에 따른 균주를 적합한 액체 배지에서 배양한 배양액 자체(조 배양물), 상기 배양액을 여과 또는 원심분리하여 균주를 제거한 여액(여과액 또는 원심분리한 상등액), 상기 배양액을 초음파 처리하거나 상기 배양액에 용해효소(lysozyme)를 처리하여 수득한 세포 파쇄액, 상기 배양액, 여액, 파쇄액을 추출한 추출액, 상기 배양액, 여액, 파쇄액, 추출액 등을 농축한 농축액 및 이들의 건조물 등을 포함하나 이에 한정되는 것은 아니다.In this specification, “culture” refers to the result obtained by inoculating a strain into a medium and culturing it for a certain period of time, including the culture medium itself (crude culture) obtained by culturing the strain according to the present invention in a suitable liquid medium, filtering or centrifuging the culture medium. Filtrate from which strains were removed (filtrate or centrifuged supernatant), cell lysate obtained by sonicating the culture or treating the culture with lysozyme, extract from the culture, filtrate, and lysate, and the culture. , filtrate, crushed liquid, extract, etc., and dried products thereof, but are not limited thereto.
본 명세서에서 용어 "농축액"은 상기 배양액을 농축한 것을 의미한다.As used herein, the term “concentrate” refers to concentrated culture medium.
본 명세서에서 용어 "추출액"은 상기 배양액 또는 그의 농축액로부터 추출한 것을 의미하며, 본 발명의 항균 효과를 나타낼 수 있는 추출물인 한, 이에 제한되지는 않고, 추출액, 추출액의 희석액 또는 농축액, 추출액을 건조하여 얻어지는 건조물, 또는 이들 조정제물 또는 정제물, 이를 분획한 분획물을 모두 포함할 수 있다.In this specification, the term "extract" means extracted from the culture medium or its concentrate, and is not limited thereto, as long as it is an extract that can exhibit the antibacterial effect of the present invention, and is not limited to extract, diluted or concentrated liquid of the extract, or dried extract. It may include all of the dried product obtained, or these crude or purified products, and the fractions thereof.
본 발명의 항균용 조성물은 유해 미생물에 감염된 부분에 직접 투여하거나 적절한 형태의 조성물로 제조하여 투여할 수 있다. 본 발명의 항균용 조성물의 투여량은 투여 대상에서 유해 미생물을 사멸시키는 효과를 발휘하기 위해 요구되는 정도의 양을 의미하는데, 제형의 종류 및 투여 대상의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여 경로, 투여 시간 및 기간을 비롯한 다양한 인자에 따라 조절될 수 있다. 가축 또는 어류에 사용하는 경우는 수의학에서 허용하는 범위 내에서 제제화하고 그에 따라 투여경로를 정할 수 있다.The antibacterial composition of the present invention can be administered directly to areas infected with harmful microorganisms, or can be prepared and administered as a composition in an appropriate form. The dosage of the antibacterial composition of the present invention refers to the amount required to exert the effect of killing harmful microorganisms in the administration subject, depending on the type of formulation and the age, weight, general health, gender, and diet of the administration subject. , can be adjusted according to various factors, including route of administration, time and period of administration. When used on livestock or fish, it can be formulated within the range permitted by veterinary medicine and the administration route can be determined accordingly.
본 발명의 항균용 조성물은 용액, 분말, 현탁액, 분산액, 에멀젼, 유성 분산액, 페이스트, 분진, 분사제 또는 과립제로 제조할 수 있으나, 이에 제한되는 것은 아니다. 본 발명의 구체적인 실시예에 의하면, 본 발명에 따른 항균용 조성물은 농약, 의약, 화장품, 생활용품, 식품 등에서 항균, 살균, 소독, 방부 등의 목적을 달성하기 위해 광범위하게 사용될 수 있다. 구체적으로, 농업에 있어서는 항균, 살균, 소독의 목적으로, 의약에 있어서는 항생제나 오염방지제와 같은 목적으로, 식품에 있어서는 방부나 항균목적으로, 화장품이나 생활용품에 있어서는 비듬억제용, 무좀방지용, 겨드랑이 채취억제용, 항여드름용 등 미생물과 직접 연관된 제품에 사용되거나 청소용 세정제나 식기세척용 세정제 등에 방부나 항균 또는 살균목적으로 사용되어 질 수 있으며, 이러한 목적으로만 한정되는 것은 아니다. The antibacterial composition of the present invention can be prepared as a solution, powder, suspension, dispersion, emulsion, oily dispersion, paste, dust, propellant or granule, but is not limited thereto. According to a specific embodiment of the present invention, the antibacterial composition according to the present invention can be widely used in pesticides, medicines, cosmetics, household goods, food, etc. to achieve purposes such as antibacterial, sterilizing, disinfecting, and preservative purposes. Specifically, in agriculture, for antibacterial, sterilization, and disinfection purposes, in medicine, for purposes such as antibiotics or anti-pollution agents, in food, for preservative or antibacterial purposes, and in cosmetics and household goods, for anti-dandruff, athlete's foot, and armpit use. It can be used in products directly related to microorganisms, such as for anti-acne collection or for anti-acne, or for preservative, antibacterial, or sterilizing purposes such as cleaning detergents or dishwashing detergents, but is not limited to these purposes.
또한, 본 발명의 조성물은 또한 식품 조성물일 수 있는데, 이러한 식품 조성물은 상술한 유효성분을 함유하는 것 외에 통상의 식품 조성물과 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 본 발명이 적용될 수 있는 상기 식품의 예로는 특별한 제한은 없으며, 육류, 소세지, 빵, 쵸컬릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함하는 낙농제품, 각종 수프, 음료수, 차, 드 링크제, 알코올 음료 및 비타민 복합제 등이 있으며, 바람직하게는 치즈일 수 있고, 통상적인 의미에서의 식품, 특히 건강식품을 모두 포함한다. 본 발명에 따른 조성물을 함유하는 건강식품으로는 상기 균주, 이의 배양물, 또는 이로부터 분리된 항균물질을 유효성분으로 하는 차, 젤리, 즙, 엑기스, 음료 등의 유해균 생장 억제를 목적으로 하는 민간요법제를 들 수 있다.In addition, the composition of the present invention may also be a food composition, and in addition to containing the above-mentioned active ingredients, such a food composition may contain various flavoring agents or natural carbohydrates as additional ingredients like a typical food composition. Examples of the food to which the present invention can be applied are not particularly limited and include meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, Beverages, teas, drinks, alcoholic beverages and vitamin complexes are included, preferably cheese, and include foods in the conventional sense, especially health foods. Health foods containing the composition according to the present invention include tea, jelly, juice, extract, beverage, etc., which contain the above-mentioned strain, its culture, or an antibacterial substance isolated therefrom as an active ingredient, and are used in the private sector for the purpose of inhibiting the growth of harmful bacteria. A therapeutic agent may be mentioned.
본 발명의 조성물은 또한 식품 첨가제 조성물일 수 있다. 본 발명에 따른 조성물을 식품 첨가제로 사용할 경우, 상술한 유효 성분 각각을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용할 수 있고, 통상적인 방법에 따라 적절하게 사용할 수 있다. 본 발명의 식품은 통상의 식품와 같이 여러 가지 향미제, 감미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드, 말토오스, 슈크로오스와 같은 디사카라이드, 및 덱스트린, 사이클로덱스트린과 같은 폴리사카라이드, 자일리톨, 소르비톨, 에리트리톨 등의 당알코올이 있다. 감미제로서는 타우마틴, 스테비아 추출물과 같은 천연 감미제나 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다. 상기 외의 본 발명의 식품에는 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다.The composition of the present invention may also be a food additive composition. When using the composition according to the present invention as a food additive, each of the above-mentioned active ingredients can be added as is or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. The food of the present invention, like conventional foods, may contain various flavoring agents, sweeteners, or natural carbohydrates as additional ingredients. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a sweetener, natural sweeteners such as thaumatin and stevia extract or synthetic sweeteners such as saccharin and aspartame can be used. In addition to the above, the food of the present invention includes various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, It may contain carbonating agents used in carbonated drinks.
본 발명자들은 구체적인 실시예를 통해 본 발명의 미생물이 치즈 부패곰팡이에 대해 우수한 항균활성을 가지고 있음을 확인하였다.Through specific examples, the present inventors confirmed that the microorganism of the present invention has excellent antibacterial activity against cheese spoilage mold.
본 발명의 일실시예에 있어서, 본 발명의 미생물인 페디오코쿠스 펜토사세우스 M132-2가 치즈 부패곰팡이균으로 알려진 아스퍼질러스 나이거(Aspergillus niger), 클라도스포리움 클라도스포리오이데스(Cladosporium cladosporioides) 및 페니실린 코뮨(Penicillium commune)의 생육을 억제하는 것을 확인하였고, 이는 실제 치즈에서 실험을 수행하였을 때에도 마찬가지로 확인되었다(실시예 2 참조).In one embodiment of the present invention, the microorganism of the present invention, Pediococcus pentosaceus M132-2, is Aspergillus niger , Cladosporium Cladosporioides, known as cheese spoilage fungi. It was confirmed that the growth of ( Cladosporium cladosporioides ) and penicillium commune ( Penicillium commune ) was inhibited, and this was also confirmed when the experiment was performed on actual cheese (see Example 2).
상기와 같은 결과를 토대로 본 발명자들은 본 발명의 미생물이 치즈 부패 곰팡이균에 대하여 현저한 항균효과를 가지고 있으며, 실제 치즈에 실험을 수행하였을 때도, 갓 만든 치즈 및 숙성 치즈 모두에서도 우수한 항균효과를 보여줌을 확인하였다. 이를 통해 본 발명의 미생물은 치즈를 비롯한 낙농업 관련 제품의 부패 방지를 위해 유용하게 활용될 수 있을 것으로 예상된다.Based on the above results, the present inventors have shown that the microorganism of the present invention has a remarkable antibacterial effect against cheese spoilage fungi, and that when experiments were performed on actual cheese, it showed excellent antibacterial effect on both freshly made cheese and aged cheese. Confirmed. Through this, it is expected that the microorganism of the present invention can be usefully used to prevent spoilage of dairy products, including cheese.
본 발명의 또 다른 양태로서, 본 발명은 페디오코쿠스 펜토사세우스(Pediococcus pentosaceus) M132-2 균주(기탁번호: KACC 92316P)를 처리하는 단계를 포함하는, 유해균 제거 또는 활성 억제 방법을 제공한다. 구체적으로, 상기 단계는 상기 균주를 3일에 한번씩 15일 동안 처리하는 것일 수 있다.In another aspect of the present invention, the present invention provides a method for removing or inhibiting the activity of harmful bacteria, comprising the step of treating Pediococcus pentosaceus M132-2 strain (Accession number: KACC 92316P). . Specifically, the step may involve treating the strain once every three days for 15 days.
상기 유해균은 치즈 부패곰팡이 일 수 있으며, 상기 치즈 부패곰팡이는 아스퍼질러스 나이거(Aspergillus niger), 푸자리움 옥시스포럼(Fusarium oxysporum), 클라도스포리움 클라도스포리오이데스(Cladosporium cladosporioides) 및 페니실린 코뮨(Penicillium commune)으로 이루어진 군에서 선택되는 하나 이상인 것일 수 있다.The harmful bacteria may be cheese rot mold, and the cheese spoilage mold is Aspergillus niger , Fusarium oxysporum , Cladosporium cladosporioides , and penicillin. It may be one or more selected from the group consisting of communes ( Penicillium commune ).
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Below, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are provided only to make the present invention easier to understand, and the content of the present invention is not limited by the following examples.
실시예 1. 본 발명 미생물의 분리 및 계통 확인Example 1. Isolation and strain confirmation of microorganisms of the present invention
청국장 1g을 취하여 멸균생리식염수 (0.85% NaCl) 9mL과 혼합하여 균질화 한 후 연속 희석법에 의해 10-4~ 10-7배 희석하였다. 희석액은 BCP 고체배지(Yeast extract 2.5g, peptone 5g, glucose 1g, tween 80 1g, L-cystein 0.1g, Bromocresol purple 0.1%(w/v), agar 15g, distilled water 1L)에 도말하여 37도의 온도로, 72시간 동안 배양하였다. 배양 후 배지의 색이 노랗게 변화 되어 형성된 집락(colony)을 대항으로 선택한 후 순수배양하였다. 순수 분리시에는 Lactobacillus MRS 고형배지에 계대 배양 후 미생물을 순수 분리하였다.1g of Cheonggukjang was taken, mixed with 9mL of sterilized saline solution (0.85% NaCl), homogenized, and then diluted 10 -4 to 10 -7 times by serial dilution. The diluted solution was spread on BCP solid medium (2.5g yeast extract, 5g peptone, 1g glucose, 1g tween 80, 0.1g L-cysteine, 0.1% (w/v) bromocresol purple, 15g agar, 1L distilled water) and incubated at a temperature of 37 degrees. and cultured for 72 hours. After culturing, the color of the medium changed to yellow, and the formed colonies were selected as controls and then pure cultured. In pure isolation, the microorganisms were isolated after subculturing on Lactobacillus MRS solid medium.
본 발명 미생물의 염기서열을 구체적으로 확인하였을 때, 본 발명 미생물은 서열번호 1로 표시되는 16S rRNA 염기서열을 포함하는 것으로 확인되었으며, GenBenk의 BLAST 데이터와 비교하여 분자 생물학적 연관성을 나타내는 계통도(도 1)를 얻었다. 이 결과에 따라 본 발명의 미생물은 선발 미생물인 페디오코쿠스 펜토사세우스(Pediococcus pentosaceus)와 99%의 상동성으로 가장 가까운 유연관계를 나타내었다. 따라서 선발미생물을 페디오코쿠스 펜토사세우스로 동정하였고, 본 발명의 미생물을 페디오코쿠스 펜토사세우스(P. pentosaceus) M132-2로 명명하였다.When the base sequence of the microorganism of the present invention was specifically confirmed, the microorganism of the present invention was confirmed to contain the 16S rRNA base sequence represented by SEQ ID NO: 1, and compared to GenBenk's BLAST data, a systematic diagram showing the molecular biological relationship (Figure 1 ) was obtained. According to these results, the microorganism of the present invention showed the closest affinity with the selected microorganism , Pediococcus pentosaceus , with 99% homology. Therefore, the selection microorganism was identified as Pediococcus pentosaceus, and the microorganism of the present invention was named Pediococcus pentosaceus ( P. pentosaceus ) M132-2.
실시예 2. 본 발명 미생물의 항균 효과 확인Example 2. Confirmation of antibacterial effect of microorganism of the present invention
본 발명의 곰팡이인 페디오코쿠스 펜토사세우스(P. pentosaseus) M132-2가 치즈에서 주로 분리되는 유해 곰팡이를 저해할 수 있는지 여부를 확인하기 위하여 치즈에서 주로 분리되는 것으로 알려진 곰팡이인 아스퍼질러스 나이거(Aspergillus niger), 클라도스포리움 클라도스포리오이데스(Cladosporium cladosporioides) 및 페니실린 코뮨(Penicillium commune) 각각에 대한 생육억제 저해 효과를 확인하였다. In order to determine whether the mold of the present invention, Pediococcus pentosaseus ( P. pentosaseus ) M132-2, can inhibit harmful molds mainly isolated from cheese, Aspergillus, a mold known to be mainly isolated from cheese, The growth inhibition inhibitory effect on each of Aspergillus niger , Cladosporium cladosporioides and Penicillium commune was confirmed.
① 상기 유해 곰팡이들은 PDA 배지에서 25℃, 3일 배양한 각 곰팡이 균은 포자를 긁어 0.75% PDA soft agar에 넣어 잘 섞고 PDA agar plate에 부어주었다. 유해 곰팡이에 항균 효과를 가지는 상등액은 배양된 페디오코쿠스 펜토사세우스 M132-2를 원심분리(9,950Хg, 4℃ 15 min)하여 회수한 상등액을 막 필터(0.45 ㎛ pore size, Millipore, Beverly, USA)로 제균하여 준비하였고, 항균활성은 agar well diffusion assay 방법을 이용하였다. 항균활성은 저해환의 반직경 크기에 따라 하기와 같이 구분하였다. 그 결과 본 발명의 미생물은 도 2에 나타낸 바와 같이 아스퍼질러스 나이거(도 2의 A) 클라도스포리움 클라도스포리오이데스(도 2의 B) 및 페니실린 코뮨(도 2의 C)에 대해 우수한 항균활성을 가지고 있음을 확인하였다.① The above harmful molds were cultured in PDA medium at 25°C for 3 days. The spores of each fungus were scraped, added to 0.75% PDA soft agar, mixed well, and poured onto a PDA agar plate. The supernatant, which has an antibacterial effect against harmful molds, was obtained by centrifuging cultured Pediococcus pentosaceus M132-2 (9,950Хg, 4℃ 15 min) and filtering the supernatant through a membrane filter (0.45 ㎛ pore size, Millipore, Beverly, USA), and the antibacterial activity was determined using the agar well diffusion assay method. Antibacterial activity was classified as follows according to the size of the radius of the inhibitory ring. As a result, as shown in Figure 2, the microorganism of the present invention is effective against Aspergillus niger (Figure 2A), Cladosporium cladosporioides (Figure 2B), and Penicillin commune (Figure 2C). It was confirmed to have excellent antibacterial activity.
② 또한, 상기와 같은 항균효과가 실제 치즈에서도 적용되는지 여부를 확인하기 위하여, 실제로 제조된 고다 치즈(Gouda cheese)를 사용하여 부패균 저해활성을 확인하였다. 갓 제조된 치즈는 숙성 온도인 15℃에서 보관되었고 코팅하지 않은 상태로 실험을 진행하였다. 실험은 치즈 표면에 페디오코쿠스 펜토사세우스 M132-2 상등액을 3일에 한번씩 분무하였고, 총 15일에 걸쳐서 진행되었다. 그 결과 도 3에 나타낸 바와 같이, 갓 제조된 치즈(도 3의 A) 및 제조 후 3개월 동안 숙성된 치즈(도 3의 B) 모두에서 본원발명의 미생물이 포함된 항균물질을 처리한 경우, 대조군(무처리 대조군)에 비해 곰팡이의 성장이 현저하게 저해된 것을 확인할 수 있었다.② In addition, in order to confirm whether the above-mentioned antibacterial effect also applies to actual cheese, the spoilage inhibitory activity was confirmed using actually manufactured Gouda cheese. Freshly manufactured cheese was stored at the ripening temperature of 15°C and the experiment was conducted without coating. The experiment was conducted over a total of 15 days, spraying Pediococcus pentosaceus M132-2 supernatant on the cheese surface once every three days. As a result, as shown in Figure 3, when both freshly manufactured cheese (A in Figure 3) and cheese aged for 3 months after manufacture (B in Figure 3) were treated with an antibacterial material containing the microorganism of the present invention, It was confirmed that the growth of mold was significantly inhibited compared to the control group (untreated control).
실시예 3. 본 발명 미생물의 특성 확인Example 3. Confirmation of characteristics of microorganisms of the present invention
3-1. 염분내성 확인3-1. Check salt tolerance
본 발명 미생물의 내염성 실험을 위해 미생물을 24시간 37℃에서 전배양한 다음, 9 log CFU/mL로 조정하여 1%, 2%, 3%, 5%, 7%, 10%의 NaCl을 첨가한 MRS 액체 배지에 전배양한 균주 배양액을 1% 접종하여 37℃에서 24시간 배양하였다. NaCl을 첨가하지 않은 대조군의 생균수와 비교하여 생존율을 계산한 결과 도 4에 나타낸 바와 같이 NaCl 1~7%까지는 90% 이상의 높은 생존율을 유지하였고, 10%의 NaCl 첨가한 배지에서는 약간 감소하여 75.1%의 생존율을 보였으며 구체적인 수치는 하기 표 1에 나타내었다.To test the salt tolerance of the microorganism of the present invention, the microorganism was pre-cultured at 37°C for 24 hours, then adjusted to 9 log CFU/mL and 1%, 2%, 3%, 5%, 7%, and 10% of NaCl was added. 1% of the pre-cultured strain culture medium was inoculated into MRS liquid medium and cultured at 37°C for 24 hours. As a result of calculating the survival rate compared to the number of viable cells in the control group without adding NaCl, as shown in Figure 4, a high survival rate of more than 90% was maintained for 1 to 7% NaCl, and slightly decreased to 75.1 in the medium with 10% NaCl added. % survival rate was observed, and specific figures are shown in Table 1 below.
기존에 내염성 유산균으로 알려진 류코노스톡 메센테로이데스(Leuconostoc mensenteroides) DSR1310은 2~4%에서 내염성 특성을 보인 반면 본 연구에서 분리한 유산균은 10%에서도 높은 생존율을 보여 넓은 범위의 염분에도 매우 높은 저항성을 가지고 있다고 판단된다. 또한 실제 자연숙성치즈는 염도가 2% 내외이기에 본 연구에서 분리한 균주의 생존율(99.0% 이상)을 고려할 때, 치즈 부패곰팡이에 대한 우수한 항균효과를 보여줄 것으로 예상된다. Leuconostoc mensenteroides DSR1310, previously known as a salt-tolerant lactic acid bacterium, showed salt-tolerant properties at 2 to 4%, while the lactic acid bacteria isolated in this study showed a high survival rate even at 10%, showing very high resistance even to a wide range of salts. It is judged to have In addition, since actual naturally ripened cheese has a salinity of around 2%, considering the survival rate (over 99.0%) of the strain isolated in this study, it is expected to show excellent antibacterial effect against cheese rot mold.
3-2. 건조내성 확인3-2. Check drying resistance
본 발명 미생물의 장기적 건조노출 분석을 위해 24시간 37℃에서 전배양시킨 후 9 log CFU/mL로 조정하여 12-well culture plate의 각 well에 200ul씩 접종하여 상온에서 10일간 방치하면서 건조시켰다. 각 시간별로 각각의 well에서 회수된 균체액을 MRS 고체 배지에 도말한 다음, 37℃에서 24시간 배양하고 계수하였다. 12-Well plate의 플라스틱 표면상에서 10일의 장기간 상온 방치 시에 따른 생균수의 변화를 측정한 결과, 도 5에 나타낸 바와 같이, 3일 건조 후 생균수는 유지되는 경향을 보였으며, 8일 건조 후에는 약 log 2 CFU/mL(생존율, 45%)의 균주가 생존해있었다. 일반적으로 세균은 2일 건조 후 생균수가 반 이하로 떨어지는데에 반해, 본 발명의 균주는 4일 건조까지는 매우 높은 수준의 생존율을 유지하고 있기에, 일반적인 균에 비해 우수한 건조 내성을 가지고 있는 것으로 판단된다.To analyze long-term drying exposure of the microorganism of the present invention, the culture was pre-cultured at 37°C for 24 hours, then adjusted to 9 log CFU/mL, and 200 ul was inoculated into each well of a 12-well culture plate and left at room temperature for 10 days to dry. The bacterial fluid recovered from each well at each time was plated on MRS solid medium, cultured at 37°C for 24 hours, and counted. As a result of measuring the change in the number of viable bacteria on the plastic surface of a 12-well plate when left at room temperature for a long period of 10 days, as shown in Figure 5, the number of viable cells tended to be maintained after 3 days of drying, and 8 days of drying showed a tendency to maintain the number of viable bacteria. Afterwards, about log 2 CFU/mL (survival rate, 45%) of the strain survived. In general, the number of viable bacteria drops to less than half after 2 days of drying, but the strain of the present invention maintains a very high level of survival rate until 4 days of drying, and is therefore considered to have excellent drying tolerance compared to general bacteria.
3-3. 저온내성 확인3-3. Check low temperature resistance
본 발명 미생물의 저온내성 실험을 위해 24시간 37℃에서 전배양시킨 후 9log CFU/mL로 조정하여 5℃, 15℃에서 24시간 배양한 후 35℃, 25℃에서 배양한 생균수와 비교하여 생존율을 계산하였다. 그 결과 도 6에 나타낸 바와 같이, 15℃ 내지 35℃의 온도에서 생존율의 변화가 확인되지 않았으며, 5℃까지 온도를 낮춘 경우에 생존율이 조금 떨어지는 것을 확인할 수 있었다. 구체적인 수치는 하기 표 2에 나타내었다.To test the low temperature tolerance of the microorganism of the present invention, the microorganism of the present invention was pre-cultured at 37°C for 24 hours, then adjusted to 9 log CFU/mL, cultured at 5°C and 15°C for 24 hours, and survival rate compared to the number of viable cells cultured at 35°C and 25°C. was calculated. As a result, as shown in FIG. 6, no change in survival rate was confirmed at a temperature of 15°C to 35°C, and it was confirmed that the survival rate decreased slightly when the temperature was lowered to 5°C. Specific values are shown in Table 2 below.
3-4. 바이오필름 형성능 확인3-4. Confirmation of biofilm formation ability
본 발명 미생물의 바이오필름 형성능력을 확인하기 위하여, crystal violet의 OD를 측정하는 microplate plate assay법을 이용하였고, 각 그룹간 평균과 표준편차를 계산하여 표기하였다. 37℃하루 밤 전배양된 페디오코쿠스 펜토사세우스 M132-2 배양액을 12-well tissue culture test plate의 각 well에 2mL씩 접종하여 3일 배양하였다. 배양 후 부유세균과 배양액을 제거한 후 2.5mL의 생리식염수로 3번 세척하였다. 잔여 식염수를 제거한 후 2mL의 99% methanol을 각 well에 분주하고 15분 동안 방치하여 biofilm을 표면에 부착시켰다. Methanol을 완전히 건조시키고 각 well마다 2mL의 0.2% crystal violet (Hucker, sigma) solution 분주 후 5분동안 방치하여 biofilm을 염색하였다. crystal violet solution을 제거하고 건조한 후 2.5mL 증류수로 1회 세척하고 33%(v/v) glacial acetic acid를 1.6mL씩 분주하여 착색된 crystal violet를 용출시켜 570nm에서 microtiter plate reader(Tecan, Sunrise)로 흡광도를 측정하였다. 본 발명의 미생물은 높은 OD값을 보여 주었고, 도 7a에 나타낸 바와 같이, 우수한 바이오필름 형성능을 가지고 있음을 확인할 수 있었다. 본원에 바이오필름 우수 균주로고 알려진 Bifidobacteria spp. 의 경우, porcine 1%로 바이오 필름을 유도했을 때, 2 내지 3 수준의 OD 값을 보여주는데, 본원발명의 박테리아 또한, 약 2.2 수준의 OD 값을 보여주어 높은 수준으로 바이오 필름을 형성하는 것을 확인할 수 있었다. 또한, 바이오 필름 생존율을 확인했을 때에도, 도 7b에 나타낸 바와 같이, 접종 후 6일 가량의 시간이 경과해도 높은 수준의 바이오 필름 생존율을 유지하고 있음을 확인할 수 있었다.In order to confirm the biofilm-forming ability of the microorganism of the present invention, a microplate plate assay method was used to measure the OD of crystal violet, and the average and standard deviation between each group were calculated and expressed. Pediococcus pentosaceus M132-2 culture pre-cultured overnight at 37°C was inoculated at 2 mL into each well of a 12-well tissue culture test plate and cultured for 3 days. After culturing, floating bacteria and culture medium were removed and washed three times with 2.5 mL of physiological saline. After removing the remaining saline solution, 2 mL of 99% methanol was dispensed into each well and left for 15 minutes to allow biofilm to attach to the surface. The methanol was completely dried, and 2 mL of 0.2% crystal violet (Hucker, Sigma) solution was dispensed into each well and left for 5 minutes to stain the biofilm. After removing and drying the crystal violet solution, washing once with 2.5 mL distilled water, dispensing 1.6 mL of 33% (v/v) glacial acetic acid to elute the colored crystal violet, and dissolving at 570 nm using a microtiter plate reader (Tecan, Sunrise). Absorbance was measured. The microorganism of the present invention showed a high OD value, and as shown in Figure 7a, it was confirmed that it had excellent biofilm forming ability. Bifidobacteria spp., known as an excellent biofilm strain herein. In the case of , when a biofilm is induced with 1% porcine, an OD value of 2 to 3 is shown, and the bacteria of the present invention also show an OD value of about 2.2 level, confirming that they form a biofilm at a high level. there was. In addition, when checking the biofilm survival rate, it was confirmed that a high level of biofilm survival rate was maintained even after about 6 days after inoculation, as shown in Figure 7b.
상기와 같은 결과를 통해 본 발명자들은 본원발명의 미생물이 우수한 수준의 치즈 부패곰팡이에 대한 항균효과를 가지고 있으며, 우수한 수준의 염분내성, 건조내성, 저온내성 및 바이오필름 형성능을 가지고 있어 지속적으로 우수한 항균효과를 유지할 수 있음을 확인하였다.Through the above results, the present inventors have shown that the microorganism of the present invention has an excellent level of antibacterial effect against cheese spoilage mold, and has excellent levels of salt tolerance, drying resistance, low temperature resistance, and biofilm formation ability, thereby continuously showing excellent antibacterial activity. It was confirmed that the effect could be maintained.
상기 진술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.The description of the present invention stated above is for illustrative purposes, and a person skilled in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. There will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive.
<110> REPUBLIC OF KOREA(MANAGEMENT : RURAL DEVELOPMENT ADMINISTRATION) <120> Microorganisms having antibacterial activity against spoilage moulds and uses thereof <130> PD20-333 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1170 <212> DNA <213> 16S rDNA of Pediococcus pentosaceus M132-2 <400> 1 gaagkkgcgg gcggctaata catgcagtcg aacgaacttc cgttaattga ttatgacgta 60 cttgtactga ttgagatttt aacacgaagt gagtggcgaa cgggtgagta acacgtgggt 120 aacctgccca gaagtagggg ataacacctg gaaacagatg ctaataccgt ataacagaga 180 aaaccgcatg gttttctttt aaaagatggc tctgctatca cttctggatg gacccgcggc 240 gtattagcta gttggtgagg taaaggctca ccaaggcagt gatacgtagc cgacctgaga 300 gggtaatcgg ccacattggg actgagacac ggcccagact cctacgggag gcagcagtag 360 ggaatcttcc acaatggacg caagtctgat ggagcaacgc cgcgtgagtg aagaagggtt 420 tcggctcgta aagctctgtt gttaaagaag aacgtgggta agagtaactg tttacccagt 480 gacggtattt aaccagaaag ccacggctaa ctacgtgcca gcagccgcgg taatacgtag 540 gtggcaagcg ttatccggat ttattgggcg taaagcgagc gcaggcggtc ttttaagtct 600 aatgtgaaag ccttcggctc aaccgaagaa gtgcattgga aactgggaga cttgagtgca 660 gaagaggaca gtggaactcc atgtgtagcg gtgaaatgcg tagatatatg gaagaacacc 720 agtggcgaag gcggctgtct ggtctgcaac tgacgctgag gctcgaaagc atgggtagcg 780 aacaggatta gataccctgg tagtccatgc cgtaaacgat gattactaag tgttggaggg 840 tttccgccct tcagtgctgc agctaacgca ttaagtaatc cgcctgggga gtacgaccgc 900 aaggttgaaa ctcaaaagaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa 960 ttcgaagcta cgcgaagaac cttaccaggt cttgacatct tctgacagtc taagagatta 1020 gaggttccct tcggggacag atgacagtgt gcatggttgt cgtcagctcg tgtcgtgaga 1080 tgttgggtta agtccccgca cgagcgcacc cttattacta gttgccagca ttaagttggg 1140 cacttctagt gaagactgcc gggtgaacaa 1170 <110> REPUBLIC OF KOREA(MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Microorganisms having antibacterial activity against spoilage molds and uses thereof <130>PD20-333 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1170 <212> DNA <213> 16S rDNA of Pediococcus pentosaceus M132-2 <400> 1 gaagkkgcgg gcggctaata catgcagtcg aacgaacttc cgttaattga ttatgacgta 60 cttgtactga ttgagatttt aacacgaagt gagtggcgaa cgggtgagta acacgtgggt 120 aacctgccca gaagtagggg ataacacctg gaaacagatg ctaataccgt ataacagaga 180 aaaccgcatg gttttctttt aaaagatggc tctgctatca cttctggatg gacccgcggc 240 gtattagcta gttggtgagg taaaggctca ccaaggcagt gatacgtagc cgacctgaga 300 gggtaatcgg ccacattggg actgagacac ggcccagact cctacgggag gcagcagtag 360 ggaatcttcc acaatggacg caagtctgat ggagcaacgc cgcgtgagtg aagaagggtt 420 tcggctcgta aagctctgtt gttaaagaag aacgtgggta agagtaactg tttacccagt 480 gacggtattt aaccagaaag ccacggctaa ctacgtgcca gcagccgcgg taatacgtag 540 gtggcaagcg ttatccggat ttattgggcg taaagcgagc gcaggcggtc ttttaagtct 600 aatgtgaaag ccttcggctc aaccgaagaa gtgcattgga aactgggaga cttgagtgca 660 gaagaggaca gtggaactcc atgtgtagcg gtgaaatgcg tagatatatg gaagaacacc 720 agtggcgaag gcggctgtct ggtctgcaac tgacgctgag gctcgaaagc atgggtagcg 780 aacaggatta gataccctgg tagtccatgc cgtaaacgat gattactaag tgttggaggg 840 tttccgccct tcagtgctgc agctaacgca ttaagtaatc cgcctgggga gtacgaccgc 900 aaggttgaaa ctcaaaagaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa 960 ttcgaagcta cgcgaagaac cttaccaggt cttgacatct tctgacagtc taagagatta 1020 gaggttccct tcggggacag atgacagtgt gcatggttgt cgtcagctcg tgtcgtgaga 1080 tgttgggtta agtccccgca cgagcgcacc cttattacta gttgccagca ttaagttggg 1140 cacttctagt gaagactgcc gggtgaacaa 1170
Claims (14)
상기 조성물은 아스퍼질러스 나이거(Aspergillus niger), 클라도스포리움 클라도스포리오이데스(Cladosporium cladosporioides) 및 페니실린 코뮨(Penicillium commune)으로 이루어진 군에서 선택되는 하나 이상의 치즈부패 곰팡이에 대해서 항곰팡이 활성을 가지는 것을 특징으로 하는, 항곰팡이 조성물.
An anti-fungal composition containing Pediococcus pentosaceus M132-2 strain (accession number: KACC 92316P) as an active ingredient,
The composition has antifungal activity against one or more cheese spoilage molds selected from the group consisting of Aspergillus niger , Cladosporium cladosporioides , and Penicillium commune . An anti-fungal composition, characterized in that it has.
상기 균주는 서열번호 1로 표시되는 16S rRNA 염기서열을 갖는 것을 특징으로 하는, 항곰팡이 조성물.
According to paragraph 1,
An anti-fungal composition, characterized in that the strain has a 16S rRNA base sequence represented by SEQ ID NO: 1.
상기 균주는 염분내성, 건조내성 및 저온내성으로 이루어진 군에서 선택되는 특성 하나 이상을 가지는 것을 특징으로 하는, 항곰팡이 조성물.
According to paragraph 1,
An anti-fungal composition, characterized in that the strain has at least one characteristic selected from the group consisting of salt tolerance, desiccation tolerance, and low temperature tolerance.
상기 균주는 바이오필름(biofilm) 형성능을 가지는 것을 특징으로 하는, 항곰팡이 조성물.
According to paragraph 1,
An anti-fungal composition, characterized in that the strain has the ability to form a biofilm.
An anti-fungal probiotic comprising the anti-fungal composition according to claim 1 as an active ingredient.
The anti-fungal composition according to claim 1 is applied to one or more cheese rots selected from the group consisting of Aspergillus niger , Cladosporium cladosporioides and Penicillium commune . A method of removing or inhibiting the activity of cheese spoilage mold, comprising the step of treating the mold.
상기 단계는 상기 항곰팡이 조성물을 상기 치즈부패 곰팡이에 3일에 한번씩 15일 동안 처리하는 것을 특징으로 하는, 방법.
According to clause 11,
The method is characterized in that the step is treating the cheese spoilage mold with the anti-mold composition once every three days for 15 days.
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