KR102167513B1 - Method for increasing astaxanthin production of Haematococcus pluvialis by drying culture - Google Patents
Method for increasing astaxanthin production of Haematococcus pluvialis by drying culture Download PDFInfo
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Abstract
본 발명은 건조 배양 방법을 통한 헤마토코쿠스 플루비알리스의 아스타잔틴의 생산 증대방법에 관한 것으로, 배양 과정 중 건조 스트레스를 유도함으로써 헤마토코쿠스 플루비알리스로부터 아스타잔틴의 생산량을 효과적으로 증대시킬 수 있으므로, 본 발명의 방법은 산업적으로 유용하게 활용될 수 있을 것이다.The present invention relates to a method for increasing the production of astaxanthin from Hematococcus fluvialis through a dry culture method, and by inducing dry stress during the culture process, the production amount of astaxanthin from Hematococcus fluvialis is effectively reduced. Since it can be increased, the method of the present invention will be industrially useful.
Description
본 발명은 건조 배양 방법을 통한 헤마토코쿠스 플루비알리스의 아스타잔틴의 생산 증대방법에 관한 것이다.The present invention relates to a method for increasing the production of astaxanthin of Hematococcus fluvialis through a dry culture method.
자연계에 존재하는 지용성 색소인 아스타잔틴(astaxanthin, 3,3'-dihydroxy-β,β'-carotene-4,4'-dione)은 적색을 띄는 케토카로티노이드(ketocarotenoid) 계통의 비식물성 색소로, 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 가지고 있다. 아스타잔틴은 이러한 독특한 분자 구조적 특성 때문에 기존의 항산화 물질보다 월등히 높은 항산화 활성을 갖는다(비타민 E보다 500배, 베타-카로틴보다 20배). 이러한 높은 항산화 활성기능으로 인해 아스타잔틴은 의약품, 식품 첨가제, 착색제 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 fat-soluble pigment that exists in nature, is a non-vegetable pigment of the red ketocarotenoid family. It is a type of carotenoid pigment that has the same chemical structure as beta-carotene. It is an antioxidant functional substance that removes harmful active oxygen. Compared to beta-carotene, hydroxyl group (-OH) and ketone group ( =O) one more. Astaxanthin has significantly higher antioxidant activity than conventional antioxidants due to these unique molecular structural properties (500 times more than vitamin E and 20 times more than beta-carotene). Due to such high antioxidant activity, astaxanthin is widely used as a pharmaceutical, food additive, colorant, and feed additive for animals and fry, and its demand and range of application are expected to rapidly expand.
미국 캘리포니아주 샌프란시스코에 위치한 시장조사 및 컨설팅기관인 그랜드 뷰 리서치사는 '소재별, 제품별, 용도별 아스타잔틴 마켓 및 2014~2025년 전망' 보고서를 통해, 2016년 5억 5,540만 달러 규모를 형성했던 글로벌 아스타잔틴 시장이 연평균 18.9%에 달하는 고속성장을 거듭해 오는 2025년 25억 7,000만 달러 규모에 도달할 수 있을 것이라 보고한 바 있다.Grand View Research, a market research and consulting firm located in San Francisco, California, USA, has formed a global market of $555.4 million in 2016 through the report'Astaxanthin Market by Material, Product, and Application and Forecast for 2014-2025'. It has been reported that the astaxanthin market will be able to reach $2.57 billion by 2025, with rapid growth at an annual average of 18.9%.
그러나, 아스타잔틴은 자가합성을 하지 못하여 먹이사슬을 통한 섭취로 획득하게 되며, 새우나 가재 등의 갑각류에서 추출할 수도 있으나 그 함량이 적고, 추출 과정 또한 어려워 적용되지 않고 있으며, 아스타잔틴을 생산하는 균주를 배양시켜 얻을 수 있으나, 그 또한 함량이 낮아 아스타잔틴의 생산량을 증대시키기 위한 배양 공정의 개발이 요구되고 있다. 또한 돌연변이체의 개발로 아스타잔틴의 생산량을 증대시키는 보고가 일부 있으나 균주의 성장과 아스타잔틴의 생산에 대한 안정화가 이루어지지 않은 상태이다.However, astaxanthin is not self-synthesizing and is obtained through ingestion through the food chain, and it can be extracted from crustaceans such as shrimp and crayfish, but its content is small and the extraction process is also difficult to apply. It can be obtained by culturing the strain to be produced, but its content is also low, so development of a culture process for increasing the production of astaxanthin is required. In addition, there have been reports of increasing the production of astaxanthin due to the development of mutants, but the growth of the strain and stabilization of the production of astaxanthin have not been achieved.
헤마토코쿠스 플루비알리스(Haematococcus pluvialis)는 담수에서 서식하는 미세조류로 카로테노이드인 아스타잔틴을 건중량의 3~4%로 축적하는 능력이 있다. 헤마토코쿠스 플루비알리스는 영양 고갈, 강한 광에 노출, 염도 증가, 너무 낮거나 높은 온도와 같은 다양한 스트레스에 대항하기 위해 포자 구조를 생성한다. 이 과정에서 스트레스에 저항하기 위해 지질, 카로테노이드 등 2차 대사 산물을 축적하는데, 그 중 적색을 띄는 케토카로티노이드인 아스타잔틴은 항산화 효과를 지닌 기능성 물질로 주목받고 있다. 지구상 생물들 중에서 헤마토코쿠스 플루비알리스가 아스타잔틴의 축적함량과 수율이 가장 높으며, 특히 이 균주는 광합성을 하기 때문에 빛에 의한 이산화탄소의 고정화와 동시에 아스타잔틴의 생산성을 높이기 위한 다양한 연구가 수행되고 있다. Haematococcus pluvialis is a microalgae that lives in freshwater and has the ability to accumulate astaxanthin, a carotenoid, to 3-4% of its dry weight. Hematococcus fluvialis produces spore structures to combat various stresses such as nutrient depletion, exposure to intense light, increased salinity, and too low or too high temperatures. In this process, secondary metabolites such as lipids and carotenoids are accumulated to resist stress. Among them, astaxanthin, a ketocarotenoid that has a red color, is attracting attention as a functional substance with an antioxidant effect. Among the living organisms on Earth, Hematococcus fluvialis has the highest accumulation content and yield of astaxanthin. In particular, since this strain is photosynthesized, various studies to increase the productivity of astaxanthin while immobilizing carbon dioxide by light Is being carried out.
한편, 한국공개특허 제2014-0075869호에는 영양세포 단계의 미세조류 세포를 퍼퓨전 공정에 의하여 연속적으로 재순환시킴으로써 세포를 고농도로 광배양하고, 광도를 단계적으로 증가시켜 아스타잔틴의 유발을 최적화 시키는 배양방법에 관한 '아스타잔틴 생산을 위한 헤마토코쿠스 플루비알리스의 퍼퓨전 배양법'이 개시되어 있고, 한국등록특허 제1745589호에는 성숙한 포자형태의 헤마토코쿠스 플루비알리스에 높은 광도와 질소원을 제공하여 발아를 유도하고 세포 내 C/N(탄소/질소)비 조절을 통해 세포벽을 약화시켜 아스타잔틴 수득을 높이는 방법에 관한 '헤마토코쿠스 플루비알리스의 세포발아 유도를 통한 아스타잔틴의 제조방법'이 개시되어 있으나, 본 발명의 건조 배양 방법을 통한 헤마토코쿠스 플루비알리스의 아스타잔틴의 생산 증대방법에 대해서는 기재된 바가 없다.On the other hand, Korean Patent Laid-Open Publication No. 2014-0075869 discloses that microalgal cells in the feeder cell stage are continuously recycled by a perfusion process to optimize the induction of astaxanthin by cultivating the cells at a high concentration and increasing the light intensity step by step. A'perfusion culture method of Hematococcus fluvialis for the production of astaxanthin' related to the culture method is disclosed, and Korean Patent No. 1745589 discloses high luminosity in mature spore-type Hematococcus fluvialis. 'Astaxanthin through induction of cell germination of Hematococcus fluvialis on a method of increasing the astaxanthin yield by providing a nitrogen source to induce germination and weakening the cell wall through regulation of the intracellular C/N (carbon/nitrogen) ratio. A method for producing taxanthin is disclosed, but there is no description of a method for increasing the production of astaxanthin by Hematococcus fluvialis through the dry culture method of the present invention.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명자들은 헤마토코쿠스 플루비알리스(Haematococcus pluvialis)로부터 아스타잔틴 생산효율을 높이는 방법을 개발하기 위해, 헤마토코쿠스 플루비알리스 균주의 배양 과정 중 습도를 조절하여 헤마토코쿠스 플루비알리스에 건조 스트레스를 처리한 결과, 건조 스트레스 무처리 조건과 비교하여 아스타잔틴의 함량이 증가되는 것을 확인함으로써, 본 발명을 완성하였다.The present invention was derived from the above requirements, in order to develop a method for increasing the astaxanthin production efficiency from Haematococcus pluvialis , the present inventors of the Hematococcus fluvialis strain The present invention was completed by confirming that the content of astaxanthin was increased as a result of treating the dry stress on Hematococcus fluvialis by controlling the humidity during the cultivation process, and that the content of astaxanthin was increased compared to the conditions without dry stress treatment.
상기 과제를 해결하기 위해, 본 발명은 헤마토코쿠스 플루비알리스(Haematococcus pluvialis)에 건조 스트레스를 유도하며 배양하는 단계를 포함하는 헤마토코쿠스 플루비알리스의 아스타잔틴 생산량 증대방법을 제공한다.In order to solve the above problems, the present invention provides a method for increasing the amount of astaxanthin produced by Hematococcus fluvialis , comprising the step of inducing and culturing dry stress in Haematococcus pluvialis . .
본 발명은 배양 과정 중 건조 스트레스를 가함으로써 헤마토코쿠스 플루비알리스로부터 아스타잔틴의 생산량을 효과적으로 증대시킬 수 있으므로, 저렴한 가격으로 판매할 수 있는 천연 아스타잔틴의 산업적 경쟁력을 확보하는 효과가 있다.The present invention can effectively increase the production of astaxanthin from Hematococcus fluvialis by applying dry stress during the cultivation process, thus securing the industrial competitiveness of natural astaxanthin that can be sold at a low price. have.
본 발명의 목적을 달성하기 위하여, 본 발명은 헤마토코쿠스 플루비알리스(Haematococcus pluvialis)에 건조 스트레스를 유도하며 배양하는 단계를 포함하는 헤마토코쿠스 플루비알리스의 아스타잔틴 생산량 증대방법을 제공한다.In order to achieve the object of the present invention, the present invention provides a method for increasing the amount of astaxanthin production of Hematococcus fluvialis comprising the step of inducing and culturing dry stress in Haematococcus pluvialis . to provide.
본 발명에 따른 아스타잔틴 생산량 증대방법은, 헤마토코쿠스 플루비알리스의 배양 과정 중에, 건조 스트레스 조건을 유도하여 비스트레스 조건에서 배양된 헤마토코쿠스 플루비알리스에 비해 아스타잔틴 생산량의 증대를 유도하는 방법으로, 상기 건조 스트레스의 유도는 배양 조건의 상대습도를 감소시켜 유도되는 것일 수 있으나, 이에 제한되지 않는다.The method of increasing the production of astaxanthin according to the present invention, during the cultivation process of Hematococcus fluvialis, induces dry stress conditions to increase the amount of astaxanthin production compared to Hematococcus fluvialis cultured in non-stress conditions. As a method of inducing an increase, the induction of the drying stress may be induced by reducing the relative humidity of the culture condition, but is not limited thereto.
본 발명의 일 구현 예에 따른 아스타잔틴 생산량 증대방법은 보다 구체적으로는,The method for increasing astaxanthin production according to an embodiment of the present invention is more specifically,
(a) 헤마토코쿠스 플루비알리스(Haematococcus pluvialis)를 액체 배지에서 배양하는 단계;(a) culturing Haematococcus pluvialis in a liquid medium;
(b) 상기 (a) 단계에서 배양한 헤마토코쿠스 플루비알리스를 고체 배지에 접종하고 22~28℃에서 65~75%의 상대습도로 5~7일간 배양하는 단계; 및(b) inoculating the Hematococcus fluvialis cultured in step (a) in a solid medium and incubating for 5 to 7 days at 22 to 28°C with a relative humidity of 65 to 75%; And
(c) 상기 (b) 단계의 배양 후, 1일 10%씩 상대습도를 순차적으로 10%까지 감소시켜 건조 스트레스를 유도하며 추가 배양하는 단계;를 포함할 수 있고, 더욱 구체적으로는,(c) after the cultivation of step (b), by sequentially reducing the relative humidity by 10% per day to 10% to induce dry stress and further culturing; may include, and more specifically,
(a) 헤마토코쿠스 플루비알리스(Haematococcus pluvialis)를 고바야시 액체 배지에서 배양하는 단계;(a) culturing Haematococcus pluvialis in Kobayashi liquid medium;
(b) 상기 (a) 단계에서 배양한 헤마토코쿠스 플루비알리스를 고바야시 고체 배지에 1.0×108 세포/㎖의 농도로 접종하고 25℃에서 70%의 상대습도로 6일간 배양하는 단계; 및(b) inoculating the Hematococcus fluvialis cultured in step (a) at a concentration of 1.0×10 8 cells/ml in Kobayashi solid medium, and incubating for 6 days at 25° C. with a relative humidity of 70%; And
(c) 상기 (b) 단계의 배양 후, 1일 10%씩 상대습도를 순차적으로 10%까지 감소시켜 건조 스트레스를 유도하며 6일간 추가 배양하는 단계;를 포함할 수 있으나, 이에 제한되지 않는다.
(c) after the cultivation of step (b), the step of sequentially reducing the relative humidity by 10% per day to 10% to induce dry stress and further culturing for 6 days; may include, but is not limited thereto.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.
Hereinafter, the present invention will be described in detail by examples. However, the following examples are only illustrative of the present invention, and the contents of the present invention are not limited to the following examples.
1. 아스타잔틴 생산량 측정1. Measurement of astaxanthin production
실험구 또는 대조구의 균주 배양액 2㎖을 원심분리기를 사용하여 13,000rpm에서 10분간 원심분리하여 상층액을 제거한 후, 세포 펠릿에 메탄올(methanol) : 디클로로메탄(dichloromethane)이 3 : 1 부피비로 혼합된 용액을 1㎖ 첨가하여 현탁시킨 후 20분간 초음파처리(sonication)하여 세포벽을 파쇄하고 색소가 용매에 추출되도록 한 다음, 13,000rpm에서 10분간 원심분리하여 불순물이 제거된 용매층을 취하였다. 그 후, 획득한 용매층을 Agilent사의 HPLC(high performance liquid chromatography, 1200 series)를 이용하여 분석하였다. 이동상으로는 디클로로메탄, 메탄올, 아세토니트릴(acetonitrile) 및 증류수의 비율을 달리하여 용매 A(dichloromethane : methanol : acetonitrile : water = 5 : 85 : 5.5 : 4.5(v/v))와 용매 B(dichloromethane : methanol : acetonitrile : water = 22 : 28 : 45.5 : 4.5(v/v))를 사용하였다. 각각의 용매는 0~5분까지 용매 A를 100％로 하였고, 8분까지 용매 B를 100％가 되도록 점차적으로 올렸다. 그 후 5분 동안 용매 B의 비율을 100％로 유지한 다음, 2분 동안 시작할 때의 용매 비율과 같게 하였다. 유속은 1㎖/min이었으며, DAD(Diode Array Detector) UV 검출기로 480nm에서 시료를 분석하였다. 표준곡선은 아스타잔틴(Sigma Chemical Co., 미국)을 이용하여 검량곡선을 작성한 후 함량 계산에 활용하였다.
After removing the supernatant by centrifuging 2 ml of the strain culture solution of the experimental or control group at 13,000 rpm for 10 minutes using a centrifuge, the cell pellet was mixed with methanol in a volume ratio of 3: 1 1 ml of the solution was added and suspended, the cell walls were crushed by sonication for 20 minutes, and the pigment was extracted into the solvent, followed by centrifugation at 13,000 rpm for 10 minutes to obtain a solvent layer from which impurities were removed. Then, the obtained solvent layer was analyzed using Agilent's high performance liquid chromatography (HPLC, 1200 series). As the mobile phase, solvent A (dichloromethane: methanol: acetonitrile: water = 5: 85: 5.5: 4.5 (v/v)) and solvent B (dichloromethane: methanol) by varying the ratio of dichloromethane, methanol, acetonitrile, and distilled water. : acetonitrile: water = 22: 28: 45.5: 4.5 (v/v)) was used. For each solvent, the solvent A was set to 100% from 0 to 5 minutes, and the solvent B was gradually increased to 100% by 8 minutes. After that, the proportion of solvent B was kept at 100% for 5 minutes, and then the same as the solvent proportion at the start for 2 minutes. The flow rate was 1 ml/min, and the sample was analyzed at 480 nm with a DAD (Diode Array Detector) UV detector. The standard curve was used to calculate the content after preparing a calibration curve using astaxanthin (Sigma Chemical Co., USA).
2. 헤마토코쿠스 플루비알리스(2. Hematococcus fluvialis ( Haematococcus pluvialisHaematococcus pluvialis ) 배양) Culture
본 발명에 사용한 헤마토코쿠스 플루비알리스(Haematococcus pluvialis) 균주는 한국원자력연구원에서 분양받아 사용하였다. 헤마토코쿠스 플루비알리스의 배양은 고바야시 배지(Kobayashi M. et al., 1992, J. Ferment. Bioeng. 74:17-20)를 이용하여 배양하였다. 고바야시 배지에 한천 1.5%를 첨가하고, 액체 배양된 헤마토코커스 플루비알리스 1.0×108 세포를 도말하여, 일반배양과 비교되는 건조 배양 후 아스타잔틴의 농도를 측정하였다. Hematococcus fluvialis ( Haematococcus pluvialis ) strain used in the present invention was sold and used by Korea Atomic Energy Research Institute. Culture of Hematococcus fluvialis was cultured using Kobayashi medium (Kobayashi M. et al., 1992, J. Ferment. Bioeng. 74:17-20). 1.5% of agar was added to the Kobayashi medium, and 1.0×10 8 cells of Hematococcus fluvialis cultured in a liquid were plated, and the concentration of astaxanthin was measured after dry culture compared to the normal culture.
본 발명에 따른 상기 건조 배양은 고체 배양 배지에 아스타잔틴을 생산하는 헤마토코쿠스 플루비알리스를 접종하고 25℃에서 5일 동안 생장시킨 후 습도를 70%에서 시작하여 하루에 10%씩 낮춰가며 건조한 바람을 이용하여 생장된 헤마토코커스 균주에 건조 스트레스를 유발시켰다. In the dry culture according to the present invention, after inoculation of hematococcus fluvialis producing astaxanthin in a solid culture medium and growing at 25° C. for 5 days, the humidity was lowered by 10% per day starting at 70%. Drying stress was induced in the grown Hematococcus strain by using dry wind.
각각의 건조 정도에 따른 고체배양 배지에서 생장하고 있는 헤마토코커스 균주를 수거하고 무게를 측정하고 동일양이 되게 시료들의 양을 조절한 후, 전술한 방법으로 아스타잔틴을 추출하여 생산량을 분석하였다.Hematococcus strains growing in the solid culture medium according to the degree of drying were collected, weighed, and the amount of samples was adjusted to be the same amount, and then astaxanthin was extracted by the above method to analyze the production amount. .
분석 균주는 습도 70%을 1일차로 해서 10%까지 감소한 7일에 걸처 수분을 유지할 수 있도록 조절하였으며, 최종적으로 습도가 조절된 7일 후 건조 배양한 헤마토코커스 균주와 보통 배양배지에서 생장한 헤마토코커스 균주를 비교하였다.
The analyzed strain was controlled to maintain moisture over 7 days, which decreased to 10% with a humidity of 70% as the first day. Finally, the Hematococcus strains dried and cultured after 7 days when the humidity was controlled and grown in normal culture medium. Hematococcus strains were compared.
실시예 1. 건조 스트레스 처리에 따른 아스타잔틴 생산량 분석Example 1. Analysis of astaxanthin production according to dry stress treatment
총 7일 동안 건조 스트레스를 처리한 헤마토코커스 플루비알리스로부터 분석된 아스타잔틴 생산량은 하기 표에서 확인되는 것과 같이 대조군(건조 스트레스 무처리; 표 2)과 비교하여 실험군(표 3)의 아스타잔틴 생산량이 6일차 이후부터 약 40% 정도 증가된 것을 알 수 있었다.Astaxanthin production analyzed from Hematococcus fluvialis treated with dry stress for a total of 7 days was compared with the control group (no dry stress treatment; Table 2) as shown in the following table. It was found that the production of taxanthin increased by about 40% from the 6th day.
상기 결과를 통해, 배양 과정 중 건조 스트레스를 유도하는 간단한 처리만으로 헤마토코커스 플루비알리스로부터 아스타잔틴의 생산량을 증대시킬 수 있음을 알 수 있었다.From the above results, it was found that the production of astaxanthin from Hematococcus fluvialis could be increased only by a simple treatment that induces dry stress during the culture process.
Claims (3)
(b) 상기 (a) 단계에서 배양한 헤마토코쿠스 플루비알리스를 고체 배지에 접종하고 22~28℃에서 65~75%의 상대습도로 5~7일간 배양하는 단계; 및
(c) 상기 (b) 단계의 배양 후, 1일 10%씩 상대습도를 순차적으로 10%까지 감소시켜 건조 스트레스를 유도하며 추가 배양하는 단계;를 포함하는 헤마토코쿠스 플루비알리스의 아스타잔틴 생산량 증대방법.(a) culturing Haematococcus pluvialis in a liquid medium;
(b) inoculating the Hematococcus fluvialis cultured in step (a) in a solid medium and incubating for 5 to 7 days at 22 to 28°C with a relative humidity of 65 to 75%; And
(c) after the cultivation of step (b), the step of sequentially reducing the relative humidity by 10% per day to 10% to induce dry stress and further culturing; containing astaxane of Hematococcus fluvialis containing How to increase tin production.
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