WO2015160154A1 - Method for increasing production of astaxanthin in haematococcus pluvialis by mature spore inoculation and iron ion-mediated harber-weiss reaction at high temperature - Google Patents

Method for increasing production of astaxanthin in haematococcus pluvialis by mature spore inoculation and iron ion-mediated harber-weiss reaction at high temperature Download PDF

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WO2015160154A1
WO2015160154A1 PCT/KR2015/003668 KR2015003668W WO2015160154A1 WO 2015160154 A1 WO2015160154 A1 WO 2015160154A1 KR 2015003668 W KR2015003668 W KR 2015003668W WO 2015160154 A1 WO2015160154 A1 WO 2015160154A1
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astaxanthin
production
conditions
haematococcus
high temperature
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Korean (ko)
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심상준
홍민의
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고려대학교산학협력단
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Priority to JP2016541591A priority Critical patent/JP6276862B2/en
Priority to CN201580004953.8A priority patent/CN105916993B/en
Publication of WO2015160154A1 publication Critical patent/WO2015160154A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes

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  • the present invention relates to a method for enhancing the production of astaxanthin in haematococcus fluvilis by a two-stage photoculture process, specifically, inoculation of mature spores under high temperature autotrophic conditions and
  • the present invention relates to a method for enhancing the production of astaxanthin in haematococcus fluvialis cells by addition of iron ions.
  • Astaxanthin (3,3'-dihydroxy- ⁇ , ⁇ '-carotene-4,4'-dione), a ketocarotenoid with a red color, is commonly used in chemicals such as beta-carotene.
  • astaxanthins are produced by yeast strains Phaffia rthodozyma and Bervibacterium, and are also widely distributed in marine and freshwater animals, but are extracted from shellfish such as shrimp and crayfish. Tin is low in content and difficult to extract and is not applied.
  • Papia Rodrigo strain has a high growth rate but a low yield of astaxanthin.
  • KR2005-0005341 A a method for producing a high content of astaxanthin by culturing a strain capable of producing astaxanthin and increasing the light irradiation dose to increase the production of astaxanthin by microalgae
  • KR2010-0105193 A discloses a method for producing astaxanthin using radiation, but there is a problem in that the radiation cost cannot be reduced.
  • the most important astaxanthin synthesis promoter of Haematococcus fluvialis to induce astaxanthin production under autotrophic conditions using only carbon dioxide as a carbon source for cell growth and astaxanthin synthesis is powerful light.
  • the sun When the sun is used as a light source, it can provide more economics to the astaxanthin conversion process of carbon dioxide, which is the main cause of greenhouse gases, and astaxanthin synthesis is promoted by strong light, while the microalgal light that absorbs sunlight
  • the rise of the temperature of the medium in the incubator causes a variety of problems, such as accelerated contamination by external bacteria.
  • the present inventors intensively sought to find a method for improving astaxanthin production yield by culturing haematococcus fluvialis in outdoor sunlight, and as a result of inoculating mature spores (cyst) under high temperature autotrophic conditions, iron ions were added.
  • the two-stage photoculture process confirmed that the production of astaxanthin in Haematococcus fluvialis cells was remarkably increased to complete the present invention.
  • the present invention comprises the steps of: (a) inoculating and growing (vegetative growth) of mature spores (cyst) of Haematococcus fluvialis; And (b) inducing the production of astaxanthin in haematococcus fluvialis by irradiating with a luminous intensity of 100-300 ⁇ E / m 2 / s in autotrophic conditions in which nitrogen is deficient and iron ions are added.
  • a luminous intensity of 100-300 ⁇ E / m 2 / s in autotrophic conditions in which nitrogen is deficient and iron ions are added.
  • Provided are methods for enhancing the production of astaxanthin by culturing Haematococcus pluvialis.
  • FIG. 1 is a schematic diagram illustrating an induction process for increasing the astaxanthin production capacity of haematococcus fluvialis via iron ion-mediated Haber-Vice reaction under autotrophic conditions at high temperature (30 ° C., 36 ° C.).
  • FIG. 2 shows the biomass and astaxanthin biosynthesis capacity of haematococcus fluvialis at various temperature conditions (23 ° C, 30 ° C, 36 ° C) and addition of iron ions during astaxanthin synthesis in autotrophic conditions. This is an effect.
  • Figure 4 shows the DCF content, SOD activity and MDA content of haematococcus fluvilis according to the temperature conditions (23 °C, 30 °C, 36 °C) and the addition of iron ions for 18 days astaxanthin synthesis in autotrophic conditions This is a result showing the effect.
  • FIG. 5 shows the astaxanthin of haematococcus fluvilis according to the concentration of Methyl viologen releasing O2- and increasing iron ions (Fe 2+ ) when promoting astaxanthin synthesis under autotrophic conditions at 23 ° C.
  • the results show that the accumulation capacity decreases and, in contrast, the maintenance and increase of astaxanthin accumulation capacity by the metal ion mediated Fenton reaction.
  • Figure 6 is a result of confirming the astaxanthin production of haematococcus fluvialis by adding iron ions to the cells cultured in the green stage at 23 °C in autotrophic conditions of high temperature (23.4-33.5 °C).
  • FIG. 9 shows the increase in the astaxanthin production capacity of haematococcus fluvilis through two-stage (green and red stage) photoculture at outdoor mid- and high temperature (23-28 ° C. and 28-33 ° C.) conditions. It shows the process and result derived.
  • inoculated mature spores and iron ions were added to Haematococcus fluvilis cells in autotrophic conditions.
  • H. pluvialis a microalgae used in the present invention, has the highest accumulation of astaxanthin among living organisms in nature, but has a disadvantage of low astaxanthin productivity under autotrophic conditions using carbon dioxide as the only carbon source.
  • the present invention provides a method for integrating the mature spores of Haematococcus fluvialis by inoculating vegetative growth; And (b) inducing the production of astaxanthin in haematococcus fluvialis by irradiating with a luminous intensity of 100-300 ⁇ E / m 2 / s in autotrophic conditions in which nitrogen is deficient and iron ions are added.
  • the present invention relates to a method for enhancing the production of astaxanthin by culturing Haematococcus pluvialis.
  • the incubation temperature is preferably 25 ⁇ 40 °C, but is not limited thereto.
  • the moderate temp of the present invention is 23-28 °C, high temp is preferably 28-33 °C, but is not limited thereto.
  • the autotrophic condition is preferably supplied with 3 to 4% carbon dioxide as an inorganic carbon source for photosynthesis, but is not limited thereto.
  • the step (a) is preferably irradiated with luminous intensity of less than 35 ⁇ E / m 2 / s, but is not limited thereto.
  • Step (a) of the present invention means “green stage”, wherein the “green stage” is irradiated with luminous intensity of 35 ⁇ E / m 2 / s or less while supplying 3-4% carbon dioxide to autotrophic condition containing nitrogen. It means low stress culture condition.
  • Step (b) of the present invention means “red stage”, and the “red stage” is irradiated with luminous intensity of 100-350 ⁇ E / m 2 / s while supplying 3-4% carbon dioxide to nitrogen-deficient autotrophic conditions. It means high stress culture condition.
  • the iron ion is preferably at least one selected from the group consisting of Fe 2 SO 4 , FeCl 2 , FeCl 3 and Fe 2 (SO 4 ) 3 , but is not limited thereto.
  • the concentration of the iron ion is preferably 40-80 ⁇ M, but is not limited thereto.
  • the iron ion is preferably added in a molar ratio of 100-600 to the active oxygen O 2 - and H 2 O 2 content, but is not limited thereto.
  • the content of astaxanthin may be characterized in that by the conversion of active oxygen O 2 - and H 2 O 2 in Haematococcus fluvialis cells into active oxygen O 2 and OH. .
  • microalgae when exposed to high temperature environments, leak thylakoids in the chloroplasts responsible for photosynthesis and, in severe cases, disintegration. Subsequently, the oxygen uptake rate of the thylakoid is amplified, and electrons that are not reduced through the Calvin cycle are combined with oxygen by Mehler Reaction (O 2 uptake + electron ⁇ O 2 ⁇ ) to form O 2 ⁇ . It is produced in large quantities in thylakoids.
  • the cells are O 2 excess production - and thereby express the SOD (superoxide dismutase) to protect the cell components from the produced O 2 - is converted to H 2 O 2 by the in-SOD (superoxide dismutase) chloroplasts.
  • SOD superoxide dismutase
  • H 2 O 2 is O 2 - because the membrane permeation is easy through the diffusion contrast to H 2 O 2 derived from the chloroplast engine is to pass the cellular oxidation-related signals to other organelles such as nucleic acids, mitochondria, vacuoles, wherein Caro Martino it is possible to Genesis directly disabled by a sensitive enzyme genes involved in the H 2 O 2 of (carotenogenesis) is consequently hippocampus Toko kusu flat ruby Alice astaxanthin synthesis is inhibited.
  • haematococcus fluvialis contains carotenoids containing astaxanthin in lipid vesicles (globules) outside the plastids due to stress reactions.
  • H 2 O 2 is O 2 - than the diffusion membrane permeable to the iron ions easily mediated Fenton reaction (Fe 2+ + H 2 O 2 ⁇ Fe 3+ + OH ⁇ ) is also generated, because, unlike with enough outside the plastid do. Therefore, it is likely that Haematococcus cells promoted astaxanthin synthesis in order to protect themselves from excessive lipid oxidation due to excessive production of OH ⁇ through iron ion mediated Haber-Weiss reaction.
  • O 2 , OH ⁇ was reported to be more reactive than O 2 ⁇ , H 2 O 2 .
  • the high-temperature culture conditions (30 - 40 °C) an intracellular large amount generated by the LROS (O 2 -, H 2 O 2) O ( an iron ion mediated by inducing the Haber-Weiss reaction largely diverted from MROS 2, OH ⁇ ) Is likely to oxidize various cellular components, including PUFA (unsaturated fatty acids), resulting in increased MDA content.
  • FeSO 4 FeSO 4 further cell LROS (O 2 -, H 2 O 2) can be accelerated through the modulation of, the iron unregulated on and LROS content (ratio control) self to the astaxanthin of the hippocampus Toko kusu flat ruby Alice cell production in a nutrient conditions It must be a very important factor, and thus it is possible to closely control the H 2 O 2 content of O 2 ⁇ in the cell.
  • the iron ion is the active oxygen O 2 - and H 2 O preferably added at 100 to 600.
  • the molar ratio for the second content, and, out of the range radicals O 2 - and H 2 O 2 If it is added in less than 100 molar ratio with respect to the content, there may be a problem that the astaxanthin accumulation efficiency is lowered, and when added in excess of 600 molar ratio with respect to the active oxygen O 2 - and H 2 O 2 content, side reactions occur There may be a problem that taraxanthin synthesis is inhibited.
  • the content of astaxanthin may be characterized in that the active oxygen O 2 - and H 2 O 2 in the Haematococcus fluvialis cells are enhanced by conversion to the active oxygen O 2 and OH. .
  • 'autotrophic condition' of the present invention refers to a state of a medium in which a plant ingests minerals from the outside of the body as a nutrient and synthesizes them as organic matter, and is also referred to as a 'self-nourishing condition', and is generally a self-nourishing condition.
  • Carbon dioxide is supplied as an inorganic carbon source for photosynthesis, and the medium composition is Ca (NO 3 ) 2 or CaCl 2 .2H 2 O, KNO 3 or KCl, Na 2 Glycerophosphate.5H 2 O, MgSO 4 .7H 2 O, Tris-aminomethane, Thiamine, Biotin, Vitamin B12, PIV metal solution, Na 2 EDTA, FeCl 3 ⁇ 6H 2 O, MnCl 2 ⁇ 4H 2 O, ZnSO 4 ⁇ 7H 2 O, CoCl 2 ⁇ 6H 2 O and Na 2 MoO It may include 4 ⁇ 2H 2 O, but is not limited thereto.
  • the strain used in the present invention was purchased from National Institute for Environmental Studies, Tsukuba, Japan as Haematococcus pluvialis NIES-144.
  • the types of medium used in this example were both NIES-C medium and NIES-N medium, and their components are shown in Table 1 below.
  • NIES-C medium autotrophic medium (autotrophic medium, purpose: growth)
  • -NIES-N medium autotrophic medium (autotrophic medium, purpose: growth inhibition, photoinduction, astaxanthin production)
  • MV methyl viologen: 10-11M, 10-9M, 10-7M (artificial O2-generator)
  • H2DCFDA carboxy-2 ', 7'-dichlorofluorescein diacetate
  • H2DCFDA is oxidized by active oxygen (O2-, H2O2)
  • SOD Superoxide dismuase
  • MDA malondialdehyde
  • PUFAs polyunsaturated fatty acids
  • It is a kind of oxidative indicator that can indirectly check the degree of oxidation of cells through the amount of intracellular MDA.
  • Example 1 Effects of Various High Temperature Culture Conditions and Iron Ion-Mediated Haber-Weiss Reaction on Biomass and Astaxanthin Accumulation in Haematococcus fluvialis Under Autotrophic Conditions
  • NIES-C medium lacking organic carbon sources
  • Biomass increase and astaxanthin accumulation were analyzed during 18 days of culture by dividing FeSO 4 (450 ⁇ M) with or without the substance that induces the mediated Haber-Weiss reaction.
  • the biomass production capacity of Haemacococcus fluvialis due to the increase in temperature in autotrophic conditions is confirmed to decrease by 30% at 30 °C, 57% at 36 °C compared to 23 °C
  • the incubation temperature is 30 °C and Fe 2+ is added to induce the Haber Weiss reaction
  • the biomass production capacity of Haemacococcus fluvialis does not add Fe 2+
  • the culture temperature is 23 °C It was confirmed that it increased by 9% compared with the case, and it was confirmed that 41% increased compared with the case where the incubation temperature was 30 ° C without adding Fe 2+ .
  • the culture temperature is 36 °C
  • Fe 2+ is added to induce the Haber-Weiss reaction
  • the biomass production capacity of Haemacococcus fluvialis does not add Fe 2+
  • the culture temperature is 23 °C It was confirmed that it increased by 3% compared with the case, and it was confirmed that 77% increased compared to the case where the culture temperature is 30 °C without adding Fe 2+ .
  • the astaxanthin production capacity of Haematococcus fluvialis due to the increase in temperature under autotrophic conditions was 23% at 30 ° C and 36 ° C compared to the case where the culture temperature was 23 ° C. While the incubation temperature was 30 ° C. and Fe 2+ was added to induce the Haber Weiss reaction, the astaxanthin production capacity of haematococcus fluvialis was increased by Fe 2+ . In addition, it was confirmed that the culture temperature increased by 17% compared with the case of 23 °C, it was confirmed that 66% increased compared to the case of the culture temperature of 30 °C without adding Fe 2+ .
  • astaxanthin synthesis was carried out for 2 days and confirmed by optical micrographs showing the astaxanthin accumulation of haematococcus cells. As shown in FIG. It was found that it was inadequate for astaxanthin production of ruby lice and that the introduction of Haber-Weiss reaction at high temperature was effective in maintaining the astaxanthin production ability of Haemacococcus fluvialis.
  • Example 2 Various high-temperature culture conditions and iron ion-mediated Haber-Weiss reactions in autotrophic conditions on the free radicals, SOD activity, lipid oxidation and carotenoid content in the early cell of astaxanthin synthesis Impact
  • iron ions at normal room temperature (23 ° C) and high temperature culture conditions (30 ° C, 36 ° C) and with each temperature Astaxanthin synthesis conditions were established by dividing with or without FeSO 4 (450 ⁇ M), a substance that induces a mediated Haber-Weiss reaction, and then, during the two days of culture, active oxygen content (DCF content), SOD activity, and lipid oxidation The degree (MDA content) and the degree of carotenoid accumulation were analyzed.
  • A H 2 O 2 active oxygen content - - O 2 is, but rapidly increased in the condition (30 °C, 36 °C) of high-temperature, high-temperature culture conditions, as a result, FIG. 4, as shown in (A), DCF content When FeSO 4 (450 ⁇ M) was added to induce the Haber-Weiss reaction, the active oxygen content decreased drastically.
  • the MDA (malodialdhyde) content was elevated in direct proportion to the DCF content (O 2 ⁇ , H 2 O 2 ) under high temperature conditions (30 ° C., 36 ° C.), but at high temperature culture. It was confirmed that the addition of FeSO 4 (450 ⁇ M) to the conditions (30 ° C., 36 ° C.) induced the Haber-Weiss reaction to increase the MDA content.
  • Inhibits proliferation artificially generates O 2 ⁇ by intensity by adding Methyl viologen at various concentrations (10-11 to 10-7 M) with strong light (150 ⁇ E / m 2 / s), and at room temperature Astaxanthin in the cells on day 4 after the astaxanthin synthesis conditions were established by adding FeSO 4 (450 ⁇ M), which is an iron-induced Haber-Weiss reaction, to and without the addition of FeSO 4 (450 ⁇ M). The degree of accumulation was analyzed.
  • Example 4-1 the astaxanthin production of hematococcus cells was investigated through inoculation of mature red spores in the green stage of the high temperature autotrophic condition in summer and the addition of iron ions in the red stage. .
  • Green stage culture was performed for 15 days, and red stage culture was performed for 63 days after addition of iron ions.
  • the astaxanthin production (mg / L / day) of hematococcus cells (mg / L / day) was 2.24 mg / L / day in spring and mid-temperature conditions due to the inoculation of mature red cyst and the addition of 50 ⁇ M of iron ion. In the high temperature culture conditions, it was found to increase by 147% to 3.29 mg / L / day (FIG. 8).
  • a two-stage photoculture process in which iron ions are added after inoculating mature spores (cyst) in the production of astaxanthin using the sun as an autotrophic condition during outdoor culture is generated in a large amount at high temperature conditions.

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Abstract

The present invention relates to a method for increasing the production of astaxanthin in Haematococcus pluvialis by a two-stage light cultivation process and, more specifically, to a method for increasing the production of astaxanthin in Haematococcus pluvialis cells by inoculating mature spores (cyst) and adding iron ions under high-temperature autotrophic conditions. The two-stage light cultivation process for the production of astaxanthin under autotrophic conditions using the sun during outdoor cultivation comprising inoculating mature spore (cyst) cells and adding iron ions, according to the present invention, can solve the problem that synthesis of astaxanthin is inhibited by excess generation of LROS(O2 -, H2O2) by effectively converting LROS(O2 -, H2O2) that is generated in a large amount under high-temperature conditions to MROS(O2, OH·) and amplifying an intracellular lipid oxidation signal, thereby more economically increasing the production of astaxanthin. Such astaxanthin is useful in a variety of industrial fields as a potent antioxidant.

Description

고온에서 성숙 포자 접종 및 철이온매개 하버-바이스 반응에 의한 해마토코쿠스 플루비알리스 내 아스타잔틴 생산량 증진 방법 Method for Enhancing Astaxanthin Production in Haematococcus fluvialis by Mature Spore Inoculation and Iron Ion-Mediated Harbor-Vice Reaction
본 발명은 이단(two-stage) 광배양 공정으로 해마토코쿠스 플루비알리스 내 아스타잔틴의 생산량을 증진시키는 방법에 관한 것으로서, 구체적으로는 고온의 자가영양조건에서 성숙 포자(cyst)의 접종 및 철이온 첨가로 해마토코쿠스 플루비알리스 세포 내 아스타잔틴의 생산량을 증진시키는 방법에 관한 것이다.The present invention relates to a method for enhancing the production of astaxanthin in haematococcus fluvilis by a two-stage photoculture process, specifically, inoculation of mature spores under high temperature autotrophic conditions and The present invention relates to a method for enhancing the production of astaxanthin in haematococcus fluvialis cells by addition of iron ions.
일반적으로 적색을 띄는 케토카로티노이드(Ketocarotenoid)인 아스타잔틴(Astaxanthin, 3,3'-dihydroxy-β, β'-carotene-4,4'-dione)은 베타-카로틴(β-carotene)과 같은 화학적 구조를 가진 카로티노이드계 색소의 일종으로, 유해 활성산소를 없애는 항산화 기능성 물질로 베타-카로틴에 비해 양쪽 말단기에 하이드록실기(-OH)와 케톤기(=O)를 하나씩 더 가지는 독특한 분자 구조적 특성 때문에 기존의 항산화 물질보다 월등히 높은 항산화 활성을 갖는다. 아스타잔틴은 대표적인 항산화제인 비타민 E보다 500배, 베타-카로틴보다 20배 정도 높은 항산화 활성을 지닌다. 이러한 높은 항산화 활성기능으로 인해 아스타잔틴은 의약품, 식품 첨가제 및 동물과 치어의 사료 첨가제로 널리 사용되고 있고, 그 수요량 및 활용 범위가 급격히 확대될 것으로 예상되고 있다.Astaxanthin (3,3'-dihydroxy-β, β'-carotene-4,4'-dione), a ketocarotenoid with a red color, is commonly used in chemicals such as beta-carotene. A type of carotenoid pigment with a structure, which is an antioxidant functional substance that eliminates harmful free radicals, and has a unique molecular structural characteristic that has one hydroxyl group (-OH) and one ketone group (= O) at both terminal groups as compared to beta-carotene. Because of this, it has much higher antioxidant activity than conventional antioxidants. Astaxanthin has 500 times higher antioxidant activity than vitamin E, a typical antioxidant, and 20 times higher than beta-carotene. Due to its high antioxidant activity, astaxanthin is widely used as a pharmaceutical, food additive, and feed additive for animals and fry, and its demand and application range are expected to expand rapidly.
이러한 아스타잔틴은 효모 균주인 파피아 로드지마(Phaffia rthodozyma)와 버비박테리아(Bervibacterium)에서 생성되며, 또한, 해양 동물과 담수 동물에 많이 분포되어 있지만, 새우나 가재 등의 갑각류에서 추출된 아스타잔틴은 함량이 적고, 추출 과정이 어려워 적용되지 않고 있으며, 파피아 로드지마 균주는 성장률이 높으나 아스타잔틴의 수율이 낮다는 문제점을 가지고 있다.These astaxanthins are produced by yeast strains Phaffia rthodozyma and Bervibacterium, and are also widely distributed in marine and freshwater animals, but are extracted from shellfish such as shrimp and crayfish. Tin is low in content and difficult to extract and is not applied. Papia Rodrigo strain has a high growth rate but a low yield of astaxanthin.
이에, 지구상에서 아스타잔틴의 축적함량과 수율측면에서 가장 우수한 미세조류인 해마토코쿠스 플루비알리스(Haematococcus pluvialis)를 사용하여 빛에 의한 이산화탄소의 고정화와 동시에 아스타잔틴의 생산성을 높이기 위한 다양한 연구가 수행되고 있다.Therefore, various studies have been made to increase the productivity of astaxanthin while immobilizing carbon dioxide by light using Haematococcus pluvialis, the best microalgae in terms of accumulation and yield of astaxanthin on the planet. Is being performed.
종래 KR2005-0005341 A에서는 아스타잔틴을 생산할 수 있는 균주를 배양시켜 이러한 균주를 이용하여 고함량의 아스타잔틴을 생산하기 위한 방법으로 광 조사량을 증가시켜서 미세조류에 의한 아스타잔틴의 생산량을 증가시키기 위한 연구가 이루어졌지만, 이 경우 특별한 형태의 반응기가 필요하고 높은 광 조사량을 위한 에너지 비용이 크다는 단점이 있다. 또한, KR2010-0105193 A에서는 방사선 조사를 이용하여 아스타잔틴의 생산방법을 개시하고 있으나, 방사선 조사 비용을 절감할 수 없는 문제점이 있다.In the conventional KR2005-0005341 A, a method for producing a high content of astaxanthin by culturing a strain capable of producing astaxanthin and increasing the light irradiation dose to increase the production of astaxanthin by microalgae Although studies have been made for this purpose, there are disadvantages in that a special type of reactor is required and the energy cost for high light irradiation is high. In addition, KR2010-0105193 A discloses a method for producing astaxanthin using radiation, but there is a problem in that the radiation cost cannot be reduced.
한편, 세포 성장 및 아스타잔틴 합성에 필요한 탄소원으로 오로지 이산화탄소만을 이용하는 자가영양조건에서 아스타잔틴 생산 유도를 위한 해마토코쿠스 플루비알리스의 가장 핵심적인 아스타잔틴 합성 촉진 인자는 바로 강력한 빛인데, 광원으로 태양을 활용했을 경우, 온실가스의 주범인 이산화탄소의 아스타잔틴 전환공정에 보다 경제성을 부여할 수 있고, 강력한 빛에 의해 아스타잔틴 합성이 촉진되는 반면, 태양광을 흡수하는 미세조류 광배양기 내 배지의 온도 상승에 따라 외부 박테리아에 의한 오염이 가속화되는 등의 다양한 문제점들이 야기된다.On the other hand, the most important astaxanthin synthesis promoter of Haematococcus fluvialis to induce astaxanthin production under autotrophic conditions using only carbon dioxide as a carbon source for cell growth and astaxanthin synthesis is powerful light. When the sun is used as a light source, it can provide more economics to the astaxanthin conversion process of carbon dioxide, which is the main cause of greenhouse gases, and astaxanthin synthesis is promoted by strong light, while the microalgal light that absorbs sunlight The rise of the temperature of the medium in the incubator causes a variety of problems, such as accelerated contamination by external bacteria.
따라서, 아스타잔틴 축적 능력이 우수한 해마토코쿠스 플루비알리스를 옥외에서 태양을 활용하여 산업적으로 이산화탄소 전환 공정에 이용하기 위해서는 고온의 자가영양조건에서의 느린 아스타잔틴 생산성 문제를 해결하는 것이 무엇보다 시급히 필요한 실정이다.Therefore, in order to use the haematococcus fluvialis, which has a high astaxanthin accumulation ability, in the outdoors using the sun for the industrial carbon dioxide conversion process, it is more important to solve the slow astaxanthin productivity problem in the high temperature autotrophic condition. It is urgently needed.
이에, 본 발명자들은 해마토코쿠스 플루비알리스를 옥외 태양광에서 배양하여 아스타잔틴 생산수율 향샹 방법을 찾고자 예의 노력한 결과, 고온의 자가영양조건에서 성숙 포자(cyst)를 접종한 후 철이온을 첨가하는 이단(two-stage) 광배양 공정으로 해마토코쿠스 플루비알리스 세포 내 아스타잔틴의 생산량이 현저히 증가하는 것을 확인하고 본 발명을 완성하게 되었다. Therefore, the present inventors intensively sought to find a method for improving astaxanthin production yield by culturing haematococcus fluvialis in outdoor sunlight, and as a result of inoculating mature spores (cyst) under high temperature autotrophic conditions, iron ions were added The two-stage photoculture process confirmed that the production of astaxanthin in Haematococcus fluvialis cells was remarkably increased to complete the present invention.
발명의 요약Summary of the Invention
본 발명의 목적은, (a) 해마토코쿠스 플루비알리스의 성숙 포자(cyst)를 접종하여 증식(vegetative growth)시키는 단계; 및 (b) 질소가 결핍되고 철이온이 첨가된 자가영양조건에서 100-300μE/m2/s의 광도를 조사하여 해마토코쿠스 플루비알리스 내 아스타잔틴의 생성을 유도하는 단계를 포함하는 해마토코쿠스 플루비알리스(Haematococcus pluvialis)의 배양에 의한 아스타잔틴의 생산량을 증진시키는 방법을 제공하는데 있다.It is an object of the present invention, (a) inoculating mature growth spores (cyst) of Haematococcus fluvialis (vegetative growth); And (b) inducing the production of astaxanthin in haematococcus fluvialis by irradiating with a luminous intensity of 100-300 μE / m 2 / s in autotrophic conditions in which nitrogen is deficient and iron ions are added. It is to provide a method for enhancing the production of astaxanthin by culturing Haematococcus pluvialis.
상기 목적을 달성하기 위하여, 본 발명은 (a) 해마토코쿠스 플루비알리스의 성숙 포자(cyst)를 접종하여 증식(vegetative growth)시키는 단계; 및 (b) 질소가 결핍되고 철이온이 첨가된 자가영양조건에서 100-300μE/m2/s의 광도를 조사하여 해마토코쿠스 플루비알리스 내 아스타잔틴의 생성을 유도하는 단계를 포함하는 해마토코쿠스 플루비알리스(Haematococcus pluvialis)의 배양에 의한 아스타잔틴의 생산량을 증진시키는 방법을 제공한다.In order to achieve the above object, the present invention comprises the steps of: (a) inoculating and growing (vegetative growth) of mature spores (cyst) of Haematococcus fluvialis; And (b) inducing the production of astaxanthin in haematococcus fluvialis by irradiating with a luminous intensity of 100-300 μE / m 2 / s in autotrophic conditions in which nitrogen is deficient and iron ions are added. Provided are methods for enhancing the production of astaxanthin by culturing Haematococcus pluvialis.
도 1은 고온(30℃, 36℃)의 자가영양조건에서 철이온 매개 하버-바이스 반응을 통한 해마토코쿠스 플루비알리스의 아스타잔틴 생산 능력 증대를 위한 유도과정을 도식화한 개략도이다.1 is a schematic diagram illustrating an induction process for increasing the astaxanthin production capacity of haematococcus fluvialis via iron ion-mediated Haber-Vice reaction under autotrophic conditions at high temperature (30 ° C., 36 ° C.).
도 2는 자가영양조건에서 아스타잔틴 합성 동안 각각의 온도 조건(23 ℃, 30 ℃, 36 ℃)과 철이온의 첨가에 따른 해마토코쿠스 플루비알리스의 바이오매스 및 아스타잔틴 생합성 능력에 미치는 영향을 나타내는 결과이다.FIG. 2 shows the biomass and astaxanthin biosynthesis capacity of haematococcus fluvialis at various temperature conditions (23 ° C, 30 ° C, 36 ° C) and addition of iron ions during astaxanthin synthesis in autotrophic conditions. This is an effect.
도 3은 자가영양조건에서 아스타잔틴 합성 18일째 각각의 온도 조건(23 ℃, 30 ℃, 36 ℃)과 철이온의 첨가에 따른 해마토코쿠스 플루비알리스의 아스타잔틴 축적 정도를 나타낸 광학현미경 이미지 결과이다. (Scale bars = 40 μm)FIG. 3 is an optical microscope showing the accumulation of astaxanthin of haematococcus fluvialis on the 18th day of astaxanthin synthesis under autotrophic conditions and the addition of iron ions The image results. (Scale bars = 40 μm)
도 4는 자가영양조건에서 아스타잔틴 합성 18일 동안 각각의 온도 조건(23 ℃, 30 ℃, 36 ℃)과 철이온의 첨가에 따른 해마토코쿠스 플루비알리스의 DCF 함량, SOD 활성 및 MDA 함량에 미치는 영향을 나타내는 결과이다. Figure 4 shows the DCF content, SOD activity and MDA content of haematococcus fluvilis according to the temperature conditions (23 ℃, 30 ℃, 36 ℃) and the addition of iron ions for 18 days astaxanthin synthesis in autotrophic conditions This is a result showing the effect.
도 5는 23 ℃ 온도의 자가영양조건에서 아스타잔틴 합성 촉진시, O2-를 인위적으로 방출하는 Methyl viologen의 농도와 철이온(Fe2+) 증가에 따른 해마토코쿠스 플루비알리스의 아스타잔틴 축적 능력의 감소 및 이와 대조적으로 금속 이온매개 Fenton 반응에 의한 아스타잔틴 축적능력의 유지 및 증가 정도를 나타내는 결과이다. FIG. 5 shows the astaxanthin of haematococcus fluvilis according to the concentration of Methyl viologen releasing O2- and increasing iron ions (Fe 2+ ) when promoting astaxanthin synthesis under autotrophic conditions at 23 ° C. The results show that the accumulation capacity decreases and, in contrast, the maintenance and increase of astaxanthin accumulation capacity by the metal ion mediated Fenton reaction.
도 6은 23 ℃에서 그린 스테이지로 배양된 세포를 고온 (23.4 - 33.5 ℃)의 자가영양조건에서 철이온을 첨가하여 해마토코쿠스 플루비알리스의 아스타잔틴 생산을 확인한 결과이다.Figure 6 is a result of confirming the astaxanthin production of haematococcus fluvialis by adding iron ions to the cells cultured in the green stage at 23 ℃ in autotrophic conditions of high temperature (23.4-33.5 ℃).
도 7은 고온 (23 ℃, 30 ℃, 33 ℃) 조건에서 성숙 포자(red cyst) 접종을 통한 그린 스테이지에서 해마토코쿠스 플루비알리스의 vegetative growth를 확인한 결과이다.7 is a result of confirming the vegetative growth of haematococcus fluvialis at the green stage through the inoculation of mature red spores at high temperature (23 ℃, 30 ℃, 33 ℃) conditions.
도 8은 여름철 고온의 자가영양조건에서 그린 스테이지로 15일간 배양 후 철이온을 첨가하여 레드 스테이지로 63일간 배양으로 해마토코쿠스 플루비알리스의 아스타잔틴 생산량 증가를 확인한 결과이다.8 is a result of confirming the increase in the astaxanthin production of haematococcus fluvialis by incubating for 63 days in the red stage by adding iron ions after 15 days incubation in the green stage under high temperature autotrophic conditions in summer.
도 9는 옥외의 중온 및 고온(23-28℃ 및 28-33℃) 조건에서, 이단(two-stage: 그린 및 레드 스테이지) 광배양을 통한 해마토코쿠스 플루비알리스의 아스타잔틴 생산 능력 증대를 유도한 과정 및 결과를 나타낸 것이다.FIG. 9 shows the increase in the astaxanthin production capacity of haematococcus fluvilis through two-stage (green and red stage) photoculture at outdoor mid- and high temperature (23-28 ° C. and 28-33 ° C.) conditions. It shows the process and result derived.
발명의 상세한 설명 및 바람직한 구현예Detailed Description of the Invention and Preferred Embodiments
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술 분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로, 본 명세서에서 사용된 명명법은 본 기술 분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.
본 발명에서는 고온의 자가영양조건에서 H.pluvialis의 생산량을 증가시켜 아스타잔틴을 대량 생산하기 위하여, 성숙 포자(cyst)를 접종하고 철이온이 첨가된 자가영양조건의 해마토코쿠스 플루비알리스 세포에 100-300μE/m2/s의 광도로 조사함으로써, 해마토코쿠스 플루비알리스 내 아스타잔틴의 생성을 유도하였다.In the present invention, in order to increase the production of H.pluvialis under high temperature autotrophic conditions, to produce astaxanthin in a large amount, inoculated mature spores and iron ions were added to Haematococcus fluvilis cells in autotrophic conditions. Was irradiated at a light intensity of 100-300 μE / m 2 / s to induce the production of astaxanthin in haematococcus fluvilis.
본 발명에서 사용한 미세 조류인 H. pluvialis는 자연계에 존재하는 생물체 중 아스타잔틴의 축적량이 가장 높으나, 이산화탄소를 유일한 탄소원으로 사용하는 자가영양조건에서는 아스타잔틴 생산성이 낮은 단점이 있다. H. pluvialis , a microalgae used in the present invention, has the highest accumulation of astaxanthin among living organisms in nature, but has a disadvantage of low astaxanthin productivity under autotrophic conditions using carbon dioxide as the only carbon source.
특히, 한국과 같은 여름철 태양은 강한 빛과 더불어 높은 온도(30 - 40℃)를 동반하게 되는데, 이러한 고온의 자가영양조건에서 해마토코쿠스 플루비알리스 세포에서 아스타잔틴 합성 시 아스타잔틴 생합성 능력이 현저히 감소하는 현상이 일어난다. 아스타잔틴 농도 및 생산성의 지속적인 감소로 세포가 극심한 환경적 스트레스에 견디지 못하고 결국 세포의 사멸이 초래되며, 고온(30 - 40℃)에서 생육이 활발한 박테리아에 의한 오염은 이런 문제를 더욱 심각하게 한다.In particular, the summer sun like Korea is accompanied by strong light and high temperature (30-40 ℃), astaxanthin biosynthesis capacity during astaxanthin synthesis in Haematococcus fluvialis cells under high temperature autotrophic conditions This markedly decreasing phenomenon occurs. Continued decreases in astaxanthin concentration and productivity cause the cells to withstand extreme environmental stresses and eventually lead to cell death, and contamination by bacteria that grow viable at high temperatures (30-40 ° C) exacerbate this problem. .
따라서, 본 발명은 일관점에서 (a) 해마토코쿠스 플루비알리스의 성숙 포자(cyst)를 접종하여 증식(vegetative growth) 시키는 단계; 및 (b) 질소가 결핍되고 철이온이 첨가된 자가영양조건에서 100-300μE/m2/s의 광도를 조사하여 해마토코쿠스 플루비알리스 내 아스타잔틴의 생성을 유도하는 단계를 포함하는 해마토코쿠스 플루비알리스(Haematococcus pluvialis)의 배양에 의한 아스타잔틴의 생산량을 증진시키는 방법에 관한 것이다.Accordingly, the present invention provides a method for integrating the mature spores of Haematococcus fluvialis by inoculating vegetative growth; And (b) inducing the production of astaxanthin in haematococcus fluvialis by irradiating with a luminous intensity of 100-300 μE / m 2 / s in autotrophic conditions in which nitrogen is deficient and iron ions are added. The present invention relates to a method for enhancing the production of astaxanthin by culturing Haematococcus pluvialis.
본 발명에 있어서, 상기 배양온도는 25 ~ 40℃ 인 것이 바람직하나, 이에 한정되는 것은 아니다. In the present invention, the incubation temperature is preferably 25 ~ 40 ℃, but is not limited thereto.
일반적인 봄의 중온 조건은 17.5 - 27.7℃이며, 여름의 고온조건은 23.4 ? 33.5℃이다. 따라서, 본 발명의 moderate temp는 23 - 28℃이며, high temp는 28 - 33℃인 것이 바람직하나, 이에 한정되는 것은 아니다.Typical mid-temperature conditions for spring are 17.5-27.7 ° C, while high-temperature conditions for summer are 23.4? 33.5 ° C. Therefore, the moderate temp of the present invention is 23-28 ℃, high temp is preferably 28-33 ℃, but is not limited thereto.
본 발명에 있어서, 상기 자가영양조건은 광합성을 위한 무기 탄소원으로 3~4% 이산화탄소를 공급하는 것이 바람직하나, 이에 한정되는 것은 아니다.In the present invention, the autotrophic condition is preferably supplied with 3 to 4% carbon dioxide as an inorganic carbon source for photosynthesis, but is not limited thereto.
본 발명에 있어서, 상기 (a) 단계는 35μE/m2/s 이하의 광도를 조사하는 것이 바람직하나, 이에 한정되는 것은 아니다.In the present invention, the step (a) is preferably irradiated with luminous intensity of less than 35μE / m 2 / s, but is not limited thereto.
본 발명의 (a) 단계는“그린 스테이지”를 의미하며, 상기 “그린 스테이지”는 질소가 포함된 자가영양조건에 3~4% 이산화탄소를 공급하면서 35μE/m2/s 이하의 광도를 조사하는 낮은 스트레스의 배양조건을 의미한다.Step (a) of the present invention means “green stage”, wherein the “green stage” is irradiated with luminous intensity of 35 μE / m 2 / s or less while supplying 3-4% carbon dioxide to autotrophic condition containing nitrogen. It means low stress culture condition.
본 발명의 (b) 단계는 “레드 스테이지”를 의미하며, 상기 “레드 스테이지”는 질소가 결핍된 자가영양조건에 3~4% 이산화탄소를 공급하면서 100-350μE/m2/s의 광도를 조사하는 높은 스트레스의 배양조건을 의미한다.Step (b) of the present invention means “red stage”, and the “red stage” is irradiated with luminous intensity of 100-350 μE / m 2 / s while supplying 3-4% carbon dioxide to nitrogen-deficient autotrophic conditions. It means high stress culture condition.
본 발명에 있어서, 상기 철이온은 Fe2SO4, FeCl2, FeCl3 및 Fe2(SO4)3로 이루어지는 군으로부터 선택되는 1종 이상인 것이 바람직하나, 이에 한정되는 것은 아니다. 또한, 상기 철이온의 농도는 40-80μM인 것이 바람직하나, 이에 한정되는 것은 아니다.In the present invention, the iron ion is preferably at least one selected from the group consisting of Fe 2 SO 4 , FeCl 2 , FeCl 3 and Fe 2 (SO 4 ) 3 , but is not limited thereto. In addition, the concentration of the iron ion is preferably 40-80 μM, but is not limited thereto.
본 발명에 있어서, 상기 철이온은 활성산소 O2 - 및 H2O2 함량에 대하여 100-600 몰비로 첨가되는 것이 바람직하나, 이에 한정되는 것은 아니다.In the present invention, the iron ion is preferably added in a molar ratio of 100-600 to the active oxygen O 2 - and H 2 O 2 content, but is not limited thereto.
본 발명에 있어서, 상기 아스타잔틴의 함량은 해마토코쿠스 플루비알리스 세포내 활성산소 O2 - 및 H2O2가 활성산소 O2 및 OH·로 전환에 의해 증진되는 것을 특징으로 할 수 있다.In the present invention, the content of astaxanthin may be characterized in that by the conversion of active oxygen O 2 - and H 2 O 2 in Haematococcus fluvialis cells into active oxygen O 2 and OH. .
일반적으로 미세조류는 고온의 환경에 노출 시, 광합성을 담당하는 엽록체 안에서 틸라코이드(thylakoids)의 누출(leak), 심할 경우에는 분해(disintegration)가 발생한다. 이어서 틸라코이드의 산소흡수률(oxygen uptake rate)이 증폭되고, 캘빈회로(calvin cycle)를 통해 환원되지 못한 전자들이 Mehler Reaction(O2 uptake + electron → O2 -)에 의해 산소와 결합하여 O2 -가 틸라코이드 안에 다량으로 생성된다. 이어서 세포는 과량 생산된 O2 -로부터 세포 성분을 보호하기 위해 SOD(superoxide dismutase)를 발현시키게 되고, 생성된 O2 -는 엽록체 내 SOD(superoxide dismutase)에 의해 H2O2로 전환된다. 결과적으로 고온의 환경은 미세조류 세포 내 다량의 H2O2를 발생시킨다. In general, microalgae, when exposed to high temperature environments, leak thylakoids in the chloroplasts responsible for photosynthesis and, in severe cases, disintegration. Subsequently, the oxygen uptake rate of the thylakoid is amplified, and electrons that are not reduced through the Calvin cycle are combined with oxygen by Mehler Reaction (O 2 uptake + electron → O 2 − ) to form O 2 − . It is produced in large quantities in thylakoids. Then the cells are O 2 excess production - and thereby express the SOD (superoxide dismutase) to protect the cell components from the produced O 2 - is converted to H 2 O 2 by the in-SOD (superoxide dismutase) chloroplasts. As a result, high temperature environments generate large amounts of H 2 O 2 in microalgal cells.
H2O2는 O2 -와는 달리 확산을 통한 세포막 투과가 용이하기 때문에 엽록체 기관으로부터 나온 H2O2는 핵산, 미토콘드리아, 액포 등 다른 세포기관으로 세포 산화 관련 시그널을 전달하게 되며, 이때 카로티노제네시스(carotenogenesis)에 관련된 유전인자들 중 H2O2에 민감한 효소들을 직접적으로 비활성화시켜 결과적으로 해마토코쿠스 플루비알리스의 아스타잔틴 합성이 저해되었을 가능성이 있다.H 2 O 2 is O 2 - because the membrane permeation is easy through the diffusion contrast to H 2 O 2 derived from the chloroplast engine is to pass the cellular oxidation-related signals to other organelles such as nucleic acids, mitochondria, vacuoles, wherein Caro Martino it is possible to Genesis directly disabled by a sensitive enzyme genes involved in the H 2 O 2 of (carotenogenesis) is consequently hippocampus Toko kusu flat ruby Alice astaxanthin synthesis is inhibited.
한편, 식물체에서 카로티노이드는 색소체(plastid)안에서 배타적으로 합성된다고 보고된 바 있으나, 해마토코쿠스 플루비알리스는 스트레스 반응에 의하여 색소체 밖의 지질 소포(lipid vesicles, globules)에 아스타잔틴을 포함하는 카로티노이드를 축적한다. 앞서 언급하였듯이 H2O2는 O2 -와는 달리 확산을 통한 세포막 투과가 용이하기 때문에 철이온 매개 펜톤 반응(Fe2+ + H2O2 → Fe3+ + OH·)은 색소체 밖에서도 충분히 발생한다. 그러므로 해마토코쿠스 세포는 철이온 매개 Haber-Weiss 반응을 통하여 과량 생성된 OH·로 인한 극심한 지질 산화로부터 자신을 방어하기 위하여 아스타잔틴 합성을 촉진 시켰을 가능성이 크다.On the other hand, carotenoids in plants have been reported to be exclusively synthesized in plastids, but haematococcus fluvialis contains carotenoids containing astaxanthin in lipid vesicles (globules) outside the plastids due to stress reactions. To accumulate. As mentioned above H 2 O 2 is O 2 - than the diffusion membrane permeable to the iron ions easily mediated Fenton reaction (Fe 2+ + H 2 O 2 → Fe 3+ + OH ·) is also generated, because, unlike with enough outside the plastid do. Therefore, it is likely that Haematococcus cells promoted astaxanthin synthesis in order to protect themselves from excessive lipid oxidation due to excessive production of OH · through iron ion mediated Haber-Weiss reaction.
또한, O2, OH·는 O2 -, H2O2 보다 반응성이 높은 것으로 보고되었다. OH·는 0.3 msec(diffusion lengths of 1.7 - 20μm)의 매우 짧은 수명(lifetime)을 가지는 반면, H2O2는 E = 1.77 V, pKa 11.6의 강력한 2개의 전자 산화제(electron-oxidant)이지만, 생물학적 분자들(biological molecules)에 대한 반응성이 낮기 때문에 대부분의 H2O2에 의한 세포 손상은 Fe2+와 같은 전이 금속(transition metal)이나 효소(enzyme)의 매개로 하여 H2O2의 O2 및 OH·전환을 유도하여 발생한다. O2 - 역시 적당히 높은 reduction potential(E = 0.94 V)을 가지고 있음에도 불구하고 여전히 생물학적 분자들(biological molecules)에 대한 반응성이 낮다.In addition, O 2 , OH · was reported to be more reactive than O 2 − , H 2 O 2 . OH · has a very short lifetime of 0.3 msec (diffusion lengths of 1.7-20 μm), while H 2 O 2 is a powerful two electron-oxidant with E = 1.77 V, pKa 11.6, but biologically due to the low reactivity of the molecules (biological molecules), most of the cell damage caused by H 2 O 2 is the H 2 O 2 by the medium of a transition metal (transition metal) or enzymes (enzyme), such as O 2 Fe 2+ And OH induction. Even though O 2 - also has a moderately high reduction potential (E = 0.94 V), it is still less responsive to biological molecules.
따라서, 고온의 배양조건(30 - 40℃)에서 세포 내 다량 생성된 LROS(O2 -, H2O2)로부터 철이온 매개 Haber-Weiss 반응을 유도하여 상당 부분 전환된 MROS(O2, OH·)는 PUFA(불포화 지방산)을 비롯한 각종 세포성분들을 산화시켜 결과적으로 MDA 함량이 증가하였을 가능성이 크다. 더욱이 실온(23℃)의 조건에서 FeSO4을 추가적으로 첨가하지 않은 세포가 FeSO4을 추가적으로 첨가한 세포에 비하여 MDA, 카로티노이드 함량이 더 높은 점으로 미루어보아 지질 산화는 Haber-Weiss 반응 시 철이온 농도와 LROS(O2 -, H2O2)의 조절을 통하여 가속화될 수 있으며, 철이온과 LROS 함량비 조절(ratio control)이 자가영양조건에서 해마토코쿠스 플루비알리스 세포의 아스타잔틴 생산을 위해서 매우 중요한 요소임에 틀림이 없으며, 이에 따라, 세포 내 O2 -의 H2O2 함량의 면밀하게 제어할 수 있다.Therefore, the high-temperature culture conditions (30 - 40 ℃) an intracellular large amount generated by the LROS (O 2 -, H 2 O 2) O ( an iron ion mediated by inducing the Haber-Weiss reaction largely diverted from MROS 2, OH ·) Is likely to oxidize various cellular components, including PUFA (unsaturated fatty acids), resulting in increased MDA content. Moreover, the iron ion concentration during MDA, oxidized lipids Having said the higher the carotenoid content that the Haber-Weiss reaction compared with the cells did not further addition of the FeSO 4 under the conditions of room temperature (23 ℃) was added FeSO 4 further cell LROS (O 2 -, H 2 O 2) can be accelerated through the modulation of, the iron unregulated on and LROS content (ratio control) self to the astaxanthin of the hippocampus Toko kusu flat ruby Alice cell production in a nutrient conditions It must be a very important factor, and thus it is possible to closely control the H 2 O 2 content of O 2 − in the cell.
본 발명의 일 실시예에서는, 상기 철이온은 활성 산소 O2 - 및 H2O2 함량에 대하여 100 내지 600 몰비로 첨가되는 것이 바람직하며, 상기 범위를 벗어나 활성 산소 O2 - 및 H2O2 함량에 대하여 100 몰비 미만으로 첨가되는 경우에는 아스타잔틴 축적 효율이 떨어지는 문제점이 있을 수 있고, 활성 산소 O2 - 및 H2O2 함량에 대하여 600 몰비를 초과하여 첨가되는 경우에는 부반응이 일어나 아스타잔틴 합성이 저해되는 문제점이 있을 수 있다.In one embodiment, the iron ion is the active oxygen O 2 - and H 2 O preferably added at 100 to 600. The molar ratio for the second content, and, out of the range radicals O 2 - and H 2 O 2 If it is added in less than 100 molar ratio with respect to the content, there may be a problem that the astaxanthin accumulation efficiency is lowered, and when added in excess of 600 molar ratio with respect to the active oxygen O 2 - and H 2 O 2 content, side reactions occur There may be a problem that taraxanthin synthesis is inhibited.
본 발명에 있어서, 상기 아스타잔틴의 함량은 해마토코쿠스 플루비알리스 세포 내 활성산소 O2 - 및 H2O2가 활성산소 O2 및 OH·로 전환에 의해 증진되는 것을 특징으로 할 수 있다.In the present invention, the content of astaxanthin may be characterized in that the active oxygen O 2 - and H 2 O 2 in the Haematococcus fluvialis cells are enhanced by conversion to the active oxygen O 2 and OH. .
본 발명의 다른 실시예에서는, 더욱 효율적인 아스타잔틴의 생산량 증진을 위하여, 고온의 배양 조건에서 그린 스테이지에서 성숙 포자 (red cyst) 접종을 통한 헤마토코쿠스 세포의 생산량을 조사하였으며, 이에 28-30℃의 고온자가배양조건의 그린 스테이지에서 성숙 포자 (red cyst) 접종 및 레드 스테이지에서 철이온의 첨가는 헤마토코쿠스 세포의 생산량과 생산수율을 현저히 증진시킬 수 있다.In another embodiment of the present invention, in order to more efficiently improve the production of astaxanthin, the production of hematococcus cells through the inoculation of mature red spores at the green stage under high temperature culture conditions was investigated. Inoculation of the red cysts at the green stage of the high temperature self-cultivation condition at 30 ° C. and the addition of iron ions at the red stage can significantly improve the production and yield of hematococcus cells.
본 발명의 용어 '자가영양조건'은 식물이 몸 밖에서 무기물을 양분으로 섭취하여 그것을 유기물로 합성할 수 있도록 하는 배지의 상태를 의미하며, '자가영양배양조건'이라고도 표현하며, 일반적으로 자가영양조건에서 광합성을 위한 무기 탄소원으로는 이산화탄소를 공급하고, 배지 조성은 Ca(NO3)2 또는 CaCl2·2H2O, KNO3 또는 KCl, Na2Glycerophosphate·5H2O, MgSO4·7H2O, Tris-aminomethane, Thiamine, Biotin, Vitamin B12, PIV metal solution, Na2EDTA, FeCl3·6H2O, MnCl2·4H2O, ZnSO4·7H2O, CoCl2·6H2O 및 Na2MoO4·2H2O 을 포함할 수 있으나, 이에 한정되는 것은 아니다.The term 'autotrophic condition' of the present invention refers to a state of a medium in which a plant ingests minerals from the outside of the body as a nutrient and synthesizes them as organic matter, and is also referred to as a 'self-nourishing condition', and is generally a self-nourishing condition. Carbon dioxide is supplied as an inorganic carbon source for photosynthesis, and the medium composition is Ca (NO 3 ) 2 or CaCl 2 .2H 2 O, KNO 3 or KCl, Na 2 Glycerophosphate.5H 2 O, MgSO 4 .7H 2 O, Tris-aminomethane, Thiamine, Biotin, Vitamin B12, PIV metal solution, Na 2 EDTA, FeCl 3 · 6H 2 O, MnCl 2 · 4H 2 O, ZnSO 4 · 7H 2 O, CoCl 2 · 6H 2 O and Na 2 MoO It may include 4 · 2H 2 O, but is not limited thereto.
실시예Example
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 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 in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .
재료material
본 발명에 사용된 균주는 Haematococcus pluvialis NIES-144로서 National Institute for Environmental Studies, Tsukuba, Japan으로부터 구입하여 사용하였다.The strain used in the present invention was purchased from National Institute for Environmental Studies, Tsukuba, Japan as Haematococcus pluvialis NIES-144.
본 실시예에 사용된 배지의 종류는 모두 NIES-C 배지 및 NIES-N 배지 2가지이고, 이들의 구성성분을 하기 표 1에 나타내었다.The types of medium used in this example were both NIES-C medium and NIES-N medium, and their components are shown in Table 1 below.
하기 표 1에서,In Table 1 below,
- NIES-C 배지: autotrophic medium (자가영양배지, 목적: 생장)NIES-C medium: autotrophic medium (autotrophic medium, purpose: growth)
- NIES-N 배지: autotrophic medium (자가영양배지, 목적: 생육억제, 광유발, 아스타잔틴 생산)-NIES-N medium: autotrophic medium (autotrophic medium, purpose: growth inhibition, photoinduction, astaxanthin production)
- CO2 supply: 3%(v/v)CO 2 supply: 3% (v / v)
- 광조건: 20 μE/m2/s (for vegetative growth); 150 μE/m2/s (for inductive growth)Light conditions: 20 μE / m 2 / s (for vegetative growth); 150 μE / m 2 / s (for inductive growth)
- FeSO4: 450 μMFeSO 4 : 450 μM
- MV (methyl viologen): 10-11M, 10-9M, 10-7M(artificial O2-generator)MV (methyl viologen): 10-11M, 10-9M, 10-7M (artificial O2-generator)
- H2DCFDA (carboxy-2',7'-dichlorofluorescein diacetate) 5 μM-5 μM of H2DCFDA (carboxy-2 ', 7'-dichlorofluorescein diacetate)
- DCF: H2DCFDA가 활성산소(O2-, H2O2)에 의해 산화되어 생성되는 물질DCF: H2DCFDA is oxidized by active oxygen (O2-, H2O2)
- SOD(superoxide dismuase): SOD는 세포 내에서 O2 -를 H2O2로 전환하는 효소Superoxide dismuase (SOD): SOD is an enzyme that converts O 2 - into H 2 O 2 in cells.
- MDA(malondialdehyde): MDA는 세포 내 PUFA(polyunsaturated fatty acids)가 산소(O2) 및 활성산소(O2 -, H2O2 및 OH·)에 의해 산화되어 전환되는 2차 대사 물질로서, 세포 내 MDA 양을 통하여 세포의 산화 정도를 간접적으로 확인할 수 있는 일종의 산화 지표이다.MDA (malondialdehyde): MDA is a secondary metabolite in which polyunsaturated fatty acids (PUFAs) in cells are oxidized and converted by oxygen (O 2 ) and free radicals (O 2 − , H 2 O 2 and OH ·), It is a kind of oxidative indicator that can indirectly check the degree of oxidation of cells through the amount of intracellular MDA.
표 1
NIES-C NIES-N
성분 함량(/L) 성분 함량(/L)
Ca(NO3)2 0.15 g CaCl2·2H2O 0.13 g
KNO3 0.10 g KCl 0.07 g
Na2Glycerophosphate·5H2O 0.05 g Na2Glycerophosphate·5H2O 0.05 g
MgSO4·7H2O 0.04 g MgSO4·7H2O 0.04 g
Tris-aminomethane 0.05 g Tris-aminomethane 0.05 g
Thiamine 0.01 ㎎ Thiamine 0.01 ㎎
Biotin 0.10 ㎍ Biotin 0.10 ㎍
Vitamin B12 0.01 ㎍ Vitamin B12 0.01 ㎍
PIV metal solution(per liter)Na2EDTA 1 gFeCl3·6H2O 0.196 gMnCl2·4H2O 0.036 gZnSO4·7H2O 0.022 gCoCl2·6H2O 4 ㎎Na2MoO4·2H2O 2.5 ㎎ 3 ㎖ PIV metal solution(per liter)Na2EDTA 1 gFeCl3·6H2O 0.196 gMnCl2·4H2O 0.036 gZnSO4·7H2O 0.022 gCoCl2·6H2O 4 ㎎Na2MoO4·2H2O 2.5 ㎎ 3 ㎖
Table 1
NIES-C NIES-N
ingredient Content (/ L) ingredient Content (/ L)
Ca (NO 3 ) 2 0.15 g CaCl 2 · 2H 2 O 0.13 g
KNO3 0.10 g KCl 0.07 g
Na 2 Glycerophosphate5H 2 O 0.05 g Na 2 Glycerophosphate5H 2 O 0.05 g
MgSO 4 7 H 2 O 0.04 g MgSO 4 7 H 2 O 0.04 g
Tris-aminomethane 0.05 g Tris-aminomethane 0.05 g
Thiamine 0.01 mg Thiamine 0.01 mg
Biotin 0.10 μg Biotin 0.10 μg
Vitamin b12 0.01 μg Vitamin b12 0.01 μg
PIV metal solution (per liter) Na 2 EDTA 1 gFeCl 3 6H 2 O 0.196 gMnCl 2 4H 2 O 0.036 gZnSO 4 7H 2 O 0.022 gCoCl 2 6H 2 O 4 mgNa 2 MoO 4 2H 2 O 2.5 mg 3 ml PIV metal solution (per liter) Na 2 EDTA 1 gFeCl 3 6H 2 O 0.196 gMnCl 2 4H 2 O 0.036 gZnSO 4 7H 2 O 0.022 gCoCl 2 6H 2 O 4 mgNa 2 MoO 4 2H 2 O 2.5 mg 3 ml
실시예 1: 자가영양조건에서 다양한 고온의 배양조건 및 철이온 매개 Haber-Weiss 반응이 해마토코쿠스 플루비알리스의 바이오매스 및 아스타잔틴 축적에 미치는 영향Example 1 Effects of Various High Temperature Culture Conditions and Iron Ion-Mediated Haber-Weiss Reaction on Biomass and Astaxanthin Accumulation in Haematococcus fluvialis Under Autotrophic Conditions
자가영양조건에서 다양한 고온의 배양조건 및 철이온 매개 Haber-Weiss 반응이 해마토코쿠스 플루비알리스의 아스타잔틴 축적에 미치는 영향을 알아보기 위하여, 하기 실험을 수행하였다.In order to investigate the effects of various high temperature culture conditions and iron ion-mediated Haber-Weiss reaction on autotrophic conditions on astaxanthin accumulation of Haemacococcus fluvialis, the following experiment was performed.
유기 탄소원이 결핍된 NIES-C 배지에서 낮은 광도의 빛(20μE/m2/s)과 오직 이산화탄소만을 유일 탄소원으로 생육하여 대수기에 이른 세포 배양액(OD680 = 약 0.8) 내 세포를 NIES-N 배지로 옮겨서 증식을 억제하고, 강한 빛(150μE/m2/s)과 함께 일반적인 실온의 배양 온도(23℃)와 고온의 배양 온도(30℃, 36℃) 그리고 이와 더불어 각각의 온도 조건에 철이온 매개 Haber-Weiss 반응을 유도하는 물질 FeSO4 (450μM)을 첨가하였을 경우와 하지 않았을 경우를 나누어 배양 18일 동안 바이오매스 증가량 및 아스타잔틴 축적 정도를 분석하였다. In NIES-C medium lacking organic carbon sources, cells in low cell light (20 μE / m 2 / s) and only carbon dioxide as the only carbon source were grown in cell culture (OD 680 = about 0.8), which reached NIES-N. Transfer to the medium to inhibit growth and, with strong light (150 μE / m 2 / s), iron at normal room temperature (23 ° C.) and high temperature (30 ° C., 36 ° C.) Biomass increase and astaxanthin accumulation were analyzed during 18 days of culture by dividing FeSO 4 (450 μM) with or without the substance that induces the mediated Haber-Weiss reaction.
1-1: 바이오매스1-1: Biomass
도 2(A)에 나타낸 바와 같이, 자가영양 조건에서 온도의 증가로 인한 해마토코쿠스 플루비알리스의 바이오매스 생산 능력은 23℃에 비해서 30℃에서 30%, 36℃에서 57% 감소하는 것이 확인된 반면에, 배양 온도가 30℃이고, Fe2+를 첨가하여 Haber Weiss 반응을 유도하였을 경우, 해마토코쿠스 플루비알리스의 바이오매스 생산 능력은 Fe2+을 첨가하지 않고, 배양 온도가 23℃인 경우에 비해서 9% 증가한 것이 확인되었고, Fe2+를 첨가하지 않고, 배양 온도가 30℃인 경우에 비해서 41% 증가한 것이 확인되었다. As shown in Figure 2 (A), the biomass production capacity of Haemacococcus fluvialis due to the increase in temperature in autotrophic conditions is confirmed to decrease by 30% at 30 ℃, 57% at 36 ℃ compared to 23 ℃ On the other hand, if the incubation temperature is 30 ℃ and Fe 2+ is added to induce the Haber Weiss reaction, the biomass production capacity of Haemacococcus fluvialis does not add Fe 2+ , the culture temperature is 23 ℃ It was confirmed that it increased by 9% compared with the case, and it was confirmed that 41% increased compared with the case where the incubation temperature was 30 ° C without adding Fe 2+ .
또한, 배양 온도가 36℃인 경우, Fe2+를 첨가하여 Haber-Weiss 반응을 유도하였을 때, 해마토코쿠스 플루비알리스의 바이오매스 생산 능력은 Fe2+을 첨가하지 않고, 배양 온도가 23℃인 경우에 비해서 3% 증가한 것이 확인되었고, Fe2+를 첨가하지 않고, 배양 온도가 30℃인 경우에 비해서 77% 증가한 것이 확인되었다. In addition, when the culture temperature is 36 ℃, when Fe 2+ is added to induce the Haber-Weiss reaction, the biomass production capacity of Haemacococcus fluvialis does not add Fe 2+ , and the culture temperature is 23 ℃ It was confirmed that it increased by 3% compared with the case, and it was confirmed that 77% increased compared to the case where the culture temperature is 30 ℃ without adding Fe 2+ .
1-2 : 아스타잔틴 축적1-2: Astaxanthin accumulation
도 2(B)에 나타낸 바와 같이, 자가 영양의 조건에서 온도의 증가로 인한 해마토코쿠스 플루비알리스의 아스타잔틴 생산 능력은 배양온도가 23℃인 경우에 비해서 30℃에서 23%, 36℃에서 42% 감소하는 것이 확인된 반면에, 배양온도가 30℃이고, Fe2+를 첨가하여 Haber Weiss 반응을 유도하였을 경우, 해마토코쿠스 플루비알리스의 아스타잔틴 생산 능력은 Fe2+을 첨가하지 않고, 배양 온도가 23℃인 경우에 비해서 17% 증가한 것이 확인되었고, Fe2+를 첨가하지 않고, 배양온도가 30℃인 경우에 비해서 66% 증가한 것이 확인되었다.As shown in Fig. 2 (B), the astaxanthin production capacity of Haematococcus fluvialis due to the increase in temperature under autotrophic conditions was 23% at 30 ° C and 36 ° C compared to the case where the culture temperature was 23 ° C. While the incubation temperature was 30 ° C. and Fe 2+ was added to induce the Haber Weiss reaction, the astaxanthin production capacity of haematococcus fluvialis was increased by Fe 2+ . In addition, it was confirmed that the culture temperature increased by 17% compared with the case of 23 ℃, it was confirmed that 66% increased compared to the case of the culture temperature of 30 ℃ without adding Fe 2+ .
또한, 배양 온도가 36℃인 경우, Fe2+를 첨가하여 Haber-Weiss 반응을 유도하였을 때, 해마토코쿠스 플루비알리스의 아스타잔틴 생산 능력은 Fe2+를 첨가하지 않고, 배양 온도가 23℃인 경우에 비해서 7% 감소하였지만, Fe2+를 첨가하지 않고, 배양 온도가 30℃인 경우에 비해서 152% 증가한 것이 확인되었다.On the contrary, if the incubation temperature is 36 ℃, when by the addition of Fe 2+ hayeoteul induce Haber-Weiss reaction, hippocampus Flags Tokoro kusu ruby astaxanthin production capacity of Alice is without the addition of Fe 2+, incubation at 23 Although it decreased by 7% compared with the case of ° C, it was confirmed that 152% increased without the addition of Fe 2+ and the culture temperature was 30 ° C.
나아가, 2일 동안 아스타잔틴 합성을 수행하여 해마토코쿠스 세포의 아스타잔틴 축적 정도를 나타내는 광학현미경 사진으로 확인한 결과, 도 3에 나타낸 바와 같이, 고온의 조건이 자가영양조건에서 해마토코쿠스 플루비알리스의 아스타잔틴 생산에 부적합하다는 것을 의미함과 동시에 고온에서 Haber-Weiss 반응의 도입이 해마토코쿠스 플루비알리스의 아스타잔틴 생산 능력의 유지에 효과적임을 확인하였다.Furthermore, astaxanthin synthesis was carried out for 2 days and confirmed by optical micrographs showing the astaxanthin accumulation of haematococcus cells. As shown in FIG. It was found that it was inadequate for astaxanthin production of ruby lice and that the introduction of Haber-Weiss reaction at high temperature was effective in maintaining the astaxanthin production ability of Haemacococcus fluvialis.
실시예 2: 자가영양조건에서 다양한 고온의 배양조건 및 철이온 매개 Haber-Weiss 반응이 해마토코쿠스 플루비알리스의 아스타잔틴 합성 초기 세포 내 활성산소 함량, SOD 활성도, 지질산화도 및 카로티노이드 함량에 미치는 영향Example 2: Various high-temperature culture conditions and iron ion-mediated Haber-Weiss reactions in autotrophic conditions on the free radicals, SOD activity, lipid oxidation and carotenoid content in the early cell of astaxanthin synthesis Impact
고온의 자가영양조건에서 아스타잔틴 합성 시, 해마토코쿠스 플루비알리스의 아스타잔틴 생합성 저해 현상을 규명하기 위하여, 하기 실험을 수행하였다. In order to investigate the phenomenon of inhibition of astaxanthin biosynthesis of haematococcus fluvialis during the synthesis of astaxanthin under high temperature autotrophic conditions, the following experiment was performed.
먼저 유기 탄소원이 결핍된 NIES-C 배지에서 낮은 광도의 빛(20μE/m2/s)과 오직 이산화탄소만을 유일 탄소원으로 생육하여 대수기에 이른 세포 배양액(OD680 =약 0.8)을 NIES-N 배지로 옮겨서 증식을 억제하고, 강한 빛 (150μE/m2/s)과 함께 일반적인 실온의 배양 온도(23℃)와 고온의 배양 조건(30℃, 36℃) 그리고 이와 더불어 각각의 온도 조건에 철이온매개 Haber-Weiss 반응을 유도하는 물질 FeSO4 (450μM)을 첨가하였을 경우와 하지 않았을 경우를 나누어 아스타잔틴 합성 조건을 조성한 후 배양 이틀 동안 세포 내 활성산소 함량(DCF 함량), SOD 활성도, 지질산화도(MDA 함량) 및 카로티노이드 축적 정도를 분석하였다. First, low-light light (20 μE / m 2 / s) and only carbon dioxide were grown on the NIES-C medium that lacked organic carbon sources, and the cell culture medium (OD 680 = about 0.8) that reached the logarithmic season was NIES-N medium. To inhibit growth, and with strong light (150 μE / m 2 / s), iron ions at normal room temperature (23 ° C) and high temperature culture conditions (30 ° C, 36 ° C) and with each temperature Astaxanthin synthesis conditions were established by dividing with or without FeSO 4 (450 μM), a substance that induces a mediated Haber-Weiss reaction, and then, during the two days of culture, active oxygen content (DCF content), SOD activity, and lipid oxidation The degree (MDA content) and the degree of carotenoid accumulation were analyzed.
그 결과, 도 4(A)에 나타낸 바와 같이, DCF 함량(활성산소 함량 - O2 -, H2O2)은 고온의 조건(30℃, 36℃)에서 급격히 상승하였으나, 고온의 배양 조건에 FeSO4 (450μM)을 첨가하여 Haber-Weiss 반응을 유도하였을 경우 활성산소 함량은 급격히 감소하였다. A (, H 2 O 2 active oxygen content - - O 2) is, but rapidly increased in the condition (30 ℃, 36 ℃) of high-temperature, high-temperature culture conditions, as a result, FIG. 4, as shown in (A), DCF content When FeSO 4 (450 μM) was added to induce the Haber-Weiss reaction, the active oxygen content decreased drastically.
또한, 도 4(B)에 나타낸 바와 같이, 아스타잔틴 합성 과정 중 SOD(superoxide dismutase) 활성은 전체적으로 증가하는 양상을 보였지만, 고온의 배양 조건(30℃, 36℃)에서 SOD 활성은 급격히 상승한 반면 고온의 배양 조건에 FeSO4 (450μM)을 첨가하여 Haber-Weiss 반응을 유도하였을 경우 SOD 활성은 현저히 감소하였다. 이것은 세포 내 과량 발생한 O2 -가 철이온 매개 Haber-Weiss 반응을 통하여 O2로 효과적으로 전환되었기 때문인 것으로 볼 수 있다.In addition, as shown in Figure 4 (B), during the astaxanthin synthesis SOD (superoxide dismutase) activity was shown to increase as a whole, while the SOD activity is rapidly increased in the high temperature culture conditions (30 ℃, 36 ℃) SOD activity was remarkably reduced when FeSO 4 (450μM) was added to incubate at high temperature to induce Haber-Weiss reaction. This may be because the excess O 2 − in the cell was effectively converted to O 2 through the iron ion mediated Haber-Weiss reaction.
나아가, 도 4(C)에 나타낸 바와 같이, MDA(malodialdhyde) 함량은 고온의 조건(30℃, 36℃)에서 DCF함량(O2 -, H2O2)과 정비례하여 상승하였으나, 고온의 배양조건(30 ℃, 36 ℃)에 FeSO4 (450μM)을 첨가하여 Haber-Weiss 반응을 유도하였을 경우 MDA 함량은 더욱 증가하는 것이 확인되었다. 이는 고온에서 생산된 활성산소 O2 -, H2O2가 철이온 매개 Haber-Weiss 반응에 의하여 신속히 O2, OH·로 전환되어 세포 내 PUFA(불포화지방산)와 빠르게 반응하여 MDA 형성을 가속화하였기 때문인 것으로 보인다.Furthermore, as shown in FIG. 4 (C), the MDA (malodialdhyde) content was elevated in direct proportion to the DCF content (O 2 − , H 2 O 2 ) under high temperature conditions (30 ° C., 36 ° C.), but at high temperature culture. It was confirmed that the addition of FeSO 4 (450 μM) to the conditions (30 ° C., 36 ° C.) induced the Haber-Weiss reaction to increase the MDA content. This is the production of active oxygen at a high temperature O 2 -, H 2 O 2 the iron ion mediated Haber-Weiss is converted by the reaction rapidly to O 2, OH · cells to respond quickly and within the PUFA (polyunsaturated fatty acids) hayeotgi accelerate MDA formation Seems to be.
실시예 3: 실온(23 ℃)의 자가영양조건에서 O2 -의 인위적인 발생과 철이온매개 Haber-Weiss 반응이 해마토코쿠스 플루비알리스의 아스타잔틴 합성 초기 세포내 아스타잔틴 축적에 미치는 영향Example 3 Effect of Artificial Generation of O 2 − and Iron Ion-mediated Haber-Weiss Reaction on Astaxanthin Synthesis of Astaxanthin Synthesis in Early Cellular Astaxanthin Accumulation in Autotrophic Conditions at Room Temperature (23 ° C)
실온(23℃)의 자가영양조건에서 아스타잔틴 합성 시 해마토코쿠스 플루비알리스 세포 내 LROS(O2 -, H2O2)의 증가로 인한 아스타잔틴 생합성 저해 현상을 규명하기 위하여, 유기 탄소원이 결핍된 NIES-C 배지에서 낮은 광도의 빛(20μE/m2/s)과 오직 이산화탄소만을 유일 탄소원으로 생육하여 대수기에 이른 세포 배양액(OD680 =약 0.8)을 NIES-N 배지로 옮겨서 증식을 억제하고, 강한 빛 (150μE/m2/s)과 함께 다양한 농도(10-11 내지 10-7 M)의 Methyl viologen을 첨가하여 O2 -를 인위적으로 강도 별로 발생시키고, 이와 더불어 일반적인 실온의 배양 온도(23℃)에서 철이온매개 Haber-Weiss 반응을 유도하는 물질 FeSO4 (450μM)을 첨가하였을 경우와 하지 않았을 경우를 나누어 아스타잔틴 합성 조건을 조성한 후 배양 4일째 세포 내 아스타잔틴 축적 정도를 분석하였다. Astaxanthin synthesis hippocampus Toko kusu flat ruby Alice intracellular LROS Self nutritional condition of room temperature (23 ℃) - In order to examine the astaxanthin biosynthesis inhibition due to the increase in the (O 2, H 2 O 2 ), the organic In low-light light (20 μE / m 2 / s) and carbon dioxide-deficient NIES-C medium, only high-carbon cell cultures (OD 680 = 0.8) were transferred to NIES-N medium. Inhibits proliferation, artificially generates O 2 − by intensity by adding Methyl viologen at various concentrations (10-11 to 10-7 M) with strong light (150 μE / m 2 / s), and at room temperature Astaxanthin in the cells on day 4 after the astaxanthin synthesis conditions were established by adding FeSO 4 (450 μM), which is an iron-induced Haber-Weiss reaction, to and without the addition of FeSO 4 (450 μM). The degree of accumulation was analyzed.
그 결과, 도 5에 나타낸 바와 같이, 해마토코쿠스 플루비알리스는 매우 적은 양의 Methyl viologen에도 민감하게 반응하여 과량의 O2 -가 발생되는 조건에서 아스타잔틴 축적 능력이 감소하는 것이 확인되었는데, 반면에 철이온의 첨가에 의한 LROS(O2 -, H2O2)의 MROS(O2, OH·)로 신속한 전환은 해마토코쿠스 플루비알리스의 아스타잔틴 축적 능력을 증진시켰다. 이는 실온(23℃)의 자가영양조건에서 해마토코쿠스 플루비알리스세포의 효과적인 아스타잔틴 생산을 위해서는 아스타잔틴 합성시, 세포내 O2 -의 H2O2 함량의 면밀한 제어가 필요함을 의미한다.As a result, as shown in Figure 5, Haematococcus fluvialis is sensitive to very small amount of Methyl viologen, it was confirmed that astaxanthin accumulation capacity decreases under the condition that excessive O 2 - occurs, On the other hand, the rapid conversion of LROS (O 2 − , H 2 O 2 ) to MROS (O 2 , OH ·) by the addition of iron ions enhanced the astaxanthin accumulation ability of haematococcus fluvialis. This means that for astaxanthin synthesis, effective control of H 2 O 2 content of intracellular O 2 - is required for the effective astaxanthin production of Haematococcus fluvialis cells at room temperature (23 ℃). do.
실시예 4: 고온의 자가영양조건에서 성숙 포자(cyst)의 접종 및 철이온 첨가에 의한 아스타잔틴 생산 증가Example 4 Increasing Astaxanthin Production by Inoculation of Mature Spores and Iron Ion in High Temperature Autotrophic Conditions
4-1: 그린 스테이지에서 성숙 포자(cyst)의 접종4-1: Inoculation of Mature Cysts at the Green Stage
본 실시예에서는 이단(two-stage) 광배양 공정으로 해마토코쿠스 플루비알리스 내 아스타잔틴의 생산량이 증진되는 것을 확인하였다.In this example, it was confirmed that the production of astaxanthin in the haematococcus fluvialis is enhanced by a two-stage photoculture process.
봄 (중온 조건: 17.5-27.3℃) 및 여름 (고온 조건: 23.4-33.5℃)의 평균 광도와 온도의 변화를 조사하여, 봄과 여름의 광도는 동일하게 유지하고 온도는 특별히 조절하지 않은 채, 옥외에서 헤마토코쿠스를 활용한 보다 경제적인 생물학적 이산화탄소 제거를 위한 공정의 조건을 조사하였다. By investigating changes in average brightness and temperature in spring (medium temperature: 17.5-27.3 ° C) and summer (high temperature condition: 23.4-33.5 ° C), the spring and summer brightness remain the same and the temperature is not specifically controlled, The conditions of the process for more economical biological CO2 removal using hematococcus outdoors were investigated.
그린 스테이지 (질소가 포함된 자가영양조건에 3~4% 이산화탄소를 공급하면서 35μE/m2/s 이하의 광도를 조사하는 낮은 스트레스의 배양조건) 동안 23℃에서 배양된 green vegetative 세포를 옥외 여름철 고온 조건인 레드 스테이지 (질소가 결핍된 자가영양조건에 3~4% 이산화탄소를 공급하면서 100-350μE/m2/s의 광도를 조사하는 높은 스트레스의 배양조건)에서 철이온을 첨가하여 36일 동안 아스타잔틴의 생산량을 조사하였다.Green vegetative cells cultured at 23 ° C. during outdoor stages at high temperatures during the green stage (low stress culture conditions irradiating 3μ4 /% CO2 to nitrogen containing autotrophic conditions and irradiating luminous intensity below 35μE / m 2 / s) Conditions for 36 days with the addition of iron ions in the red stage (high stress culture conditions that irradiate 100-350 μE / m 2 / s of light while supplying 3-4% carbon dioxide to nitrogen-deficient autotrophic conditions) The yield of taraxanthin was investigated.
그 결과, 50μM의 철이온 농도에서 효과적으로 헤마토코쿠스 세포의 아스타잔틴 생합성이 효과적으로 진행됨을 확인하였으며(도 6), 반면 철이온이 첨가되지 않은 광반응기 내 헤마토코쿠스 세포는 아스타잔틴 생합성이 저해되었다.As a result, it was confirmed that astaxanthin biosynthesis of hematococcus cells effectively proceeded at an iron ion concentration of 50 μM (FIG. 6), while hematococcus cells in the photoreactor without addition of iron ions were astaxanthin. Biosynthesis was inhibited.
더욱 효과적인 아스타잔틴의 생산량 증진을 위하여, 고온의 배양 조건에서 그린 스테이지에서 성숙 포자(red cyst) 접종을 통한 헤마토코쿠스 세포의 vegetative growth 가능성 여부를 21일간 인도어 랩 스케일에서 확인하였다. 그 결과, 비록 고온의 배양조건에서 성숙 포자 (red cyst) 접종 시 23℃ 배양 온도 조건보다 lag phase는 이틀 정도 길어졌지만, 성공적으로 그린 스테이지가 진행되었다(도 7).In order to enhance the production of astaxanthin more effectively, the possibility of vegetative growth of hematococcus cells through the inoculation of mature red spores at the green stage under the high temperature culture conditions was confirmed on the Indian lab scale for 21 days. As a result, although the lag phase was longer by two days than the 23 ° C. culture temperature when the red cyst inoculated at high culture conditions, the green stage was successfully progressed (FIG. 7).
4-2: 그린 스테이지에서 성숙 포자(cyst) 세포의 접종 및 레드 스테이지에서 철이온 첨가4-2: Inoculation of Mature Cyst Cells at Green Stage and Iron Ion Addition at Red Stage
실시예 4-1의 결과에 따라, 여름철 고온의 자가영양조건의 그린 스테이지에서 성숙 포자(red cyst) 접종 및 레드 스테이지에서 철이온 첨가를 통하여, 헤마토코쿠스 세포의 아스타잔틴 생산량을 조사하였다. 그린 스테이지 배양은 15일간 수행하였으며, 철이온 첨가 후 레드 스테이지 배양은 63일간 수행하였다.According to the results of Example 4-1, the astaxanthin production of hematococcus cells was investigated through inoculation of mature red spores in the green stage of the high temperature autotrophic condition in summer and the addition of iron ions in the red stage. . Green stage culture was performed for 15 days, and red stage culture was performed for 63 days after addition of iron ions.
그 결과, 성숙 포자(red cyst) 접종 및 50μM의 철이온 첨가에 의해 헤마토코쿠스 세포의 아스타잔틴 생산량(mg/L/day)은 봄철 중온의 조건에서 옥외배양은 2.24 mg/L/day이고, 고온 배양조건에서는 3.29 mg/L/day로 147% 증가한 것으로 나타났다(도 8).As a result, the astaxanthin production (mg / L / day) of hematococcus cells (mg / L / day) was 2.24 mg / L / day in spring and mid-temperature conditions due to the inoculation of mature red cyst and the addition of 50 μM of iron ion. In the high temperature culture conditions, it was found to increase by 147% to 3.29 mg / L / day (FIG. 8).
도 9는 옥외의 중온 및 고온(23-28℃ 및 28-33℃) 조건에서, 이단(two-stage: 그린 및 레드 스테이지) 광배양을 통한 해마토코쿠스 플루비알리스의 아스타잔틴 생산능 증대를 유도한 과정 및 결과를 나타낸 것으로, 중온의 그린 스테이지 및 철이온이 첨가되지 않은 레드 스테이지 배양조건 (57일간 2.24mg/L/day)에 비해 고온의 자가영양 그린 스테이지에서 성숙 포자(red cyst) 접종 및 레드 스테이지의 철이온 첨가 배양조건은 아스타잔틴 생산량 및 생산수율의 현저한 증가(27일간 5.53mg/L/day)를 나타냈다.9 shows the increase in astaxanthin production of haematococcus fluvilis through two-stage (green and red stage) photoculture in outdoor medium and high temperature (23-28 ° C and 28-33 ° C) conditions. It shows the process and the result of the development, and the mature green spore (red cyst) in the high temperature autotrophic green stage compared to the medium temperature green stage and the red stage culture condition without iron ion (57 days 2.24mg / L / day) Incubation conditions of iron inoculation and red stage showed a significant increase in astaxanthin production and yield (5.53 mg / L / day for 27 days).
본 발명에 따르면, 옥외배양 시 자가영양조건으로 태양을 이용한 아스타잔틴 생산에서 성숙 포자(cyst)를 접종한 후 철이온을 첨가하는 이단(two-stage) 광배양 공정은 고온의 조건에서 다량 발생되는 LROS(O2 -, H2O2)를 효과적으로 MROS(O2, OH·)로 전환하여 세포 내 지질 산화 신호를 증폭하여 LROS(O2 -, H2O2)의 과량 발생에 의한 아스타잔틴 합성을 저해하는 문제점을 해결할 수 있으므로, 보다 경제적으로 아스타잔틴 생산을 증진시킬 수 있고 이러한 아스타잔틴은 강력한 항산화물질로서 다양한 산업분야에서 유용하다.According to the present invention, a two-stage photoculture process in which iron ions are added after inoculating mature spores (cyst) in the production of astaxanthin using the sun as an autotrophic condition during outdoor culture is generated in a large amount at high temperature conditions. Ars by excess generation of - (, H 2 O 2 O 2) - LROS being (O 2, H 2 O 2 ) to effectively MROS (O 2, OH ·) to switch to amplify the lipid oxidation signal cell LROS As it can solve the problem of inhibiting taraxanthin synthesis, it is possible to improve the production of astaxanthin more economically and such astaxanthin is a powerful antioxidant and useful in various industries.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

  1. 다음 단계를 포함하는 해마토코쿠스 플루비알리스(Haematococcus pluvialis)의 배양에 의한 아스타잔틴의 생산량을 증진시키는 방법:A method for enhancing the production of astaxanthin by culturing Haematococcus pluvialis comprising the following steps:
    (a) 해마토코쿠스 플루비알리스의 성숙 포자(cyst)를 접종하여 증식(vegetative growth) 시키는 단계; 및(a) inoculating and propagating mature spores of Haematococcus fluvialis (vegetative growth); And
    (b) 질소가 결핍되고 철이온이 첨가된 자가영양조건에서 100-300μE/m2/s의 광도를 조사하여 해마토코쿠스 플루비알리스 내 아스타잔틴의 생성을 유도하는 단계.(b) inducing the production of astaxanthin in haematococcus fluvialis by irradiating with luminous intensity of 100-300 μE / m 2 / s under autotrophic conditions with nitrogen deficiency and iron ions.
  2. 제1항에 있어서, 상기 배양 온도는 25∼40℃ 인 것을 특징으로 하는 아스타잔틴의 생산량을 증진시키는 방법.The method of claim 1, wherein the incubation temperature is 25 to 40 ° C. 3.
  3. 제1항에 있어서, 상기 자가영양조건은 광합성을 위한 무기 탄소원으로 3~4% 이산화탄소를 공급하는 것을 특징으로 하는 아스타잔틴의 생산량을 증진시키는 방법.The method of claim 1, wherein the autotrophic condition is to supply 3 to 4% carbon dioxide as an inorganic carbon source for photosynthesis.
  4. 제1항에 있어서, 상기 (a) 단계는 35μE/m2/s 이하의 광도를 조사하는 것을 특징으로 하는 아스타잔틴의 생산량을 증진시키는 방법.According to claim 1, wherein the step (a) is a method of increasing the production of astaxanthin, characterized in that irradiating a light intensity of less than 35μE / m 2 / s.
  5. 제1항에 있어서, 상기 철이온은 Fe2SO4, FeCl2, FeCl3 및 Fe2(SO4)3로 이루어지는 군으로부터 선택되는 1종 이상인 것을 특징으로 하는 아스타잔틴의 생산량을 증진시키는 방법.The method of claim 1, wherein the iron ions are at least one selected from the group consisting of Fe 2 SO 4 , FeCl 2 , FeCl 3 and Fe 2 (SO 4 ) 3 . .
  6. 제1항에 있어서, 상기 철이온의 농도는 40-80μM인 것을 특징으로 하는 아스타잔틴의 생산량을 증진시키는 방법.The method of claim 1, wherein the iron ion concentration is 40-80 μM.
  7. 제1항에 있어서, 상기 철이온은 활성산소 O2- 및 H2O2 함량에 대하여 100-600 몰비로 첨가되는 것을 특징으로 하는 아스타잔틴의 생산량을 증진시키는 방법.The method of claim 1, wherein the iron ions are added in an amount of 100-600 molar ratio based on the contents of the active oxygen O 2 -and H 2 O 2 .
  8. 제1항에 있어서, 상기 아스타잔틴의 함량은 해마토코쿠스 플루비알리스 세포내 활성산소 O2 - 및 H2O2가 활성산소 O2 및 OH·로 전환에 의해 증진되는 것을 특징으로 하는 아스타잔틴의 생산량을 증진시키는 방법.The method of claim 1, wherein the content of astaxanthin is enhanced by the conversion of free radicals O 2 - and H 2 O 2 in Haematococcus fluvialis cells into free radicals O 2 and OH. How to increase the production of taraxanthin.
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