KR20110122426A - Method for culturing microalgae thraustochytrid - Google Patents

Method for culturing microalgae thraustochytrid Download PDF

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KR20110122426A
KR20110122426A KR1020100041944A KR20100041944A KR20110122426A KR 20110122426 A KR20110122426 A KR 20110122426A KR 1020100041944 A KR1020100041944 A KR 1020100041944A KR 20100041944 A KR20100041944 A KR 20100041944A KR 20110122426 A KR20110122426 A KR 20110122426A
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thraustochytrid
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서정우
김철호
홍원경
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한국생명공학연구원
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Priority to PCT/KR2011/002999 priority patent/WO2011139040A2/en
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Abstract

PURPOSE: A method for culturing Thraustochytrid microalgae is provided to efficiently and cheaply obtain bio-oil using soybean powder as a nitrogen source. CONSTITUTION: A method for culturing Thraustochytrid microalgae comprises a step of culturing the microalgae in a medium containing soybean powder. The Thraustochytrid microalgae is KRS101(KCTC11686BP). A method for preparing bio-oil comprises: a step of culturing the microalgae in the medium containing soybean powder; and a step of collecting bio-oil from the cultured strain or medium. The bio oil is DHA(docosahexaenoic acid).

Description

Ｔｈｒａｕｓｔｏｃｈｔｒｉｄ계 미세조류의 배양방법{Method for Culturing Microalgae Thraustochytrid}Method for Culturing Microalgae Thraustochytrid Method of Cultured Ｔｈｒａｕｓｔｏｃｈｔｒｉｄ

본 발명은 Thraustochytrid계 미세조류의 배양방법에 관한 것으로, 더욱 자세하게는 바이오오일 생성능을 가지는 Thraustochytrid계 미세조류를 콩분말이 함유된 배지에서 배양하는 것을 특징으로 하는 Thraustochytrid계 미세조류의 배양방법에 관한 것이다.
The present invention relates to a method for culturing Thraustochytrid microalgae, and more particularly, to a method for culturing Thraustochytrid microalgae, characterized in that the cultivation of Thraustochytrid microalgae having a bio-oil generating ability in a medium containing soybean powder. .

최근 각광 받고 있는 바이오디젤을 제조하는데 사용되는 바이오오일의 주요한 공급원은 식물성 유지 혹은 미세조류 오일 등 광합성 오일이다. 바이오오일의 대량 생산 방법으로 최근에는 유기영양 미생물의 발효배양법이 주목을 받고 있다. 폐기물, 부산물 및 잉여 바이오매스 자원으로부터 생산된 미생물 발효오일은 광합성오일과 함께 바이오디젤의 원료로 활용될 것으로 기대된다. Chlorella protothecoides, Yarrowia lipolytica, Rhodosporidium toruloides, Rhodotorula glutinis 등이 대표적인 유지성 미생물이며, 이들의 발효 공정 연구가 활발하게 진행 중이다.The main source of biooil used to make biodiesel, which is in the spotlight in recent years, is photosynthetic oil such as vegetable oil or microalgal oil. Recently, the fermentation culture method of organic nutrition microorganism has attracted attention as a mass production method of bio oil. Microbial fermentation oils produced from waste, by-products and surplus biomass resources are expected to be used as raw materials for biodiesel with photosynthetic oil. Chlorella protothecoides, Yarrowia lipolytica, Rhodosporidium toruloides, and Rhodotorula glutinis are representative oil-retaining microorganisms, and their fermentation process studies are actively conducted.

이들 유지성 미생물 중, Thraustochytrid 계열 미세조류는 DHA(docosahexaenoic acid)와 같은 고도불포화지방산 (polyunsaturated fatty acid)를 고농도로 함유하는 유지성 미생물이다. DHA는 두뇌, 안구조직 및 신경계에 필수적인 지방산으로 특히 유아의 시력 및 운동신경능력 개발에 중요한 기능을 하는 것으로 알려졌다. 또한 치매 환자 뇌에서는 그 양이 현저하게 줄어드는 것으로 보고되었으며, 노안의 황반변성 억제 등 다양한 항노화 기능들이 새롭게 밝혀지고 있다. 이러한 유용한 생리적 기능에도 불구하고, 인체는 자체적으로 충분한 량의 DHA를 합성할 수 없기 때문에 외부로부터 공급되어야 하는 필수 영양소로 인식되어 세계보건기구를 비롯한 각국의 공인 기관들이 DHA를 하루 1 g 이상 꾸준히 섭취할 것을 권장하고 있다. 때문에 DHA는 건강기능성 식품 등 다양한 제품으로 상용화되고 있으며, 의약품 원료로도 활용 가능성 높아 DHA의 상업적 가치는 매우 높다고 할 수 있다. 따라서 DHA를 고농도로 함유하는 Thraustochytrid 미세조류의 오일은 식품, 사료 등 고부가가치 산업원료로 주목을 받고 있으며, Thraustochytrid 미세조류에서 고효율로 DHA를 생산할 수 있는 방법들이 요구되고 있다.Among these oleaginous microorganisms, the Thraustochytrid family of microalgae is an oleaginous microorganism containing a high concentration of polyunsaturated fatty acid such as doxahexaenoic acid (DHA). DHA is an essential fatty acid for the brain, eye tissue, and nervous system, and is known to play an important role in the development of vision and motor capacity, especially in infants. In addition, the amount of dementia has been reported to be significantly reduced in the brain, and various anti-aging functions such as suppressing macular degeneration of presbyopia are newly revealed. Despite these useful physiological functions, the human body is unable to synthesize sufficient amounts of DHA on its own and is recognized as an essential nutrient that must be supplied from the outside world. I recommend doing it. Therefore, DHA has been commercialized as a variety of products, such as health functional food, and can be used as a raw material for pharmaceuticals, so the commercial value of DHA is very high. Therefore, the oil of Thraustochytrid microalgae containing a high concentration of DHA is attracting attention as a high value-added industrial raw materials such as food, feed, etc., and there is a need for a method for producing DHA with high efficiency in Thraustochytrid microalgae.

한편, 콩은 식물성 단백질 및 지방의 공급원으로 오래 전부터 된장, 두부, 간장 등의 식품 원료로 활용되어 왔다. 최근에는 암 예방을 비롯한 다양한 생리적 효능을 지닌 이소플라본이 풍부하게 함유되어 있는 것이 밝혀져 건강기능성 식품으로 각광을 받고 있으며, 대량재배를 통한 대량생산이 가능하여, 함유하규 영양성분에 비해 제조 단가가 낮은 작물이기도 하다.On the other hand, soybeans have been used as a source of vegetable protein and fat for a long time as a food raw material such as miso, tofu, soy sauce. Recently, it has been found to contain abundant isoflavones with various physiological effects, including cancer prevention, and has been spotlighted as a health functional food, and it is possible to mass-produce through mass cultivation, so that the manufacturing cost is lower than that contained It is also a crop.

이에, 본 발명에서는 Thraustochytrid 계열 미세조류의 고효율 배양방법을 개발하고자 예의 노력한 결과, 콩 분말을 영양원으로 활용하여, Thraustochytrid 계열 미세조류를 배양할 경우, DHA를 고농도로 함유한 Thraustochytrid 계열 미세조류를 배양할 수 있다는 것을 확인하고 본 발명을 완성하게 되었다.
Therefore, in the present invention, as a result of intensive efforts to develop a high-efficiency culture method of Thraustochytrid-based microalgae, when cultivating Thraustochytrid-based microalgae using soybean powder as a nutrient source, Thraustochytrid-based microalgae containing a high concentration of DHA It was confirmed that the present invention can be completed.

본 발명의 목적은 Thraustochytrid 계열 미세조류를 고효율로 배양하여 경제적으로 바이오오일을 생산할 수 있는 방법을 제공하는데 있다.
An object of the present invention is to provide a method for economically producing bio-oil by culturing Thraustochytrid-based microalgae with high efficiency.

상기 목적을 달성하기 위하여, 본 발명은 바이오오일 생성능을 가지는 Thraustochytrid계 미세조류를 콩분말이 함유된 배지에서 배양하는 것을 특징으로 하는 Thraustochytrid계 미세조류의 배양방법을 제공한다.In order to achieve the above object, the present invention provides a method for culturing Thraustochytrid microalgae, characterized in that the culture of Thraustochytrid microalgae having the ability to produce bio-oil in a medium containing soybean powder.

본 발명은 또한, (a) 바이오오일 생성능을 가지는 Thraustochytrid계 미세조류를 콩분말이 함유된 배지에서 배양하는 단계; 및 (b) 상기 배양된 균체 또는 배지에서 바이오오일을 회수하는 단계를 포함하는 바이오오일의 제조방법을 제공한다.
The present invention also comprises the steps of: (a) culturing a Thraustochytrid-based microalgae having a biooil production ability in a medium containing soybean powder; And (b) provides a method for producing a bio-oil comprising the step of recovering the bio-oil from the cultured cells or media.

본 발명에 따르면, 바이오오일 생성능을 가지는 Thraustochytrid계 미세조류를 콩분말을 질소원으로 사용하여 배양하여, 효율적이며, 경제적으로 바이오오일을 제조할 수 있다. 또한, 식용가능한 콩분말을 배지로 사용함으로, 배양산물을 식품 및 사료 등의 제조원료로 활용할 수 있다.
According to the present invention, by culturing the Thraustochytrid microalgae having the ability to produce bio-oil soybean powder as a nitrogen source, it is possible to efficiently and economically produce bio-oil. In addition, by using an edible soybean powder as a medium, the culture product can be utilized as a raw material for food and feed.

도 1은 신규 Thraustochytrid 계열 미세조류 KRS101의 현미경 관찰 사진을 나타낸 것이다.Figure 1 shows a microscopic picture of the novel Thraustochytrid family of microalgae KRS101.

일 관점에서, 본 발명은 바이오오일 생성능을 가지는 Thraustochytrid계 미세조류를 콩분말이 함유된 배지에서 배양하는 것을 특징으로 하는 Thraustochytrid계 미세조류의 배양방법에 관한 것이다.In one aspect, the present invention relates to a method for culturing Thraustochytrid microalgae, characterized in that the culture of Thraustochytrid microalgae having the ability to produce bio-oil in a medium containing soybean powder.

본 발명에 있어서, 상기 Thraustochytrid계 미세조류는 KRS101(KCTC11686BP)인 것을 특징으로 할 수 있고, 상기 콩분말을 단일 질소원으로 함유하는 배지에서 배양하는 것을 특징으로 할 수 있다.In the present invention, the Thraustochytrid microalgae may be characterized in that KRS101 (KCTC11686BP), it may be characterized in that the culture in a medium containing the soy powder as a single nitrogen source.

다른 관점에서, 본 발명은 (a) 바이오오일 생성능을 가지는 Thraustochytrid계 미세조류를 콩분말이 함유된 배지에서 배양하는 단계; 및 (b) 상기 배양된 균체 또는 배지에서 바이오오일을 회수하는 단계를 포함하는 바이오오일의 제조방법에 관한 것이다.In another aspect, the present invention comprises the steps of (a) culturing a Thraustochytrid microalgae having a bio-oil production ability in a medium containing soybean powder; And (b) relates to a method for producing a bio-oil comprising the step of recovering the bio-oil from the cultured cells or media.

본 발명에 있어서, Thraustochytrid계 미세조류는 KRS101(KCTC11686BP)인 것을 특징으로 할 수 있고, 상기 콩분말을 단일 질소원으로 함유하는 배지에서 배양하는 것을 특징으로 할 수 있다.In the present invention, Thraustochytrid microalgae may be characterized in that KRS101 (KCTC11686BP), it may be characterized in that the culture in a medium containing the soy powder as a single nitrogen source.

본 발명에 있어서, 바이오오일은 DHA(docosahexaenoic acid)인 것을 특징으로 할 수 있다.In the present invention, the biooil may be characterized as DHA (docosahexaenoic acid).

본 발명의 일 양태에서, 사용된 미세조류 KRS101은 말레이시아 맹그로브지역의 나뭇잎, 토양 시료에서 채취한 것으로, Thraustochytrid 미세조류 분리용 B1 배지 (1 g/L yeast extract, 1 g/L peptone, 10 g/L agar를 300 mg/L Penicillin G와 500 mg/L streptomycin sulfate를 첨가한 천연해수 1 L에 녹임) 에서 배양하여, 순수분리한 후, Thrasustochytrid 미세조류의 전형적인 특징인 유주자 (zoospore)낭을 형성하는 균주를 분리하여 수득하였다.In one embodiment of the present invention, the microalgae KRS101 used is taken from the leaves and soil samples of the mangrove area of Malaysia, B1 medium (1 g / L yeast extract, 1 g / L peptone, 10 g / for separating Thraustochytrid microalgae) Lagare was dissolved in 1 L of natural seawater with 300 mg / L Penicillin G and 500 mg / L streptomycin sulfate), followed by pure separation to form a zoospore sac, which is typical of Thrasustochytrid microalgae. Strains were obtained by isolation.

상기 KRS101는 균체 성장은 질소원(예를 들면, yeast extract)의 농도가 높을수록 우수하지만, 균체내에 축적되는 바이오오일의 함량은 반대로 질소원(예를 들면, yeast extract)의 농도가 낮을수록 우수하며, 최대 건조균체량의 70%까지 오일의 함량이 증가하였다. The KRS101 cell growth is superior to the concentration of a nitrogen source (for example, yeast extract), but the content of bio oil accumulated in the cell is in contrast to a low concentration of the nitrogen source (for example, yeast extract), The oil content increased up to 70% of the maximum dry cell mass.

본 발명의 일 양태에서, 미세조류 KRS101 균주는 질소원으로 콩분말을 함유한 배지에서 높은 균체성장율을 나타내었으며, DHA를 포함하는 바이오오일의 생산율 또한 높았다.
In one embodiment of the present invention, the microalgal KRS101 strain showed a high cell growth rate in the medium containing soybean powder as a nitrogen source, the production rate of bio-oil containing DHA was also high.

이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.
Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

실시예Example 1 : 신규  1: new DHADHA 함유  contain 유지성Maintainability 미세조류의 분리, 동정 Isolation and Identification of Microalgae

말레이시아 맹그로브지역의 나뭇잎, 토양 시료를 50ml falcon tube를 이용하여 채취하고, 생리식염수 10ml을 가하여 현탁한 후 적당히 희석하여 Thraustochytrid 미세조류 분리용 B1 배지 (1 g/L yeast extract, 1 g/L peptone, 10 g/L agar를 300 mg/L Penicillin G와 500 mg/L streptomycin sulfate를 첨가한 천연해수 1 L에 녹임) (Burja et al. 2006)에 도말하였다. 28℃에서 200rpm으로 2-4일간 배양하여 얻어진 콜로니들을 B1 배지에 재접종하여 순수분리한 후 현미경으로 관찰하여 Thrasustochytrid 미세조류의 전형적인 특징인 유주자 (zoospore)낭을 형성하는 30개의 콜로니를 분리하였다 (도 1).Samples of leaves and soils from the mangrove area of Malaysia were collected using 50ml falcon tube, suspended in 10ml of physiological saline, and diluted appropriately to separate Thraustochytrid microalgae B1 medium (1 g / L yeast extract, 1 g / L peptone, 10 g / L agar is dissolved in 1 L of natural seawater with 300 mg / L Penicillin G and 500 mg / L streptomycin sulfate) (Burja et. al . 2006). Colonies obtained by incubating at 200 rpm for 2-4 days at 28 ° C were re-inoculated in B1 medium and purified after separation. Thirty colonies forming zoospore sacs, typical of Thrasustochytrid microalgae, were isolated ( 1).

분리한 30개의 콜로니들을 50 mL marine broth (Sigma-Aldrich) (250-mL 플라스크)를 이용하여 28℃에서 120rpm으로 3일간 배양한 후, 균체를 회수하여 60℃에서 12시간 동안 감압원심분리기로 건조하였다. 건조된 균체를 5% 메탄올-황산 (methanolic sulfuric acid) 용액 3 mL에 재현탁하여 90℃에서 1시간 동안 반응시킨 후 생성된 지방산에스테르를 0.6 mL의 핵산으로 추출하여 기체크로마토그래피로 분석하였다. The 30 colonies isolated were incubated at 120 rpm at 28 ° C. for 3 days using 50 mL marine broth (Sigma-Aldrich) (250-mL flask), and the cells were recovered and dried at 60 ° C. for 12 hours under reduced pressure centrifuge. It was. The dried cells were resuspended in 3 mL of 5% methanol-sulfuric acid solution and reacted at 90 ° C. for 1 hour, and the resulting fatty acid esters were extracted with 0.6 mL of nucleic acid and analyzed by gas chromatography.

그 결과, 표 1에 나타낸 바와 같이 균체에는 고농도의 고도불화지방산이 함유되어 있는 것으로 나타났으며, 특히 DHA의 함량은 전체 지방산의 49.5%에 달하는 것으로 나타났다. 조사한 30개의 콜로니가 유사한 지방산 조성을 보이는 것으로 확인되었다.
As a result, as shown in Table 1, the cells contained high concentrations of highly fluorinated fatty acids, in particular, the content of DHA was found to reach 49.5% of the total fatty acids. Thirty colonies examined were found to have similar fatty acid compositions.

신규 Thraustochytrid 계열 미세조류 KRS101의 지방산 조성 Fatty Acid Composition of New Thraustochytrid Microalgae KRS101 Fatty acidsFatty acids 14:014: 0 15:015: 0 16:116: 1 16:016: 0 17:017: 0 18:318: 3 18:218: 2 18:118: 1 20:520: 5 22:5
(n6)22: 5
(n6) 22:622: 6 22:5
(n3)22: 5
(n3) MarineMarine -- 17.3917.39 -- 8.898.89 3.13.1 -- 0.930.93 -- 2.182.18 8.128.12 49.5949.59 0.860.86 BasalBasal 2.732.73 14.0914.09 0.430.43 24.6024.60 2.722.72 0.260.26 -- 0.910.91 0.480.48 10.0010.00 39.4939.49 0.350.35

분리한 콜로니의 분자생물학적 동정을 위하여 18S rRNA 유전자 서열을 분석하였다. 하나의 콜로니로부터 전형적인 Phenol-chloroform법으로 염색체 DNA를 분리한 후, 이로부터 Thraustochytrid 미세조류 18S rRNA 유전자 증폭용 프라이머 5'-ATGAACATCAAAAA-3'(P1, 서열번호 2)와 5'-ATGAACATCAAAAA-3'(P2, 서열번호 3) 을 이용하여 PCR법으로 18S rRNA 유전자 DNA를 증폭하였다. PCR 증폭은 EF Taq polymerase(Takara)(2.5 U), polymerase buffer, 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. 형질 전환된 재조합 대장균들로부터 플라스미드 DNA를 추출(Qiagen, USA)하고, 제한효소 EcoRI을 처리하여 원하는 크기의 DNA 단편이 클로닝된 것을 확인하였으며, 염기서열을 결정하였다 (서열번호 1). 염기서열 상동성 분석 결과, Aurantiochytrium sp. BL11 및 Schizochytrium sp. BURABQ 133의 18S rRNA 유전자와 99%의 상동성을 보이는 새로운 Thraustochytrid계열 미세조류로 확인되어 KRS101로 명명하고, 한국생명공학연구원 유전자은행에 2010년 4월 22일자로 기탁번호 KCTC11686BP로 기탁하였다.
The 18S rRNA gene sequence was analyzed for molecular biology of the isolated colonies. Chromosomal DNA was isolated from a single colony using a typical Phenol-chloroform method, and then primers 5'-ATGAACATCAAAAA-3 '(P1, SEQ ID NO: 2) and 5'-ATGAACATCAAAAA-3' for Thraustochytrid microalgae 18S rRNA gene amplification. 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 (Takara) (2.5 U), polymerase buffer, dNTP mixture (1 mM each), 1 μl of each primer (100 pmol), and 500 ng of template DNA. An amplifier (Takara, Japan) was performed 30 times at 96 ° C for 30 seconds, 43 ° C for 1 minute, and 72 ° C for 3 minutes. 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 Escherichia coli (Qiagen, USA) and treated with restriction enzyme Eco RI to confirm that the DNA fragment of the desired size was cloned, and the nucleotide sequence was determined (SEQ ID NO: 1). Sequence homology analysis revealed that Aurantiochytrium sp. BL11 and Schizochytrium sp. A new Thraustochytrid family of microalgae that showed 99% homology with the 18S rRNA gene of BURABQ 133 was identified as KRS101, and was deposited with KCTC11686BP on April 22, 2010 to the Korea Biotechnology Research Institute Gene Bank.

실시예Example 2: 콩 분말을 첨가한 배지에서 신규  2: New in medium supplemented with soybean powder DHADHA 고함유High content ThraustochytridThraustochytrid 미세조류의 배양 Microalgae Cultivation

다양한 농도의 콩 분말가루 (5g/L, 10g/L 및 20 g/L)를 첨가한 기본배지 (포도당 60 g/L, Corn steep solid 5 g/L, Ammonium acetate 5 g/L, KH2PO4 3 g/L, 인공해수염 15 g/L)를 사용하여 신규 DHA 고함유 Thraustochytrid 미세조류 KRS101을 배양하였다. 단일콜로니를 기본배지 15 mL을 이용하여 28℃에서 120 rpm으로 3일간 전배양한 후 배양액 1 mL을 상기의 배지에 각각 접종하여 28℃에서 120 rpm으로 3일간 배양하였다. Basic medium supplemented with various concentrations of soy flour (5 g / L, 10 g / L and 20 g / L) (Glucose 60 g / L, Corn steep solid 5 g / L, Ammonium acetate 5 g / L, KH 2 PO 4 3 g / L, artificial sea salt 15 g / L) was used to incubate the new DHA-rich Thraustochytrid microalgae KRS101. Single colonies were precultured at 120 rpm at 28 ° C. for 3 days using 15 mL of the basic medium, and then 1 mL of the culture solution was inoculated into the medium, followed by incubation at 120 rpm at 28 ° C. for 3 days.

상기 배양액을 원심분리법으로 회수하여 PBS 버퍼 (phosphate buffered saline, pH 7.2)로 3회 세척한 후 60℃에서 12시간 건조하여 건조중량을 측정하였다. 하기의 수정된 Bligh-Dyer법 (Burja et al., 2007)에 의해 오일 중량을 분석하였다. The culture was recovered by centrifugation, washed three times with PBS buffer (phosphate buffered saline, pH 7.2), and dried at 60 ° C. for 12 hours to measure dry weight. The oil weight was analyzed by the following modified Bligh-Dyer method (Burja et al., 2007).

먼저, 건조 중량 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 하였다. 미리 무게를 측정해둔 알루미늄 접시로 클로로포름 층을 조심스럽게 옮긴 후 80℃에서 30분 동안 건조한 후 오일의 무게를 측정하였다. 전체 오일 함량은 아래와 같이 산출하였다.First, 6.25 mL of chloroform, 12.5 mL of methanol, 5 mL of 50 mM K 2 HPO 4 buffer (pH 7.4) was added to 125 mg of dry weight, and the reaction was performed at 28 rpm at 200 rpm for 1 hour, followed by 6.25 mL of chloroform, K 2 HPO 4 6.25 mL of buffer was added and mixed 30 times, and left for 30 minutes to separate the aqueous layer and the organic solvent layer containing the oil. The chloroform layer was carefully transferred to a pre-weighed aluminum dish, dried at 80 ° C. for 30 minutes, and weighed for oil. The total oil content was calculated as follows.

총 오일함량 (%, 오일 g /건조균체량 100 g) = (WL-WD)xVCx100/VPxWS
Total oil content (%, oil g / dry cell weight 100 g) = (W L -W D ) xV C x100 / V P xW S

WL: 알루미늄 접시의 무게W L : weight of aluminum plate

WD: 알루미늄 접시 + 지질의 무게W D : weight of aluminum plate + lipid

VC: Chloroform의 총 부피V C : Total Volume of Chloroform

VP: 알루미늄 접시에 옮긴 Chloroform의 부피V P : Volume of Chloroform transferred to aluminum plate

WS: 사용한 균체의 무게 (건조중량)
W S : Weight of used cells (dry weight)

한편, 오일중에 함유된 DHA의 함량은 기체크로마토그래피법으로 측정하였다. 적당량의 건조된 균체를 5% 메탄올-황산 용액 3 mL에 현탁하여 90℃에서 1시간동안 반응하여 지방산에스테르를 생성한 다음 핵산 0.6 mL로 추출하여 기체크로마토그래피로 분석하였다.On the other hand, the content of DHA contained in the oil was measured by gas chromatography. An appropriate amount of dried cells was suspended in 3 mL of a 5% methanol-sulfuric acid solution to react at 90 ° C. for 1 hour to produce a fatty acid ester, followed by extraction with 0.6 mL of nucleic acid and analyzed by gas chromatography.

콩 분말가루를 첨가한 기본 배지에서 미세조류를 배양한 결과는 표 2에 보인 바와 같으며, 미세조류를 접종하지 않은 대조구의 경우는 표 3에 보인 바와 같다.
The results of culturing the microalgae in the basal medium to which the soy flour was added are shown in Table 2, and the control group not inoculated with the microalgae is shown in Table 3.

콩 분말을 첨가한 배지에서 신규 Thraustochytrid 계열 미세조류 KRS101배양산물의 분석 결과Analysis of New Thraustochytrid Microalgae KRS101 Culture Products in Soybean Powder-Added Medium 콩 분말 (g/L)Soybean Powder (g / L) 건조중량 (g/L)Dry weight (g / L) 오일 함량 (%)Oil content (%) 지방산 조성Fatty acid composition 14:014: 0 15:015: 0 16:016: 0 18:018: 0 18:118: 1 18:218: 2 20:520: 5 22:6
(n6)22: 6
(n6) 22:6
(n3)22: 6
(n3) 22:5
(n3)22: 5
(n3) 00 29.4929.49 22.6622.66 3.753.75 0.900.90 44.1244.12 0.760.76 ndnd 0.330.33 1.101.10 6.176.17 32.3832.38 1.541.54 55 31.2131.21 24.9424.94 3.743.74 0.900.90 42.1242.12 1.361.36 4.974.97 6.256.25 1.011.01 5.765.76 29.8329.83 1.481.48 1010 35.2835.28 25.3225.32 3.583.58 0.830.83 41.3741.37 2.042.04 7.777.77 9.439.43 1.181.18 4.734.73 26.6126.61 ndnd 2020 41.2441.24 27.2227.22 2.892.89 0.630.63 37.7237.72 2.862.86 11.3311.33 13.4713.47 1.061.06 3.833.83 19.4419.44 ndnd

20:5, EPA; 22:6(n6), DPA; 22:6(n3), DHA; 22:5(n3), DPA. nd, not detected
20: 5, EPA; 22: 6 (n 6), DPA; 22: 6 (n 3), DHA; 22: 5 (n 3), DPA. nd, not detected

미세조류 미접종 콩 분말 기본 배지의 성분 분석 결과Component Analysis of Microalgae Uninoculated Soybean Powder Basic Medium 콩 분말 (g/L)Soybean Powder (g / L) 건조중량 (g/L)Dry weight (g / L) 오일 함량 (%)Oil content (%) 지방산 조성Fatty acid composition 14:014: 0 15:015: 0 16:016: 0 18:018: 0 18:118: 1 18:218: 2 20:520: 5 22:6
(n6)22: 6
(n6) 22:6
(n3)22: 6
(n3) 22:5
(n3)22: 5
(n3) 55 2.822.82 25.1025.10 ndnd ndnd 11.2011.20 4.044.04 29.8529.85 54.2554.25 ndnd ndnd ndnd ndnd 1010 5.355.35 24.7624.76 ndnd ndnd 11.5011.50 3.763.76 28.6628.66 55.2855.28 ndnd ndnd ndnd ndnd 2020 12.7012.70 24.1624.16 ndnd ndnd 12:0812:08 3.603.60 25.2125.21 27.6027.60 ndnd ndnd ndnd ndnd

20:5, EPA; 22:6(n6), DPA; 22:6(n3), DHA; 22:5(n3), DPA. nd, not detected 20: 5, EPA; 22: 6 (n 6), DPA; 22: 6 (n 3), DHA; 22: 5 (n 3), DPA. nd, not detected

실시예Example 3:  3: 탄소원으로As a carbon source 콩 분말을 이용한 배지에서 신규  New in Medium with Soy Powder DHADHA 고함유High content Thraustochytrid 미세조류의 배양 Culture of Thraustochytrid Microalgae

실시예 2의 결과를 토대로 기본배지의 탄소원 혹은 질소원을 콩 분말로 대체한 배지에서 KRS101 미세조류를 배양하였다. Based on the results of Example 2, KRS101 microalgae were cultured in a medium in which the carbon source or nitrogen source of the base medium was replaced with soybean powder.

먼저 탄소원인 포도당을 배제한 기본배지에 콩 분말을 각각 5g/L, 10g/L 및 20 g/L을 첨가한 배지에서 신규 DHA 고함유 Thraustochytrid 미세조류 KRS101을 상기와 동일한 방법으로 배양한 후, 건조중량, 오일 함량 및 지방산 조성을 분석하였다. First, the new DHA-rich Thraustochytrid microalgae KRS101 was cultured in a medium containing 5g / L, 10g / L and 20g / L of soybean powder in a basic medium excluding glucose as a carbon source, and then dried. , Oil content and fatty acid composition were analyzed.

그 결과, 표 4에 나타난 바와 같이, KRS101의 균체 성장이 크게 감소하는 것으로 나타나 탄소원으로서 콩 분말은 적합하지 않은 것으로 확인되었다.
As a result, as shown in Table 4, cell growth of KRS101 was found to be greatly reduced, so it was confirmed that soybean powder was not suitable as a carbon source.

탄소원으로 포도당 대신에 콩 분말을 첨가한 배지에서 신규 Thraustochytrid 계열 미세조류 KRS101 배양 산물의 분석 결과Analysis of New Thraustochytrid Microalgae KRS101 Culture Products in Medium Added Soybean Powder Instead of Glucose as a Carbon Source 콩 분말 (g/L)Soybean Powder (g / L) 건조중량 (g/L)Dry weight (g / L) 오일 함량 (%)Oil content (%) 지방산 조성Fatty acid composition 14:014: 0 15:015: 0 16:016: 0 18:018: 0 18:118: 1 18:218: 2 20:520: 5 22:6
(n6)22: 6
(n6) 22:6
(n3)22: 6
(n3) 22:5
(n3)22: 5
(n3) 00 6.206.20 3.963.96 0.940.94 1.211.21 18.3218.32 8:368:36 4.644.64 1.471.47 2.542.54 11.4411.44 47.3447.34 2.412.41 55 8.248.24 3.943.94 3.743.74 0.560.56 16.7216.72 23.7523.75 20.5920.59 4.534.53 1.131.13 5.805.80 21.4621.46 1.161.16 1010 10.3410.34 12.5912.59 ndnd 0.360.36 15.5615.56 22.6622.66 21.1921.19 7.917.91 1.361.36 5.275.27 18.5818.58 1.241.24 2020 16.5416.54 6.816.81 ndnd ndnd 16.3616.36 23.6123.61 22.0422.04 6.176.17 1.741.74 4.844.84 18.7018.70 4.284.28

20:5, EPA; 22:6(n6), DPA; 22:6(n3), DHA; 22:5(n3), DPA. nd, not detected
20: 5, EPA; 22: 6 (n 6), DPA; 22: 6 (n 3), DHA; 22: 5 (n 3), DPA. nd, not detected

실시예Example 4: 질소원으로 콩 분말을 이용한 배지에서 신규  4: New in Medium Using Soybean Powder as Nitrogen Source DHADHA 고함유High content Thraustochytrid 미세조류의 배양 Culture of Thraustochytrid Microalgae

유기 질소원인 Corn steep solid를 배제한 기본배지에 콩 분말을 5, 10, 20 g/L을 첨가한 배지에서 신규 DHA 고함유 Thraustochytrid 미세조류 KRS101을 상기와 동일한 방법으로 배양한 후, 건조중량, 오일 함량 및 지방산 조성을 분석하였다. After culturing the new DHA-rich Thraustochytrid microalgae KRS101 in a medium containing 5, 10, 20 g / L of soybean powder in a basic medium excluding Corn steep solid, an organic nitrogen source, the dry weight and oil content And fatty acid composition.

그 결과, 표 5에 나타난 바와 같이 KRS101는 매우 우수한 균체 성장을 보였으며, 예상대로 콩 분말이 효과적인 질소원으로 활용될 수 있는 것으로 확인되었다.As a result, as shown in Table 5, KRS101 showed very good cell growth, and it was confirmed that the soybean powder could be used as an effective nitrogen source as expected.

표 5는 전체 지방산중에 차지하는 DHA의 함량을 퍼센트로 나타낸 값으로, 즉, 콩분말의 함량이 증가할수록 콩에 포함된 다른 지방산의 농도가 증가하여 DHA의 비율은 차츰 감소하는 것으로 나타났다.
Table 5 shows the percentage of DHA in the total fatty acids. That is, as the content of soybean powder increases, the concentration of other fatty acids in soybeans increases and the proportion of DHA gradually decreases.

질소원으로 콩 분말을 첨가한 배지에서 신규 Thraustochytrid 계열 미세조류 KRS101 배양 산물의 분석 결과Analysis of New Thraustochytrid Microalgae KRS101 Culture Products in Medium Added Soybean Powder as Nitrogen Source 콩 분말 (g/L)Soybean Powder (g / L) 건조중량 (g/L)Dry weight (g / L) 오일 함량 (%)Oil content (%) 지방산 조성Fatty acid composition 14:014: 0 15:015: 0 16:016: 0 18:018: 0 18:118: 1 18:218: 2 20:520: 5 22:6
(n6)22: 6
(n6) 22:6
(n3)22: 6
(n3) 22:5
(n3)22: 5
(n3) 00 2.572.57 6.716.71 1.301.30 4.044.04 31.5131.51 ndnd ndnd 1.401.40 3.243.24 10.9210.92 44.8644.86 1.391.39 55 29.8029.80 27.2127.21 2.012.01 0.460.46 35.3535.35 7.767.76 6.836.83 1.761.76 1.421.42 6.356.35 30.6830.68 1.161.16 1010 32.0232.02 32.2632.26 3.093.09 0.640.64 42.0742.07 9.959.95 8.168.16 2.662.66 0.940.94 4.924.92 24.0324.03 ndnd 2020 42.9942.99 35.2035.20 2.482.48 ndnd 34.0234.02 9.559.55 7.807.80 2.882.88 0.720.72 2.942.94 15.7915.79 ndnd

20:5, EPA; 22:6(n6), DPA; 22:6(n3), DHA; 22:5(n3), DPA. nd, not detected
20: 5, EPA; 22: 6 (n 6), DPA; 22: 6 (n 3), DHA; 22: 5 (n 3), DPA. nd, not detected

한국생명공학연구원Korea Research Institute of Bioscience and Biotechnology KCTC11686BPKCTC11686BP 2010042220100422

<110> Korea Institute Bioscience and Biotechnology <120> Method for Culturing Microalgae Thraustochytrid <130> P10-B125 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 1779 <212> DNA <213> Thraustochytrid KRS101 <400> 1 tacctggttg atcctgccag tagtcatatg ctcgtctcaa agattaagcc atgcatgtgt 60 aagtataagc gattgtactg tgagactgcg aacggctcat tatatcagta ataatttctt 120 cggtagtttc ttttatatgg atacctgcag taattctgga aataatacat gctgtaagag 180 ccctgtatgg ggctgcactt attagattga agccgatttt attggtgaat catgataatt 240 gagcagattg acatttttgt cgatgaatcg tttgagtttc tgccccatca gttgtcgacg 300 gtagtgtatt ggactacggt gactataacg ggtgacggag agttagggct cgactccgga 360 gagggagcct gagagacggc taccatatcc aaggatagca gcaggcgcgt aaattaccca 420 ctgtggactc cacgaggtag tgacgagaaa tatcgatgcg aagcgtgtat gcgttttgct 480 atcggaatga gagcaatgta aaaccctcat cgaggatcaa ctggagggca agtctggtgc 540 cagcagccgc ggtaattcca gctccagaag catatgctaa agttgttgca gttaaaaagc 600 tcgtagttga atttctggca tgggcgaccg gtgctttccc tgaatgggga ttgattgtct 660 gtgttgcctt ggccatcttt ctcatgctgt tattggtatg agatctttca ctgtaatcaa 720 agcagagtgt tccaagcagg tcgtatgacc ggtatgttta ttatgggatg ataagatagg 780 acttgggtgc tattttgttg gtttgcacgc ctgagtaatg gttaatagga acagttgggg 840 gtattcgtat ttaggagcta gaggtgaaat tcttggattt ccgaaagacg aactagagcg 900 aaggcattta ccaagcatgt tttcattaat caagaacgaa agtctgggga tcgaagatga 960 ttagatacca tcgtagtcta gaccgtaaac gatgccgact tgcgattgtt gggtgcttta 1020 ttaatgggcc 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 agtacgtttt taacttctta gagggacatg tccggtttac gggcaggaag 1380 ttcgaggcaa taacaggtct gtgatgccct tagatgttct gggccgcacg cgcgctacac 1440 tgatgggttc atcgggtttt aattctgatt tttggaattg agtgcttggt cggaaggcct 1500 ggctaatcct tggaacgctc atcgtgctgg ggctagattt ttgcaattat taatctccaa 1560 cgaggaattc ctagtaaacg caagtcatca gcttgcattg aatacgtccc tgccctttgt 1620 acacaccgcc cgtcgcacct accgattgaa cggtccgatg aaaccatggg atgtttctgt 1680 ttggattaat ttttggacag aggcagaact cgggtgaatc ttattgttta gaggaaggtg 1740 aagtcgtaac aaggtttccg taggtgaacc tgcggaagg 1779 <210> 2 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 atgaacatca aaaa 14 <210> 3 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 atgaacatca aaaa 14 <110> Korea Institute Bioscience and Biotechnology <120> Method for Culturing Microalgae Thraustochytrid <130> P10-B125 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 1779 <212> DNA <213> Thraustochytrid KRS101 <400> 1 tacctggttg atcctgccag tagtcatatg ctcgtctcaa agattaagcc atgcatgtgt 60 aagtataagc gattgtactg tgagactgcg aacggctcat tatatcagta ataatttctt 120 cggtagtttc ttttatatgg atacctgcag taattctgga aataatacat gctgtaagag 180 ccctgtatgg ggctgcactt attagattga agccgatttt attggtgaat catgataatt 240 gagcagattg acatttttgt cgatgaatcg tttgagtttc tgccccatca gttgtcgacg 300 gtagtgtatt ggactacggt gactataacg ggtgacggag agttagggct cgactccgga 360 gagggagcct gagagacggc taccatatcc aaggatagca gcaggcgcgt aaattaccca 420 ctgtggactc cacgaggtag tgacgagaaa tatcgatgcg aagcgtgtat gcgttttgct 480 atcggaatga gagcaatgta aaaccctcat cgaggatcaa ctggagggca agtctggtgc 540 cagcagccgc ggtaattcca gctccagaag catatgctaa agttgttgca gttaaaaagc 600 tcgtagttga atttctggca tgggcgaccg gtgctttccc tgaatgggga ttgattgtct 660 gtgttgcctt ggccatcttt ctcatgctgt tattggtatg agatctttca ctgtaatcaa 720 agcagagtgt tccaagcagg tcgtatgacc ggtatgttta ttatgggatg ataagatagg 780 acttgggtgc tattttgttg gtttgcacgc ctgagtaatg gttaatagga acagttgggg 840 gtattcgtat ttaggagcta gaggtgaaat tcttggattt ccgaaagacg aactagagcg 900 aaggcattta ccaagcatgt tttcattaat caagaacgaa agtctgggga tcgaagatga 960 ttagatacca tcgtagtcta gaccgtaaac gatgccgact tgcgattgtt gggtgcttta 1020 ttaatgggcc 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 agtacgtttt taacttctta gagggacatg tccggtttac gggcaggaag 1380 ttcgaggcaa taacaggtct gtgatgccct tagatgttct gggccgcacg cgcgctacac 1440 tgatgggttc atcgggtttt aattctgatt tttggaattg agtgcttggt cggaaggcct 1500 ggctaatcct tggaacgctc atcgtgctgg ggctagattt ttgcaattat taatctccaa 1560 cgaggaattc ctagtaaacg caagtcatca gcttgcattg aatacgtccc tgccctttgt 1620 acacaccgcc cgtcgcacct accgattgaa cggtccgatg aaaccatggg atgtttctgt 1680 ttggattaat ttttggacag aggcagaact cgggtgaatc ttattgttta gaggaaggtg 1740 aagtcgtaac aaggtttccg taggtgaacc tgcggaagg 1779 <210> 2 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 atgaacatca aaaa 14 <210> 3 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 atgaacatca aaaa 14

Claims (7)

바이오오일 생성능을 가지는 Thraustochytrid계 미세조류를 콩분말이 함유된 배지에서 배양하는 것을 특징으로 하는 Thraustochytrid계 미세조류의 배양방법.
A method for culturing Thraustochytrid microalgae, characterized in that the culture of Thraustochytrid microalgae having bio-oil generating ability in a medium containing soy powder.
제1항에 있어서, Thraustochytrid계 미세조류는 KRS101(KCTC11686BP)인 것을 특징으로 하는 Thraustochytrid계 미세조류의 배양방법.
The method according to claim 1, wherein the Thraustochytrid microalgae is KRS101 (KCTC11686BP).
제1항에 있어서, 콩분말을 단일 질소원으로 함유하는 배지에서 배양하는 것을 특징으로 하는 Thraustochytrid계 미세조류의 배양방법.
The method of culturing Thraustochytrid microalgae according to claim 1, wherein the soybean powder is cultured in a medium containing a single nitrogen source.
다음 단계를 포함하는 바이오오일의 제조방법:
(a) 바이오오일 생성능을 가지는 Thraustochytrid계 미세조류를 콩분말이 함유된 배지에서 배양하는 단계; 및
(b) 상기 배양된 균체 또는 배지에서 바이오오일을 회수하는 단계.
Biooil manufacturing method comprising the following steps:
(a) culturing the Thraustochytrid microalgae having bio-oil-producing ability in a medium containing soybean powder; And
(b) recovering the biooil from the cultured cells or media.
제4항에 있어서, Thraustochytrid계 미세조류는 KRS101(KCTC11686BP)인 것을 특징으로 하는 바이오오일의 제조방법.
The method according to claim 4, wherein the Thraustochytrid microalgae is KRS101 (KCTC11686BP).
제4항에 있어서, 콩분말을 단일 질소원으로 함유하는 배지에서 배양하는 것을 특징으로 하는 바이오오일의 제조방법.
The method of producing biooil according to claim 4, wherein the soybean powder is cultured in a medium containing a single nitrogen source.
제4항에 있어서, 바이오오일은 DHA(docosahexaenoic acid)인 것을 특징으로 하는 바이오오일의 제조방법.The method of claim 4, wherein the biooil is DHA (docosahexaenoic acid).
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GB2571506A (en) * 2017-11-07 2019-09-04 Strameno Ltd Microorganism
KR20220080593A (en) * 2020-12-07 2022-06-14 씨제이제일제당 (주) Method for preparing biomass comprising protein and omega-3 fatty acids from single microalgae, and the biomass prepared therefrom

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WO2007068997A2 (en) * 2005-12-16 2007-06-21 Avestha Gengraine Technologies Pvt.Ltd. Docosahexaenoic acid (dha) producing thraustochytrid strain - sc1

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Publication number Priority date Publication date Assignee Title
GB2571506A (en) * 2017-11-07 2019-09-04 Strameno Ltd Microorganism
GB2571506B (en) * 2017-11-07 2022-04-06 Strameno Ltd Microorganism
KR20220080593A (en) * 2020-12-07 2022-06-14 씨제이제일제당 (주) Method for preparing biomass comprising protein and omega-3 fatty acids from single microalgae, and the biomass prepared therefrom

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