技术领域technical field
本发明属于微藻生物技术领域，涉及一种微藻培养生产叶黄素的方法。The invention belongs to the field of microalgae biotechnology, and relates to a method for producing lutein by culturing microalgae.
背景技术Background technique
叶黄素(Lutein)又名“植物黄体素”，是广泛存在于蔬菜、花卉、水果等植物和藻类中的一种天然活性物质。因其对人体具有保护视力、延缓动脉硬化、抗癌、抗氧化、保护皮肤等多个重要生理功效，所以叶黄素目前广泛作为食品和饲料添加剂，同时叶黄素鲜亮的橙黄色使其作为家禽畜、水产养殖动物和动物组织的增色剂。Lutein, also known as "plant lutein", is a natural active substance widely present in vegetables, flowers, fruits and other plants and algae. Lutein is currently widely used as a food and feed additive because it has many important physiological effects on the human body, such as protecting eyesight, delaying arteriosclerosis, anti-cancer, anti-oxidation, and protecting the skin. Color enhancer for poultry, aquaculture animals and animal tissue.
叶黄素在医药和保健品、食品和饲料、化妆品及水产养殖行业等方面广泛的应用前景，使许多国内外研究机构和公司对叶黄素的生物合成、分离提取、生理生化功能等进行研究。目前，叶黄素主要从万寿菊、金盏花等植物中提取获得，但是利用这些植物生产叶黄素存在着某些缺点。首先，叶黄素在这些植物中主要是酯化形式存在，因此从万寿菊、金盏花等植物中提取叶黄素时需要进行皂化处理，该步骤不但降低了效率和收率，同时残余的皂化剂容易污染叶黄素产品，给纯化过程增加了难度。其次，种植万寿菊、金盏花等植物需要大量的土地，这些植物的生长周期一般为3～4个月(相对于可快速生长的微藻而言，周期太长)。The wide application prospect of lutein in medicine and health care products, food and feed, cosmetics and aquaculture industry has made many domestic and foreign research institutions and companies conduct research on the biosynthesis, separation and extraction, physiological and biochemical functions of lutein, etc. . At present, lutein is mainly extracted from plants such as marigolds and marigolds, but there are some disadvantages in using these plants to produce lutein. First of all, lutein mainly exists in the form of esterification in these plants, so when extracting lutein from plants such as marigolds and marigolds, saponification treatment is required. This step not only reduces the efficiency and yield, but also the residual saponification agent It is easy to contaminate the lutein product, which increases the difficulty of the purification process. Secondly, planting plants such as marigolds and marigolds requires a large amount of land, and the growth cycle of these plants is generally 3 to 4 months (compared to the microalgae that can grow rapidly, the cycle is too long).
相比较而言，利用微藻来生产叶黄素具有某些优势。首先叶黄素在微藻中主要是以游离的形式存在，因此在生产过程中不需要皂化步骤；其次，微藻的生长周期短、生产设备占地面积小，同时利用微藻生产叶黄素不受季节、气候及地域条件限制，产品质量和产量相对稳定；最后，微藻(如小球藻等)本身就是一种高价值的产品，含有大量的蛋白质、油脂、多糖等活性成分，可以分离提取这些物质，实现微藻细胞的综合利用。In comparison, the use of microalgae to produce lutein has certain advantages. First of all, lutein mainly exists in the free form in microalgae, so no saponification step is required in the production process; secondly, the growth cycle of microalgae is short, the production equipment occupies a small area, and at the same time, microalgae is used to produce lutein Not limited by seasons, climate and geographical conditions, product quality and output are relatively stable; finally, microalgae (such as chlorella, etc.) itself is a high-value product, containing a large amount of active ingredients such as protein, oil, polysaccharide, etc. These substances are separated and extracted to realize the comprehensive utilization of microalgae cells.
至今，利用微藻产叶黄素的培养模式主要有光自养和异养两种。微藻光自养培养的缺点是微藻细胞生长速度慢、细胞密度及叶黄素产率低。目前光自养培养微藻的叶黄素最高体积产率为Sanchez J F等在2L圆柱式光生物反应器中培养Scenedesmus almeriensis所获得的4.8mg/L/d(Sanchez J F，Fernandez-Sevilla J M，Acien F G.，et al.Biomass and lutein productivity ofScenedesmus almeriensis：influence of irradiance，dilution rate and temperature.Appl Microbiol Biotechnol，2008，79：719～729)，叶黄素的最高面积产率为Fernandez等在4000L管道式反应器中培养Scenedesmus almeriensis所达到的290mg/m2/d(Del Campo J A，Mercedes Garcia González，Guerrero M G.Outdoorcultivation of microalgae for carotenoid production：current state and perspectives.Appl Microbiol Biotechnol，2007，74：1163～1174)。So far, there are two main culture modes for microalgae to produce lutein: photoautotrophic and heterotrophic. The disadvantages of microalgae photoautotrophic culture are the slow growth rate of microalgae cells, low cell density and low lutein yield. At present, the highest volumetric yield of lutein in photoautotrophic cultured microalgae is 4.8 mg/L/d obtained by Sanchez J F et al. in a 2L cylindrical photobioreactor (Sanchez J F, Fernandez-Sevilla J M, Acien F G., et al. Biomass and lutein productivity of Scenedesmus almeriensis: influence of irradiance, dilution rate and temperature. Appl Microbiol Biotechnol, 2008, 79: 719～729), the highest area yield of lutein is Fernandez et al. in the 4000L pipeline The 290 mg/m2 /d achieved by cultivating Scenedesmus almeriensis in a type reactor (Del Campo J A, Mercedes Garcia González, Guerrero M G. Outdoor cultivation of microalgae for carotenoid production: current state and perspectives. Appl Microbiol Biotechnol, 2007, 74: 1163 ~1174).
微藻异养培养的优点是微藻能够在生物反应器中进行高密度培养，细胞生长速率快，但存在细胞内叶黄素、叶绿素等色素和蛋白质含量低等缺点。异养培养能够获得高细胞密度和高细胞生长速度，从而使得叶黄素的产率相对于光自养培养有很大提高。The advantage of heterotrophic culture of microalgae is that microalgae can be cultured at high density in a bioreactor, and the cell growth rate is fast, but there are disadvantages such as low content of pigments and proteins such as lutein and chlorophyll in the cells. Heterotrophic culture can obtain high cell density and high cell growth rate, so that the yield of lutein is greatly improved compared with photoautotrophic culture.
微藻除异养培养和光自养培养模式外，还有一种不常用的培养模式即混合营养培养。迄今，微藻培养产叶黄素的最高体积产率(145mg/L/d)和胞内叶黄素的最高含量(7.6mg/g)均是在连续光照的混合营养培养条件下获得的(Martin，Lucia.Process for obtaining lutein from algae.EP180843A1，2007)。但该培养模式只能在可蒸汽灭菌的封闭式光生物反应器中进行，且培养过程必须保证绝对的无菌，同时需要光源的合理配置，这在实际生产中无法实现。因此，利用混合营养模式培养微藻生产叶黄素不具有产业化价值。In addition to the heterotrophic culture and photoautotrophic culture mode of microalgae, there is also an uncommon culture mode, that is, mixed trophic culture. So far, the highest volumetric yield (145mg/L/d) and the highest intracellular lutein content (7.6mg/g) of lutein produced by microalgae culture were obtained under the condition of mixed nutrient culture with continuous light ( Martin, Lucia. Process for obtaining lutein from algae. EP180843A1, 2007). However, this culture mode can only be carried out in a closed photobioreactor that can be steam sterilized, and the culture process must ensure absolute sterility, and requires a reasonable configuration of light sources, which cannot be realized in actual production. Therefore, it is not of industrial value to cultivate microalgae to produce lutein in mixed nutrition mode.
由上述可见，无论是采用光自养培养模式还是异养培养模式，较低的胞内叶黄素含量和叶黄素产率，同时加上微藻大规模培养较高的成本，制约了应用微藻培养来生产叶黄素的产业化进程。因此，有必要探索新的微藻培养工艺或方法使叶黄素产率及含量大幅度提高，同时又使微藻大规模培养的成本大幅度下降，这样才能够满足利用微藻培养产叶黄素的产业化要求。It can be seen from the above that whether the photoautotrophic culture mode or the heterotrophic culture mode is adopted, the low intracellular lutein content and lutein yield, coupled with the high cost of large-scale culture of microalgae, restrict the application. Industrial process of microalgae culture to produce lutein. Therefore, it is necessary to explore a new microalgae culture process or method to greatly increase the yield and content of lutein, and at the same time greatly reduce the cost of large-scale microalgae culture, so that the use of microalgae to produce lutein can be satisfied. The industrialization requirements of the element.
综合上述几种产叶黄素微藻培养模式的优缺点，本发明设计了一种用于产叶黄素微藻培养的“异养-稀释-光诱导串联培养”模式，其流程如下：首先利用生物反应器异养培养产叶黄素微藻以获得高密度细胞，待培养液中有机碳源消耗完毕后，用不含有机碳源的培养基稀释藻液，经短时间内(8～24h)的光照诱导，使藻细胞内的叶黄素快速大量积累。该模式中的异养阶段是在摇瓶、机械搅拌式、气升式、鼓泡式等可异养培养的生物反应器中进行，目的是为了在短时间内获得较高密度的藻细胞；光诱导阶段可在任何可用于微藻光自养培养的系统中进行，目的是通过光诱导作用提高胞内叶黄素含量；异养阶段与光诱导阶段分别独立进行，异养阶段放出的藻液先用光诱导培养基进行稀释后再转入光诱导培养阶段。过程中需确保进入光诱导阶段的藻液中不含有机碳源，这样可避免光诱导阶段滋生过多的杂菌；而通过稀释作用可以确保光诱导阶段的藻细胞能获得充足的光照，实现胞内叶黄素含量的快速提高。Based on the advantages and disadvantages of the above-mentioned several lutein-producing microalgae culture modes, the present invention designs a "heterotrophic-dilution-light-induced series culture" mode for the lutein-producing microalgae culture. The process is as follows: first, use a bioreactor Heterotrophic culture of lutein-producing microalgae to obtain high-density cells. After the organic carbon source in the culture solution is consumed, the algae solution is diluted with a medium without organic carbon source, and induced by light for a short period of time (8-24h). Rapid and massive accumulation of lutein in algae cells. The heterotrophic stage in this model is carried out in bioreactors such as shake flask, mechanical stirring type, airlift type, bubbling type, etc., which can be cultured heterotrophically, in order to obtain higher density of algae cells in a short time; The light-induced stage can be carried out in any system that can be used for microalgae photoautotrophic culture, the purpose is to increase the intracellular lutein content through light-induced effect; the heterotrophic stage and the light-induced stage are carried out independently, and the algae released from the heterotrophic stage The solution was first diluted with light-induced culture medium and then transferred to the light-induced culture stage. In the process, it is necessary to ensure that the algae liquid entering the light induction stage does not contain organic carbon sources, so as to avoid the excessive growth of bacteria in the light induction stage; and through dilution, it can ensure that the algae cells in the light induction stage can receive sufficient light to achieve Rapid increase in intracellular lutein content.
本发明将微藻培养法生产叶黄素分成藻体生长和产物(叶黄素)积累两个阶段，即异养培养和光诱导培养。通过异养培养可在短时间内获得大量的可产叶黄素的微藻细胞，藻液稀释后转入光诱导培养，藻体内叶黄素含量迅速提升至初始的两倍甚至两倍以上。本发明具有如下优势：(1)光诱导的藻细胞密度很高(2～10g/L)，是常规光自养培养藻细胞密度(约0.2～1g/L)的10倍左右；(2)光诱导时间很短(约1d～2d)，而微藻光自养培养时间很长(约7d～14d)，因此，叶黄素的生产效率大为提高；(3)相对于微藻光自养培养，光诱导时较高的藻细胞密度使得光诱导所需的占地面积很小，同时高细胞密度使得采收成本大幅度降低；(4)异养培养几乎不受气候、天气的影响，光诱导培养可以在玻璃房内，自然光照或人工光照条件下进行，因而，采用本发明的方法可以实现叶黄素的连续生产；(5)分离提取叶黄素后的剩余藻体的综合利用，可获得额外的产品和经济效益，降低了叶黄素的综合生产成本。The invention divides the production of lutein by the microalgae culture method into two stages of algae body growth and product (lutein) accumulation, that is, heterotrophic culture and light-induced culture. Through heterotrophic culture, a large number of lutein-producing microalgal cells can be obtained in a short period of time. After the algae liquid is diluted, it is transferred to light-induced culture, and the lutein content in the algae rapidly increases to twice or even more than the initial value. The present invention has the following advantages: (1) the light-induced algae cell density is very high (2-10g/L), which is about 10 times that of conventional photoautotrophic cultured algae cell density (about 0.2-1g/L); (2) The light induction time is very short (about 1d~2d), while the photoautotrophic culture time of microalgae is very long (about 7d~14d), so the production efficiency of lutein is greatly improved; (3) Compared with microalgae photoautotrophic Higher algae cell density during light induction makes the area required for light induction very small, and the high cell density greatly reduces the harvesting cost; (4) Heterotrophic culture is almost not affected by climate and weather , light-induced culture can be carried out in a glass house under natural light or artificial light conditions, therefore, the continuous production of lutein can be realized by adopting the method of the present invention; (5) synthesis of remaining algal bodies after separating and extracting lutein Utilization, additional products and economic benefits can be obtained, and the comprehensive production cost of lutein is reduced.
综述所述，本发明的培养模式合理地结合了异养和光自养培养两种方式的各自优势，与其它模式相比，具有生产效率高、培养系统的组合方式灵活和生产成本低等优点，可充分发挥异养模式可获得高密度藻液和光诱导阶段叶黄素快速积累的优势，为解决源于微藻的叶黄素产业化问题提供重要的技术手段。In summary, the culture mode of the present invention rationally combines the respective advantages of heterotrophic and photoautotrophic culture modes. Compared with other modes, it has the advantages of high production efficiency, flexible combination of culture systems and low production costs. The heterotrophic model can give full play to the advantages of high-density algae liquid and rapid accumulation of lutein in the light-induced stage, providing an important technical means for solving the industrialization of lutein from microalgae.
发明内容Contents of the invention
本发明一方面提供一种快速积累微藻胞内叶黄素的方法，该方法包括微藻的异养培养步骤，将所获得的微藻异养培养液稀释后进行光诱导培养的步骤，以及任选的藻细胞采收、叶黄素分离提取的步骤。One aspect of the present invention provides a method for rapidly accumulating intracellular lutein in microalgae, the method comprising a step of heterotrophic culture of microalgae, a step of diluting the obtained heterotrophic culture solution of microalgae and performing light-induced culture, and Optional steps of harvesting algae cells and separating and extracting lutein.
本发明另一方面提供一种源于微藻的叶黄素生产方法，该方法包括微藻异养的步骤，将微藻的异养培养液稀释后进行光诱导培养的步骤，以及藻细胞采收、叶黄素分离提取的步骤。Another aspect of the present invention provides a method for producing lutein derived from microalgae. The method includes the step of microalgae heterotrophy, the step of diluting the heterotrophic culture solution of microalgae and then performing light-induced culture, and collecting algae cells. The steps of collecting and separating and extracting lutein.
本发明的方法能够实现胞内叶黄素的快速积累，大大提高了生产效率，降低了生产成本，且提供了高品质的叶黄素。The method of the invention can realize rapid accumulation of intracellular lutein, greatly improves production efficiency, reduces production cost, and provides high-quality lutein.
在一个具体实施方式中，所述的微藻选自：蛋白核小球藻(Chlorellapyrenoidosa)、普通小球藻(Chlorella vulgaris)、海水小球藻(Marine chlorella)、原壳小球藻(Chlorella protothecoides)、椭圆小球藻(Chlorella ellipsoidea)、Chlorella zofingiensis、Chlorella regularis、Chlorella sorokina、Spongiococcumexcentricum和Chlamydomonas agloeformis。In a specific embodiment, the microalgae are selected from the group consisting of Chlorella pyrenoidosa, Chlorella vulgaris, Marine chlorella, Chlorella protothecoides ), Chlorella ellipsoidea, Chlorella zofingiensis, Chlorella regularis, Chlorella sorokina, Spongiococcumexcentricum and Chlamydomonas agloeformis.
在一个具体实施方式中，所述微藻异养培养的步骤包括：在生物反应器中加入pH为4.0～9.0的培养基，按工作体积的0.1～30％接入微藻藻种进行分批培养、补料分批培养、半连续培养或连续培养，培养温度为10～40℃，控制pH小于9.0，控制溶氧在1％以上。In a specific embodiment, the step of heterotrophic culture of microalgae includes: adding a medium with a pH of 4.0 to 9.0 into the bioreactor, inserting microalgae species according to 0.1 to 30% of the working volume for batch For culture, fed-batch culture, semi-continuous culture or continuous culture, the culture temperature is 10-40°C, the pH is controlled to be less than 9.0, and the dissolved oxygen is controlled to be above 1%.
在一个具体实施方式中，所述光诱导培养包括将稀释后的藻液转入光诱导装置中进行光诱导，连续光照或间歇光照，培养温度为5～50℃，光照强度为0.1～150klx，光诱导培养周期为1～150小时。In a specific embodiment, the light-induced culture includes transferring the diluted algae liquid into a light-induced device for light-induced induction, continuous or intermittent light, the culture temperature is 5-50°C, and the light intensity is 0.1-150klx. The light-induced culture period is 1-150 hours.
在一个具体实施方式中，异养培养基由氮源、有机碳源以及少量无机盐、微量元素和水组成；光诱导培养基由氮源、无机盐和水组成。In a specific embodiment, the heterotrophic medium is composed of nitrogen source, organic carbon source, a small amount of inorganic salt, trace elements and water; the light-induced medium is composed of nitrogen source, inorganic salt and water.
在一个具体实施方式中，所述异养步骤在摇瓶、机械搅拌式、气升式或鼓泡式可异养培养的生物反应器中进行，所述光诱导培养步骤在摇瓶或敞开式的跑道池或圆池、封闭式的平板式光生物反应器或管道式光生物反应器或柱式光生物反应器或薄膜立袋与吊袋光生物反应器等任何可用于微藻光自养培养的装置中进行，光源为自然光或各种人工光。In a specific embodiment, the heterotrophic step is carried out in a shake flask, mechanically stirred, air-lift or bubbling bioreactor capable of heterotrophic culture, and the light-induced culture step is carried out in a shake flask or an open The runway pool or round pool, closed flat plate photobioreactor or pipeline photobioreactor or column photobioreactor or film vertical bag and hanging bag photobioreactor, etc. can be used for microalgae photoautotrophy The cultivation is carried out in a device, and the light source is natural light or various artificial lights.
在一个具体实施方式中，当产叶黄素藻种为普通小球藻时，异养所使用的培养基基本上由以下成分组成：KNO3 5～15克/升、葡萄糖10～60克/升、KH2PO40.3～0.9克/升、Na2HPO4·12H2O 1.0～10.0克/升、MgSO4·7H2O 0.2～1.0克/升、CaCl20.05～0.3克/升、FeSO4·7H2O 0.01～0.05克/升、微量元素0.5～4ml和水，其中微量元素的组成为H3BO3 5～15克/升、ZnSO4·7H2O 5.0～10.0克/升、MnCl2·H2O1.0～2.0克/升、(NH4)6Mo7O24·4H2O 0.5～1.5克/升、CuSO4·5H2O 1.0～2.0克/升和Co(NO3)2·6H2O 0.1～0.9克/升。In a specific embodiment, when the xanthophyll-producing algae species is Chlorella vulgaris, the culture medium used for heterotrophy basically consists of the following components: KNO3 5-15 g/L, glucose 10-60 g/L, KH2 PO4 0.3～0.9 g/L, Na2 HPO4 12H2 O 1.0～10.0 g/L, MgSO4 7H2 O 0.2～1.0 g/L, CaCl2 0.05～0.3 g/L, FeSO4 ·7H2 O 0.01～0.05g/L, trace elements 0.5～4ml and water, wherein the composition of trace elements is H3 BO3 5～15g/L, ZnSO4 ·7H2 O 5.0～10.0g/L, MnCl2 ·H2 O 1.0～2.0 g/L, (NH4 )6 Mo7 O24 ·4H2 O 0.5～1.5 g/L, CuSO4 ·5H2 O 1.0～2.0 g/L and Co(NO3 )2 ·6H2 O 0.1-0.9 g/L.
在一个具体实施方式中，当小球藻为蛋白核小球藻时，异养所使用的培养基基本上由以下成分组成：葡萄糖10～60克/升、尿素2～8克/升、KH2PO4 1～2克/升、Na2HPO4·12H2O 1.0～10.0克/升、MgSO4·7H2O 1～2克/升、CaCl2 0.05～0.1克/升、柠檬酸三钠0.1～2.0克/升、Fe-EDTA溶液0.5～1mL、A5溶液1～5mL和水；其中Fe-EDTA溶液配方为FeSO4·7H2O 20～30克/升和EDTA 20～40克/升；A5溶液配方为H3BO3 2.5～4.0克/升、MnCl2·4H2O 1.0～2.0克/升、ZnSO4·7H2O0.1～0.6克/升、CuSO4·5H2O 5～10克/升和Na2MoO4 0.01～0.05克/升。In a specific embodiment, when the chlorella is Chlorella pyrenoidosa, the culture medium used for heterotrophy basically consists of the following components: glucose 10-60 g/L, urea 2-8 g/L, KH2 PO4 1~2 g/L, Na2 HPO4 12H2 O 1.0~10.0 g/L, MgSO4 7H2 O 1~2 g/L, CaCl2 0.05~0.1 g/L, tricitric acid Sodium 0.1-2.0 g/L, Fe-EDTA solution 0.5-1mL, A5 solution 1-5mL and water; the formula of Fe-EDTA solution is FeSO4 7H2 O 20-30 g/L and EDTA 20-40 g/L liter; A5 solution formula is H3 BO3 2.5-4.0 g/L, MnCl2 4H2 O 1.0-2.0 g/L, ZnSO4 7H2 O 0.1-0.6 g/L, CuSO4 5H2 O 5-10 g/L and Na2 MoO4 0.01-0.05 g/L.
在一个具体实施方式中，采用超临界CO2萃取法、有机溶剂提取法或超声辅助溶剂提取法提取叶黄素。In a specific embodiment, the lutein is extracted by supercritical CO2 extraction, organic solvent extraction or ultrasonic-assisted solvent extraction.
在一个具体实施方式中，本发明的方法还包括：将提取叶黄素后的藻体与其他色素混合进行喷雾干燥制成藻粉，或对藻体中的生物活性物质进行分离提取。In a specific embodiment, the method of the present invention further includes: mixing the algal body after extracting lutein with other pigments and spray drying to make algal powder, or separating and extracting the biologically active substances in the algae body.
在一个具体实施方式中，所述其他色素包括叶绿素。在一个具体实施方式中，所述生物活性物质包括蛋白质、叶绿素和多糖。In a specific embodiment, said other pigments include chlorophyll. In a specific embodiment, the biologically active substances include proteins, chlorophyll and polysaccharides.
附图说明Description of drawings
图1和图2显示5L生物反应器/3L平板式光生物反应器串联系统中采用异养-稀释-光诱导串联培养蛋白核小球藻生产叶黄素的过程。其中，图1显示蛋白核小球藻在5L生物反应器异养培养过程；图2显示蛋白核小球藻在3L平板式光生物反应器内光诱导培养过程。Figure 1 and Figure 2 show the process of producing lutein by using heterotrophic-dilution-light-induced serial culture of Chlorella pyrenoidosa in a 5L bioreactor/3L flat-plate photobioreactor series system. Wherein, Fig. 1 shows the heterotrophic culture process of Chlorella pyrenoidosa in a 5L bioreactor; Fig. 2 shows the light-induced culture process of Chlorella pyrenoidosa in a 3L flat-plate photobioreactor.
图3和图4显示50L生物反应器/10L圆柱式光生物反应器串联系统中采用异养-稀释-光诱导串联培养蛋白核小球藻生产叶黄素的过程。其中，图3显示蛋白核小球藻在50L生物反应器异养培养过程；图4显示蛋白核小球藻在10L圆柱式光生物反应器内光诱导培养过程。Figure 3 and Figure 4 show the process of producing lutein by heterotrophic-dilution-light-induced serial culture of Chlorella pyrenoidosa in a 50L bioreactor/10L cylindrical photobioreactor series system. Wherein, Fig. 3 shows the heterotrophic culture process of Chlorella pyrenoidosa in a 50L bioreactor; Fig. 4 shows the light-induced culture process of Chlorella pyrenoidosa in a 10L cylindrical photobioreactor.
图5和图6显示50L生物反应器/30L平板式光生物反应器串联系统中采用异养-稀释-光诱导串联培养普通小球藻生产叶黄素的过程。其中，图5显示普通小球藻在50L生物反应器中异养培养过程；图6显示普通小球藻在户外30L平板式光生物反应器内光诱导培养。Figure 5 and Figure 6 show the process of producing lutein by heterotrophic-dilution-light-induced serial culture of Chlorella vulgaris in a 50L bioreactor/30L flat-plate photobioreactor series system. Among them, Figure 5 shows the heterotrophic culture process of Chlorella vulgaris in a 50L bioreactor; Figure 6 shows the light-induced culture of Chlorella vulgaris in an outdoor 30L flat-plate photobioreactor.
图7和图8显示5L生物反应器/3L平板式生物光生物反应器串联系统中采用异养-稀释-光诱导串联培养椭圆小球藻生产叶黄素的过程。其中，图7显示椭圆小球藻在5L生物反应器异养培养过程；图8显示椭圆小球藻在3L平板式光生物反应器中光诱导过程。Figure 7 and Figure 8 show the process of producing lutein by using heterotrophic-dilution-light-induced serial culture of Chlorella ellipsoides in a 5L bioreactor/3L flat-plate biophotobioreactor series system. Wherein, Fig. 7 shows the process of heterotrophic culture of Chlorella ellipsoid in a 5L bioreactor; Fig. 8 shows the process of photoinduction of Chlorella ellipsoid in a 3L flat photobioreactor.
具体实施方案specific implementation plan
适用于本申请的微藻包括但不限于蛋白核小球藻(Chlorella pyrenoidosa)、普通小球藻(Chlorella vulgaris)、海水小球藻(Marine chlorella)、原壳小球藻(Chlorella protothecoides)、椭圆小球藻(Chlorella ellipsoidea)、Chlorellazofingiensis、Chlorella regularis、Chlorella sorokina、Spongiococcum excentricum和Chlamydomonas agloeformis。Microalgae suitable for this application include, but are not limited to, Chlorella pyrenoidosa, Chlorella vulgaris, Marine chlorella, Chlorella protothecoides, Chlorella ellipsoidea, Chlorella zofingiensis, Chlorella regularis, Chlorella sorokina, Spongiococcum excentricum, and Chlamydomonas agloeformis.
在优选的实施方式中，本发明采用蛋白核小球藻、普通小球藻和椭圆小球藻来生产叶黄素。In a preferred embodiment, the present invention uses Chlorella pyrenoidosa, Chlorella vulgaris and Chlorella ellipsoides to produce lutein.
1.微藻在生物反应器中的高密度异养培养1. High-density heterotrophic culture of microalgae in bioreactors
此步骤的目的是为了快速获得大量藻细胞，以供光诱导阶段快速合成积累叶黄素。The purpose of this step is to quickly obtain a large number of algal cells for the rapid synthesis and accumulation of lutein in the light-induced stage.
可采用本领域熟知的各种培养基来进行微藻异养培养。通常，异养培养基含有氮源、有机碳源、少量无机盐、微量元素和水。Various media well known in the art can be used for heterotrophic culture of microalgae. Typically, a heterotrophic medium contains a nitrogen source, an organic carbon source, a small amount of inorganic salts, trace elements and water.
这类培养基包括HA-SK培养基(中国专利ZL 200610024004.9)、Endo培养基(Ogbonna J.C.，Masui.H.，Tanaka.H.Sequential heterotrophic：autotrophiccultivation-an efficient method of producing Chlorella biomass for health foodand animal feed.J.Appl.Phycol.1997，9，359～366)等。This type of medium includes HA-SK medium (Chinese patent ZL 200610024004.9), Endo medium (Ogbonna J.C., Masui.H., Tanaka.H. Sequential heterotrophic: autotrophiccultivation-an efficient method of producing Chlorella biomass for health food and animal feed .J.Appl.Phycol.1997, 9, 359-366) and so on.
本发明所用的HA-SK培养基基本上由KNO3、葡萄糖以及少量无机盐、微量元素和水组成。在所述技术方案中，所述微量元素宜选自H3BO3、ZnSO4·7H2O、MnCl2·H2O，(NH4)6Mo7O24·4H2O、CuSO4·5H2O和Co(NO3)2·6H2O中的一种、多种或全部。The HA-SK medium used in the present invention basically consists of KNO3 , glucose, a small amount of inorganic salts, trace elements and water. In the technical solution, the trace elements are preferably selected from H3 BO3 , ZnSO4 ·7H2 O, MnCl2 ·H2 O, (NH4 )6 Mo7 O24 ·4H2 O, CuSO4 · One, more or all of 5H2 O and Co(NO3 )2 ·6H2 O.
本文所用的术语“基本上由……组成”表示本发明的组合物中除了含有主要组分KNO3、葡萄糖以及少量无机盐、微量元素和水外，还可包含一些对于组合物的基本特性或新的特性(即可维持微藻在较短的培养周期内细胞密度达到较高的水平，同时活性物质含量与常规异养培养相比有较大幅度提高)没有实质上影响的组分。本文所用的术语“由……组成”表示本发明的组合物由所指出的具体组分组成，没有其他组分，但是可以带有含量在通常范围内的杂质。The term "consisting essentially of" as used herein means that in addition to the main components KNO3 , glucose and a small amount of inorganic salts, trace elements and water, the composition of the present invention may also contain some essential properties or The new characteristics (i.e. maintain the cell density of microalgae to reach a higher level in a shorter culture period, and at the same time, the active substance content is greatly increased compared with conventional heterotrophic culture) have no substantial influence on the components. As used herein, the term "consisting of" means that the composition of the present invention consists of the specified components indicated, and no other components, but may contain impurities in the usual range.
在该培养基中，培养基的各组分可在一定范围内变化而不会对微藻细胞密度和品质有很大的实质影响。因此，这些组分的用量不应受实施例的严格限制。如本领域技术人员所熟知的，培养基中还可加入少量无机盐，例如硫酸镁、氯化钙、硫酸亚铁和磷酸盐等，以及少量微量元素如Mn、Zn、B、I、M、Cu、Co等。In this culture medium, each component of the culture medium can be varied within a certain range without substantial influence on the microalgae cell density and quality. Therefore, the amounts of these components used should not be strictly limited by Examples. As well known to those skilled in the art, a small amount of inorganic salts, such as magnesium sulfate, calcium chloride, ferrous sulfate and phosphate, etc., and a small amount of trace elements such as Mn, Zn, B, I, M, Cu, Co, etc.
在本发明中，较佳的微量元素组分宜选自H3BO3、ZnSO4·7H2O、MnCl2·H2O、(NH4)6Mo7O24·4H2O、CuSO4·5H2O和Co(NO3)2·6H2O中的一种或多种。无机盐和微量元素的用量可根据常规知识确定。In the present invention, the preferred trace element components are preferably selected from H3 BO3 , ZnSO4 ·7H2 O, MnCl2 ·H2 O, (NH4 )6 Mo7 O24 ·4H2 O, CuSO4 • One or more of 5H2 O and Co(NO3 )2 •6H2 O. The amount of inorganic salts and trace elements can be determined according to conventional knowledge.
本发明所采用的HA-SK培养基基本上由以下成分组成：KNO3 5～15克/升、葡萄糖10～60克/升、KH2PO4 0.3～0.9克/升、Na2HPO4·12H2O 1.0～10.0克/升、MgSO4·7H2O 0.2～1.0克/升、CaCl2 0.05～0.3克/升、FeSO4·7H2O 0.01～0.05克/升；微量元素0.5～4ml，其中微量元素的组成为H3BO3 5～15克/升、ZnSO4·7H2O5.0～10.0克/升、MnCl2·H2O 1.0～2.0克/升、(NH4)6Mo7O24·4H2O 0.5～1.5克/升、CuSO4·5H2O 1.0～2.0克/升和Co(NO3)2·6H2O 0.1～0.9克/升；水。The HA-SK medium used in the present invention basically consists of the following components: KNO3 5-15 g/L, glucose 10-60 g/L, KH2 PO4 0.3-0.9 g/L, Na2 HPO4 · 12H2 O 1.0～10.0 g/L, MgSO4 7H2 O 0.2～1.0 g/L, CaCl2 0.05～0.3 g/L, FeSO4 7H2 O 0.01～0.05 g/L; trace elements 0.5～4ml , wherein the composition of trace elements is H3 BO3 5-15 g/L, ZnSO4 ·7H2 O 5.0-10.0 g/L, MnCl2 ·H2 O 1.0-2.0 g/L, (NH4 )6 Mo7 O24 ·4H2 O 0.5-1.5 g/l, CuSO4 ·5H2 O 1.0-2.0 g/l and Co(NO3 )2 ·6H2 O 0.1-0.9 g/l; water.
本发明所用的Endo培养基基本上由以下成分组成：葡萄糖10～60克/升、尿素2～8克/升、KH2PO4 1～2克/升、Na2HPO4·12H2O 1.0～10.0克/升、MgSO4·7H2O1～2克/升、CaCl2 0.05～0.1克/升、柠檬酸三钠0.1～2.0克/升、Fe-EDTA溶液0.5～1mL，A5溶液1～5mL；其中Fe-EDTA溶液配方为FeSO4·7H2O 20～30克/升和EDTA 20～40克/升；A5溶液配方为H3BO3 2.5～4.0克/升、MnCl2·4H2O 1.0～2.0克/升、ZnSO4·7H2O 0.1～0.6克/升、CuSO4·5H2O 5～10克/升和Na2MoO4 0.01～0.05克/升；水。The Endo medium used in the present invention basically consists of the following components: glucose 10-60 g/L, urea 2-8 g/L, KH2 PO4 1-2 g/L, Na2 HPO4 ·12H2 O 1.0 ~10.0 g/L, MgSO4 7H2 O1~2 g/L, CaCl2 0.05~0.1 g/L, trisodium citrate 0.1~2.0 g/L, Fe-EDTA solution 0.5~1mL, A5 solution 1~ 5mL; the formula of Fe-EDTA solution is FeSO4 7H2 O 20-30 g/L and EDTA 20-40 g/L; the formula of A5 solution is H3 BO3 2.5-4.0 g/L, MnCl2 4H2 O 1.0-2.0 g/L, ZnSO4 ·7H2 O 0.1-0.6 g/L, CuSO4 ·5H2 O 5-10 g/L and Na2 MoO4 0.01-0.05 g/L; water.
在根据上述配方配制培养基后，可用常规手段如酸或碱将所述培养基的pH调为4.0～9.0，并在115～120℃下高压灭菌15～20分钟。可采用分批、补料分批、半连续和连续培养等多种方式实施异养培养。After preparing the culture medium according to the above formula, the pH of the culture medium can be adjusted to 4.0-9.0 by conventional means such as acid or alkali, and autoclaved at 115-120° C. for 15-20 minutes. Heterotrophic culture can be implemented in various ways such as batch, fed-batch, semi-continuous and continuous culture.
当异养培养采用补料分批培养方式时，将相应配制好的培养基加入到生物反应器中，补加水至工作体积，通常装料系数为0.6～0.8，然后蒸汽灭菌(121℃，维持约20分钟)，当温度降至30～35℃时，按工作体积的1～15％接入微藻藻种开始异养培养。When the fed-batch culture method is adopted for heterotrophic culture, the correspondingly prepared medium is added to the bioreactor, and water is added to the working volume, usually with a filling coefficient of 0.6-0.8, and then steam sterilized (121°C, maintain for about 20 minutes), when the temperature drops to 30-35° C., insert microalgae and algal species according to 1-15% of the working volume to start heterotrophic culture.
在异养培养一段时间后，当培养基中葡萄糖被消耗完(通常为27～45小时)时需要进行补料，补加碳源(如葡萄糖)、氮源(如，培养普通小球藻的氮源为KNO3、培养蛋白核小球藻的氮源为尿素)和无机盐等营养盐，补加的营养盐是经浓缩后的上述相应培养基，促使微藻继续生长。可每隔5～8小时补料，葡萄糖的补加浓度可为15～25克/升，氮源溶液的补加浓度可为2～5克/升。当补加一定次数(一般为4～7次)的营养盐后，微藻细胞密度达到最高时，异养培养阶段结束。After a period of heterotrophic culture, when the glucose in the medium is consumed (usually 27 to 45 hours), feeding needs to be carried out, and a carbon source (such as glucose) and a nitrogen source (such as, for cultivating Chlorella vulgaris) need to be added. The nitrogen source is KNO3 , and the nitrogen source for cultivating Chlorella pyrenoidosa is urea) and inorganic salts and other nutrient salts, and the added nutrient salts are the concentrated above-mentioned corresponding medium to promote the continued growth of microalgae. Feeds can be fed every 5-8 hours, the supplementary concentration of glucose can be 15-25 grams/liter, and the supplementary concentration of nitrogen source solution can be 2-5 grams/liter. When the nutrient salt is supplemented for a certain number of times (generally 4 to 7 times), the microalgae cell density reaches the highest level, and the heterotrophic culture stage ends.
无论采用何种培养方式，在培养过程中，须控制适合的培养条件使微藻正常生长。通常，控制温度为20～35℃，例如28～30℃，溶氧不低于5％的空气饱和浓度，pH不高于9.0。在优选的实施例中，溶氧不低于10％的空气饱和浓度，pH不高于8.5。Regardless of the culture method adopted, during the culture process, suitable culture conditions must be controlled to allow the normal growth of microalgae. Usually, the controlled temperature is 20-35°C, for example, 28-30°C, the dissolved oxygen is not lower than 5% of the air saturation concentration, and the pH is not higher than 9.0. In a preferred embodiment, the dissolved oxygen is not lower than 10% of the air saturation concentration, and the pH is not higher than 8.5.
通常，当采用分批培养、补料分批培养时，异养培养阶段结束时生物反应器中有机碳源需完全消耗完。当采用半连续培养方式时，带放操作是在生物反应器中有机碳源完全消耗完时进行。Generally, when batch culture or fed-batch culture is adopted, the organic carbon source in the bioreactor needs to be completely consumed at the end of the heterotrophic culture stage. When the semi-continuous culture method is adopted, the belt release operation is carried out when the organic carbon source in the bioreactor is completely consumed.
异养可以在摇瓶、机械搅拌式、气升式、鼓泡式等可异养培养的生物反应器中进行。Heterotrophy can be carried out in shake flasks, mechanically stirred, airlift, bubbling and other bioreactors that can be cultured heterotrophically.
2.高浓度藻液的稀释2. Dilution of high concentration algae liquid
此步骤的目的是为了使转入光诱导培养的产叶黄素微藻高效地吸收光能，提高光能利用效率，同时降低藻细胞的死亡率。因为光强在藻液中呈“时、空、非线性”衰减，所以在高细胞密度下，反应器中藻细胞大部分处于暗区，几乎接受不到光照，这样藻细胞很容易死亡而且会影响光诱导的效率。The purpose of this step is to make the xanthophyll-producing microalgae transferred to light-induced culture absorb light energy efficiently, improve light energy utilization efficiency, and reduce the death rate of algae cells. Because the light intensity decays in "time, space, and nonlinear" in the algae liquid, under high cell density, most of the algae cells in the reactor are in the dark area and hardly receive light, so the algae cells are easy to die and will be damaged. Affects photoinduced efficiency.
异养培养获得的高密度藻液应进行稀释操作，用水和不含有机碳源的培养基对高密度的藻液进行稀释，使细胞密度维持在0.1～10克/升，调节pH至5.0～8.0。在其它实施例中，稀释藻液，使细胞密度维持在1～8克/升。在优选的实施例中，使细胞密度维持在1.0～5.0克/升。The high-density algae liquid obtained from heterotrophic culture should be diluted, and the high-density algae liquid should be diluted with water and a medium without organic carbon source to maintain the cell density at 0.1-10 g/L and adjust the pH to 5.0- 8.0. In other embodiments, the algae fluid is diluted to maintain a cell density of 1-8 g/L. In a preferred embodiment, the cell density is maintained at 1.0-5.0 g/L.
可采用各种已知的稀释培养基来稀释藻液。通常，光诱导培养基由氮源、无机盐和水组成，相对于异养培养基不含有有机碳源。在优选的实施方案中，所述培养基的氮源浓度为2～10克/升，较佳为2～8克/升。所述氮源可以与异养培养步骤中所用的氮源相同或不同。Various known dilution media can be used to dilute the algae solution. Typically, light-induced media consist of nitrogen sources, inorganic salts, and water, and contain no organic carbon sources relative to heterotrophic media. In a preferred embodiment, the nitrogen source concentration of the medium is 2-10 g/L, preferably 2-8 g/L. The nitrogen source may be the same as or different from that used in the heterotrophic culture step.
在一个优选的具体方案中，异养培养获得的高密度藻细胞宜用不含有机碳源、氮源浓度为2～10克/升的初始培养基(例如，培养普通小球藻时采用不含葡萄糖的HA-SK培养基，培养蛋白核小球藻时采用不含葡萄糖的Endo培养基)进行适当稀释。In a preferred specific scheme, the high-density algae cells obtained by heterotrophic culture should use an initial medium that does not contain organic carbon sources and has a nitrogen source concentration of 2-10 g/liter (for example, when cultivating Chlorella vulgaris, different Glucose-containing HA-SK medium, when culturing Chlorella pyrenoidosa, use Endo medium without glucose) for appropriate dilution.
稀释采用的培养基无需高压灭菌，配制好后调节pH至5.0～8.0即可使用。The culture medium used for dilution does not need to be sterilized by autoclaving, and it can be used after adjusting the pH to 5.0-8.0 after preparation.
在具体实施方式中，对普通小球藻，稀释培养基(光诱导培养基)由以下成分组成：KNO3 1～8克/升、MgSO4·7H2O、0.5～1.0克/升、CaCl2 0.01～0.06克/升、FeSO4·7H2O 0.01～0.06克/升、EDTA 0.020～0.052克/升。In a specific embodiment, for Chlorella vulgaris, the diluted medium (light-induced medium) consists of the following components: KNO3 1-8 g/L, MgSO4 ·7H2 O, 0.5-1.0 g/L, CaCl2 0.01-0.06 g/L, FeSO4 ·7H2 O 0.01-0.06 g/L, EDTA 0.020-0.052 g/L.
对蛋白核小球藻，稀释培养基(光诱导培养基)由以下成分组成：尿素0.1～2.0克/升、MgSO4·7H2O、0.5～1.0克/升、CaCl2 0.01～0.06克/升、FeSO4·7H2O0.01～0.06、EDTA 0.020～0.052克/升、柠檬酸钠0.08～0.5克/升。For Chlorella pyrenoidosa, the diluted medium (light-induced medium) consists of the following components: urea 0.1-2.0 g/L, MgSO4 ·7H2 O, 0.5-1.0 g/L, CaCl2 0.01-0.06 g/L liter, FeSO4 ·7H2 O0.01～0.06, EDTA 0.020～0.052g/liter, sodium citrate 0.08～0.5g/liter.
3.光诱导培养3. Light-induced culture
该步骤的目的是让产叶黄素微藻接受充足的光照，通过光诱导使藻细胞快速大量合成积累叶黄素。The purpose of this step is to allow the lutein-producing microalgae to receive sufficient light, and induce the algal cells to rapidly synthesize and accumulate lutein in large quantities through light induction.
如上所述高密度微藻培养液经稀释后，将所得稀释液转入光诱导装置中进行光诱导培养。温度控制在5～50℃，光照强度为0.11～150klx，连续光照或间歇光照，光诱导培养周期为1～150小时，通气量为0.1～2.0vvm。其中所述的光生物反应器包括所有的封闭式光生物反应器(摇瓶、管道式、平板式、柱式、薄膜立袋与吊袋等)和所有的开放式光生物反应器(跑道池、圆池和鼓泡式大盆等)。After the above-mentioned high-density microalgae culture solution is diluted, the resulting dilution is transferred to a light-induced device for light-induced culture. The temperature is controlled at 5-50°C, the light intensity is 0.11-150klx, continuous light or intermittent light, the light-induced culture period is 1-150 hours, and the ventilation rate is 0.1-2.0vvm. The photobioreactors described therein include all closed photobioreactors (shake flask, pipeline, flat plate, column, film stand-up bag and hanging bag, etc.) and all open photobioreactors (runway pool) , round pool and bubbling large basin, etc.).
通常，培养温度可控制在15～35℃的范围内，例如18～35℃、20～35℃、20～30℃等。通常，光照强度为1～70klx，例如，1～60、1～50、1～40、1～30、1～20、1～10klx等，可视具体生产情况而定。通常，如通过气体造成藻液充分混合，则通气量可控制为0.15～2.0vvm，例如，0.2～1.8、0.5～1.5、0.8～1.5、1.0～1.5vvm等。在其它实施方式中，培养温度控制在10～50℃，光照强度为1～10klx，通气量为0.05～2.0vvm。Usually, the culture temperature can be controlled within the range of 15-35°C, such as 18-35°C, 20-35°C, 20-30°C, etc. Usually, the light intensity is 1-70klx, for example, 1-60, 1-50, 1-40, 1-30, 1-20, 1-10klx, etc., depending on the specific production situation. Usually, if the algae liquid is fully mixed by gas, the ventilation rate can be controlled to 0.15-2.0vvm, for example, 0.2-1.8, 0.5-1.5, 0.8-1.5, 1.0-1.5vvm, etc. In other embodiments, the culture temperature is controlled at 10-50° C., the light intensity is 1-10 klx, and the ventilation rate is 0.05-2.0 vvm.
在其它实施例中，光诱导培养周期为8～100小时，例如，根据实际的天气情况，光诱导培养周期可以为8～90小时、8～80小时、8～60小时、8～48小时、8～24小时不等；或者，光诱导培养周期可以为12～72小时、12～60小时、12～48小时、12～36小时、12～24小时不等或24～60小时、24～48小时不等。In other embodiments, the light-induced culture period is 8-100 hours, for example, according to the actual weather conditions, the light-induced culture period can be 8-90 hours, 8-80 hours, 8-60 hours, 8-48 hours, ranging from 8 to 24 hours; or, the light-induced culture period can be 12 to 72 hours, 12 to 60 hours, 12 to 48 hours, 12 to 36 hours, 12 to 24 hours, or 24 to 60 hours, 24 to 48 hours Hours vary.
在本申请中，“光诱导培养周期”包括了整个光诱导培养过程，例如，户外培养时光诱导培养周期包括夜晚没有光照的时间。In the present application, the "light-induced culture period" includes the entire light-induced culture process, for example, the light-induced culture period for outdoor culture includes the time when there is no light at night.
在本申请中，“光照时间”指使用本申请所述光照强度对微藻实施光诱导培养的时间，即该时间不包括夜晚没有光照的时间。在一些实施例中，光诱导培养步骤的光照时间为8～48小时，例如8～36小时、8～24小时、8～18小时、8～12小时、12～36小时、12～24小时不等，以及上述范围内的任意时长。In the present application, "illumination time" refers to the time when microalgae are subjected to light-induced culture using the light intensity described in the application, that is, the time does not include the time without light at night. In some embodiments, the illumination time of the light-induced culturing step is 8-48 hours, such as 8-36 hours, 8-24 hours, 8-18 hours, 8-12 hours, 12-36 hours, 12-24 hours, etc. etc., and any duration within the above range.
因此，本申请的光诱导培养步骤也包括光照时间为8～48小时范围内的光诱导培养步骤。可采用人工光照的方式进行光诱导培养，也可在户外利用自然光照的方式进行光诱导培养。Therefore, the light-induced culturing step in the present application also includes the light-induced culturing step with the light time in the range of 8 to 48 hours. The light-induced culture can be carried out by means of artificial light, or light-induced culture can be carried out outdoors by using natural light.
在具体实施方式中，当培养液中叶黄素浓度达到最高时，结束光诱导培养，收获藻细胞进行叶黄素的分离提取或直接采收藻细胞进行藻粉制备。In a specific embodiment, when the concentration of lutein in the culture solution reaches the highest level, the light-induced culture is terminated, and the algal cells are harvested for separation and extraction of lutein, or the algal cells are directly harvested for preparation of algal powder.
4.藻细胞采收、叶黄素分离提取及藻体综合利用4. Harvesting of algae cells, separation and extraction of lutein and comprehensive utilization of algae bodies
光诱导培养结束后，对小球藻进行离心采收，获得湿藻体。藻细胞的采收方法包括但不限于高速离心、絮凝，气浮或过滤等技术；藻细胞破壁方法包括但不限于藻体自溶、高压匀浆、酶水解、水相热解等湿法破壁方法。After the light-induced cultivation, the chlorella was harvested by centrifugation to obtain wet algal bodies. Algal cell harvesting methods include but not limited to high-speed centrifugation, flocculation, air flotation or filtration and other technologies; algal cell wall breaking methods include but not limited to algae autolysis, high-pressure homogenization, enzyme hydrolysis, aqueous phase pyrolysis and other wet methods Breaking method.
采用传统的有机溶剂提取法对小球藻进行叶黄素的提取。首先将有机溶剂加入到藻泥中进行萃取，然后进行搅拌离心获得上清液和藻体沉淀，对上清液进行减压浓缩、搅拌加水、过滤获得叶黄素晶体。Lutein was extracted from Chlorella algae by traditional organic solvent extraction. First, adding an organic solvent into the algae mud for extraction, then performing stirring and centrifugation to obtain a supernatant and algae precipitates, concentrating the supernatant under reduced pressure, stirring, adding water, and filtering to obtain lutein crystals.
上清液中的其他成分可逐步分离提取获得脂肪酸、叶绿素等，或直接将上清液中的所有成分与藻体沉淀混合喷雾干燥获得小球藻粉。Other components in the supernatant can be gradually separated and extracted to obtain fatty acids, chlorophyll, etc., or directly mix all the components in the supernatant with the algae sediment and spray dry to obtain chlorella powder.
在一个较佳的方案中，采用超临界CO2萃取技术对小球藻进行叶黄素的分离提取。在一个更佳的方案中，将获得的小球藻液浓缩后直接喷雾干燥获得小球藻粉。In a preferred scheme, the lutein is separated and extracted from Chlorella by supercritical CO2 extraction technology. In a better solution, the obtained chlorella liquid is concentrated and then directly spray-dried to obtain chlorella powder.
本发明中，可对培养所得的微藻进行综合利用，提取其中的多不饱和脂肪酸、蛋白质、叶绿素、多糖等各种活性成分。活性成分的提取顺序并无特殊限制，但通常要满足先提取的步骤不能导致后提取的成分损失这一前提。In the present invention, the cultured microalgae can be comprehensively utilized to extract various active components such as polyunsaturated fatty acids, proteins, chlorophyll, polysaccharides and the like. The order of extraction of the active ingredients is not particularly limited, but it is generally necessary to meet the premise that the first extraction step cannot cause the loss of the later extracted ingredients.
本文中涉及到藻细胞干重和叶黄素含量测定方法如下：In this paper, the methods for determining the dry weight of algae cells and the content of lutein are as follows:
藻细胞干重测定：在微藻(如小球藻)培养过程中取培养液V毫升，8000rpm离心10分钟，将离心后的藻体用去离子水洗涤3次，转移至称量瓶(W1(克))中，在105℃烘箱中烘干至恒重W2(克)。藻体干重Cx可根据下式计算：Cx(克/升)＝(W2-W1)/V/1000。Algae cell dry weight determination: in microalgae (as Chlorella) cultivation process, get culture solution V milliliter, 8000rpm centrifugation 10 minutes, the algal body after centrifugation is washed 3 times with deionized water, is transferred to weighing bottle (W1 (g)), and dried in an oven at 105°C to a constant weight W2 (g). The dry weight Cx of algae can be calculated according to the following formula: Cx (g/L)=(W2 -W1 )/V/1000.
叶黄素测定：采用高效液相色谱法(HPLC)，具体操作步骤见文献(ZhengYun Wu，Chun Lei Shi et al.Modeling of lutein production by heterotrophicChlorella in batch and fed-batch cultures.World Journal of Microbiology andBiotechnology，2007，23：1233-1238)。Determination of lutein: using high performance liquid chromatography (HPLC), see the literature (ZhengYun Wu, Chun Lei Shi et al. Modeling of lutein production by heterotrophic Chlorella in batch and fed-batch cultures. World Journal of Microbiology and Biotechnology, 2007, 23:1233-1238).
实施例1Example 1
在5L生物反应器中加入下述异养培养基和水至2.8L后进行蒸汽灭菌，然后当温度降到30℃时接入蛋白核小球藻，开始异养培养。Add the following heterotrophic medium and water to 2.8L in a 5L bioreactor, then steam sterilize it, then insert Chlorella pyrenoidosa when the temperature drops to 30°C, and start heterotrophic culture.
异养培养条件：温度为30±1℃，空气流量为1vvm，pH小于8.0，控制溶氧15％以上。Heterotrophic culture conditions: the temperature is 30±1°C, the air flow rate is 1vvm, the pH is less than 8.0, and the dissolved oxygen is controlled above 15%.
在接种后53.9h后第一次补料，之后每隔5～8h进行补料，共补加4次，培养至88.40h细胞干重达到132.2g/L(见图1)，此时异养培养结束转入光诱导培养。Feed was fed for the first time after 53.9 hours after inoculation, and then fed every 5 to 8 hours for a total of 4 times, and the dry weight of cells reached 132.2g/L at 88.40 hours after cultivation (see Figure 1). At this time, heterotrophic At the end of the culture, they were transferred to light-induced culture.
将异养培养结束后的高密度藻液稀释到2.55g/L，并加入下述光诱导培养基，转入到3L平板式光生物反应器中进行光诱导培养。光诱导培养条件：温度维持在28～33℃，空气流量为1vvm，双侧光照，每侧光强为15klx。光诱导培养27h后，细胞干重从2.55g/L降低到1.82g/L，叶黄素从1.57mg/gDcw上升至3.64mg/gDcw，光诱导27h时叶黄素的产率为5.88mg/L/d；若光诱导12h时叶黄素的产率可达12.1mg/L/d(为目前微藻光自养生产叶黄素最高产率4.8mg/L/d的2.5倍)(见图2)。Dilute the high-density algae liquid after the heterotrophic culture to 2.55g/L, add the following light-induced medium, and transfer it to a 3L flat-plate photobioreactor for light-induced culture. Light-induced culture conditions: the temperature is maintained at 28-33°C, the air flow rate is 1vvm, both sides are illuminated, and the light intensity on each side is 15klx. After 27 hours of light-induced culture, the dry weight of cells decreased from 2.55g/L to 1.82g/L, lutein increased from 1.57mg/gDcw to 3.64mg/gDcw, and the yield of lutein at 27h of light-induced induction was 5.88mg/ L/d; if light-induced 12h, the yield of lutein can reach 12.1mg/L/d (2.5 times of the highest yield of 4.8mg/L/d for microalgae photoautotrophic production of lutein) (see figure 2).
异养及补料培养基：Heterotrophic and fed media:
葡萄糖60.0克 尿素8.0克 MgSO4·7H2O 2.0克Glucose 60.0 grams Urea 8.0 grams MgSO4 7H2 O 2.0 grams
KH2PO41.1克 Na2HPO4·12H2O 9.0克 CaCl20.02克KH2 PO4 1.1 g Na2 HPO4 12H2 O 9.0 g CaCl2 0.02 g
柠檬酸三钠1.8克Trisodium citrate 1.8g
Fe-EDTA溶液1.0ml 微量元素溶液4.5ml 水1000mlFe-EDTA solution 1.0ml Trace element solution 4.5ml Water 1000ml
其中Fe-EDTA溶液配方为FeSO4·7H2O 15克/升和EDTA1.4克/升，微量元素溶液配方为H3BO3 2.11克/升，MnCl2·4H2O 0.81克/升，ZnSO4·7H2O 0.11克/升，CuSO4·5H2O 10.0克/升，Na2MoO4 0.05克/升。Among them, the formula of Fe-EDTA solution is FeSO4 7H2 O 15 g/L and EDTA 1.4 g/L, the formula of trace element solution is H3 BO3 2.11 g/L, MnCl2 4H2 O 0.81 g/L, ZnSO4 ·7H2 O 0.11 g/liter, CuSO4 ·5H2 O 10.0 g/liter, Na2 MoO4 0.05 g/liter.
光诱导培养基：Light-induced medium:
尿素0.5克 MgSO4·7H2O 1.0克 柠檬酸三钠0.05克Urea 0.5g MgSO4 7H2 O 1.0g Trisodium citrate 0.05g
CaCl20.01克 Fe-EDTA溶液0.4ml 水1000mlCaCl2 0.01g Fe-EDTA solution 0.4ml water 1000ml
其中Fe-EDTA溶液配方为FeSO4·7H2O 15克/升和EDTA1.4克/升。The formula of Fe-EDTA solution is FeSO4 ·7H2 O 15 g/L and EDTA 1.4 g/L.
实施例2Example 2
在50L生物反应器中加入下述异养培养基和自来水至25L后在121℃灭菌20min，然后当温度降至30℃左右时按工作体积的10％接入蛋白核小球藻，开始异养培养。Add the following heterotrophic medium and tap water to a 50L bioreactor to 25L, then sterilize at 121°C for 20min, then insert Chlorella pyrenoidosa according to 10% of the working volume when the temperature drops to about 30°C, and start heterotrophic Raise and cultivate.
异养培养条件：温度为30℃，空气流量为1vvm，pH在6.0～8.0，控制溶氧15％以上。在培养过程中，当葡萄糖消耗完后，进行补加碳源，当尿素消耗完时，补加氮源。通过6次补加碳源，3次补加氮源后在98.89h藻细胞密度达130.5g/L(见图3)。Heterotrophic culture conditions: the temperature is 30°C, the air flow rate is 1vvm, the pH is 6.0-8.0, and the dissolved oxygen is controlled above 15%. During the cultivation process, when the glucose was consumed, the carbon source was supplemented, and when the urea was consumed, the nitrogen source was supplemented. After adding carbon source 6 times, the density of algae cells reached 130.5g/L at 98.89h after adding nitrogen source 3 times (see Figure 3).
将异养培养结束后的高密度藻稀释到2.01g/L，并加入光诱导培养基，转入到10L圆柱光生物反应器内进行光诱导。Dilute the high-density algae after heterotrophic culture to 2.01g/L, add light-induced medium, and transfer to a 10L cylindrical photobioreactor for light-induced induction.
光诱导培养条件：自然温度，温度在30℃，光强在3klx，空气流量为1vvm。光诱导培养30h后，细胞干重从2.01g/L降低到1.66g/L，叶黄素从2.55mg/gDcw上升至3.47mg/gDcw，光诱导30h，叶黄素产率为4.61mg/L/d；若只光诱导24h，叶黄素的产率达5.73mg/L/d(见图4)。培养基与实施例1的培养基一致。Light-induced culture conditions: natural temperature, temperature at 30°C, light intensity at 3klx, air flow at 1vvm. After 30 hours of light-induced culture, the dry weight of cells decreased from 2.01g/L to 1.66g/L, and the lutein increased from 2.55mg/gDcw to 3.47mg/gDcw. After 30h of light-induced induction, the yield of lutein was 4.61mg/L /d; if only light-induced 24h, the yield of lutein reached 5.73mg/L/d (see Figure 4). The culture medium is consistent with that of Example 1.
实施例3Example 3
在50L生物反应器中加入下述异养培养基和自来水至25L后在121℃灭菌20min，然后当温度降至30℃左右时按工作体积的13％接入普通小球藻，开始异养培养。Add the following heterotrophic medium and tap water to a 50L bioreactor to 25L, then sterilize at 121°C for 20 minutes, then insert Chlorella vulgaris into 13% of the working volume when the temperature drops to about 30°C, and start heterotrophy nourish.
异养培养条件：温度为30℃，空气流量为1vvm，pH小于9.0。在培养过程中，当碳源消耗完后，进行补加葡萄糖，当氮源消耗完时，补加硝酸钾。通过5次补加碳源和氮源，58.20h藻细胞密度最高达54.5g/L(见图5)。Heterotrophic culture conditions: temperature is 30°C, air flow is 1vvm, pH is less than 9.0. During the cultivation process, when the carbon source was exhausted, glucose was supplemented, and when the nitrogen source was exhausted, potassium nitrate was supplemented. After adding carbon source and nitrogen source 5 times, the algae cell density reached 54.5g/L at 58.20h (see Figure 5).
将异养培养的高密度藻稀释到3.2g/L左右，并加入光诱导培养基，转入到30L平板式光生物反应器在户外进行光诱导培养。光诱导培养条件：自然温度，自然光照，空气流量为1.0vvm。光诱导28h，叶黄素从初始的1.10mg/gDcw上升至1.82mg/gDcw；光诱导23h时，叶黄素含量为1.67mg/gDcw，叶黄素产率达5.23mg/L/d(见图6)。Dilute the heterotrophically cultured high-density algae to about 3.2g/L, add light-induced medium, and transfer to a 30L flat-plate photobioreactor for light-induced culture outdoors. Light-induced culture conditions: natural temperature, natural light, and air flow of 1.0vvm. After 28 hours of light induction, lutein increased from the initial 1.10mg/gDcw to 1.82mg/gDcw; at 23 hours of light induction, the content of lutein was 1.67mg/gDcw, and the yield of lutein reached 5.23mg/L/d (see Figure 6).
异养和补料培养基：Heterotrophic and fed media:
葡萄糖60.0克 硝酸钾10.0克 MgSO4·7H2O 0.2克Glucose 60.0 grams Potassium nitrate 10.0 grams MgSO4 7H2 O 0.2 grams
KH2PO40.3克 Na2HPO4·12H2O 8.8克 CaCl20.02克KH2 PO4 0.3g Na2 HPO4 12H2 O 8.8g CaCl20.02g
Fe-EDTA溶液1.0ml 微量元素溶液3.5ml 水1000mlFe-EDTA solution 1.0ml Trace element solution 3.5ml Water 1000ml
其中Fe-EDTA溶液配方为FeSO4·7H2O 15克/升和EDTA1.4克/升，微量元素溶液配方为H3BO3 2.86克/升、MnCl2·4H2O 0.11克/升、ZnSO4·7H2O 9.22克/升、CuSO4·5H2O 1.00克/升、(NH4)6Mo7O24·4H2O 0.1克/升和Co(NO3)2·6H2O0.9克/升。Among them, the Fe-EDTA solution formula is FeSO4 7H2 O 15 g/L and EDTA 1.4 g/L, the trace element solution formula is H3 BO3 2.86 g/L, MnCl2 4H2 O 0.11 g/L, ZnSO4 7H2 O 9.22 g/L, CuSO4 5H2 O 1.00 g/L, (NH4 )6 Mo7 O24 4H2 O 0.1 g/L and Co(NO3 )2 6H2 O0 .9 g/l.
光诱导培养基：Light-induced medium:
硝酸钾0.5克 MgSO4·7H2O 0.6克Potassium nitrate 0.5g MgSO4 7H2 O 0.6g
CaCl20.03克 Fe-EDTA溶液1.5ml 水1000mlCaCl2 0.03 grams Fe-EDTA solution 1.5ml water 1000ml
其中Fe-EDTA溶液配方为FeSO4·7H2O 8克/升和EDTA 10.4克/升。The formula of the Fe-EDTA solution is FeSO4 ·7H2 O 8 g/L and EDTA 10.4 g/L.
实施例4Example 4
在5L生物反应器中加入下述异养培养基和水至2.8L后进行蒸汽灭菌，然后当温度降到30℃时按工作体积的8％接入椭圆小球藻，开始异养培养。Add the following heterotrophic medium and water to 2.8L in a 5L bioreactor, then steam sterilize it, then insert Chlorella ellipsoides into 8% of the working volume when the temperature drops to 30°C, and start heterotrophic culture.
异养培养条件：温度为30±1℃，空气流量为1vvm，pH小于8.5，控制溶氧5％以上。Heterotrophic culture conditions: the temperature is 30±1°C, the air flow is 1vvm, the pH is less than 8.5, and the dissolved oxygen is controlled above 5%.
补料两次后在66h，藻细胞密度为53.0g/L(见图7)，此时异养培养结束转入光诱导培养。At 66 hours after feeding twice, the algae cell density was 53.0g/L (see Figure 7), at this time the heterotrophic culture was completed and transferred to light-induced culture.
将异养培养结束后的高密度藻液稀释到4.0g/L，并加入下述光诱导培养基，转入到3L平板式光生物反应器中进行光诱导培养。光诱导培养条件：温度维持在28～33℃，空气流量为1.0vvm，双侧光照，每侧光强为15klx。光诱导培养48h后，细胞干重从4.0g/L降低到2.8g/L，叶黄素从1.5mg/gDcw上升至3.6mg/gDcw，叶黄素产率为5.04mg/L/d；光诱导24h，叶黄素产率可达9.24mg/L/d(见图8)。培养基与实施例3的培养基一致。Dilute the high-density algae liquid after the heterotrophic culture to 4.0g/L, add the following light-induced medium, and transfer it to a 3L flat-plate photobioreactor for light-induced culture. Light-induced culture conditions: the temperature is maintained at 28-33°C, the air flow rate is 1.0vvm, both sides are illuminated, and the light intensity on each side is 15klx. After 48 hours of light-induced culture, the dry weight of cells decreased from 4.0g/L to 2.8g/L, lutein increased from 1.5mg/gDcw to 3.6mg/gDcw, and the yield of lutein was 5.04mg/L/d; After 24 hours of induction, the yield of lutein can reach 9.24 mg/L/d (see Figure 8). The culture medium is consistent with that of Example 3.
尽管上面已经描述了本发明的具体例子，但是有一点对于本领域技术人员来说是明显的，即在不脱离本发明的精神和范围的前提下可对本发明作各种变化和改动。因此，所附权利要求覆盖了所有这些在本发明范围内的变动。Although specific examples of the present invention have been described above, it will be apparent to those skilled in the art that various changes and modifications can be made in the present invention without departing from the spirit and scope of the present invention. It is therefore intended to cover in the appended claims all such changes which are within the scope of this invention.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010567920.3ACN102094061B (en) | 2010-12-01 | 2010-12-01 | Method for producing lutein from microalgae |
| PCT/CN2011/082261WO2012065545A1 (en) | 2010-11-16 | 2011-11-16 | Microalgae culturing method for oil and lutein rapid accumulation |
| Application Number | Priority Date | Filing Date | Title |
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| CN201010567920.3ACN102094061B (en) | 2010-12-01 | 2010-12-01 | Method for producing lutein from microalgae |
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| CN201010567920.3AActiveCN102094061B (en) | 2010-11-16 | 2010-12-01 | Method for producing lutein from microalgae |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012065545A1 (en)* | 2010-11-16 | 2012-05-24 | 华东理工大学 | Microalgae culturing method for oil and lutein rapid accumulation |
| CN103103128A (en)* | 2011-11-10 | 2013-05-15 | 中国石油化工股份有限公司 | Method for high efficiency enrichment culture of microalgae |
| CN102517218B (en)* | 2011-12-28 | 2013-05-29 | 广东海洋大学 | A method for cultivating microalgae on sea surface |
| CN103571906B (en)* | 2012-07-27 | 2018-12-11 | 上海泽元海洋生物技术有限公司 | A kind of new method efficiently producing astaxanthin using microalgae |
| CN104662162A (en)* | 2013-09-26 | 2015-05-27 | 华东理工大学 | Method using micro-algae for high-efficiency production of astaxanthin |
| WO2021009781A1 (en)* | 2019-07-16 | 2021-01-21 | Bio-P S.R.L. | Process for extraction of nutraceutical compounds from microalgae by using co2 in supercritical conditions |
| CN111500464A (en)* | 2020-05-21 | 2020-08-07 | 福州大学 | Method for producing lutein by first mixotrophic-later autotrophic microalgae |
| CN114214202B (en)* | 2021-12-27 | 2024-02-13 | 广西源藻生物科技有限公司 | Method for co-producing lutein and protein by heterotrophic-photoinduction tandem culture of microalgae |
| CN116790382B (en)* | 2023-08-17 | 2023-11-03 | 藻辰(山东)生物工程有限公司 | High-yield lutein mutant chlorella, and preparation method and application thereof |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1837351A (en)* | 2006-04-12 | 2006-09-27 | 华东理工大学 | Method for cultivating chlorella with high density and high quality |
| CN1878872A (en)* | 2004-05-26 | 2006-12-13 | 雅马哈发动机株式会社 | Method of producing xanthophyll |
| CN101555454A (en)* | 2009-05-15 | 2009-10-14 | 华南理工大学 | Method for synchronously improving biomass and lutein of heterotrophic chlorella |
| CN102325874A (en)* | 2008-12-19 | 2012-01-18 | α-J研究有限合伙公司 | Optimization of algal product production through uncoupling cell proliferation and algal product production |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1878872A (en)* | 2004-05-26 | 2006-12-13 | 雅马哈发动机株式会社 | Method of producing xanthophyll |
| CN1837351A (en)* | 2006-04-12 | 2006-09-27 | 华东理工大学 | Method for cultivating chlorella with high density and high quality |
| CN102325874A (en)* | 2008-12-19 | 2012-01-18 | α-J研究有限合伙公司 | Optimization of algal product production through uncoupling cell proliferation and algal product production |
| CN101555454A (en)* | 2009-05-15 | 2009-10-14 | 华南理工大学 | Method for synchronously improving biomass and lutein of heterotrophic chlorella |
| Publication number | Publication date |
|---|---|
| CN102094061A (en) | 2011-06-15 |
| Publication | Publication Date | Title |
|---|---|---|
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