KR20190110186A - Microalgae Schizochytrium sp. ABC-101 strain producing bio-oil containing high concentration of DHA and method for producing DHA using the strain - Google Patents
Microalgae Schizochytrium sp. ABC-101 strain producing bio-oil containing high concentration of DHA and method for producing DHA using the strain Download PDFInfo
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- KR20190110186A KR20190110186A KR1020180031814A KR20180031814A KR20190110186A KR 20190110186 A KR20190110186 A KR 20190110186A KR 1020180031814 A KR1020180031814 A KR 1020180031814A KR 20180031814 A KR20180031814 A KR 20180031814A KR 20190110186 A KR20190110186 A KR 20190110186A
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Abstract
Description
본 발명은 DHA를 고농도로 포함한 바이오오일 생산 미세조류인 스키조키트리움 속 ABC-101 균주 및 상기 균주를 이용한 DHA 생산 방법에 관한 것이다.The present invention relates to a strain of ABC-101 in the genus Szozoquitium, a bio-oil producing microalgae containing a high concentration of DHA, and a method of producing DHA using the strain.
미세조류는 지구 상에 약 300,000종이 있다고 추정되고 있고, 종에 따라 담수, 해수, 기수에서 생장할 수 있을 뿐 아니라 미세조류 바이오매스(Microalgal biomass)를 이용하여 바이오 연료 및 고부가가치 물질을 생산할 수 있는 미생물이다. 일반적으로 미세조류를 배양하는 방식에는 지구적 기후변화의 주범이라 이야기하는 온실가스인 이산화탄소를 에너지원과 탄소영양원으로 이용하는 광자가배양(Phototrophic cultivation)과 이산화탄소를 이용하여 얻은 초본계나 목질계 바이오매스를 재가공해 얻어지는 다양한 유기탄소원(예를 들면, 포도당, 복합당)들을 이용하는 종속배양(Heterotrophic cultivation) 및 위의 두 가지 배양방식을 모두 이용하는 혼합배양(Mixotrophic cultivation)으로 나눠진다. 이 중 미세조류를 이용하여 생산되는 고부가가치 물질들을 경제적으로 대량 생산하기 위해서는 기후 및 환경 영향을 적게 받으며 좁은 면적에서도 빠르게 생장할 수 있어 대량으로 생산할 수 있는 유기영양 미생물의 배양방법이 주로 사용되며, 이를 위해서는 미세조류의 다양한 유기탄소원 사용 여부가 선행적으로 연구되어야할 뿐 아니라 목적 산물을 고수율로 고생산하는 균주이면서 그에 맞는 배양 방법의 최적화도 필요하다.It is estimated that there are about 300,000 species of microalgae, and depending on the species, they can grow in freshwater, seawater and brackish water, and can produce biofuels and high value-added substances using microalgal biomass. It is a microorganism. In general, microalgae cultivation includes phototrophic cultivation using carbon dioxide, a greenhouse gas that is considered to be the culprit of global climate change, as an energy source and carbon nutrient source, and herbal or woody biomass obtained using carbon dioxide. Heterotrophic cultivation using a variety of organic carbon sources (eg, glucose, complex sugars) obtained by reprocessing and mixotrophic cultivation using both of the above cultures. Among them, in order to economically mass-produce high value-added materials produced using microalgae, cultivation methods of organic nutrients that can be produced in large quantities are mainly used because they are less affected by climate and environment and can grow quickly in a small area. For this purpose, not only the use of various organic carbon sources of microalgae should be studied in advance, but also the optimization of the cultivation method suitable for the high yield of the desired product in high yield.
대량 생산된 미세조류 바이오매스는 그 자체로도 건강기능식품, 가축 및 어류의 사료와 같은 고부가가치물질로 이용될 수 있지만 현재 대표적으로 미세조류가 이용되는 방법으로는 미세조류 유래 지질을 이용하는 방법이고, 이를 통해 바이오연료 및 그의 부산물인 글리세롤의 생산, 프로세싱 오일로서의 활용, 의약품 및 화장품의 원료로도 사용된다. 특히, 지질 추출 후 남은 잔사 바이오매스(Algal biomass residue)는 추가 공정 및 생물학적 전환을 통해 또 다른 공정의 원료 및 부산물로 사용되기도 한다.The mass-produced microalgae biomass can be used as a high value-added substance such as dietary supplements of food, livestock and fish, but the microalgae is currently used as a microalgae-derived lipid. It is also used as a raw material for the production of biofuels and its by-product glycerol, as a processing oil, and as a raw material for pharmaceuticals and cosmetics. In particular, algal biomass residues remaining after lipid extraction may be used as raw materials and by-products of other processes through further processes and biological conversions.
이처럼 미세조류 유래 지질은 여러 방면으로 다양하게 사용되고 있는데 이 중 그 중요성이 더욱더 커지는 것이 미세조류 유래 오메가-3 다중불포화지방산이다. 오메가-3 다중불포화지방산은 북유럽 에스키모의 식생활 연구를 통해 옥스퍼드 대학의 싱클레어 교수에 의해 1952년 처음 발견되었으며, 대표적인 예로는 DHA(Docosahexaenoic acid, C22:6n-3) 다중불포화지방산이 알려져 있다. 일반적으로 DHA 다중불포화지방산의 공급원으로 현재 많이 사용되고 있는 것은 참치, 연어, 고등어와 같은 심해성 어류들에서 추출한 어류기반 DHA 다중불포화지방산이다. 하지만 심해성 어류들은 먹이사슬의 상위 계층으로서 중금속, 내분비계 교란 물질, 방사성물질 등의 오염물질들의 환경축적으로 인한 오염 문제가 심각하며 더욱이 생선의 비린내가 섭취 의욕을 떨어 트릴수도 있어 이를 제거하기 위한 분리/정제 공정들이 복잡하다. 특히 무엇보다 DHA 다중불포화지방산을 필요로 하는 인류의 요구량은 해마다 증가하고 있지만 이의 공급 원료가 되는 어유의 양은 세계적으로 포화 상태에 이르렀으며 앞으로 증가할 DHA 다중불포화지방산의 요구량에 맞추어서 안정적으로 원료를 공급할 수 있고 기존의 어류 유래 오일보다 환경오염물질에 안전하며 분리/정제 공정도 간단한 새로운 수단으로 미세조류 유래 DHA 다중불포화지방산이 주목을 받고 있다.As described above, microalgae-derived lipids are used in various ways, and among them, the importance of microalgae-derived omega-3 polyunsaturated fatty acids is increased. Omega-3 polyunsaturated fatty acids were first discovered in 1952 by Professor Sinclair at Oxford University through a dietary study of Nordic Eskimos, and a representative example is Docosahexaenoic acid (C22: 6n-3) polyunsaturated fatty acids. In general, the most widely used source of DHA polyunsaturated fatty acid is fish-based DHA polyunsaturated fatty acid extracted from deep-sea fish such as tuna, salmon and mackerel. However, deep-sea fishes are the upper tier of the food chain, which is a serious pollution problem due to environmental accumulation of contaminants such as heavy metals, endocrine disruptors, and radioactive substances. Moreover, fishy fish may lower the intake of fish. Separation / purification processes are complex. In particular, human demand for DHA polyunsaturated fatty acids is increasing year by year, but the amount of fish oil that is used as a feedstock has reached saturation in the world, and it is possible to supply raw materials stably to meet the demand of DHA polyunsaturated fatty acids. Microalgae-derived DHA polyunsaturated fatty acids are attracting attention as a new means that can be more environmentally contaminated than conventional fish-derived oils and that the separation / purification process is simple.
DHA 다중불포화지방산의 효능으로 신경의 발달, 전달 및 병리, 대사 조절 및 선천적 면역체계인 염증반응의 조절에서 중요한 역할을 하는 것으로 밝혀지고 있다. 신경 발달, 전달 및 병리에 대한 영향으로 오메가-3 다중불포화지방산은 사람의 생애 전주기 동안 뇌구조 및 기능에 밀접한 관계가 있다. 특히 유아의 두뇌 및 안구조직 발달에 중요할뿐 아니라 신경 세포의 생존 및 염증반응 조절을 통한 중추신경계의 발달, 기능 및 노화에 중요한 역할과 ADHD(Attention deficit hyperactivity disorder), 조현증, 우울증, 알쯔하이머 치매 등 다양한 정신질환과의 연관성이 규명되고 있다. 대사조절 기능으로는 오메가-3 다중불포화지방산은 비만세포의 대사 조절과 당 섭취, 인슐린 조절에 관여하며, 중성지질의 함량 제어에 중요한 역할을 하는 것으로 알려져 있다. 염증반응 조절 기능으로는 각종 염증에 영향을 미치는 유전자를 발현을 억제하는데 효과적인 것으로 밝혀졌다.The efficacy of DHA polyunsaturated fatty acids has been shown to play an important role in the development, transmission and pathology of nerves, regulation of metabolism and regulation of the inflammatory response, the innate immune system. Omega-3 polyunsaturated fatty acids are closely related to brain structure and function throughout human life cycle due to their effects on neural development, transmission and pathology. In particular, it is important for the development of brain and eye tissues in infants, and plays an important role in the development, function, and aging of central nervous system through the regulation of survival and inflammatory response of nerve cells, Attention deficit hyperactivity disorder (ADHD), schizophrenia, depression, Alzheimer's dementia. It has been linked to various mental diseases. Omega-3 polyunsaturated fatty acid is known to play an important role in metabolic regulation, glucose uptake, and insulin regulation of mast cells and to control the content of neutral lipids. Inflammatory response control function has been shown to be effective in suppressing the expression of genes that affect various inflammation.
한편, 트라우스토키트리드(Thraustochytrid) 계열 미세조류는 앞서 이야기한 오메가-3 지방산으로 알려진 DHA(Docosahexaenoic acid) 다중불포화지방산을 비롯한 다양한 다중불포화지방산(Polyunsaturated fatty acid, PUFA)을 고농도로 생산하면서 경제적인 대량 생산이 가능한 유기종속영양 미세조류이다. 이는 초본계 및 목질계 또는 비식용작물의 당화 및 정제 공제 공정에서 얻어지는 유기탄소원을 사용하여 생장이 가능하며 이를 이용한 경제적인 대량생산이 가능하다. 트라우스토키트리드(Thraustochytrid) 계열 미세조류는 다른 미세조류와 특징적으로 세포벽이 얇다고 알려져 있으며, 이는 미세조류 배양 후 습식 및 건식의 형태로 유기용매 또는 유기용매 사용 없이도 지질의 추출이 가능하다. 이에 따라 DHA 불포화지방산을 고농도로 함유하는 트라우스토키트리드(Thraustochytrid) 계열 미세조류는 목적 산물을 생산하기 위한 최적의 배양 조건을 탐색하여 적용한다면 고부가가치 산업(건강기능식품, 의약품, 화장품, 프로세싱 오일, 사료 등)과 바이오연료 산업의 시장 경쟁력을 제공할 수 있을 것으로 기대된다.On the other hand, the Thraustochytrid family of microalgae is economical by producing various polyunsaturated fatty acids (PUFAs) at high concentrations, including DHA (Docosahexaenoic acid) polyunsaturated fatty acids, known as omega-3 fatty acids. It is an organic nutrient microalgae capable of mass production. It is possible to grow using the organic carbon source obtained in the saccharification and purification deduction process of herbaceous, woody or non-edible crops and economical mass production using the same. The Thraustochytrid family of microalgae is known to be thinner than other microalgae and is characterized by a thin cell wall. The microalgae can be extracted in wet and dry forms without the use of organic solvents or organic solvents. Therefore, the Thraustochytrid family of microalgae containing high concentrations of DHA unsaturated fatty acids can be applied to high value-added industries (health functional foods, pharmaceuticals, cosmetics, processing oils) by searching for and applying the optimal culture conditions to produce the desired product. , Feed, etc.) and the biofuel industry.
한편, 한국등록특허 제1732062호에서는 '트라우스토키트리드 균주로부터 무용매 추출을 통한 바이오오일 추출 방법'이 개시되어 있고, 한국등록특허 제1780298호에서는 '트라우스토키트리드계 미세조류를 이용한 섬유질계 바이오매스로부터 바이오오일의 제조방법'이 개시되어 있으나, 본 발명에서와 같이, 'DHA를 고농도로 포함한 바이오오일 생산 미세조류인 스키조키트리움 속 ABC-101 균주 및 상기 균주를 이용한 DHA 생산 방법'에 대해서는 밝혀진 바가 전혀 없다.Meanwhile, Korean Patent No. 1732062 discloses a method for extracting bio-oil through solvent-free extraction from Trastutoquitrid strains, and Korean Patent No. 1780298 discloses a fibrous system using 'Taustokitrid microalgae'. Although a method for producing bio-oil from biomass has been disclosed, as in the present invention, in the 'Schizoquitrium strain ABC-101, which is a bio-oil-producing microalgae containing a high concentration of DHA, and a method for producing DHA using the strain' Nothing is known about it.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명은 위의 요구를 맞춘 다양한 종류의 탄소원과 배지 조성에서 고 DHA 생산성을 가지면서 높은 생장성과 지질 생산성을 보이는 신규한 균주 스키조키트리움 ABC-101 균주를 분리하였고, 특히, 55~65 g/ℓ 포도당, 8~27g/ℓ 효모 추출물, 5~50 g/ℓ 해수염 및 0.1~2.0 ㎖/ℓ 비타민이 포함된 배지에서 배양한 경우, DHA 함량이 높은 고수율의 바이오오일 생산성을 확인할 수 있었다. 따라서, 본 발명의 균주는 바이오연료나 의약품, 건강기능식품, 화장품, 사료, 타이어 드레드 및 고무 제조에 필요한 프로세싱 오일, 기타 소재 등의 원료에 사용될 수 있음을 확인하여 본 발명을 완성하였다.The present invention is derived from the above requirements, the present invention is a novel strain Schizochytrium ABC- exhibiting high growth and lipid productivity while having high DHA productivity in various kinds of carbon source and medium composition meeting the above needs. 101 strains were isolated, especially when cultured in medium containing 55-65 g / l glucose, 8-27 g / l yeast extract, 5-50 g / l sea salt and 0.1-2.0 ml / l vitamin. High yield of high yield biooil productivity was confirmed. Therefore, the strain of the present invention has been confirmed that the present invention can be used in raw materials such as biofuel or pharmaceuticals, health functional foods, cosmetics, feed, tire thread and processing oil, other materials required for rubber production, and completed the present invention.
상기 목적을 달성하기 위하여, 본 발명은 DHA(Docosahexaenoic acid, 22:6)를 고농도로 포함한 바이오오일을 생산하며, 기탁번호가 KCTC13455BP로 기탁된 미세조류인 스키조키트리움 속(Schizochytrium sp.) ABC-101 균주를 제공한다.In order to achieve the above object, the present invention produces a bio-oil containing a high concentration of DHA (Docosahexaenoic acid, 22: 6), Schizochytrium sp. Of the microalgae deposited with KCTC13455BP accession number ABC- 101 strains are provided.
또한, 본 발명은 상기 균주 또는 상기 균주를 포함하는 배양액을 유효성분으로 포함하는 DHA(Docosahexaenoic acid, 22:6) 제조용 미생물 제제를 제공한다.In addition, the present invention provides a microbial preparation for producing DHA (Docosahexaenoic acid, 22: 6) comprising the strain or a culture solution containing the strain as an active ingredient.
또한, 본 발명은 상기 균주를 배양하고, 상기 배양한 균주로부터 DHA(Docosahexaenoic acid, 22:6)를 분리하는 것을 특징으로 하는 DHA의 제조 방법을 제공한다.The present invention also provides a method for producing DHA, comprising culturing the strain and separating DHA (Docosahexaenoic acid, 22: 6) from the cultured strain.
또한, 본 발명은 상기 균주 또는 상기 균주를 포함하는 배양액을 유효성분으로 포함하는 DHA(Docosahexaenoic acid, 22:6) 및 팔미트산 고함유 바이오오일 제조용 미생물 제제를 제공한다.In another aspect, the present invention provides a microbial preparation for producing bio-oil containing DHA (Docosahexaenoic acid, 22: 6) and palmitic acid containing the strain or the culture medium containing the strain as an active ingredient.
또한, 본 발명은 상기 균주를 배양하는 단계;In addition, the present invention comprises the steps of culturing the strain;
상기 배양된 균주로부터 지질을 분리하는 단계;Separating lipids from the cultured strains;
상기 지질을 트랜스에스테르화시켜 지방산 에스테르를 생산하는 단계를 포함하는 바이오디젤의 생산 방법을 제공한다.It provides a method for producing biodiesel comprising transesterifying the lipid to produce a fatty acid ester.
또한, 본 발명은 상기 균주를 배양하는 단계;In addition, the present invention comprises the steps of culturing the strain;
상기 배양된 균주로부터 바이오오일을 분리하는 단계; 및Separating the biooil from the cultured strain; And
상기 분리된 바이오오일을 트랜스에스테르화시켜 지방산 에스테르 및 글리세롤을 생성하는 단계를 포함하는 글리세롤의 제조 방법을 제공한다.It provides a method for producing glycerol comprising transesterifying the separated bio-oil to produce fatty acid esters and glycerol.
또한, 본 발명은 상기 균주, 상기 균주에 의해 생산된 바이오오일, 상기 균주의 배양액, 상기 배양액의 농축액 또는 상기 배양액의 건조물을 유효성분으로 함유하는 식품, 사료 조성물 또는 화장료 조성물을 제공한다.The present invention also provides a food, feed composition or cosmetic composition containing the strain, the bio-oil produced by the strain, the culture of the strain, the concentrate of the culture or the dried product of the culture as an active ingredient.
또한, 본 발명은 상기 균주를 배양하는 단계;In addition, the present invention comprises the steps of culturing the strain;
상기 배양된 균주로부터 지질을 분리하는 단계; 및Separating lipids from the cultured strains; And
상기 분리한 지질을 촉매전환시켜 항공유로 전환하는 단계를 포함하는 바이오항공유의 생산 방법을 제공한다.It provides a method for producing bio-air fuel comprising the step of catalytic conversion of the separated lipids to aviation oil.
본 발명에서는 특정 배지 조건에서 배양한 경우, DHA 함량이 높은 고수율의 바이오오일 생산성을 가지는 스키조키트리움 속(Schizochytrium sp.) ABC-101을 새롭게 분리하였다. 본 발명의 신규한 스키조키트리움 속 ABC-101 균주는 바이오매스 생산 및 이를 원료로 사용하여 유용한 물질을 생산하는 공정에 큰 기여를 할 수 있어 관련 산업에 매우 유용하다.In the present invention, when cultured under specific medium conditions, Schizochytrium sp. ABC-101 having high yield of high oil biofuel DHA content was newly isolated. ABC-101 strain of the genus Suzukikytrium of the present invention is very useful for the related industry because it can make a significant contribution to the production of biomass and the production of useful substances using it as a raw material.
도 1은 본 발명에서 분리한 스키조키트리움 속(Schizochytrium sp.) ABC-101의 18S rDNA 염기서열을 나타낸다.
도 2는 본 발명에서 분리한 스키조키트리움 속(Schizochytrium sp.) ABC-101의 분류학적 계통도를 나타낸다.
도 3은 5L 배양기를 이용한 본 발명의 스키조키트리움 속(Schizochytrium sp.) ABC-101 균주의 회분식 배양 결과이다.Figure 1 shows the 18S rDNA sequence of the Schizochytrium sp. ABC-101 isolated in the present invention.
Figure 2 shows a taxonomic diagram of the genus Schizochytrium sp. Isolated from the present invention ABC-101.
Figure 3 is a batch culture result of the Schizochytrium sp. ( Schizochytrium sp.) Strain of the present invention using a 5L incubator.
본 발명의 목적을 달성하기 위하여, 본 발명은 DHA(Docosahexaenoic acid, 22:6)를 고농도로 포함한 바이오오일을 생산하고, 고수율의 바이오오일 생산성을 가지는 미세조류인 스키조키트리움 속(Schizochytrium sp.) ABC-101 균주를 제공한다.In order to achieve the object of the present invention, the present invention produces a bio-oil containing a high concentration of DHA (Docosahexaenoic acid, 22: 6), Schizochytrium sp. ) ABC-101 strain.
상기 스키조키트리움 속 ABC-101 균주는 해수로부터 미세조류 분리용 한천 배지에 접종하여 성장이 빠른 미세조류 순수 콜로니로 분리되었으며, 상기 분리한 균주의 분자생물학적 동정을 위해 18S rRNA 유전자 서열 상동성을 분석한 결과 스키조키트리움 속 균주로 동정되었고, 이를 스키조키트리움 속 ABC-101으로 명명하였다. 본 발명에서 분리한 상기 스키조키트리움 속(Schizochytrium sp.) ABC-101 균주를 한국생명공학연구원(KCTC)에 2018년 1월 4일자로 기탁하였다(기탁번호: KCTC13455BP).ABC-101 strains of the genus Szozoquitium were inoculated in agar medium for microalgae separation from seawater and separated into fast microalgae pure colonies, and analyzed for 18S rRNA gene sequence homology for molecular biological identification of the isolated strains. As a result, it was identified as a strain of the genus Sjozukitrium, which was named ABC-101 in the genus Szukikitrium. The Schizochytrium sp. ABC-101 strain isolated from the present invention was deposited to the Korea Research Institute of Bioscience and Biotechnology (KCTC) on January 4, 2018 (Accession No .: KCTC13455BP).
본 발명의 일 구현 예에 따른 스키조키트리움 속 ABC-101 균주는 DHA 함량이 균주 건중량당 40~45%이고, 바이오오일 생산성은 10~20 g/ℓ·일이고, 바이오오일 생산 수율은 0.3~0.4g/g 글루코스일 수 있고, 바람직하게는 DHA 함량이 균주 건조중량당 43%이고, 바이오오일 생산 수율은 0.33g/g 글루코스이고, 바이오오일 생산성은 15.2 g/ℓ·일일 수 있으나, 이에 제한되지 않는다.ABC-101 strain of the genus Schizoquitrium according to an embodiment of the present invention has a DHA content of 40-45% per dry weight of the strain, bio oil productivity is 10-20 g / ℓ, bio-oil production yield is 0.3 ~ 0.4 g / g glucose, preferably DHA content of 43% per strain dry weight, biooil production yield 0.33 g / g glucose, biooil productivity may be 15.2 g / L It doesn't work
또한, 본 발명은 상기 균주 또는 상기 균주를 포함하는 배양액을 유효성분으로 포함하는 DHA(Docosahexaenoic acid, 22:6) 제조용 미생물 제제를 제공한다. 상기 미생물 제제는 스키조키트리움 속(Schizochytrium sp.) ABC-101 균주를 유효성분으로 포함할 수 있으며, DHA의 생산에 효과적으로 이용될 수 있다.In addition, the present invention provides a microbial preparation for producing DHA (Docosahexaenoic acid, 22: 6) comprising the strain or a culture solution containing the strain as an active ingredient. The microbial agent may include Schizochytrium sp. ABC-101 strain as an active ingredient and may be effectively used for the production of DHA.
또한, 본 발명은 상기 균주를 배양하고, 상기 배양한 균주로부터 DHA(Docosahexaenoic acid, 22:6)를 분리하는 것을 특징으로 하는 DHA의 제조 방법을 제공한다.The present invention also provides a method for producing DHA, comprising culturing the strain and separating DHA (Docosahexaenoic acid, 22: 6) from the cultured strain.
본 발명의 일 구현 예에 따른 방법에서, 상기 배양은 55~65 g/ℓ 포도당, 8~27g/ℓ 효모 추출물, 5~50 g/ℓ 해수염 및 0.1~2.0 ㎖/ℓ 비타민이 포함된 배지에서 배양할 수 있으나, 이에 제한되지 않는다. 또한, 상기 배양은 55~65 g/ℓ 포도당, 8~27g/ℓ 효모 추출물, 5~50 g/ℓ 해수염 및 0.1~2.0 ㎖/ℓ 비타민이 포함된 배지에 KH2PO4 8~10g/L를 추가로 포함할 수 있으나, 이에 제한되지 않는다.In the method according to an embodiment of the present invention, the culture medium containing 55-65 g / L glucose, 8-27 g / L yeast extract, 5-50 g / L sea salt and 0.1-2.0 mL / L vitamin It may be cultured in, but is not limited thereto. In addition, the cultivation is 55 ~ 65 g / ℓ glucose, 8 ~ 27g / ℓ yeast extract, 5 ~ 50 g / ℓ sea salt and 0.1 ~ KH 2 that contains the 2.0 ㎖ / ℓ vitamin medium PO 4 8 ~ 10g / It may further include L, but is not limited thereto.
상기 균주는 다양한 배양 방법 및 상기 배양한 균주로부터 DHA를 분리하는 방법은 당업계에 공지된 임의의 방법을 이용할 수 있다. The strain may be any of a variety of culture methods and methods for separating DHA from the cultured strains known in the art.
또한, 본 발명은 상기 균주 또는 상기 균주를 포함하는 배양액을 유효성분으로 포함하는 DHA(Docosahexaenoic acid, 22:6) 및 팔미트산 고함유 바이오오일 제조용 미생물 제제를 제공한다. 상기 미생물 제제는 스키조키트리움 속(Schizochytrium sp.) ABC-101 균주를 유효성분으로 포함할 수 있으며, DHA 및 팔미트산의 생산에 효과적으로 이용될 수 있다. 상기 DHA는 탄소수가 22이며 이중결합수가 6인 지방산(22:6 지방산)이며, 팔미트산은 탄소수가 16이며 이중결합수가 0인 지방산(16:0 지방산)이다.In another aspect, the present invention provides a microbial preparation for producing bio-oil containing DHA (Docosahexaenoic acid, 22: 6) and palmitic acid containing the strain or the culture medium containing the strain as an active ingredient. The microbial agent may include Schizochytrium sp. ABC-101 strain as an active ingredient, and may be effectively used for the production of DHA and palmitic acid. The DHA is a fatty acid having 22 carbon atoms and having 6 double bonds (22: 6 fatty acids), and palmitic acid is a fatty acid having 16 carbon atoms and 0 double bonds (16: 0 fatty acids).
본 발명의 일 구현 예에 따른 DHA 및 팔미트산 고함유 바이오오일 제조용 미생물 제제에서, 상기 바이오오일은 지방산 총 중량 기준으로 DHA 35~42% 및 팔미트산 45~50%를 함유하는 것일 수 있고, 바람직하게는 바이오오일 총 중량 기준으로 DHA 38~40% 및 팔미트산 48~50%를 함유하는 것일 수 있으나, 이에 제한되지 않는다.In the microbial preparation for producing DHA and palmitic acid-containing biooil according to an embodiment of the present invention, the biooil may be one containing DHA 35-42% and palmitic acid 45-50% based on the total weight of fatty acids, Preferably, it may include 38 to 40% DHA and 48 to 50% palmitic acid based on the total weight of biooil, but is not limited thereto.
또한, 본 발명은 상기 균주를 배양하고, 상기 배양한 균주로부터 바이오오일을 분리하는 것을 특징으로 하는 바이오오일의 제조 방법을 제공한다. 상기 균주는 다양한 배양 온도에서 바이오오일을 고농도로 축적할 수 있으며, 상기 배양 방법 및 상기 배양한 균주로부터 바이오오일을 분리하는 방법은 당업계에 공지된 임의의 방법을 이용할 수 있다. In another aspect, the present invention provides a method for producing a bio-oil, characterized in that for culturing the strain, separating the bio-oil from the cultured strain. The strain may accumulate high concentrations of biooil at various culture temperatures, and the culture method and the method of separating the biooil from the cultured strain may use any method known in the art.
또한, 본 발명은 In addition, the present invention
상기 균주를 배양하는 단계;Culturing the strain;
상기 배양된 균주로부터 지질을 분리하는 단계; 및Separating lipids from the cultured strains; And
상기 지질을 트랜스에스테르화시켜 지방산 에스테르를 생산하는 단계를 포함하는 바이오디젤의 생산 방법을 제공한다.It provides a method for producing biodiesel comprising transesterifying the lipid to produce a fatty acid ester.
본 발명의 방법에서, 상기 균주 배양 배지는 당업계에서 일반적으로 통용되는 배지를 이용할 수 있다. 상기 지방산 에스테르는 바람직하게는, 메틸 에스테르 또는 에틸 에스테르일 수 있으나, 이에 제한되지는 않는다. 상기 바이오디젤은 바이오매스에 포함된 지방산의 트랜스에스테르화 과정에 의해 메틸 에스테르 또는 에틸 에스테르 형태로 생산될 수 있으며, 상기 트랜스에스테르화 과정의 부산물로 글리세롤이 생성될 수 있다.In the method of the present invention, the strain culture medium may use a medium commonly used in the art. The fatty acid ester may preferably be, but is not limited to, methyl ester or ethyl ester. The biodiesel may be produced in the form of methyl ester or ethyl ester by the transesterification process of fatty acids included in the biomass, and glycerol may be produced as a by-product of the transesterification process.
상기 트랜스에스테르화 과정은 미세조류 균주 내 포함된 지질인 트리글리세라이드를 지방산 에스테르화 시키는 방법을 말한다. 상기 트렌스에스테르화 과정의 반응액으로 에탄올, 메탄올 등을 첨가할 수 있으나, 이에 제한되지는 않는다. 또한, 상기 트랜스에스테르화 과정의 반응 촉매제로서 산 또는 염기 촉매를 사용할 수 있으나 이에 제한되지는 않는다.The transesterification process refers to a method for fatty acid esterification of triglycerides, which are lipids contained in microalgal strains. Ethanol, methanol, etc. may be added as the reaction solution of the transesterification process, but is not limited thereto. In addition, an acid or a base catalyst may be used as the reaction catalyst of the transesterification process, but is not limited thereto.
상기 바이오디젤은 바이오연료로서 하기의 장점을 가질 수 있다; (1) 바이오연료는 일반적인 바이오매스 자원으로부터 손쉽게 얻을 수 있다. (2) 바이오연료는 연소에 있어서 이산화탄소의 순환을 나타낸다. (3) 바이오연료는 환경 친화적인 잠재력을 갖는다. (4) 바이오연료를 사용함으로써 환경, 경제 및 소비자에게 많은 이익을 준다. (5) 바이오연료는 자연계에서 미생물에 의해 무해한 물질로 분해될 수 있고, 지속가능성에 기여한다.The biodiesel may have the following advantages as a biofuel; (1) Biofuels can be easily obtained from common biomass resources. (2) Biofuels represent the circulation of carbon dioxide in combustion. (3) Biofuels have the potential to be environmentally friendly. (4) The use of biofuels benefits the environment, the economy and consumers. (5) Biofuels can be broken down into harmless substances by microorganisms in nature and contribute to sustainability.
또한, 본 발명은 In addition, the present invention
상기 균주를 배양하는 단계;Culturing the strain;
상기 배양된 균주로부터 바이오오일을 분리하는 단계; 및Separating the biooil from the cultured strain; And
상기 분리된 바이오오일을 트랜스에스테르화시켜 지방산 에스테르 및 글리세롤을 생성하는 단계를 포함하는 글리세롤의 제조 방법을 제공한다.It provides a method for producing glycerol comprising transesterifying the separated bio-oil to produce fatty acid esters and glycerol.
본 발명의 방법에서, 상기 균주의 배양 배지 및 바이오오일 분리 방법은 전술한 바와 같다.In the method of the present invention, the culture medium and biooil separation method of the strain is as described above.
상기 글리세롤은 윤활제, 연고나 좌약과 같은 약품의 제조, 관장제, 식품이나 화장품의 건조방지제 또는 정미제, 부동액, 냉각제, 도료, 안료, 인쇄 잉크, 투명비누의 제조, 실험실의 분석 시약, 용제, 셀로판, 접착제, 알키드 수지 또는 다이나마이트 등의 제조에 이용될 수 있으나, 이에 제한되지는 않는다.The glycerol is a lubricant, ointment or suppository, pharmaceuticals, enema, food or cosmetics drying or scouring agent, antifreeze, coolant, paint, pigment, printing ink, transparent soap, laboratory analytical reagents, solvents, cellophane , Adhesives, alkyd resins or dynamite may be used in the manufacture of, but is not limited thereto.
또한, 본 발명에서 바이오오일과 지질은 같은 의미로 혼용하여 사용될 수 있다.In the present invention, biooil and lipid may be used interchangeably in the same sense.
또한, 본 발명은 상기 균주, 상기 균주에 의해 생산된 바이오오일, 상기 균주의 배양액, 상기 배양액의 농축액 또는 상기 배양액의 건조물을 유효성분으로 함유하는 식품을 제공한다. 상기 스키조키트리움 속 ABC-101 균주는 불포화 지방산을 포함한 지질함량이 높기 때문에, 상기 균주, 상기 균주에 의해 생산된 바이오오일 또는 상기 균주를 포함하는 배양액을 유효성분으로 함유하는 식품은 영양학적으로 매우 가치가 높다. 상기 식품은 상기 균주 또는 상기 균주에 의해 생산된 바이오오일을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있으며 가공식품 형태로 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 상기 가공식품의 종류에는 특별한 제한은 없다. 상기 스키조키트리움 속 ABC-101 균주 분말을 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 가공식품을 모두 포함한다.The present invention also provides a food containing the strain, the bio-oil produced by the strain, the culture of the strain, the concentrate of the culture or the dried product of the culture as an active ingredient. Since the strain ABC-101 of the genus Szozoquitrium has a high lipid content including unsaturated fatty acids, the food containing the strain, the bio-oil produced by the strain or the culture solution containing the strain as an active ingredient is very nutritionally. High value The food may be added as is or the bio-oil produced by the strain as it is, or may be used with other foods or food ingredients, may be used in the form of processed foods, and may be appropriately used according to conventional methods. There is no particular limitation on the type of processed food. Examples of foods to which the ABC-101 strain powder in Sjozukitrium may be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various Soups, beverages, teas, drinks, alcoholic beverages and vitamin complexes, and includes all processed foods in the conventional sense.
또한, 본 발명은 상기 균주, 상기 균주에 의해 생산된 바이오오일, 상기 균주의 배양액, 상기 배양액의 농축액 또는 상기 배양액의 건조물을 유효성분으로 함유하는 사료 조성물을 제공한다. 사료 조성물 중의 상기 스키조키트리움 속 ABC-101 균주, 상기 균주에 의해 생산된 바이오오일, 상기 균주의 배양액, 상기 배양액의 농축액 또는 상기 배양액의 건조물의 함량은 급여 가축의 종, 주령, 체중, 및 사육 조건 등에 따라 적절히 선택될 수 있다. 본 발명의 사료 조성물은 기술분야에 공지된 사료 제조방법에 따라 제조될 수 있으며, 예를 들어, 각종 사료 원료 또는 배합사료와 본 발명의 상기 균주, 상기 균주에 의해 생산된 바이오오일, 상기 균주의 배양액, 상기 배양액의 농축액 또는 상기 배양액의 건조물을 혼합한 후, 추가적인 가공 공정, 예를 들어 펠렛 형태로의 성형 또는 과립 등의 형태로의 절단 단계 등을 더 수행함으로써 제조될 수 있다. 본 발명의 사료 조성물의 구성성분, 조성, 제조방법, 급여방법 등은 기술분야에 공지된 통상의 기술로부터 적절히 선택될 수 있음이 통상의 기술자에게 명백하다.In another aspect, the present invention provides a feed composition containing the strain, the bio-oil produced by the strain, the culture of the strain, the concentrate of the culture or the dried product of the culture as an active ingredient. The content of the strain ABC-101 of the genus Szozoquitrium, the bio-oil produced by the strain, the culture of the strain, the concentrate of the culture, or the dry matter of the culture in the feed composition is the species, age, weight, and breeding of the feed animals. It may be appropriately selected depending on the conditions and the like. The feed composition of the present invention may be prepared according to a feed production method known in the art, for example, various feed raw materials or blended feed and the strain of the present invention, the bio-oil produced by the strain, of the strain After mixing the culture solution, the concentrated solution of the culture solution or the dried product of the culture solution, it may be prepared by further performing an additional processing step, for example, molding in the form of pellets or cutting in the form of granules and the like. It will be apparent to those skilled in the art that the ingredients, compositions, methods of preparation, methods of feeding, etc. of the feed compositions of the present invention may be appropriately selected from conventional techniques known in the art.
또한, 본 발명은 상기 균주, 상기 균주에 의해 생산된 바이오오일, 상기 균주의 배양액, 상기 배양액의 농축액 또는 상기 배양액의 건조물을 유효성분으로 함유하는 화장료 조성물을 제공한다. 본 발명의 화장료 조성물에 포함되는 성분은 유효 성분으로서의 상기 스키조키트리움 속 ABC-101 균주, 상기 균주에 의해 생산된 바이오오일, 상기 균주의 배양액, 상기 배양액의 농축액 또는 상기 배양액의 건조물 이외에 화장품 조성물에 통상적으로 이용되는 성분들을 포함하며, 예컨대 항산화제, 안정화제, 용해화제, 비타민, 안료 및 향료와 같은 통상적인 보조제, 그리고 담체를 포함한다. 본 발명의 화장료 조성물은 당업계에서 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있으며, 예를 들어, 용액, 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 비누, 계면활성제-함유 클린싱, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션 및 스프레이 등으로 제형화될 수 있으나, 이에 한정되는 것은 아니다. 보다 상세하게는, 유연 화장수(스킨), 영양 화장수(밀크로션), 영양 크림, 마사지 크림, 에센스, 아이크림, 클렌징 크림, 클렌징 폼, 클렌징 워터, 팩, 스프레이 또는 파우더의 제형으로 제조될 수 있다.The present invention also provides a cosmetic composition containing the strain, the bio-oil produced by the strain, the culture of the strain, the concentrate of the culture or the dried product of the culture as an active ingredient. The components included in the cosmetic composition of the present invention may be used in cosmetic compositions in addition to the ABC-101 strain of the genus Szozoquitrium as the active ingredient, the bio-oil produced by the strain, the culture medium of the strain, the concentrated solution of the culture solution or the dried product of the culture solution. Commonly used ingredients include, for example, conventional auxiliaries such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavors, and carriers. The cosmetic composition of the present invention may be prepared in any formulation commonly prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing , Oils, powder foundations, emulsion foundations, wax foundations and sprays, and the like, but are not limited thereto. More specifically, it may be prepared in the form of a flexible lotion (skin), nourishing lotion (milk lotion), nourishing cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder. .
또한, 본 발명은 상기 균주를 배양하는 단계;In addition, the present invention comprises the steps of culturing the strain;
상기 배양된 균주로부터 지질을 분리하는 단계; 및Separating lipids from the cultured strains; And
상기 분리한 지질을 촉매전환시켜 항공유로 전환하는 단계를 포함하는 바이오항공유의 생산 방법을 제공한다.It provides a method for producing bio-air fuel comprising the step of catalytic conversion of the separated lipids to aviation oil.
본 발명의 상기 균주 배양 배지는 당업계에서 일반적으로 통용되는 배지를 이용할 수 있으며, 본 발명의 균주 배양액으로부터 지질을 분리하는 방법은 당업계에 공지된 임의의 방법을 이용할 수 있다. The strain culture medium of the present invention may use a medium commonly used in the art, and the method for separating lipids from the strain culture solution of the present invention may use any method known in the art.
본 발명에 따른 바이오항공유 제조방법은 통상적으로 촉매의 존재 하에서 지질로부터 탈산소(deoxygenation), 이성화(isomerization) 및 탈산소(deoxygenation), 이성화(isomerization) 및 크래킹(cracking) 반응을 통해 파라핀계 탄화수소를 형성시키고, 상기 반응생성물을 증류시켜 항공유분을 추출하는 단계를 포함하여 이루어질 수 있으나, 이에 제한되지 않는다.Biofuel production method according to the present invention is typically a paraffinic hydrocarbon through the deoxygenation, isomerization and deoxygenation, isomerization and cracking reaction from lipids in the presence of a catalyst Forming and distilling the reaction product may include the step of extracting the aviation oil, but is not limited thereto.
본 발명의 상기 촉매반응을 위해 사용되는 촉매로는 NiMo, CoMo, Pt, Pd가 사용될 수 있으나, 이에 제한되지 않는다.NiMo, CoMo, Pt, Pd may be used as the catalyst used for the catalytic reaction of the present invention, but is not limited thereto.
또한, 본 발명은 상기 균주를 배양하는 단계; 및In addition, the present invention comprises the steps of culturing the strain; And
상기 배양된 균주로부터 지질을 분리하는 단계를 포함하는 지질의 생산 방법 및 상기 방법에 의해 생산된 지질을 제공한다.It provides a method for producing a lipid comprising the step of separating the lipid from the cultured strain and the lipid produced by the method.
또한, 본 발명은 상기 방법에 의해 생산된 지질을 유효성분으로 함유하는 식품, 사료 조성물, 화장료 조성물, 타이오 트레드 또는 프로세싱 오일을 제공한다.The present invention also provides a food, feed composition, cosmetic composition, tao tread or processing oil containing the lipid produced by the method as an active ingredient.
이하, 실시예를 통하여 본 발명의 구성 및 효과를 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것일 뿐, 본 발명의 범위가 이들 실시예에 의해 한정되는 것은 아니다.Hereinafter, the configuration and effects of the present invention through the embodiments will be described in more detail. These examples are only for illustrating the present invention, but the scope of the present invention is not limited by these examples.
실시예 1. 신규 해양 미세조류 채취 및 선별Example 1 New Marine Microalgae Collection and Screening
해수 시료를 5 ml 채취하고, 생리식염수 10 ml을 가하여 현탁한 후 적당히 희석하여 해양 미세조류 분리용 한천 배지(10 g/L 글루코스, 10 g/L 해수염, 1 g/L 효모 추출물, 1 g/L 펩톤, 10 g/L 아가, 300 mg/L 페니실린 G, 500 mg/L 스트렙토마이신 설페이트)에 도말하였다. 28℃에서 200 rpm으로 4일간 배양하여 얻어진 콜로니들을 해양 미세조류 분리용 한천 배지에 재접종하여 성장이 빠른 미세조류 순수 콜로니를 분리하였다. 분리한 콜로니를 50 mL 기본배지(탄소원 포도당 60 g/L, 질소원 효모 추출물 10 g/L, 인공해수염 5 g/L, KH2PO4 9g/L)가 포함된 250 mL 배플 플라스크를 이용하여 28℃에서 120 rpm으로 3일간 배양한 후, 원심분리법으로 회수한 균체를 PBS 버퍼(phosphate buffered saline, pH 7.2)로 3회 세척한 후 동결건조기에서 건조하여 건조균체중량을 측정하였다. 건조된 균체를 5% 메탄올-황산(methanolic sulfuric acid) 용액 6 mL에 재현탁하여 90℃에서 1시간 동안 반응시킨 후 생성된 지방산에스테르를 1.0 mL의 헥산으로 추출하여 기체크로마토그래피로 분석하였다.5 ml of seawater samples are taken, suspended in 10 ml of physiological saline, and diluted appropriately. Agar medium for separation of marine algae (10 g / L glucose, 10 g / L sea salt, 1 g / L yeast extract, 1 g) / L peptone, 10 g / L agar, 300 mg / L penicillin G, 500 mg / L streptomycin sulfate). Colonies obtained by incubating at 200 rpm for 4 days at 28 ° C. were reinoculated into agar medium for marine microalgae separation to separate pure microalgae fast growth. The colonies were separated using a 250 mL baffle flask containing 50 mL base medium (60 g / L of carbon source glucose, 10 g / L of nitrogen source yeast extract, 5 g / L of artificial sea salt, 9 g / L of KH 2 PO 4 ). After incubating at 120 rpm for 3 days at 28 ° C, the cells collected by centrifugation were washed three times with PBS buffer (phosphate buffered saline, pH 7.2) and dried in a freeze dryer to measure the dry cell weight. The dried cells were resuspended in 6 mL of 5% methanol-sulfuric acid solution and reacted at 90 ° C. for 1 hour. The resulting fatty acid esters were extracted with 1.0 mL of hexane and analyzed by gas chromatography.
그 결과, 표 1에 나타낸 바와 같이 균체에 축적된 지질 중 다중불포화지방산인 DHA 함량이 37.53%로 확인되었으며, 포화지방산 팔미트산(palmitic acid)의 함량은 46.90%로 확인되었다. As a result, as shown in Table 1, the polyunsaturated fatty acid DHA content was found to be 37.53% in lipids accumulated in the cells, and the content of saturated fatty acid palmitic acid (palmitic acid) was found to be 46.90%.
실시예Example 2. 분류학적 분석 2. Taxonomic Analysis
분리한 콜로니의 분자생물학적 동정을 위하여 18S rRNA 유전자 서열을 분석하였다(도 1). 하나의 콜로니로부터 페놀-클로로포름법으로 염색체 DNA를 분리한 후, 이로부터 해양 미세조류 18S rRNA 유전자 증폭용 프라이머 5'-cctggttgatcctgccag-3'(P1, 서열번호 2)와 5'-ttgatccttctgcaggttca-3'(P2, 서열번호 3)을 이용하여 PCR법으로 18S rRNA 유전자 DNA를 증폭하였다. PCR 증폭은 EF Taq 폴리머라제(Takara)(2.5 U), 폴리머라제 버퍼, dNTP 혼합물(각 1 mM), 각 프라이머(100 pmol) 1μl, 주형 DNA 500ng을 함유하는 PCR 반응용액(50μl)을 준비한 후, 유전자 증폭기(Takara, Japan)로 96℃ 30초, 43℃ 1분, 72℃ 3분 조건으로 30회간 수행하였다. PCR 반응액을 1% 아가로즈겔에서 전기 영동하여 예상되는 크기의 DNA 단편이 증폭된 것을 확인하고 pGEM-TEasy 벡터(Promega, USA)를 이용하여 대장균 DH5α으로 형질도입하였다. 형질전환된 재조합 대장균들로부터 플라스미드 DNA를 추출(Qiagen, USA)하고, 제한효소 EcoRI을 처리하여 원하는 크기의 DNA 단편이 클로닝된 것을 확인하였으며, 염기서열을 결정하였다(서열번호 1). 염기서열 상동성 분석을 실시한 결과를 토대로 신규 분리한 미세조류를 스키조키트리움 속(Schizochytrium sp.) ABC-101으로 명명하였다 (도 2).The 18S rRNA gene sequence was analyzed for molecular biological identification of the isolated colonies (FIG. 1). Chromosomal DNA was isolated from a colony by phenol-chloroform method, and then the 5'-cctggttgatcctgccag-3 '(P1, SEQ ID NO: 2) and 5'-ttgatccttctgcaggttca-3' primers for amplification of marine microalgae 18S rRNA genes. 18S rRNA gene DNA was amplified by PCR using P2, SEQ ID NO: 3). PCR amplification was performed by preparing a PCR reaction solution (50 μl) containing EF Taq polymerase (2.5 U), polymerase buffer, dNTP mixture (1 mM each), 1 μl of each primer (100 pmol), and 500 ng of template DNA. , 30 times with a genetic amplifier (Takara, Japan) at 96 ℃ 30 seconds, 43 ℃ 1 minutes, 72 ℃ 3 minutes conditions. The PCR reaction solution was electrophoresed on 1% agarose gel to confirm that the DNA fragment of the expected size was amplified and transduced with E. coli DH5α using a pGEM-TEasy vector (Promega, USA). Plasmid DNA was extracted from the transformed recombinant E. coli (Qiagen, USA), and the restriction enzyme Eco RI was treated to confirm that the DNA fragment of the desired size was cloned, and the nucleotide sequence was determined (SEQ ID NO: 1). Based on the results of sequence homology analysis, the newly isolated microalgae was named Schizochytrium sp. ABC-101 (FIG. 2).
실시예 3. 신규 스키조키트리움 속(Example 3. A genus of new ski jokitrium SchizochytriumSchizochytrium sp.) ABC-101 균주의 균체 성장 및 DHA 함유 바이오오일 생산 능력 분석 sp.) Cell growth and analysis of DHA-containing biooil production capacity of ABC-101 strain
신규 스키조키트리움 속(Schizochytrium sp.) ABC-101 균주의 균체 성장 및 DHA 함유 오일 생산 능력을 다양한 영양원 조건에서 조사하였다. 기본배지로는 탄소원 포도당 60 g/L, 질소원 효모 추출물 10 g/L, KH2PO4 9 g/L, 인공해수염(sea salt) 5 g/L을 함유한 배지를 사용하였다. 단일콜로니를 기본배지 15 mL을 이용하여 28℃에서 120 rpm으로 36시간 전배양한 후 초기 접종 광학 밀도 (OD600nm) 0.5로 다양한 농도의 탄소원, 질소원, 해수염 및 비타민 혼합액 (티아민 9.5 g/L, 비오틴 0.2 g/L, 시아노 코발라민 B12 1.0 g/L)을 함유한 배지에 각각 접종하여 28℃에서 120 rpm으로 60시간 배양하였다. 원심분리법으로 회수한 균체를 PBS 버퍼 (phosphate buffered saline, pH 7.2)로 3회 세척한 후 동결건조기에서 24시간 건조하여 건조균체중량을 측정하였다.Cell growth and DHA-containing oil production of the novel Schizochytrium sp. ABC-101 strain were investigated under various nutrient conditions. As a basic medium, a medium containing 60 g / L of carbon source glucose, 10 g / L of nitrogen source yeast extract, 9 g / L of KH 2 PO 4 and 5 g / L of artificial sea salt was used. Single colonies were pre-incubated for 36 hours at 120 rpm at 28 ° C using 15 mL of basic medium, followed by various concentrations of carbon source, nitrogen source, sea salt and vitamin mixtures with initial optical density (OD 600 nm ) 0.5 (thiamine 9.5 g / L). , 0.2 g / L biotin and 1.0 g / L cyano cobalamin B12) were inoculated and incubated at 28 ° C. at 120 rpm for 60 hours. The cells collected by centrifugation were washed three times with PBS buffer (phosphate buffered saline, pH 7.2) and dried in a freeze dryer for 24 hours to measure the dry cell weight.
DHA 함유 오일함량은 수정된 Bligh-Dyer법을 의하여 분석되었다. 건조 균체량 125 mg에 클로로포름 6.25 mL, 메탄올 12.5 mL, 50 mM K2HPO4 버퍼 (pH 7.4) 5 mL을 가하여 28℃에서 200 rpm으로 1시간 동안 반응한 후 클로로포름 6.25 mL, K2HPO4 버퍼 6.25 mL을 첨가하여 30회 정도 섞어준 후 30분 동안 방치하여 수층과 오일이 함유된 유기용매층으로 분리되도록 했다. 알루미늄 접시에 클로로포름 층을 90℃에서 30분 동안 건조한 후 오일의 무게를 측정했다. 전체 오일 함량은 아래와 같이 산출했다.The oil content of DHA was analyzed by the modified Bligh-Dyer method. To 125 mg of dry cell weight, 6.25 mL of chloroform, 12.5 mL of methanol, and 5 mL of 50 mM K 2 HPO 4 buffer (pH 7.4) were added and reacted at 200 rpm at 28 ° C. for 1 hour, followed by 6.25 mL of chloroform and 6.25 mL of K 2 HPO 4 buffer. After adding 30 mL of the mixture and left for 30 minutes to separate the aqueous layer and the organic solvent layer containing the oil. The chloroform layer was dried in an aluminum dish at 90 ° C. for 30 minutes and the oil was weighed. The total oil content was calculated as follows.
총 오일함량 (%, 오일 g /건조균체량 100 g) = (WL-WD)×VC×100/VP×WS Total oil content (%, oil g / dry cell weight 100 g) = (W L -W D ) × V C × 100 / V P × W S
WL: 알루미늄 접시의 무게W L : weight of aluminum plate
WD: 알루미늄 접시 + 지질의 무게W D : weight of aluminum plate + lipid
VC: 클로로포름의 총 부피V C : total volume of chloroform
VP: 알루미늄 접시에 옮긴 클로로포름의 부피V P : volume of chloroform transferred to aluminum dish
WS: 사용한 균체의 무게 (건조중량)W S : Weight of used cells (dry weight)
오일중에 함유된 다양한 지방산의 함량은 기체크로마토그래피법으로 측정하였다. 미세조류 건조 균체를 4% 황산-메탄올 용액 6 mL에 현탁하여 90℃에서 1시간 동안 반응하여 지방산에스테르를 생성한 다음 헥산 1 mL로 추출하여 기체크로마토그래피로 분석하였다. 유일 탄소원인 포도당을 다양한 농도로 첨가한 기본배지에서 ABC-101 균주를 배양한 결과, 표 2와 같이 60 g/L의 농도에서 가장 높은 균체 성장을 보이는 것으로 나타났으며 (건조 균체량 20.3 g/L), 이때 오일의 함량은 건조균체량의 62.5% 이었으며, DHA의 함량은 전체지방산의 39.8%로 나타났다.The content of various fatty acids in the oil was measured by gas chromatography. The microalgae dry cells were suspended in 6 mL of 4% sulfuric acid-methanol solution, reacted at 90 ° C. for 1 hour to produce fatty acid esters, and then extracted with 1 mL of hexane and analyzed by gas chromatography. As a result of culturing ABC-101 strain in a basic medium containing glucose, which is the only carbon source at various concentrations, it showed the highest cell growth at a concentration of 60 g / L as shown in Table 2 (dry cell weight 20.3 g / L). At this time, the oil content was 62.5% of the dry cell weight, and the DHA content was 39.8% of the total fatty acid.
(g L-1)Concentration
(g L -1 )
(g L-1)Dry cell weight
(g L -1 )
(% DCW)Lipid
(% DCW)
(% TFA)DHA
(% TFA)
질소원인 효모 추출물을 다양한 농도로 첨가한 기본배지에서 ABC101 균주를 배양하였을 때, 표 3에 나타낸 바와 같이, 균체 성장은 효모 추출물의 농도가 높을수록 우수하였지만, 오일의 함량은 반대로 효모 추출물의 농도가 낮을수록 우수한 것으로 나타났으며, 최대 건조균체량의 62.5%까지 오일의 함량이 증가하였다. 반면 DHA 함량은 효모 추출물의 농도가 낮을수록 다소 감소하는 것으로 나타났다.When the ABC101 strain was cultured in the basic medium to which the nitrogen source yeast extract was added at various concentrations, as shown in Table 3, the cell growth was better as the concentration of the yeast extract was higher, but the oil content was inversely opposite to the concentration of the yeast extract. The lower the value, the better, and the oil content increased up to 62.5% of the maximum dry cell mass. On the other hand, DHA content was found to decrease slightly with lower concentration of yeast extract.
(g L-1)Concentration
(g L -1 )
(g L-1)Dry cell weight
(g L -1 )
(% DCW)Lipid
(% DCW)
(% TFA)DHA
(% TFA)
해수염의 농도에 따른 영향을 살펴본 결과, 표 4에 나타낸 바와 같이, 10 g/L의 농도에서 가장 높은 균체 성장을 보이는 것으로 나타났으며 (건조 균체량 24.3 g/L), 이때 오일의 함량은 건조균체량의 62.7% 이었으며, DHA의 함량은 전체지방산의 40.1%인 것으로 조사되었다. As a result of examining the effect of sea salt concentration, as shown in Table 4, it showed the highest cell growth at the concentration of 10 g / L (dry cell weight 24.3 g / L), where the oil content is dry cell weight The content of DHA was 40.1% of total fatty acid.
(g L-1)Concentration
(g L -1 )
(g L-1)Dry cell weight
(g L -1 )
(% DCW)Lipid
(% DCW)
(% TFA)DHA
(% TFA)
비타민 혼합액 (티아민 9.5 g/L, 비오틴 0.2 g/L, 시아노 코발라민 B12 1.0 g/L)의 농도에 따른 영향을 살펴본 결과, 표 5에 나타낸 바와 같이, 비타민 혼합액 0.1 mL/L를 첨가한 농도에서 기존의 배양 시간보다 12시간 빠른 배양 48시간에서 가장 높은 균체 성장을 보이는 것으로 나타났으며 (건조 균체량 25.0 g/L), 이때 오일의 함량은 건조균체량의 64.6% 이었으며, DHA의 함량은 전체지방산의 37.4%인 것으로 조사되었다.As a result of examining the effect of the concentration of the vitamin mixture (thiamine 9.5 g / L, biotin 0.2 g / L, cyano cobalamin B12 1.0 g / L), as shown in Table 5, the concentration of 0.1 mL / L added to the vitamin mixture The highest cell growth was observed at 48 hours of cultivation at 12 hours earlier than the conventional culture time (dry cell weight of 25.0 g / L), where the oil content was 64.6% of the dry cell weight, and the content of DHA was total fatty acid. Was found to be 37.4%.
(mL L-1)Concentration
(mL L -1 )
(g L-1)Dry cell weight
(g L -1 )
(% DCW)Lipid
(% DCW)
(% TFA)DHA
(% TFA)
실시예 4. 신규 스키조키트리움 속(Example 4 Genus Ski Jokitrium SchizochytriumSchizochytrium sp.) ABC-101 균주의 다양한 영양원 이용 능력 sp.) Ability to use various nutrients of ABC-101 strain
신규 스키조키트리움 속 ABC-101 균주의 다양한 영양원 이용 능력을 조사하였다. 기본배지에 다양한 탄소원, 질소원을 첨가하여 상기와 같은 방법으로 배양하여 균체 성장과 오일 및 DHA의 함량을 조사하였다. 포도당 대신에 탄소원으로 프럭토스(fructose), 갈락토스(galactose), 자일로스(xylose), 락토오스(lactose), 말토오스(maltose), 수크로스(sucrose), 글리세롤(glycerol) 및 전분(starch)을 60 g/L로 첨가한 경우, 표 6에 나타낸 바와 같이, 포도당에 비해 다소 감소하기는 하였지만 여전히 ABC-101 균주는 균체 성장이 가능하였다. 특히 프럭토스에서 포도당과 유사한 균체 성장을 보이는 것으로 나타났다.The use of various nutrient sources of ABC-101 strain of the novel Szozoquitrium was investigated. Various carbon sources and nitrogen sources were added to the basic medium and cultured in the same manner as above to examine cell growth and oil and DHA contents. 60 g of fructose, galactose, xylose, lactose, maltose, sucrose, glycerol and starch as carbon sources instead of glucose When added in / L, as shown in Table 6, although slightly reduced compared to glucose, the ABC-101 strain was still possible cell growth. In particular, fructose-like cell growth appeared to be similar.
(g L-1)Dry cell weight
(g L -1 )
(% DCW)Lipid
(% DCW)
(% TFA)DHA
(% TFA)
유기질소원으로 효모 추출물 대신에 옥수수 침지액, 소고기 추출물, 맥아 추출물, 펩톤, 트립톤을 10 g/L의 농도로 첨가해준 경우, 표 7에 나타낸 바와 같이, ABC-101은 균체 성장이 가능하였으며, 특히 트립톤에서는 효모 추출물과 유사한 균체 성장을 보이는 것으로 나타났다. 또한 암모늄 아세테이트, 암모늄 나이트레이트, 암모늄 설페이트, 소듐 나이트레이트, 우레아, 소듐 글루타메이트를 10 g/L의 농도로 첨가하여 다양한 무기질소원의 영향을 살펴본 결과, 표 8에 나타낸 바와 같이, ABC-101은 균체 성장이 가능하였으며, 소듐 글루타메이트에서 가장 우수한 균체 성장을 보이는 것으로 나타났다.In the case of adding corn steep liquor, beef extract, malt extract, peptone, and tryptone at a concentration of 10 g / L as an organic nitrogen source, as shown in Table 7, ABC-101 was able to grow cells. In particular, tryptone showed similar cell growth with yeast extract. In addition, ammonium acetate, ammonium nitrate, ammonium sulfate, sodium nitrate, urea and sodium glutamate were added at a concentration of 10 g / L to examine the effects of various inorganic nitrogen sources. As shown in Table 8, ABC-101 was a cell Growth was possible, showing the best cell growth in sodium glutamate.
(g L-1)Dry cell weight
(g L -1 )
(% DCW)Lipid
(% DCW)
(% TFA)DHA
(% TFA)
(g L-1)Dry cell weight
(g L -1 )
(% DCW)Lipid
(% DCW)
(% TFA)DHA
(% TFA)
실시예Example 5. 발효조를 이용한 신규 5. New using fermenter 스키조키트리움Ski Jockey Triumph 속 ABC-101 균주의 바이오오일 생산 Biooil Production of Genus ABC-101 Strains
5L 배양기를 이용한 스키조키트리움 속 ABC-101 균주의 배양을 수행하였다. 상기의 기본 배지에서 전배양한 균체를 3L의 동일배지 (5L jar fermentor)로 이식하여 28℃에서 2 vvm의 조건으로 회분식 발효를 수행하면서 12시간 간격으로 균체를 회수하여 균체 성장과 오일 및 DHA 함량을 조사하였다. 그 결과 도 3에 나타낸 바와 같이, 배양 48시간째에 오일 생산성 및 DHA 생산성은 각각 15.2 g/L/day, 6.58 g/L/day이었다. 사용된 포도당 영양원 대비 오일의 생산수율은 0.33 g/g 글루코스이었으며, DHA의 함량은 43.3%인 것으로 확인되었다. Cultivation of ABC-101 strain of genus Sjozukitrium was performed using a 5L incubator. Cells pre-cultivated in the above medium were transplanted into 3L of the same medium (5L jar fermentor) and subjected to batch fermentation under conditions of 2 vvm at 28 ° C to recover the cells at 12 hour intervals. Was investigated. As a result, as shown in FIG. 3, the oil productivity and the DHA productivity were 15.2 g / L / day and 6.58 g / L / day, respectively, at 48 hours of culture. The yield of oil was 0.33 g / g glucose and the content of DHA was 43.3%.
productivity
(g/L/day)Lipid
productivity
(g / L / day)
(g/g glucose)Lipid yield
(g / g glucose)
content
(%)DHA
content
(%)
productivity
(g/L/day)DHA
productivity
(g / L / day)
<110> ADVANCED BIOMASS R＆D CENTER <120> Microalgae Schizochytrium sp. ABC-101 strain producing bio-oil containing high concentration of DHA and method for producing DHA using the strain <130> PN18054 <160> 3 <170> KopatentIn 2.0 <210> 1 <211> 1777 <212> DNA <213> Schizochytrium sp. <400> 1 cctggttgat cctgccagta gtcatatgct cgtctcaaag attaagccat gcatgtgtaa 60 gtataagcga ttgtactgtg agactgcgaa cggctcatta tatcagtaat aatttcttcg 120 gtagtttctt ttatatggat acctgcagta attctggaaa taatacatgc tgtaagagcc 180 ctgtatgggg ctgcacttat tagattgaag ccgattttat tggtgaatca tgataattga 240 gcagattgac tattttggtc gatgaatcgt ttgagtttct gccccatcag ttgtcgacgg 300 tagtgtattg gactacggtg actataacgg gtgacggaga gttagggctc gactccggag 360 agggagcctg agagacggct accatatcca aggatagcag caggcgcgta aattacccac 420 tgtggactcc acgaggtagt gacgagaaat atcgatgcga agcgtgtatg cgttttgcta 480 tcggaatgag agcaatgtaa aaccctcatc gaggatcaac tggagggcaa gtctggtgcc 540 agcagccgcg gtaattccag ctccagaagc atatgctaaa gttgttgcag ttaaaaagct 600 cgtagttgaa tttctggcat gggcgaccgg tgctttccct gaatggggat tgattgtctg 660 cgttgccttg gccatctttc tcatgctatt tttggtatga gatctttcac tgtaatcaaa 720 gcagagtgtt ccaagcaggt cgtatgaccg gtatgtttat tatgggatga taagatagga 780 cttgggtgct attttgttgg tttgcacgcc tgagtaatgg ttaataggaa cagttggggg 840 tattcgtatt taggagctag aggtgaaatt cttggatttc cgaaagacga actagagcga 900 aggcatttac caagcatgtt ttcattaatc aagaacgaaa gtctggggat cgaagatgat 960 tagataccat cgtagtctag accgtaaacg atgccgactt gcgattgttg ggtgctttat 1020 tatatgggcc tcagcagcag cacatgagaa atcaaagtct ttgggttccg gggggagtat 1080 ggtcgcaagg ctgaaactta aaggaattga cggaagggca ccaccaggag tggagcctgc 1140 ggcttaattt gactcaacac gggaaaactt accaggtcca gacataggta ggattgacag 1200 attgagagct ctttcatgat tctatgggtg gtggtgcatg gccgttctta gttggtggag 1260 tgatttgtct ggttaattcc gttaacgaac gagacctcgg cctactaaat agtgcgtggt 1320 atggcaacat agtgcgtttt aacttcttag agggacatgt ccggtttacg ggcaggaagt 1380 tcgaggcaat aacaggtctg tgatgccctt agatgttctg ggccgcacgc gcgctacact 1440 gatgggttca tcgggtttta atttcatttt tggaattgag tgcttggtcg gaaggcctgg 1500 ctaatccttg gaacgctcat cgtgctgggg ctagattttt gcaattatta atctccaacg 1560 aggaattcct agtaaacgca agtcatcagc ttgcattgaa tacgtccctg ccctttgtac 1620 acaccgcccg tcgcacctac cgattgaacg gtccgatgaa accatgggat gtttctgttt 1680 ggattcattt ttggacagag gcagaactcg ggtgaatctt attgtttaga ggaaggtgaa 1740 gtcgtaacaa ggtttccgta ggtgaacctg cagaagg 1777 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 cctggttgat cctgccag 18 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ttgatccttc tgcaggttca 20 <110> ADVANCED BIOMASS R & D CENTER <120> Microalgae Schizochytrium sp. ABC-101 strain producing bio-oil containing high concentration of DHA and method for producing DHA using the strain <130> PN18054 <160> 3 <170> KopatentIn 2.0 <210> 1 <211> 1777 <212> DNA <213> Schizochytrium sp. <400> 1 cctggttgat cctgccagta gtcatatgct cgtctcaaag attaagccat gcatgtgtaa 60 gtataagcga ttgtactgtg agactgcgaa cggctcatta tatcagtaat aatttcttcg 120 gtagtttctt ttatatggat acctgcagta attctggaaa taatacatgc tgtaagagcc 180 ctgtatgggg ctgcacttat tagattgaag ccgattttat tggtgaatca tgataattga 240 gcagattgac tattttggtc gatgaatcgt ttgagtttct gccccatcag ttgtcgacgg 300 tagtgtattg gactacggtg actataacgg gtgacggaga gttagggctc gactccggag 360 agggagcctg agagacggct accatatcca aggatagcag caggcgcgta aattacccac 420 tgtggactcc acgaggtagt gacgagaaat atcgatgcga agcgtgtatg cgttttgcta 480 tcggaatgag agcaatgtaa aaccctcatc gaggatcaac tggagggcaa gtctggtgcc 540 agcagccgcg gtaattccag ctccagaagc atatgctaaa gttgttgcag ttaaaaagct 600 cgtagttgaa tttctggcat gggcgaccgg tgctttccct gaatggggat tgattgtctg 660 cgttgccttg gccatctttc tcatgctatt tttggtatga gatctttcac tgtaatcaaa 720 gcagagtgtt ccaagcaggt cgtatgaccg gtatgtttat tatgggatga taagatagga 780 cttgggtgct attttgttgg tttgcacgcc tgagtaatgg ttaataggaa cagttggggg 840 tattcgtatt taggagctag aggtgaaatt cttggatttc cgaaagacga actagagcga 900 aggcatttac caagcatgtt ttcattaatc aagaacgaaa gtctggggat cgaagatgat 960 tagataccat cgtagtctag accgtaaacg atgccgactt gcgattgttg ggtgctttat 1020 tatatgggcc tcagcagcag cacatgagaa atcaaagtct ttgggttccg gggggagtat 1080 ggtcgcaagg ctgaaactta aaggaattga cggaagggca ccaccaggag tggagcctgc 1140 ggcttaattt gactcaacac gggaaaactt accaggtcca gacataggta ggattgacag 1200 attgagagct ctttcatgat tctatgggtg gtggtgcatg gccgttctta gttggtggag 1260 tgatttgtct ggttaattcc gttaacgaac gagacctcgg cctactaaat agtgcgtggt 1320 atggcaacat agtgcgtttt aacttcttag agggacatgt ccggtttacg ggcaggaagt 1380 tcgaggcaat aacaggtctg tgatgccctt agatgttctg ggccgcacgc gcgctacact 1440 gatgggttca tcgggtttta atttcatttt tggaattgag tgcttggtcg gaaggcctgg 1500 ctaatccttg gaacgctcat cgtgctgggg ctagattttt gcaattatta atctccaacg 1560 aggaattcct agtaaacgca agtcatcagc ttgcattgaa tacgtccctg ccctttgtac 1620 acaccgcccg tcgcacctac cgattgaacg gtccgatgaa accatgggat gtttctgttt 1680 ggattcattt ttggacagag gcagaactcg ggtgaatctt attgtttaga ggaaggtgaa 1740 gtcgtaacaa ggtttccgta ggtgaacctg cagaagg 1777 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 cctggttgat cctgccag 18 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ttgatccttc tgcaggttca 20
Claims (14)
상기 배양된 균주로부터 지질을 분리하는 단계; 및
상기 지질을 트랜스에스테르화시켜 지방산 에스테르를 생산하는 단계를 포함하는 바이오디젤의 생산 방법.Culturing the strain according to any one of claims 1 to 3;
Separating lipids from the cultured strains; And
The method of producing biodiesel comprising transesterifying the lipid to produce a fatty acid ester.
상기 배양된 균주로부터 바이오오일을 분리하는 단계; 및
상기 분리된 바이오오일을 트랜스에스테르화시켜 지방산 에스테르 및 글리세롤을 생성하는 단계를 포함하는 글리세롤의 제조 방법.Culturing the strain of any one of claims 1 to 3;
Separating the biooil from the cultured strain; And
Transesterifying the separated biooil to produce fatty acid esters and glycerol.
상기 배양된 균주로부터 지질을 분리하는 단계; 및
상기 분리한 지질을 촉매전환시켜 항공유로 전환하는 단계를 포함하는 바이오항공유의 생산 방법.
Culturing the strain according to any one of claims 1 to 3;
Separating lipids from the cultured strains; And
The method of producing bio-air fuel comprising the step of catalytic conversion of the separated lipids to aviation oil.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022124590A1 (en) * | 2020-12-07 | 2022-06-16 | 씨제이제일제당(주) | Novel schizochytrium sp. strain and polyunsaturated fatty acid production method using same |
| WO2023080399A1 (en) * | 2021-11-08 | 2023-05-11 | 씨제이제일제당 (주) | Novel strain of schizochytrium sp. with easy intracellular oil extraction and a method for producing oil containing omega-3 using same |
| WO2023080400A1 (en) * | 2021-11-08 | 2023-05-11 | 씨제이제일제당 (주) | Novel strain of schizochytrium sp. with easy intracellular oil extraction and method for producing oil containing omega3 using same |
| WO2024128494A1 (en) * | 2022-12-15 | 2024-06-20 | 씨제이제일제당 (주) | Novel schizochytrium sp. strain and method for producing oil containing omega-3 using same |
-
2018
- 2018-03-20 KR KR1020180031814A patent/KR20190110186A/en active IP Right Grant
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022124590A1 (en) * | 2020-12-07 | 2022-06-16 | 씨제이제일제당(주) | Novel schizochytrium sp. strain and polyunsaturated fatty acid production method using same |
| WO2023080399A1 (en) * | 2021-11-08 | 2023-05-11 | 씨제이제일제당 (주) | Novel strain of schizochytrium sp. with easy intracellular oil extraction and a method for producing oil containing omega-3 using same |
| WO2023080400A1 (en) * | 2021-11-08 | 2023-05-11 | 씨제이제일제당 (주) | Novel strain of schizochytrium sp. with easy intracellular oil extraction and method for producing oil containing omega3 using same |
| WO2024128494A1 (en) * | 2022-12-15 | 2024-06-20 | 씨제이제일제당 (주) | Novel schizochytrium sp. strain and method for producing oil containing omega-3 using same |
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