KR101823073B1 - New microorganism SJP-SNU KCTC12756BP and use thereof - Google Patents
New microorganism SJP-SNU KCTC12756BP and use thereof Download PDFInfo
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
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- 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
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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- A23V2200/00—Function of food ingredients
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- A23V2200/32—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the digestive tract
- A23V2200/3204—Probiotics, living bacteria to be ingested for action in the digestive tract
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- A—HUMAN NECESSITIES
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- A23V2250/00—Food ingredients
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Abstract
Description
본 발명은 신종 SJP-SNU 효모 균주에 관한 것으로, 보다 구체적으로 장정착성, 내열성, 알코올 분해효능, 악취 저감효능 및 항인플루엔자 효능이 우수하며 병원성이 없는 SJP-SNU 균주 및, 상기 균주를 포함하는 프로바이오틱스 조성물, 식품 조성물 및 사료 조성물에 관한 것이다.The present invention relates to a novel SJP-SNU yeast strain, and more particularly to a novel SJP-SNU strain having excellent long-term fixability, heat resistance, alcohol decomposition efficacy, odor reduction efficacy and anti-influenza efficacy and no pathogenicity, Probiotic compositions, food compositions and feed compositions.
효모는 진핵세포로 구성된 단세포 미생물로서, 당을 발효시켜 에탄올과 이산화탄소를 생산하는 능력이 있으며, 주로 출아에 의해 증식하는 진균류의 총칭으로 사용되고 있다. 효모는 과일의 표면, 토양, 공기, 해수 또는 식물의 표피와 꽃술 및 동물의 체내에 이르기까지 폭넓게 분포되어 자연계로부터 쉽게 분리될 수 있다. 또한 효모는 예로부터 알코올 제조 및 제빵 등의 용도로 인간에게 매우 유용하게 이용되어 왔다.Yeast is a single cell microorganism composed of eukaryotic cells. It is capable of producing ethanol and carbon dioxide by fermenting sugars, and is generally used as a generic term for fungi that proliferate mainly by germination. Yeast can be widely distributed from the surface of fruits, soil, air, seawater or the epidermis of plants to flowers and animals and can be easily separated from the natural world. In addition, yeast has been used for human beings very usefully for alcohol production and baking.
최근에는 효모 자체가 값싼 단백질 및 비타민 영양공급원으로 사용되기도 하고, 특히 장내 미생물의 균형을 유지하고 병원성 세균의 증식을 억제하며, 면역 증진의 효과 등이 있는 것으로 보고되었다. 그에 따라 현재 효모는 프로바이오틱스 또는 보조사료 생균제로써 사료 내 영양소의 소화율 및 가축 생산성을 개선시키기 위한 사료 첨가제로 이용되고 있다.Recently, it has been reported that yeast itself is used as a cheap protein and vitamin nutrient source, especially in maintaining intestinal microorganism balance, inhibiting the growth of pathogenic bacteria, and improving immunity. As a result, yeast has been used as a feed additive to improve the digestibility of feed nutrients and livestock productivity as probiotics or supplementary feed probiotics.
효모는 식품 분야를 포함한 다양한 산업 분야에서 이용되고 있으며, 인체나 동물의 장에 서식하면서 장내 유해균의 발육을 억제함으로써 장내 건강 유지에 중요한 역할을 하고 있다. 또한, 축산분야에서 효모는 가축의 사료에 첨가되어 가축의 성장에 필요한 소화효소의 분비를 촉진하고, 장내 세균수를 안정화시키며, 대사성 질병을 예방하는 역할을 수행하여 가축의 생산성을 향상시켜, 효과적인 생균제로 널리 이용되고 있다.Yeast is used in various industrial fields including food field, and it plays an important role in maintaining intestinal health by inhibiting the development of harmful bacteria in the intestines while living in the intestines of human and animals. In addition, in the livestock sector, yeast is added to the feed of livestock to promote the secretion of digestive enzymes necessary for the growth of livestock, to stabilize intestinal bacterial counts, to prevent metabolic diseases, to improve livestock productivity, It is widely used as a probiotic agent.
최근에는 효모 세포벽의 주성분인 글루칸이 동물의 면역 기능을 강화시키는 물질로 보고되고, 동물에 급여될 때 동물의 질병 저항성을 높이는 효과를 나타내는 것으로 알려졌다. 이에 따라 효모는 가축의 생산성 향상을 위해 사용되는 항생제가 야기하는 가축 내 잔류 및 내성 문제를 해결하는 대체 물질로서 현재 주목을 받고 있다. 따라서, 장정착성 및 생체에 대한 안전성 등이 우수하여, 프로바이오틱스 조성물, 식품 또는 사료에 효과적으로 사용할 수 있는 신종 효모 균주의 개발이 필요한 실정이다.Recently, glucan, a major component of yeast cell wall, has been reported as a substance that enhances the immune function of an animal and has been shown to have an effect of increasing the disease resistance of an animal when it is fed to an animal. Therefore, yeast has been attracting attention as a substitute substance to solve the residual and tolerance problem in livestock caused by antibiotics used for improving productivity of livestock. Therefore, it is necessary to develop new yeast strains which are excellent in the fixation property and the safety to the living body, and can be effectively used for the probiotic composition, food or feed.
본 발명이 해결하고자 하는 과제는 장정착성, 내열성, 알코올 분해효능, 악취 저감효능 및 항인플루엔자 효능이 우수하며 병원성이 없는 SJP-SNU 균주를 제공하는 것이다.A problem to be solved by the present invention is to provide an SJP-SNU strain excellent in the fixation property, the heat resistance, the alcohol decomposing effect, the odor reducing effect and the anti-influenza effect and having no pathogenicity.
본 발명이 해결하고자 하는 또 다른 과제는 상기 균주를 유효성분으로 포함하는 프로바이오틱스 조성물, 식품 또는 사료를 제공하는 것이다.Another object of the present invention is to provide a probiotic composition, food or feed containing the strain as an active ingredient.
본 발명의 발명자들은 프로바이오틱스 조성물, 식품 또는 사료에 활용할 수 있는 신규 효모 균주를 발굴하기 위해 예의 연구 노력한 결과, 본 발명의 SJP-SNU 신규 효모 균주를 발견하였다. 본 발명의 발명자들은 부숙토에 존재하는 미생물을 60℃에서 항생제가 포함된 배지에 도말하고, 37℃에서 종야 배양하여 높은 증식성을 보인 효모 집락을 선발하였다. 이후, 상기 집락의 효모가 S. cerevisiae 및 S. boulardii에 비해 증식성, 장정착성 및 내열성이 우수하며, 악취 저감효능, 에탄올 분해효능 및 항인플루엔자 효능을 가지며, 병원성이 없는 것을 확인하였다. 또한, 상기 효모의 유전체를 분석한 결과 신종 효모임을 확인하고 SJP-SNU 균주로 명명하여, 본 발명을 완성하였다.The inventors of the present invention have made extensive efforts to discover new yeast strains which can be used in probiotic compositions, foods or feeds, and as a result, they have found a novel yeast strain of SJP-SNU of the present invention. The inventors of the present invention selected the yeast colonies showing high proliferation by cultivating the microorganisms present in the composted soil at 60 ° C. on a medium containing antibiotics and culturing at 37 ° C. Thereafter, it was confirmed that the colony yeast was superior to S. cerevisiae and S. boulardii in proliferation, long-term fixability and heat resistance, and had no odor reducing effect, ethanol degradation efficacy and anti-influenza efficacy, and was not pathogenic. The genome of the yeast was analyzed to confirm that it was a new yeast strain and named as SJP-SNU strain, thereby completing the present invention.
본 발명은 신종 효모인 SJP-SNU 균주에 관한 것이다. 본 발명의 SJP-SNU 균주는 장정착성, 내열성, 알코올 분해효능, 악취 저감효능 및 항인플루엔자 효능이 우수하며 병원성이 없어, 프로바이오틱스 조성물, 식품 또는 사료에 유용하게 사용될 수 있다.The present invention relates to an SJP-SNU strain which is a new yeast strain. The SJP-SNU strain of the present invention is excellent in long-term fixation property, heat resistance, alcohol decomposing effect, odor reducing effect and anti-influenza effect and is not pathogenic, and can be usefully used in probiotics composition, food or feed.
본 발명의 SJP-SNU 균주는 35 내지 40℃, 바람직하게 35 내지 37℃에서 높은 증식성(온도 저항성)을 가지며, 65℃에서 내열성을 가질 수 있다. 상기 SJP-SNU 균주는 일반적인 효모종이 25 내지 30℃에서 배양되는 것과 달리 35 내지 40℃, 바람직하게 37℃에서 매우 높은 증식성을 보이므로, 프로바이오틱스로써 우수한 장점을 가질 수 있다. 또한, 본 발명의 SJP-SNU 균주는 계태아 또는 4주령의 닭에서 병원성을 나타내지 않으며, 장정착성이 우수하여 프로바이오틱스로 사용되기에 우수한 장점을 가진다.The SJP-SNU strain of the present invention has high proliferative (temperature resistance) at 35 to 40 占 폚, preferably 35 to 37 占 폚, and may have heat resistance at 65 占 폚. The SJP-SNU strain exhibits very high proliferation at 35 to 40 ° C., preferably at 37 ° C., unlike a conventional yeast strain cultured at 25 to 30 ° C., and thus has excellent advantages as probiotics. In addition, the SJP-SNU strain of the present invention does not show pathogenicity in fetuses or 4-week-old chickens, and has excellent advantages in being used as probiotics because of its excellent long-term fixability.
본 발명의 SJP-SNU 균주는 대전에 소재하는 한국생명공학연구원 생물자원센터에 2015년 2월 5일자로 기탁하여, 기탁번호 KCTC12756BP로 기탁된 효모 균주일 수 있다.The SJP-SNU strain of the present invention may be a yeast strain deposited with the Korean Society for Biotechnology Research, Biotechnology Center, Daejeon, on February 5, 2015 and deposited with the deposit number KCTC12756BP.
또한, 본 발명은 상기 SJP-SNU 균주를 포함하는 프로바이오틱스(생균제) 조성물을 제공한다. 상기 조성물은 SJP-SNU 균주, 상기 균주의 배양액, 상기 배양액의 농축액 및 상기 배양액의 건조물 등으로 이루어진 군에서 선택된 어느 하나 이상을 유효성분으로 포함할 수 있다. 본 발명의 SJP-SNU 균주는 생체 온도와 유사한 온도(35 내지 40℃)에서 안정적으로 증식할 수 있으며, 장정착성이 우수하며 병원성이 없으므로, 프로바이오틱스 조성물의 유효성분으로 효과적으로 사용될 수 있다.The present invention also provides a probiotic (probiotic) composition comprising the SJP-SNU strain. The composition may include at least one selected from the group consisting of a strain of SJP-SNU, a culture of the strain, a concentrated solution of the culture solution, and a dried product of the culture solution. The SJP-SNU strain of the present invention can stably grow at a temperature (35 to 40 ° C) similar to a living body temperature, has excellent long-term fixability and is not pathogenic, and thus can be effectively used as an effective ingredient of a probiotic composition.
본 발명에 있어서, 배양물은 특정 미생물을 배양 배지 또는 배양액에서 배양한 것을 의미하며, 상기 배양물은 상기 특정 미생물을 포함하는 것일 수 있다. 상기 배양물은 그 제형이 한정되지 않고, 일 예로 액체 또는 고체일 수 있다. 바람직하게 본 발명의 배양액은 액체 배지에 특정 미생물을 접종하여 배양한 것을 의미할 수 있다. 또한, 본 발명에 있어서 배양액의 농축액은 상기 배양액을 농축한 것을 의미할 수 있으며, 배양액의 건조물은 상기 배양액에서 수분을 제거한 것을 의미할 수 있다.In the present invention, the culture means that a specific microorganism is cultured in a culture medium or a culture solution, and the culture may contain the specific microorganism. The culture is not limited in its formulation, and may be, for example, liquid or solid. Preferably, the culture medium of the present invention may mean that the culture medium is inoculated with a specific microorganism in a liquid medium. In the present invention, the concentrate of the culture liquid may mean that the culture liquid is concentrated, and the dried material of the culture liquid may mean that moisture is removed from the culture liquid.
본 발명에 있어서, 프로바이오틱스(probiotics)는 사람 또는 동물에 제공되어 사람 또는 동물 숙주의 장내 균총을 개선함으로써 유익한 효과를 주는 단일 또는 복합 균주 형태의 생균제일 수 있다. 일반적으로 프로바이오틱스는 장내 세균총의 이상 발효에 의하여 야기되는 제반 증상을 치료하고 개선하는 효과가 있으며, 숙주 장내의 소화관 벽에 밀집, 정착하여 유해 미생물이 정착하지 못하게 하는 작용을 한다. 생균활성제는 사람 또는 가축의 장내 미생물 균총을 개선시켜 건강에 유익한 효능을 주는 생균제를 의미할 수 있다. 미생물이 생균활성제로 활용되기 위해서는 위장을 통과하는 과정에서 일정 균수가 생존해야 하므로, 내열성 및 장내 생존력이 우수하여 섭취 후 일정기간 그 활성이 유지될 수 있어야 한다. 본 발명의 SJP-SNU 균주는 상기와 같은 생균활성제의 요구 조건을 만족시키므로, 우수한 프로바이오틱스 조성물로 사용될 수 있다.In the present invention, probiotics can be a probiotics in the form of a single or multiple strains which are provided to humans or animals to provide beneficial effects by ameliorating intestinal flora of a human or animal host. In general, probiotics have the effect of treating and ameliorating all the symptoms caused by abnormal fermentation of intestinal bacterial flora, and they function to prevent harmful microorganisms from settlement by being densely settled and fixed on the walls of the digestive tract in the host's intestines. The live bacterial active agent may mean a probiotic agent that improves intestinal microflora of a human or livestock to give health-beneficial effects. In order for the microorganism to be used as a live-active agent, a certain number of bacteria must survive the process of passing through the stomach. Therefore, the microorganism should be able to maintain its activity for a certain period of time after ingestion because of its excellent heat resistance and intestinal viability. The SJP-SNU strain of the present invention satisfies the requirement of the live microorganism activator as described above, and thus can be used as an excellent probiotic composition.
또한, 본 발명에 있어서 프로바이오틱스 조성물은 정장 작용을 가지므로, 정장용 조성물로 사용될 수 있다. 정장용은 사람 또는 동물의 장내 세균총의 이상 발효에 의하여 야기되는 제반 증상을 치료 또는 개선하는 용도를 의미할 수 있다. 상기 증상으로는 예를 들어, 설사(예를 들어 유해 미생물에 의한 감염성 설사 또는 장 급성 설사 등), 위장염, 염증성 장질환, 신경성 장염 증후군 또는 소장 미생물 과성장증 등이 포함될 수 있으나, 이에 제한되는 것은 아니다.Further, in the present invention, the probiotic composition has a dressing action and can therefore be used as a composition for dressing. Formal application may mean the treatment or amelioration of various symptoms caused by abnormal fermentation of intestinal flora of a person or an animal. Such symptoms may include, for example, diarrhea (for example, infectious diarrhea or intestinal diarrhea caused by harmful microorganisms), gastroenteritis, inflammatory bowel disease, neurodegenerative syndrome or small intestine microorganisms and growth conditions, It is not.
본 발명의 프로바이오틱스 조성물은 당업계에 공지된 방법에 따라 다양한 제형 및 방법으로 제조 및 투여될 수 있다.The probiotic compositions of the present invention can be prepared and administered in a variety of formulations and methods according to methods known in the art.
또한, 본 발명은 상기 프로바이오틱스 조성물을 포함하는 식품 또는 식품 첨가제를 제공한다. 또한, 본 발명은 상기 프로바이오틱스 조성물을 포함하는 사료 또는 사료 첨가제를 제공한다. 상기 식품 또는 사료를 통해 SJP-SNU 균주를 포함하는 프로바이오틱스 조성물이 사람 또는 동물의 장내로 효과적으로 전달될 수 있다.The present invention also provides a food or food additive comprising the probiotic composition. The present invention also provides a feed or feed additive comprising the probiotic composition. The probiotic composition comprising the SJP-SNU strain can be effectively delivered into the intestines of a human or animal through the food or feed.
본 발명에 있어서 식품은 영양소를 한 가지 또는 그 이상 함유하는 천연물 또는 가공품을 의미하며, 바람직하게 어느 정도의 가공 공정을 거쳐 직접 먹을 수 있는 상태가 된 것을 의미한다. 통상적으로, 상기 식품은 건강기능 식품, 음료, 식품 첨가제 또는 음료 첨가제 등일 수 있으나, 이에 제한되는 것은 아니다. 예를 들어, 상기 식품은 각종 식품류, 음료, 껌, 차, 비타민 복합제, 기능성 식품, 특수영양식품(예를 들어 조제유류 또는 영유아식 등), 건강보조식품, 과자류(예를 들어 스넥류) 또는 유가공식품 등일 수 있으나, 이에 제한되는 것은 아니다. 상기 식품은 당업계에 공지된 통상적인 제조 방법에 따라 제조될 수 있다.In the present invention, the term " food " means a natural product or a processed product containing one or more nutrients, preferably a state of being able to be eaten directly through a certain degree of processing. Typically, the food may be, but is not limited to, a health functional food, beverage, food additive or beverage additive. For example, the food may be selected from the group consisting of various foods, beverages, gums, tea, vitamin complex, functional foods, special nutrition foods (such as crude oil or infant formula), health supplements, confectionery Food, and the like. The food can be prepared according to a conventional manufacturing method known in the art.
상기 식품은 식품학적으로 허용 가능한 식품 보조 첨가제를 포함할 수 있으며, 기능성 식품의 제조에 통상적으로 사용되는 적절한 담체, 부형제 또는 희석제 등을 추가적으로 포함할 수 있다.The food may comprise a pharmaceutically acceptable food-aid additive and may additionally comprise suitable carriers, excipients or diluents conventionally used in the manufacture of a functional food.
본 발명에 있어서, 상기 식품은 상기 프로바이오틱스 조성물을 전체 식품 중량의 0.0001 내지 99 중량%, 바람직하게 0.001 내지 90 중량%, 더욱 바람직하게 0.1 내지 80 중량%, 가장 바람직하게 1 내지 50 중량%로 포함할 수 있으나, 이에 제한되는 것은 아니며, 식품의 종류 또는 용도에 따라서 적절하게 조절될 수 있다.In the present invention, the food contains 0.0001 to 99% by weight, preferably 0.001 to 90% by weight, more preferably 0.1 to 80% by weight, most preferably 1 to 50% by weight of the total food weight of the probiotic composition But it is not limited thereto and can be appropriately adjusted depending on the kind or use of the food.
본 발명에 있어서, 상기 사료 첨가제는 사료에 첨가되어 사용될 수 있고, 그에 따라 사료를 섭취하는 동물의 장내에서 유해 미생물에 대한 저항성 증진을 유도함으로써 가축의 생산성을 증진시킬 수 있다.In the present invention, the feed additive may be added to feed, thereby improving the productivity of livestock by inducing resistance to harmful microorganisms in the intestines of an animal that feeds the feed.
본 발명에 있어서, 상기 사료는 상기 프로바이오틱스 조성물을 전체 사료 중량의 0.0001 내지 99 중량%, 바람직하게 0.001 내지 90 중량%, 더욱 바람직하게 0.1 내지 80 중량%, 가장 바람직하게 1 내지 50 중량%로 포함할 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the feed contains 0.0001 to 99% by weight, preferably 0.001 to 90% by weight, more preferably 0.1 to 80% by weight, most preferably 1 to 50% by weight of the total feed weight of the probiotic composition But is not limited thereto.
본 발명의 사료 또는 사료 첨가제를 적용할 수 있는 동물 종류는 소, 돼지, 양, 염소, 개 또는 고양이 등의 각종 가축, 애완동물, 조류 및 어류 등으로 이루어진 군에서 선택된 1종 이상일 수 있으며, 이에 제한되는 것은 아니다.The animal species to which the feed or feed additive of the present invention can be applied may be at least one kind selected from the group consisting of cattle such as cattle, pig, sheep, goat, dog or cat, pet, But is not limited to.
본 발명의 신규 SJP-SNU 효모 균주는 장정착성, 내열성, 알코올 분해효능, 악취 저감효능, 항인플루엔자 효능이 우수하며 병원성을 나타내지 않는다.The novel SJP-SNU yeast strain of the present invention is excellent in the fixation property, the heat resistance, the alcohol decomposing effect, the odor reducing effect, the anti-influenza effect and does not show pathogenicity.
본 발명의 SJP-SNU 균주는 상기와 같은 특성을 가짐으로써, 프로바이오틱스 조성물, 식품 또는 식품 첨가제, 사료 또는 사료 첨가제 등으로 광범위하게 활용될 수 있다.The SJP-SNU strain of the present invention has the above-described characteristics, and thus can be widely used as a probiotic composition, food or food additive, feed or feed additive, and the like.
도 1은 SJP-SNU 효모 균주와 S. cerevisiae (KCTC 50757)의 장정착성을 비교한 결과를 나타낸다.
도 2는 SJP-SNU 효모 균주와 S. boulardii의 37℃에서의 증식성을 비교한 결과이다.
도 3은 SJP-SNU 효모 균주와 S. cerevisiae (KCTC 50757) 및 S. boulardii의 내열성을 비교한 결과이다.
도 4는 SJP-SNU 효모 균주의 에탄올 분해 효능을 나타낸 결과이다. SJP-SNU 효모 균주의 경우 37℃에서 에탄올을 분해하고 산을 생성해, pH 지시약인 페놀레드의 색이 적색에서 황색으로 변한 결과를 나타낸 것이다.
도 5는 SJP-SNU 효모 균주의 황화수소 저감 효능을 나타낸 결과이다.
도 6은 SJP-SNU 효모 균주의 비병원성을 나타낸 결과로 A는 SJP-SNU 신종효모를 계태아섬유아세포에 접종 시 세포변성효과가 전혀 없는 결과를 나타낸 것이다. B는 SJP-SNU 효모 균주를 10일령 SPF 발육란에 요막강 경로로 접종하고, 계태아 폐사와 병변 유무를 조사한 결과로, 인산완충용액을 접종한 대조군과 비교 시 폐사가 전혀 없었고, 병변이 관찰되지 않아 병원성이 없는 결과를 나타낸 것이다.
도 7은 게놈 분석을 통하여 SJP-SNU 효모 균주를 동정한 결과를 나타낸다.FIG. 1 shows the results of comparing the intestinal fixation of SJP-SNU yeast strains and S. cerevisiae (KCTC 50757).
Fig. 2 shows the results of comparing the proliferative activity of the SJP-SNU yeast strain and S. boulardii at 37 占 폚.
FIG. 3 shows the results of comparing heat resistance of SJP-SNU yeast strain with S. cerevisiae (KCTC 50757) and S. boulardii .
Fig. 4 shows the results of ethanol degradation of the SJP-SNU yeast strain. In the case of SJP-SNU yeast strain, ethanol was decomposed at 37 ° C to produce acid, and the color of phenol red as pH indicator changed from red to yellow.
Figure 5 shows the hydrogen sulfide-reducing activity of the SJP-SNU yeast strain.
FIG. 6 shows the results of the non-pathogenicity of the SJP-SNU yeast strain. As a result, A showed no cytopathic effect upon inoculation of SJP-SNU new yeast into fetal fibroblast. B was obtained by inoculating the SJP-SNU yeast strain into the umbilical cord at 10-day-old SPF developmental flora, and there was no mortality compared with the control group inoculated with the phosphate buffer solution, and no lesion was observed The results showed no pathogenicity.
FIG. 7 shows the results of identifying SJP-SNU yeast strains through genome analysis.
이하, 본 발명의 이해를 돕기 위하여 실시예 등을 들어 상세하게 설명하기로 한다. 그러나, 본 발명에 따른 실시예들은 여러 가지 다른 형태로 변형될 수 있으며, 본 발명의 범위가 하기 실시예들에 한정되는 것으로 해석되어서는 안 된다. 본 발명의 실시예들은 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.
실시예Example 1: One: SJPSJP -- SNUSNU 신종 효모의 분리 Isolation of new yeast
다양한 곳에서 채취한 각각 1g의 부숙토에 항생제-PBS(gentamycin, kanamycin, ampicilin, tetracyclin 및 streptomycin-50ug/ml) 9ml를 첨가하였다. 이후 부숙토를 37℃에서 2시간 배양하고, 60℃에서 10분간 열처리 하였다. 이후 항생제-PBS로 10진 희석하고, YMA에 100ul씩 골고루 도포 후 37℃에서 18시간 배양하였다. 배양된 미생물 가운데 가장 큰 집락을 클로닝하고, 이를 메틸렌 블루로 염색하여 효모를 분리하였으며, 이를 SJP-SNU로 명명하였다.9 ml of antibiotics-PBS (gentamycin, kanamycin, ampicillin, tetracyclin and streptomycin-50 ug / ml) were added to 1 g of composted soil collected at various sites. The composted soil was then incubated at 37 ° C for 2 hours and heat-treated at 60 ° C for 10 minutes. Then, 10-dilution with antibiotic-PBS was performed, 100 ul aliquots were applied to YMA and cultured at 37 째 C for 18 hours. The largest colonies of the cultured microorganisms were cloned and stained with methylene blue to isolate the yeast, which was named SJP-SNU.
실시예Example 2: 장 정착성 비교 2: Comparison of the fixing property
4주령(120913) SPF(specific pathogen free) 닭을 3마리씩 3그룹을 설정하여 107cfu/ml의 SJP-SNU, S. cerevisiae(KCTC 50757) 및 PBS(대조군, Con)를 하루 1회 500ul씩 3일간 경구 투여하였다. 이후 4일째 3마리의 맹장변을 채취하여 합친 후 무게를 측정하였다. 이후 항생제-PBS(gentamycin, kanamycin, ampicilin, tetracyclin 및 streptomycin-50ug/ml)로 10배 희석 후, 10진 희석하여 YMA에 100ul씩 골고루 도포한 후, 37℃에서 18시간 배양하여 집락 수를 측정하였다. 그 결과 대조군과 S. cerevisiae 투여군은 효모가 전혀 분리되지 않았지만(0 colony forming unit(cfu)/g), SJP-SNU는 맹장변 그램당 3.8x105cfu/g이 분리되어 장 정착성이 뛰어난 것을 확인하였다(도 1).SJP-SNU, S. cerevisiae (KCTC 50757) and PBS (control, Con) were inoculated once per day with 500ul each of 10 7 cfu / ml of 4 weeks old (120913) SPF (specific pathogen free) Orally for 3 days. On the 4th day, 3 appendices were collected and weighed. After 10-fold dilution with antibiotics-PBS (gentamycin, kanamycin, ampicillin, tetracycline and streptomycin-50 ug / ml), 10 μl of dilution was applied to each well of 100 μl of YMA and incubated at 37 ° C. for 18 hours to measure the number of colonies . As a result, the control group and the S. cerevisiae treated group showed no separation of yeast (0 colony forming unit (cfu) / g), whereas SJP-SNU separated 3.8 x 10 5 cfu / g per cecomorphogram, (Fig. 1).
S. cerevisiae에 비해 37℃에서 배양성이 좋으며 시중에 판매되고 있는 Saccharomyces boulardii를 SJP-SNU와 함께 37℃에서 배양성을 비교하였다. 그 결과 SJP-SNU는 18시간만에 매우 활발한 성장을 보인 반면, S. boulardii는 18시간 배양에서 집락을 형성하지 못하였다. 나아가, S. boulardii의 집락은 32시간 배양 후에도 SJP-SNU의 집락보다 작았다. 이를 통해 SJP-SNU가 S. boulardii보다 37℃에서의 배양성이 탁월함을 확인하였다(도 2). Saccharomyces boulardii , which is well- rounded at 37 ° C compared to S. cerevisiae and commercially available, was compared at 37 ° C with SJP-SNU. As a result, SJP-SNU showed very active growth in 18 hours, while S. boulardii did not colonize in culture for 18 hours. Furthermore, the colony of S. boulardii was smaller than that of SJP-SNU after 32 hours of incubation. Thus, it was confirmed that SJP-SNU was superior to S. boulardii at 37 ° C. (FIG. 2).
실시예Example 3: 내열성 비교 3: Heat resistance comparison
S. cerevisiae (KCTC 50757) 및 S. boulardii는 YM broth에서 30℃에서 18시간 배양하였고, SJP-SNU는 37℃에서 18시간 배양하였다. 이후 각각을 55, 60 및 65℃에서 10분간 열처리하고, YMA에 도말하여 37℃에서 배양한 후 생존 여부를 확인하였다. 그 결과 모두 60℃까지는 생존하였으나, 65℃에서는 SJP-SNU만 생존하여 뚜렷한 차이를 보였다(도 3). S. cerevisiae (KCTC 50757) and S. boulardii were cultured in YM broth at 30 ° C for 18 hours and SJP-SNU at 37 ° C for 18 hours. Then, each of them was heat-treated at 55, 60 and 65 ° C for 10 minutes, spread on YMA and cultured at 37 ° C, and then viability was confirmed. As a result, all survived up to 60 ° C., but only SJP-SNU survived at 65 ° C. (FIG. 3).
실시예Example 4: 4: SJPSJP -- SNUSNU 균주 배양액의 항 인플루엔자 바이러스 효과 Anti-influenza virus effect of strain culture
SJP-SNU을 24시간 동안 배양한 배양액(pH 4.59)을 여과하였다. A/chicken/Korea/01310/2001 (H9N2)(국립수의과학검역원 조류질병과; 등록특허 제10-0790801호) 조류 인플루엔자 바이러스 배양액을 유기물(5% BCS in PBS)로 1/20의 비율로 희석한 후, SJP-SNU 배양액과 1:1로 혼합하였다. 이를 4℃에서 30분간 반응시킨 후 단계별 희석하고, Egg-bit에 접종하여 Virus Control과 비교하였다. 그 결과 SJP-SNU 모균주 배양액에서 10배 이상의 바이러스 역가(TCID50/0.1ml)의 감소가 관찰되어 유의적인 차이를 보였다(P<0.05).SJP-SNU was cultured for 24 hours, and the culture solution (pH 4.59) was filtered. A / chicken / Korea / 01310/2001 (H9N2) (National Veterinary Research and Quarantine Service, Bird Disease Division, Registered Patent No. 10-0790801) The avian influenza virus culture was diluted 1/20 with an organic substance (5% BCS in PBS) And then mixed with the SJP-SNU culture solution at a ratio of 1: 1. They were reacted at 4 ° C for 30 minutes, diluted stepwise, and inoculated with Egg-bit and compared with Virus Control. As a result, there was a significant difference (P <0.05) in the decrease of virus titer (TCID 50 /0.1 ml) of 10 times or more in SJP-SNU parent strain culture.
a HA 양성 / 접종 well 수a HA positive / inoculated well
실시예Example 5: 에탄올 분해능 비교 5: Comparison of ethanol degradability
5%, 10% 및 15% EtOH (in PBS + phenol red 0.1%)에 SJP-SNU 106 cfu/100ul을 접종한 후 37℃에서 16시간 동안 배양하였다. 5% EtOH에서 SJP-SNU를 접종하여 배양한 경우, SJP-SNU를 접종하지 않은 대조군 대비 지시약(Phenol Red) 색이 확연하게 노란색으로 변하였다. 또한, 10% EtOH에서도 SJP-SNU를 접종하여 배양한 경우, 대조군 대비 구별 가능한 정도로 노란색으로 변하였다. 그러나, 15% EtOH의 경우 차이가 없었다(도 4A).SJP-
5%, EtOH (in PBS)를 사용하여 S. exiguus (KCTC7169) 표준주와 SJP-SNU의 에탄올(EtOH) 분해능을 비교하기 위해 각각의 균주를 106 cfu/100ul을 접종 후, 37℃에서 16시간 동안 배양하였다. 그 결과 S. exiguus (KCTC7169) 표준주의 경우 균주를 접종하지 않은 대조군 대비 변화가 없었으나, SJP-SNU의 경우 뚜렷이 구별되는 노란색을 보였다. 따라서 SJP-SNU의 경우 37℃에서 에탄올을 분해하여 아세트산으로 분해하는 능력이 탁월함을 확인하였다(도 4B).To compare the ethanol (EtOH) resolution of S. exiguus (KCTC7169) standard strain and SJP-SNU using 5% EtOH (in PBS), 10 6 cfu / 100ul of each strain was inoculated, Lt; / RTI > As a result, there was no change in S. exiguus (KCTC7169) standard compared to the control group in which the strain was not inoculated, but SJP-SNU showed distinct yellow color. Therefore, it was confirmed that the ability of SJP-SNU to decompose ethanol into acetic acid at 37 ° C was excellent (FIG. 4B).
실시예Example 6: 황화수소 분해능 비교 6: Comparison of hydrogen sulfide resolution
혐기 상태에서 유기물이 미생물에 의해 분해될 때 산생되는 황화수소(H2S)는 무색의 독성 가스로 알려져 있다. SJP-SNU 균주의 황화수소 저감 효능을 평가하기 위해, 황화수소를 산생하는 Salmonella Enteritidis (SE) 최소 균수를 측정하였다. 이를 위해, LB 육즙배지에 배양한 SE 4.3x109cfu/ml을 TSI 사면배지에 10-3 ~ 10-10 희석한 후 접종하여, 37℃에서 18시간 배양하였다. 그 결과 10-6 희석하는 경우 SE에 의해 산생된 황화수소에 의해 배지 전체가 흑변되는 것을 확인하였다(도 5A).Hydrogen sulfide (H 2 S), which is produced when organic matter is decomposed by microorganisms in an anaerobic state, is known as a colorless toxic gas. In order to evaluate the hydrogen sulfide-reducing effect of the SJP-SNU strain, Salmonella enteritidis (SE) producing the hydrogen sulfide was measured. For this, SE 4.3 × 10 9 cfu / ml cultured in LB broth was inoculated after diluting 10 -3 to 10 -10 in TSI slope medium and cultured at 37 ° C. for 18 hours. As a result, it was confirmed that the entire medium was blackened by hydrogen sulfide produced by SE when 10 -6 dilution (FIG. 5A).
YM broth에서 배양한 SJP-SNU (8.8x109cfu/ml) 및 S. boulardii (2x108cfu/ml) 원액 및 원액을 2배 희석한 것을 준비하였다. 이들을 각각 10진 희석하여 각 희석 효모 450ul와 상기 SE 10-6 희석균 50ul를 혼합하여 사면배지에 접종하였다. 이를 37℃에서 18시간 동안 배양한 결과, SJP-SNU 및 S. boulardii 모두에서 효모의 수가 많을수록 황화수소 산생에 의한 배지의 흑변이 억제되는 경향이 관찰되었다(도 5B).SJP-SNU (8.8x10 9 cfu / ml) and S. boulardii (2x10 8 cfu / ml) stock solution and stock solution were cultured in YM broth. Diluted by them respectively,
실시예Example 7: 병원성 평가 7: Pathogenicity assessment
YM broth에 SJP-SNU를 접종하고 37℃에서 24시간 배양한 배양액을 100배 희석하여 배양하였다. 이를 계태아섬유아세포에 접종하고 5일 후 관찰한 결과, 세포변성효과가 관찰되지 않았다(도 6A). SJP-SNU (107CFU/ml) 배양액을 SPF종란에 0.1 ml/개씩 접종한 후 60시간 동안 배양하며 계태아의 생존 유무를 조사하였다. 60시간후 4℃에서 종야 정치한 후 계태아의 병변을 관찰한 결과, SJP-SNU 접종 계태아는 모두 60시간동안 생존하였으며, 계태아 역시 대조군과 차이를 보이지 않아 계태아에 대한 병원성이 없음을 확인하였다(도 6B).SJP-SNU was inoculated in YM broth and incubated at 37 ° C for 24 hours. The culture was diluted 100 times and cultured. After 5 days of inoculation with fetal fibroblasts, no cytopathic effect was observed (Fig. 6A). SJP-SNU (10 7 CFU / ml) was inoculated into the SPF seedlings at a concentration of 0.1 ml / ml and incubated for 60 hours. The SJP-SNU-inoculated fetus survived for 60 hours, and the fetus did not show any pathogenicity to the fetus because it did not differ from the control group. (Fig. 6B).
면역억제에 의한 SJP-SNU의 병원성을 평가하기 위해, 4주령 SPF 시험계 3마리씩 4그룹을 설정하여 107cfu/ml의 SJP-SNU, S. cerevisiae (KCTC 50757) 및 PBS (대조군)를 하루 1회 500 ul씩 3일간 경구 투여하였다. 이후, 4일간 사료 급이를 중단(음수공급)하여 면역을 억제(Protein-energy malnutrition 모델)시킨 후, 5일째 시험계의 혈액을 채취하였다. 신장 및 간장으로부터 면봉 재료를 채취하여, YM 한천배지(항생제)에 도말하여 효모의 유무를 조사하였다. 그 결과 효모가 전혀 분리되지 않아, SJP-SNU의 내부 장기 침습성은 없는 것으로 확인되었다. In order to evaluate the pathogenicity of SJP-SNU by immunosuppression, four groups of three 4-week-old SPF test systems were set up, and 10 7 cfu / ml of SJP-SNU, S. cerevisiae (KCTC 50757) 500 μl per day for 3 days. Thereafter, the feeding was stopped (negative supply) for 4 days to inhibit the immune system (protein-energy malnutrition model), and the blood of the test system was collected on the fifth day. Cotton swabs were taken from kidneys and liver and plated on YM agar medium (antibiotics) to determine the presence or absence of yeast. As a result, yeast was not isolated at all and it was confirmed that SJP-SNU was not invasive to the internal organs.
12주령 SPF 성계 3수에 109 cfu/ml/수의 SJP-SNU를 피하 접종한 후 14일간 임상 증상을 관찰하였다. 성계 3수를 접종 14일 후 안락사시켜 병변을 관찰하였다. 그 결과 대조군 대비 임상증상을 보이지 않았고 부검 결과 병변을 보이지 않아, SJP-SNU는 병원성이 없음을 확인하였다.SJP-SNU was injected subcutaneously at 10 9 cfu / ml / water in the 3-week-old SPF group and clinical symptoms were observed for 14 days. Three days after the inoculation, eosinophilic lesions were observed. As a result, no clinical symptoms compared to the control group, no autopsy results, and no SJP-SNU pathogenicity.
실시예Example 8: 게놈분석을 이용한 균주 동정 8: Identification of strains using genome analysis
본 발명의 SJP-SNU 신종 효모의 게놈을 DNeasy® Blood & Tissue Kit (QIAGEN 사)를 사용하여 추출한 후, NGS 기법을 통해 염기서열을 결정하였다. SJP-SNU 신종 효모의 염기서열 어셈블리, ORF(open reading frame) 예측, BLAST 검색 및 효모 동정(Krona tool) 등의 게놈 분석은 인트론바이오테크놀로지에서 수행하였다. 그 결과 SJP-SNU 신종효모 균주의 게놈은 Pichia kudriavzevii와 74.40%, Candida glycerinogenes와 13.05% 및 Debaryomyces hansenii과 10.72% 유사함을 확인하였다(표 3).The genome of the SJP-SNU new yeast strain of the present invention was extracted using DNeasy Blood & Tissue Kit (QIAGEN), and the nucleotide sequence was determined by the NGS technique. Genomic analysis such as nucleotide sequence assembly of SJP-SNU new yeast, ORF (open reading frame) prediction, BLAST search and yeast identification (Krona tool) was performed by Intron Biotechnology. As a result, the genome of the SJP-SNU new yeast strain was Pichia 74.40 % with kudriavzevii, 13.05% with Candida glycerinogenes , and Debaryomyces hansenii and 10.72% (Table 3).
이러한 결과로부터 SJP-SNU 신종 효모는 주변환경에 적응하기 위해 다양한 효모 균주들과 heterospecific hybridizations을 통해 진화한 신종 효모인 것으로 확인되었다(도 7).From these results, it was confirmed that the new yeast strain SJP-SNU was a new yeast that evolved through heterospecific hybridizations with various yeast strains in order to adapt to the surrounding environment (FIG. 7).
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김재홍 등. 서울대학교 산학협력단(발행처 농림축산식품부). 신종 효모로 구성된 프로바이오틱 생균제 효능기전 연구(2015.05.28.)* |
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