KR101912299B1 - Saccharomyces exiguus having capacity of inhibiting growth and development of Saccharomyces, composition comprising the same and method for manufacturing fermented food using the same - Google Patents
Saccharomyces exiguus having capacity of inhibiting growth and development of Saccharomyces, composition comprising the same and method for manufacturing fermented food using the same Download PDFInfo
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- KR101912299B1 KR101912299B1 KR1020160071159A KR20160071159A KR101912299B1 KR 101912299 B1 KR101912299 B1 KR 101912299B1 KR 1020160071159 A KR1020160071159 A KR 1020160071159A KR 20160071159 A KR20160071159 A KR 20160071159A KR 101912299 B1 KR101912299 B1 KR 101912299B1
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- strain
- saccharomyces
- yeast
- food
- composition
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
본 명세서에는 신규한 사카로미세스 엑시구스 균주, 이를 포함하는 조성물 및 이를 이용하여 발효식품을 제조하는 방법이 개시된다. 본 발명의 일 측면인 상기 균주는, 독소를 생산하여, 다른 효모균의 생육을 억제할 수 있다. 또한, 상기 균주는 pH 환경에 대한 민감성이 적기 때문에, 광범위한 pH 환경에서 독소를 생산할 수 있다. 따라서, 본 발명의 균주, 그 배양액, 그 파쇄물 또는 그 추출물을 이용하면, 광범위한 pH 환경에서, 오염 효모를 제거하여 식품의 부패나 변질을 최소화할 수 있으므로 유용하다.Disclosed herein is a novel strain of Saccharomyces axigus, a composition comprising the same, and a method of producing a fermented food using the same. The strain, which is an aspect of the present invention, can produce toxins and inhibit the growth of other yeast bacteria. In addition, since the strain is less sensitive to the pH environment, toxins can be produced in a wide range of pH environments. Therefore, the use of the strain, the culture, the lysate thereof, or the extract thereof of the present invention is useful because it can minimize the spoilage and alteration of the food by removing the contaminated yeast in a wide pH environment.
Description
본 명세서에는 신규 사카로미세스 엑시구스 균주, 이를 포함하는 조성물 및 이를 이용하여 발효식품을 제조하는 방법이 개시된다.Disclosed herein is a novel Saccharomyces axigus strain, a composition comprising the same, and a method for producing a fermented food using the same.
효모는 식품 부패 및 변질의 주요 원인이며, 효모로 인한 부패 및 변질은 음료, 유제품, 즉석 식품, 발효 식품 등 다양한 식품업체에서 중요한 해결과제로 여기고 있다. 이러한 문제점을 해결하기 위해, 탈수 건조 또는 염장 등의 수분 조절 방법, 냉장 또는 냉동 등의 온도 조절 방법, pH 조절 방법, 가열 살균, 진공 포장을 통한 산소 제거 등의 다양한 시도가 계속되고 있으나, 아직까지 만족할 만한 효과를 얻지는 못하였다. 이외에도 항효모제(즉, 방부제)를 사용하는 방법이 꾸준히 사용되어 왔으나, 최근에는 소비자들의 건강에 대한 관심이 높아지면서, 화학 첨가제가 아닌 천연물을 이용한 효모 제어에 대한 연구가 이루어지고 있다. 그러나 천연물을 이용한 효모 제어를 위해서는 천연물의 추출, 정제 및 농축 등이 필요한데, 이에 투입되는 비용이 고가라는 문제점이 있다. 따라서, 신체에 무해하며, 경제적으로 발효식품의 부패 및 변질을 개선할 수 있는 방법에 대한 연구가 필요한 실정이다.Yeast is a major cause of food corruption and deterioration, and corruption and deterioration caused by yeast are viewed as important challenges for a variety of food companies, including beverages, dairy products, ready-to-eat foods, and fermented foods. In order to solve such problems, various attempts have been made to dehydrate and dry, such as moisture control methods such as salting, temperature control methods such as refrigeration or freezing, pH adjustment methods, heat sterilization and oxygen removal through vacuum packing, But did not achieve a satisfactory effect. In addition, the use of anti-yeast (ie, preservative) has been used regularly. However, as consumers are increasingly interested in health, studies on yeast control using natural products, rather than chemical additives, have been conducted. However, in order to control yeast using natural products, it is necessary to extract, purify and concentrate natural products. Therefore, there is a need for research on a method which is harmless to the body and can economically improve the corruption and alteration of the fermented food.
일 측면에서, 본 발명의 목적은 발효 식품의 부패 및 변질을 최소화하는 것이다.In one aspect, an object of the present invention is to minimize corruption and degeneration of fermented foods.
다른 측면에서, 본 발명의 목적은, 식품 변질의 요인이 되는 오염 효모를 제거하는 것이다.In another aspect, an object of the present invention is to remove contaminated yeast which is a factor of food deterioration.
다른 측면에서, 본 발명의 목적은, 사카로미세스 속 균주에 대하여 독성이 있는 균주를 제공하는 것이다.In another aspect, an object of the present invention is to provide a strain toxic to Saccharomyces cerevisiae.
다른 측면에서, 본 발명의 목적은, 광범위한 pH 에서 오염 효모의 생육을 억제할 수 있는 균주를 제공하는 것이다.In another aspect, an object of the present invention is to provide a strain capable of inhibiting the growth of contaminated yeast at a wide pH.
다른 측면에서, 본 발명의 목적은, 저비용으로 오염 효모의 생육을 억제하는 것이다.In another aspect, an object of the present invention is to inhibit the growth of contaminated yeast at low cost.
일 측면에서, 본 발명은 pH 3.0 내지 pH 5.0의 전체 pH 범위 내에서, 사카로미세스(Saccharomyces) 속 효모에 대해 독성이 있는 독소를 생산하며, 균주 자신은 상기 독소에 대해 저항성이 있는, 사카로미세스 엑시구스(Saccharomyces exiguus) 균주를 제공한다.In one aspect, the present invention provides a method of producing toxins that are toxic to Saccharomyces sp. Yeast within the entire pH range of pH 3.0 to pH 5.0, wherein the strain itself is resistant to the toxin, Saccharomyces exiguus strain.
다른 측면에서, 본 발명은, 상기 균주, 그 배양액, 그 파쇄물 또는 그 추출물을 포함하는 조성물을 제공한다.In another aspect, the present invention provides a composition comprising the strain, a culture thereof, a lysate thereof, or an extract thereof.
다른 측면에서, 본 발명은, 상기 균주를 배양하는 단계를 포함하는 식품 제조방법을 제공한다.In another aspect, the present invention provides a food manufacturing method comprising culturing the strain.
본 발명의 일 측면인 사카로미세스 엑시구스(Saccharomyces exiguus) 균주는, 다른 효모균의 생육을 억제할 수 있는 독소를 생산한다. 본 발명의 균주는 pH 환경에 대한 민감성이 적기 때문에, 광범위한 pH 환경에서 독소를 생산할 수 있다. 또한, 본 발명의 균주는 사카로미세스 속 효모 외의 미생물에 대해서는 독성이 없다. 따라서, 본 발명의 균주, 그 배양액, 그 파쇄물 또는 그 추출물을 이용하면, 광범위한 pH 환경에서, 유익균은 보존하면서, 오염 효모를 제거하여 식품의 부패나 변질을 최소화할 수 있으므로 유용하다. MRS to the one aspect of the invention Saccharomyces eksi Goose (Saccharomyces exiguus strains produce toxins that can inhibit the growth of other yeast strains. Since the strain of the present invention is less sensitive to the pH environment, toxins can be produced in a wide range of pH environments. In addition, the strain of the present invention is not toxic to microorganisms other than Saccharomyces cerevisiae. Therefore, the use of the strain, the culture, the lysate thereof, or the extract thereof of the present invention is useful because it can minimize the spoilage and alteration of the food by removing the contaminated yeast while preserving the beneficial bacteria in a wide pH environment.
도 1은 본 발명 효모의 지모시드 생성능을 확인한 결과를 보이는 도이다.
도 2는 본 발명 효모의 계통도이다.FIG. 1 is a graph showing the results of confirming the ability of the present invention yeast seeds to be produced.
2 is a flow diagram of the yeast of the present invention.
본 발명은 일 측면에서, pH 3.0 내지 pH 5.0의 전체 pH 범위 내에서, 사카로미세스(Saccharomyces) 속 효모에 대해 독성이 있는 독소를 생산하며, 균주 자신은 상기 독소에 대해 저항성이 있는, 사카로미세스 엑시구스(Saccharomyces exiguus) 균주이다. In one aspect, the present invention provides a method of producing toxins that are toxic to Saccharomyces sp. Yeast within the entire pH range of pH 3.0 to pH 5.0, wherein the strain itself is resistant to the toxin, Saccharomyces exiguus .
예컨대, 상기 균주는, pH 3.0 이상, pH 3.1 이상, pH 3.3 이상, pH 3.5 이상, pH 3.7 이상, pH 3.9 이상, pH 4.0 이상, pH 4.1 이상, pH 4.3 이상, pH 4.5 이상, pH 4.7 이상 또는 pH 4.9 이상에서 독소를 생산할 수 있고, pH 5.0 이하, pH 4.9 이하, pH 4.7 이하, pH 4.5 이하, pH 4.3 이하, pH 4.1 이하, pH 4.0 이하, pH 3.9 이하, pH 3.7 이하, pH 3.5 이하, pH 3.3 이하 또는 pH 3.1 이하에서 독소를 생산할 수 있다. 특히, pH 3.0 내지 pH 3.5, 또는 pH 4.5 내지 pH 5.0의 범위에서 독소를 생산할 수 있다. For example, the strain may have a pH of 3.0 or higher, a pH of 3.1 or higher, a pH of 3.3 or higher, a pH of 3.5 or higher, a pH of 3.7 or higher, a pH of 3.9 or higher, a pH of 4.0 or higher, a pH of 4.1 or higher, a pH of 4.3 or higher, pH less than 4.9, less than pH 4.9, less than pH 4.7, less than pH 4.5, less than pH 4.3, less than pH 4.1, less than pH 4.0, less than pH 3.9, less than pH 3.7, less than pH 3.5, The toxin can be produced at pH 3.3 or below or pH 3.1 or below. In particular, the toxin can be produced in the range of pH 3.0 to pH 3.5, or pH 4.5 to pH 5.0.
본 명세서에서 '독소'란, 균주에서 분비되어, 다른 생물체의 대사, 번식 또는 생장을 억제 또는 방해하거나, 그 결과 사멸에 이르게 하는 물질을 의미할 수 있다. As used herein, the term " toxin " may refer to a substance that is secreted by a strain and inhibits or prevents the metabolism, reproduction or growth of another organism, resulting in its death.
일 구현예에서, 상기 독소는 단백질성 독소일 수 있다. 구체적으로, 상기 단백질 성 독소는 지모시드(zymocide)일 수 있다. In one embodiment, the toxin may be a proteolytic toxin. Specifically, the protein toxin may be zymocide.
킬러 효모는 다른 효모를 죽이는 단백질성 독소를 생성하기도 하는데, 이 독소를 킬러 팩터 또는 킬러 독소 등으로 불렀으나, 효모에 대해서만 독성을 나타내고 다른 고등생물이나 세균에는 독성을 나타내지 않으므로, 지모시드라고 불린다. 킬러효모의 지모시드 생산능력은 효모 세포질에 존재하는 킬러 플라스미드에 의하여 결정되는 것으로 알려져 있다.Killer yeast also produces proteinaceous toxins that kill other yeast. These toxins are called killer factors or killer toxins, but they are called gemocides because they are toxic only to yeast and do not show toxicity to other higher organisms or bacteria. The production capacity of killer yeast is known to be determined by the killer plasmid present in yeast cytoplasm.
지모시드 단백질은 매우 적은 양으로도 다른 효모의 단백질 합성과 핵산 등의 유전체 합성을 저해하여 다른 효모의 생육을 억제할 수 있다. 또한, 지모시드는 효모에 대해서는 독성이 있지만, 다른 고등 생물이나 세균에는 독성이 없는 것일 수 있다.Glycoside proteins can inhibit the growth of other yeasts by inhibiting protein synthesis and nucleic acid synthesis of other yeasts even in very small amounts. In addition, it may be toxic to yeast, but not toxic to other higher organisms or bacteria.
일 구현예에서, 상기 사카로미세스 엑시구스 균주는 20 내지 30℃의 온도에서 독소를 생산할 수 있다. 예컨대, 20℃ 이상, 21℃ 이상, 23℃ 이상, 25℃ 이상, 27℃ 이상, 29℃ 이상, 또는 30℃ 이하, 29℃ 이하, 27℃ 이하, 25℃ 이하, 23℃ 이하, 또는 21℃ 이하에서 독소를 생산할 수 있다.In one embodiment, the Saccharomyces axigus strain can produce the toxin at a temperature of 20 to 30 < 0 > C. For example, at least 20 占 폚, at least 21 占 폚, at least 23 占 폚, at least 25 占 폚, at least 27 占 폚, at least 29 占 폚, or at least 30 占 폚, at least 29 占 폚, at least 27 占 폚, at least 25 占 폚, The following toxins can be produced.
일 구현예에서, 상기 사카로미세스 속 효모는, 사카로미세스 세레비지에(Saccharomyces cerevisiae)일 수 있으나, 이에 제한되는 것은 아니다. In one embodiment, the Saccharomyces sp. May be, but is not limited to, Saccharomyces cerevisiae .
상기 균주는, 사카로마세스 엑시구스 SMY76 균주 또는 사카로마세스 엑시구스 SMY77 균주일 수 있다. 일 구현예에서, 상기 균주는 사카로마세스 엑시구스 SMY76 균주일 수 있다.The strain may be Saccharomyces exigus SMY76 strain or Saccharomyces exigus SMY77 strain. In one embodiment, the strain may be S. cerevisiae SMY76 strain.
또한, 상기 균주는 기탁번호KCCM11818P인 균주일 수 있다. In addition, the strain may be a strain having the accession number KCCM11818P.
또한, 일 구현예에서, 상기 균주는 고추 및 무 중 하나 이상을 포함하는 채소의 발효물로부터 유래한 것일 수 있다. 상기 발효물은 자연 발효물일 수 있다. 본 명세서에서 '자연 발효물'이란, 시료의 발효를 위하여 인위적으로 효모 등을 첨가하지 않고 수행되는 발효를 통해 얻어지는 결과물을 의미할 수 있다. 일 구현예에서 상기 자연발효에 사용되는 시료는, 고추 및 무 중 하나 이상을 포함할 수 있다. 예컨대, 상기 자연발효물은 상기 시료와 마늘 및 소금을 첨가하고, 20 내지 30℃에서의 상온에서 2 내지 4일간 발효하여 얻어진 것일 수 있다. Also, in one embodiment, the strain may be derived from a fermentation of a vegetable comprising at least one of pepper and radish. The fermented product may be a natural fermented product. In the present specification, 'natural fermentation product' may mean the product obtained through fermentation without artificially adding yeast or the like for fermentation of the sample. In one embodiment, the sample used in the natural fermentation may include one or more of pepper and radish. For example, the natural fermented product may be obtained by adding the sample, garlic, and salt, and fermenting at 20 to 30 DEG C for 2 to 4 days at room temperature.
상기 균주는 서열번호 5의 서열 및 서열번호 6의 서열 중 하나 이상을 포함할 수 있다. 예컨대, 서열번호 5의 서열은 상기 사카로마세스 엑시구스 SMY76 균주의 ITS 서열을 포함하고, 서열번호 6의 서열은 사카로마세스 엑시구스 SMY76 균주의 26s rDNA의 서열을 포함할 수 있다. The strain may include one or more of the sequence of SEQ ID NO: 5 and the sequence of SEQ ID NO: 6. For example, the sequence of SEQ ID NO: 5 may include the ITS sequence of the SACRAMESES EXIGUSS SMY76 strain, and the sequence of SEQ ID NO: 6 may include the sequence of 26s rDNA of the SACRAMESES EXIGUS SMY76 strain.
본 발명은 다른 측면에서, 상기 균주, 그 배양액, 그 파쇄물 또는 그 추출물을 포함하는 조성물일 수 있다. 상기 배양액, 파쇄물 또는 추출물은, 제균된 것을 포함할 수 있다. 또한, 상기 배양액은, 배양액을 원심분리하여 얻은 상등액을 포함할 수 있다. 상기 추출물의 제조방법은 제한되지 않고, 업계에 알려진 주지의 방법에 의해 수행될 수 있다.In another aspect, the present invention may be a composition comprising the strain, its culture, its lysate or an extract thereof. The culture broth, crushed product, or extract may contain a sterilized product. Further, the culture liquid may include a supernatant obtained by centrifuging the culture liquid. The method for producing the above extract is not limited, and can be carried out by well-known methods known in the art.
일 구현예에서, 상기 조성물은 식품 조성물 또는 약학적 조성물일 수 있다.In one embodiment, the composition may be a food composition or a pharmaceutical composition.
일 구현예에서, 상기 식품은 발효 식품일 수 있고, 나아가, 상기 발효는 유산균 발효를 포함할 수 있다. 본 명세서에서 발효식품은 예컨대, 간장, 된장, 고추장과 같은 콩 발효식품, 치즈, 요거트와 같은 발효유제품, 김치, 젓갈과 같은 소금절임류 또는 조미료와 같은 풍미제 및 빵을 포함할 수 있으나 이에 제한되는 것은 아니다. In one embodiment, the food may be a fermented food, and further, the fermentation may include lactic acid fermentation. Fermented foods herein may include, for example, soybean fermented foods such as soy sauce, soybean paste, hot pepper paste, fermented milk products such as cheese, yogurt, flavoring agents such as salt pickles or condiments such as kimchi, It is not.
본 발명의 일 측면에 따른 식품 조성물의 제형은 특별히 한정되지 않으나, 예를 들어, 정제, 과립제, 분말제, 드링크제와 같은 액제, 캐러멜, 겔, 바 등으로 제형화될 수 있다. 식품 조성물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 증진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 포함할 수 있다. 각 제형의 식품 조성물은 유효 성분 이외에 해당 분야에서 통상적으로 사용되는 성분들을 제형 또는 사용 목적에 따라 당업자가 어려움 없이 적의 선정하여 배합할 수 있으며, 다른 원료와 동시에 적용할 경우 상승 효과가 일어날 수 있다.The formulation of the food composition according to one aspect of the present invention is not particularly limited, but may be formulated into, for example, tablets, granules, powders, liquid preparations such as drinks, caramels, gels, bars and the like. The food composition may contain various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate etc.), pectic acid and its salts, alginic acid and its salts, Colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. The food composition of each formulation can be blended with the ingredients commonly used in the field in addition to the active ingredient without difficulty by those skilled in the art depending on the purpose of formulation or use, and synergistic effect can be obtained when the composition is applied simultaneously with other ingredients.
약학 조성물은 경구 또는 비경구의 여러 가지 제형일 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 연질 또는 경질 캡슐제 등이 포함되며, 이러한 고형제제는 하나 이상의 화합물에 적어도 하나 이상의 부형제 예를 들면, 전분, 탄산칼슘, 수크로오스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 스테아린산 마그네슘, 탈크 등과 같은 윤활제들도 사용된다. 경구투여를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁용제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.The pharmaceutical composition may be of various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, soft or hard capsules, etc. These solid preparations may contain one or more excipients such as starch, calcium carbonate, sucrose, Or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.
본 발명의 상기 약학적 조성물은 목적하는 바에 따라 비경구 투여하거나 경구 투여할 수 있으며, 하루에 체중 1 ㎏당 0.1~500 ㎎, 바람직하게는 1~100 ㎎의 양으로 투여되도록 1 내지 수회에 나누어 투여할 수 있다. 특정 환자에 대한 투여용량은 환자의 체중, 연령, 성별, 건강 상태, 식이, 투여 시간, 투여 방법, 배설률, 질환의 중증도 등에 따라 변화될 수 있다.The pharmaceutical composition of the present invention may be administered parenterally or orally, and may be administered in an amount of 0.1 to 500 mg, preferably 1 to 100 mg per kg of body weight per day, Lt; / RTI > The dosage for a particular patient may vary depending on the patient's body weight, age, sex, health condition, diet, time of administration, administration method, excretion rate, severity of disease, and the like.
상기 약학 조성물은, 각각 통상의 방법에 따라 산제, 과립제, 정제, 연질 또는 경질 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 주사제 및 멸균 주사용액 등을 비롯하여 약제학적 제제에 적합한 어떠한 형태로든 제형화하여 사용될 수 있다.The pharmaceutical composition may be formulated into tablets, capsules, tablets, capsules, capsules, tablets, capsules, capsules, tablets, pills, capsules, Or may be formulated into a formulation.
또한 약학적 조성물은, 쥐, 생쥐, 가축, 인간 등의 포유동물에 비경구, 경구 등의 다양한 경로로 투여될 수 있으며, 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구, 경피(trandermally), 정맥, 근육, 피하주사에 의해 투여될 수 있다.The pharmaceutical composition may also be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes including parenteral, oral, and the like, all manner of administration being expected, for example oral, transdermal trandermally, intravenous, intramuscular, subcutaneous injection.
본 발명은 일 측면에서, 상기 균주를 배양하는 단계를 포함하는 식품 제조방법이다. 상기 식품 제조방법은, 상기 균주, 그 배양물, 그 파쇄물 또는 그 추출물을 식품에 첨가하는 단계를 더 포함할 수 있다.In one aspect, the present invention is a food manufacturing method comprising culturing the strain. The food manufacturing method may further include a step of adding the strain, the culture, the lysate thereof, or an extract thereof to the food.
또한, 상기 미생물(Saccaharomyces exiguous SMY76)은 2016년 3월 15일 한국 미생물 보존센터에 기탁번호 KCCM11818P로 기탁하였다.The microorganism (Saccaharomyces exiguous SMY76) was deposited on March 15, 2016 with the deposit number KCCM11818P at the Korean Society for Microbiological Conservation.
이하, 하기의 실시예를 통하여 본 발명을 보다 구체적으로 설명한다. 그러나, 하기 실시예는 본 발명에 대한 이해를 돕기 위해 예시의 목적으로만 제공된 것일 뿐, 본 발명의 범주 및 범위가 이에 한정되지 않는다.Hereinafter, the present invention will be described more specifically with reference to the following examples. However, the following examples are provided for illustrative purposes only in order to facilitate understanding of the present invention, and the scope and scope of the present invention are not limited thereto.
[실시예 1] 야생 효모 분리를 위한 시료의 제조[Example 1] Preparation of sample for isolation of wild yeast
지모시드를 생성하는 야생효모 분리를 위해 자연발효 시료를 제조하였다. 먼저 쌀 또는 현미 15~30%에 액당화 효소 0.001~0.01% 를 첨가한 후, 70±5℃에서 14~18시간 액당화를 진행한 후, 85에서 1시간 살균 및 냉각 후 상온(20~30)에서 7일간 자연발효를 진행하였다. 고추 또는 무 70%~90%에 마늘 2%~5%, 소금 2% 첨가 후 상온(20~30)에서 3일간 자연발효를 진행 하여 시료를 제조하였다.Natural fermentation samples were prepared for the isolation of wild yeast producing gymoside. First, 0.001 to 0.01% of a liquid saccharifying enzyme is added to 15 to 30% of rice or brown rice, followed by liquification at 70 ± 5 ° C. for 14 to 18 hours. After sterilization at 85 ° C. for 1 hour and cooling, ) For 7 days. The samples were prepared by adding 2% ~ 5% of garlic and 2% of salt to pepper or 70% ~ 90% of non - fermented soybeans, followed by natural fermentation at room temperature (20 ~ 30) for 3 days.
[실시예 2] 야생효모의 분리 및 선별[Example 2] Separation and selection of wild yeast
[실시예 2-1] 1차 분리[Example 2-1] First separation
멸균된 생리식염수(0.85% NaCl) 90ml에 실시예 1에서 제조한 시료를 10g 주입 후 30분간 균일화를 진행하였으며, 균일화된 시료는 연속희석법으로 103~106으로 희석하였다. 희석액 0.1 m을 50mM 암피실린이 첨가된 PDB(Potato Dextrose Broth) 아가배지에 도말하고, 25 항온기에서 2일간 배양하여 유백색의 집락을 형성하는 효모를 분리하였다. After injecting 10 g of the sample prepared in Example 1 into 90 ml of sterilized physiological saline (0.85% NaCl), the homogenization was performed for 30 minutes, and the homogenized sample was diluted to 10 3 to 10 6 by the continuous dilution method. 0.1m of the diluted solution was plated on a PDB (Potato Dextrose Broth) agar medium supplemented with 50mM of ampicillin, and cultured in a 25 ° C incubator for 2 days to isolate a yeast that formed milky colonies.
[실시예 2-2] 2차 분리[Example 2-2] Secondary separation
실시예 2-1에서 분리된 효모를 NaCl이 5% 첨가된 YEPD(2% yeast extract, 2% peptone, 2% glucose/dextrose) 액체 배지에 접종하여 30에서 24시간 배양하여 내염성을 가진 효모를 선별하였으며, pH를 3.5로 조절한 YEPD(2% yeast extract, 2% peptone, 2% glucose/dextrose) 액체 배지에 접종하고, 30에서 24시간 배양하여 내산성을 가진 효모를 선별하였다. 선별된 효모 총 5종(SMY 73, SMY74, SMY75, SMY76, SMY77)으로서, 아래 표 1에 기재된 바와 같다.Yeast isolated in Example 2-1 was inoculated into a liquid medium containing YEPD (2% yeast extract, 2% peptone, 2% glucose / dextrose) containing 5% NaCl and cultured for 30 to 24 hours to select yeast having salt resistance And yeast with acid resistance was selected by inoculation in a liquid medium of YEPD (2% yeast extract, 2% peptone, 2% glucose / dextrose) adjusted to pH 3.5 and culturing for 30 to 24 hours. The five yeasts selected (SMY73, SMY74, SMY75, SMY76, SMY77) are as shown in Table 1 below.
SMY 74
SMY 75SMY 73
SMY 74
SMY 75
SMY 77SMY 76
[실시예 2-3] 지모시드 생성 효모의 선별[Example 2-3] Screening of yeast producing yeast seeds
사카로미세스 세레비지에(Saccharomyces cerevisiae) KCTC7296 균주를 YEPD(2% yeast extract, 2% peptone, 2% glucose/dextrose) 액체 배지에 접종하여 25에서 24시간 배양하고, 100ml를 취해 pH 4.2±0.1으로 조절한 YEPD(2% yeast extract, 2% peptone, 2% glucose/dextrose)에 agar를 2% 첨가하여 고체 배지에 도말한 다음 멸균된 8mm 직경의 페이퍼 디스크(Toyo Roshi CO. LTD, 일본)를 얹었다. 그 후, 실시예 2-2에서 선별된 효모를 pH 4.2 ± 0.1로 조절한 YEPD(2% yeast extract, 2% peptone, 2% glucose/dextrose) 액체 배지에 접종하여 25에서 24~48시간 배양 후 13000rpm으로 원심 분리하여 상등액을 분리하고, 분리된 상등액을 0.20㎛ 필터를 통해 제균한 뒤 상기 페이퍼 디스크 위에 150㎕씩 분주하였다. 이후, 상등액이 분주된 페이퍼 디스크를 25 항온기에서 24시간 동안 배양한 후, 페이퍼 디스크 주위에 형성된 투명환 직경을 측정하였다. 그 결과, SMY76 및 SMY77 두 효모에서 지모시드 생성으로 인한 투명환이 관찰되었다. SMY76의 투명환의 크기는 약 12mm이고, SMY77의 투명환의 크기는 약 11mm 였다. 효모 생육 억제력이 가장 좋은 효모인 SMY76 및 SMY77를 최종적으로 선별하였다. Saccharomyces YEPD a cerevisiae) KCTC7296 strain (2% yeast extract, 2% peptone, 2% glucose / dextrose) YEPD for 24 hours culture at 25 were inoculated into the liquid medium, it takes a 100ml adjusted to pH 4.2 ± 0.1 (2% yeast extract , 2% peptone, 2% glucose / dextrose), 2% agar was added to the solid medium, and sterilized 8 mm diameter paper disks (Toyo Roshi CO. LTD., Japan) were placed. Thereafter, the yeast selected in Example 2-2 was inoculated into a liquid medium of YEPD (2% yeast extract, 2% peptone, 2% glucose / dextrose) adjusted to pH 4.2 ± 0.1 and cultured at 25 to 24 to 48 hours The supernatant was separated by centrifugation at 13000 rpm, the separated supernatant was sterilized by a 0.20 mu m filter, and 150 mu l was dispensed on the paper disk. Thereafter, the paper disk on which the supernatant liquid was dispensed was incubated in a 25 ° C incubator for 24 hours, and the diameter of the transparent pores formed around the paper disk was measured. As a result, transparent rings due to the generation of zymoside were observed in both SMY76 and SMY77 yeasts. The size of the transparent ring of SMY76 was about 12 mm, and the size of transparent ring of SMY77 was about 11 mm. SMY76 and SMY77, which are the best yeast growth inhibiting yeast, were finally selected.
[실시예 3] 선별 효모의 동정[Example 3] Identification of yeast sorted
지모시드 생성능이 확인된 SMY76 및 SMY77 두 효모를 동정하기 위해, ITS 영역과 26s rDNA를 증폭하여 염기서열을 얻었다. 구체적으로 유니버셜 프라이머인 ITS1 F(5'-TCCGTAGGTGAACCTGCGG-3')(서열번호 1)와 ITS4 R(5'-TCCTCCGCTTATTGATATGC-3')(서열번호 2)을 사용하여 ITS 영역을 증폭하였으며, 유니버셜 프라이머인 NL1(5'-GCATATCAATAAGCGGAGGAAAAG-3')(서열번호 3)과 NL4(5'-GGTCCGTGTTTCAAGACGG-3') (서열번호 4)를 이용하여 26s rDNA를 증폭하였다. 증폭된 ITS, 26s rDNA는 Wizard DNA 정제 키트 (Promega, Madison, Wisconsin, USA)를 사용하여 정제한 뒤, 솔젠트㈜에 의뢰하여 염기서열을 분석하였다. BLAST로 상동성을 비교하고, 메가 프로그램(MEGA program)을 이용하여 계통도 분석을 실시하였다(도 2) 그 결과, 두 효모 모두 사카로미세스 엑시구스(Saccharomyces exiguus)인 것으로 동정되었다.In order to identify two SMY76 and SMY77 yeasts with the ability to produce glycoside synthase, the ITS region and 26s rDNA were amplified to obtain nucleotide sequences. Specifically, the ITS region was amplified using the universal primer ITS1F (5'-TCCGTAGGTGAACCTGCGG-3 ') (SEQ ID NO: 1) and ITS4R (5'-TCCTCCGCTTATTGATATGC-3' 26s rDNA was amplified using NL1 (5'-GCATATCAATAAGCGGAGGAAAAG-3 ') (SEQ ID NO: 3) and NL4 (5'-GGTCCGTGTTTCAAGACGG-3') (SEQ ID NO: 4). Amplified ITS and 26s rDNA were purified using a Wizard DNA purification kit (Promega, Madison, Wisconsin, USA), and analyzed by sequencing with Solgent. Comparing the homology in BLAST, and by using the program MEGA (MEGA program) was subjected to analysis system diagram (FIG. 2) As a result, the MRS in both yeast Saccharomyces eksi Goose (Saccharomyces exiguus .
본 발명의 일 관점에 따른 조성물의 제형예를 아래에서 설명하나, 다른 여러 가지 제형으로도 응용 가능하며, 이는 본 발명을 한정하고자 함이 아닌 단지 구체적으로 설명하고자 함이다.Formulation examples of compositions according to one aspect of the present invention are described below, but may be applied to various other formulations, which are not intended to be limiting but merely illustrative of the present invention.
[제조예 1] 발효유(fermented milk)[Production Example 1] Fermented milk
[제조예 2] 발효 음료[Manufacturing Example 2] Fermented beverage
[제조예 3] 채소 발효물을 이용한 김치풍 소스[Preparation Example 3] Preparation of kimchi sprout sauce using vegetable fermented product
무 발효물 30중량%, 고추 발효물 30중량%, 식초 0.5 중량%, 양파 5 중량%, 현미 발효물 20중량%, 다진마늘 3 중량%, 산탄검 0.05중량%, 정제수 11.45중량%, SMY 76 균주의 배양액 1 중량%를 넣어 소스를 제조하였다.30% by weight of non-fermented product, 30% by weight of fermented red pepper, 0.5% by weight of vinegar, 5% by weight of onion, 20% by weight of brown rice fermentation, 3% by weight of chopped garlic, 0.05% by weight of xanthan gum, 1% by weight of the culture medium of the strain was added to prepare a sauce.
<110> SAMPIO FOOD COMPANY <120> Saccharomyces exiguous having capacity for inhibiting growth and development of Saccharomyces, composition comprising the same and method for manufacturing fermented food using the same <130> 16P175IND <160> 6 <170> KopatentIn 2.0 <210> 1 <211> 19 <212> DNA <213> primer_ITS1_foward <400> 1 tccgtaggtg aacctgcgg 19 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer_ITS4_reverse <400> 2 tcctccgctt attgatatgc 20 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer_NL1 <400> 3 gcatatcaat aagcggagga aaag 24 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer_NL4 <400> 4 ggtccgtgtt tcaagacgg 19 <210> 5 <211> 697 <212> DNA <213> ITS sequence of SMY76 <400> 5 ttcggaagga tcattaaaga aaatgatgaa taattggtag aggaggagtg atcgagcctg 60 cgcttaagtg cgcggcgagg ttgttctttt tcgttacgcc gataattctt tacacacact 120 ggagttttat ttctacagta tcggaggtag caataccgcc aaaacaaaac acaaacaatt 180 attttttatt attttaattt gtcatttcaa aatctgcttt attgcagtaa ccaaaatatt 240 caaaactttc aacaacggat ctcttggttc tcgcatcgat gaagaacgca gcgaaatgcg 300 atacgtaatg tgaattgcag aattccgtga atcatcgaat ctttgaacgc acattgcgcc 360 ccttggtatt ccagggggca tgcctgtttg agcgtcattt ccttctcaaa tacttgtatt 420 tggttgtgag tgacactcag tcttgcattg agttaacttg aaattgttgg ccgtagcggt 480 tgttgcagct tatagttttt gtgtaatggt atgatttctt tactattaaa caggatgctt 540 gagcatgtcg tattaggttt taccaactcc ggcagactcg gtatcttgga agagcgtact 600 ggcattagaa aattgagact gtcgactgtg gcaaacagta ctctttaagt ttgacctcaa 660 atcagctagg aatacccgct gaacttaagc atatcaa 697 <210> 6 <211> 972 <212> DNA <213> 26s rDNA sequence of SMY76 <400> 6 ttaagcatat caataagcgg aggaaaagaa accaaccggg attgccttag taacggcgag 60 tgaagcggca aaagctcaaa tttgaaatct ggtaccttcg gtgcccgagt tgtaatttgt 120 agagggcgac tttggggcgg ctccttgtct atgttccttg gaacaggacg tcatagaggg 180 tgagaatccc gtgtggcgag gagtgcggtt ccgtgtaaag cgctctcgaa gagtcgagtt 240 gtttgggaat gcagctctaa gtgggtggta aattccatct aaagctaaat attggcgaga 300 gaccgatagc gaacaagtac agtgatggaa agatgaaaag aactttgaaa agagagtgaa 360 aaagtacgtg aaattgttga aagggaaggg catttgatca gacatggtgt tttgtgcccc 420 tcgctccttg tgggtggggg aatctcgcag ctcactgggc cagcatcagt tttggcggtc 480 ggataaaacc aggggaacgt agcttgcttc gggaagtatt atagcctctg ggaatacggc 540 cagccgggac tgaggaacgc gattcgtcaa ggatgctggc ataatggtta tatgccgccc 600 gtcttgaaac acggaccaag gagtctaacg tctatgcgag tgtttgggtg tgaaacccat 660 acgcgtaatg aaagtgaacg taggttgggg cctgtcaaag ggtgcacaat cgaccgatcc 720 tgatgttttc agatggattt gagtaagagc atagctgttg ggacccgaaa gatggtgaac 780 tatgcctgaa tagggtgaag ccagaggaaa ctctggtgga ggctcgtagc ggttctgacg 840 tgcaaatcga tcgtcgaatt tgggtatagg ggcgaaagac taatcgaacc atctagtagc 900 tggttcctgc cgaagtttcc ctcaggatag cagaagctcg tatcagtttt atgaggtaaa 960 gcgaatgatt ag 972 <110> SAMPIO FOOD COMPANY <120> Saccharomyces exiguous capacity for inhibiting growth and development of Saccharomyces, composition comprising the same and method for manufacturing fermented food using the same <130> 16P175IND <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 19 <212> DNA <213> primer_ITS1_foward <400> 1 tccgtaggtg aacctgcgg 19 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer_ITS4_reverse <400> 2 tcctccgctt attgatatgc 20 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer_NL1 <400> 3 gcatatcaat aagcggagga aaag 24 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer_NL4 <400> 4 ggtccgtgtt tcaagacgg 19 <210> 5 <211> 697 <212> DNA <213> ITS sequence of SMY76 <400> 5 ttcggaagga tcattaaaga aaatgatgaa taattggtag aggaggagtg atcgagcctg 60 cgcttaagtg cgcggcgagg ttgttctttt tcgttacgcc gataattctt tacacacact 120 ggagttttat ttctacagta tcggaggtag caataccgcc aaaacaaaac acaaacaatt 180 attttttatt attttaattt gtcatttcaa aatctgcttt attgcagtaa ccaaaatatt 240 caaaactttc aacaacggat ctcttggttc tcgcatcgat gaagaacgca gcgaaatgcg 300 atacgtaatg tgaattgcag aattccgtga atcatcgaat ctttgaacgc acattgcgcc 360 ccttggtatt ccagggggca tgcctgtttg agcgtcattt ccttctcaaa tacttgtatt 420 tggttgtgag tgacactcag tcttgcattg agttaacttg aaattgttgg ccgtagcggt 480 tgttgcagct tatagttttt gtgtaatggt atgatttctt tactattaaa caggatgctt 540 gagcatgtcg tattaggttt taccaactcc ggcagactcg gtatcttgga agagcgtact 600 ggcattagaa aattgagact gtcgactgtg gcaaacagta ctctttaagt ttgacctcaa 660 atcagctagg aatacccgct gaacttaagc atatcaa 697 <210> 6 <211> 972 <212> DNA <213> 26s rDNA sequence of SMY76 <400> 6 ttaagcatat caataagcgg aggaaaagaa accaaccggg attgccttag taacggcgag 60 tgaagcggca aaagctcaaa tttgaaatct ggtaccttcg gtgcccgagt tgtaatttgt 120 agagggcgac tttggggcgg ctccttgtct atgttccttg gaacaggacg tcatagaggg 180 tgagaatccc gtgtggcgag gagtgcggtt ccgtgtaaag cgctctcgaa gagtcgagtt 240 gtttgggaat gcagctctaa gtgggtggta aattccatct aaagctaaat attggcgaga 300 gaccgatagc gaacaagtac agtgatggaa agatgaaaag aactttgaaa agagagtgaa 360 aaagtacgtg aaattgttga aagggaaggg catttgatca gacatggtgt tttgtgcccc 420 tcgctccttg tgggtggggg aatctcgcag ctcactgggc cagcatcagt tttggcggtc 480 ggataaaacc aggggaacgt agcttgcttc gggaagtatt atagcctctg ggaatacggc 540 cagccgggac tgaggaacgc gattcgtcaa ggatgctggc ataatggtta tatgccgccc 600 gtcttgaaac acggaccaag gagtctaacg tctatgcgag tgtttgggtg tgaaacccat 660 acgcgtaatg aaagtgaacg taggttgggg cctgtcaaag ggtgcacaat cgaccgatcc 720 tgatgttttc agatggattt gagtaagagc atagctgttg ggacccgaaa gatggtgaac 780 tatgcctgaa tagggtgaag ccagaggaaa ctctggtgga ggctcgtagc ggttctgacg 840 tgcaaatcga tcgtcgaatt tgggtatagg ggcgaaagac taatcgaacc atctagtagc 900 tggttcctgc cgaagtttcc ctcaggatag cagaagctcg tatcagtttt atgaggtaaa 960 gcgaatgatt ag 972
Claims (17)
균주 자신은 상기 독소에 대해 저항성이 있는, 기탁번호 KCCM11818P로 수탁된 사카로미세스 엑시구스(Saccharomyces exiguus) 균주.Produce toxins that are toxic to Saccharomyces sp. Yeast,
The strain itself is a strain of Saccharomyces exiguus deposited with the deposit number KCCM11818P which is resistant to the toxin.
상기 균주는, 20 내지 30℃의 온도에서 독소를 생산하는, 사카로미세스 엑시구스 균주.The method according to claim 1,
Wherein the strain produces a toxin at a temperature of 20 to 30 占 폚.
상기 독소는, 단백질성 독소인, 사카로미세스 엑시구스 균주.The method according to claim 1,
The toxin is a proteolytic toxin, Saccharomyces axigus strain.
상기 단백질성 독소는, 지모시드(zymocide)인, 사카로미세스 엑시구스 균주.The method of claim 3,
Wherein the proteolytic toxin is a zymocide, Saccharomyces axigus strain.
상기 사카로미세스 속 효모는, 사카로미세스 세레비지에(Saccharomyces cerevisiae)인, 사카로미세스 엑시구스 균주.The method according to claim 1,
The Saccharomyces cerevisiae strain is a Saccharomyces cerevisiae strain, Saccharomyces axigus strain.
상기 균주는 고추 및 무 중 하나 이상을 포함하는 채소의 발효물로부터 유래한 것인, 사카로미세스 엑시구스 균주. The method according to claim 1,
Wherein the strain is derived from a fermentation product of a vegetable comprising at least one of pepper and radish.
상기 균주는 서열번호 5의 서열 및 서열번호 6의 서열 중 하나 이상을 포함하는, 사카로미세스 엑시구스 균주.The method according to claim 1,
Wherein the strain comprises at least one of the sequence of SEQ ID NO: 5 and the sequence of SEQ ID NO: 6.
상기 독소는 pH 4.2±0.1 범위 내에서 생산되는 것을 특징으로 하는, 사카로미세스 엑시구스 균주.The method according to claim 1,
Wherein said toxin is produced in a pH range of 4.2 +/- 0.1.
상기 균주는, 사카로미세스 엑시구스 SMY76 균주인, 사카로미세스 엑시구스 균주.The method according to claim 1,
The strain is Saccharomyces axigus strain, which is a strain of Saccharomyces axigus SMY76.
상기 조성물은, 효모 활성 억제용 조성물인, 조성물.11. The method of claim 10,
Wherein the composition is a composition for inhibiting yeast activity.
상기 식품은, 발효 식품인, 조성물.11. The method of claim 10,
Wherein the food is a fermented food.
상기 발효는 유산균 발효를 포함하는, 조성물.14. The method of claim 13,
Wherein the fermentation comprises lactic acid fermentation.
상기 균주, 그 배양물, 그 파쇄물 또는 그 추출물을 식품에 첨가하는 단계를 포함하는, 식품 제조방법.16. The method of claim 15,
And adding the strain, the culture, the lysate thereof, or an extract thereof to the food.
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US20070196382A1 (en) | 2006-02-22 | 2007-08-23 | Food Industry Research And Development Institute | Saccharomyces cerevisiae strains for hyper-producing glutathione and gamma-glutamylcysteine and processes of use |
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