| Publication number | CN1392244 A |
| Publication type | Application |
| Application number | CN 02138827 |
| Publication date | Jan 22, 2003 |
| Filing date | Jul 26, 2002 |
| Priority date | Jul 26, 2002 |
| Also published as | CN1181184C |
| Publication number | 02138827.X, CN 02138827, CN 1392244 A, CN 1392244A, CN-A-1392244, CN02138827, CN02138827.X, CN1392244 A, CN1392244A |
| Inventors | 李夜光, 张宝玉 |
| Applicant | 中国科学院武汉植物研究所 |
| Export Citation | BiBTeX, EndNote, RefMan |
| Referenced by (10), Classifications (3), Legal Events (3) | |
| External Links: SIPO, Espacenet | |
技术领域 FIELD
本发明涉及一种培养雨生红球藻生产虾青素的方法，特别涉及一种用二氧化碳(CO2)调节培养液pH值控制红球藻孢子的形成、虾青素积累和循环使用培养基的方法。 The present invention relates to a method of culturing pluvialis algae produce astaxanthin, particularly to a regulating carbon dioxide (CO2) medium pH control spore formation Haematococcus astaxanthin accumulation and recycling medium Methods.
背景技术 BACKGROUND
虾青素(Astaxanthin)因具有优越的着色性能和极强的抗氧化性而备受国内外广大研究者的关注。 Astaxanthin (Astaxanthin) because of superior color performance and very strong antioxidant activity and much domestic and foreign researchers. 目前虾青素最主要的用途是在水产养殖中用作色素，特别是在三文鱼和鳟鱼的养殖业中，其次是作为食品添加剂和保健品，在医药方面的应用也具有很大的潜力。 At present, the most important use of astaxanthin is used as a pigment in aquaculture, particularly in salmon and trout farming, and secondly as a food additive and health care products, used in medicine also has great potential. 据最新报道，2001年三文鱼养殖产量已突破100万吨。 According to the latest reports, the 2001 salmon aquaculture production has exceeded 100 million tons. 另外，加拿大、智利和日本将成为太平洋三文鱼的主要生产国(BjorndaleT.1990)。 In addition, Canada, Chile and Japan will become the main producing countries (BjorndaleT.1990) Pacific salmon. 目前有95％以上的虾青素是人工合成的，其售价约为2000-2500美元/公斤，每年市场消费量大约为2亿美元(R.Todd Lorenz et al.2000)。 There are currently more than 95% of astaxanthin is a synthetic, its price is about $ 2000-2500 / kg, the annual volume of approximately $ 200 million market consumption (R.Todd Lorenz et al.2000). 但人工合成的产物是类似于类胡萝卜素的成分(Mayne STet al.1988；Storebakken T.et al.1984)，不能有效地被鱼类吸收利用。 However, the product is a synthetic ingredient similar to the carotenoid (Mayne STet al.1988; Storebakken T.et al.1984), can not be effectively absorbed by the fish. 并且美国的食品和药品管理委员会(FDA)不允许人工合成的虾青素添加在三文鱼的饲料中(Sinnot R.，1988)。 And the US Food and Drug Administration (FDA) does not allow synthetic astaxanthin added to salmon feed (Sinnot R., 1988).
目前虾青素的来源有4条途径：(1)化学合成 化学合成的虾青素产物主要是类似于类胡萝卜素的成分(Mayne STet al.1988；Storebakken T.et al.1984)。 There are sources of astaxanthin in four ways: (1) chemical synthesis of chemically synthesized astaxanthin products are mainly ingredients like carotenoids (Mayne STet al.1988; Storebakken T.et al.1984). 近年来，在鱼的饲料中使用人工合成色素受到越来越多的限制(Sinnot R.，1988)。 In recent years, the use of fish feed in synthetic pigments are more and more restrictions (Sinnot R., 1988).
(2)从甲壳类动物中提取：甲壳类动物的甲壳中含有虾青素，但含量很低。 (2) extracted from crustaceans: crustacean shells containing astaxanthin, but the content is very low. 并且这些甲壳中灰分几丁质的含量较高，限制了虾青素的提取和利用。 And these crustaceans in chitin ash content is high, limiting the extraction and use of astaxanthin.
(3)利用真菌生产：真菌类如红发夫酵母菌(Phaffia rhodozyma)野生株系中含有0.05％的虾青素；某些突变株系的虾青素含量可达细胞干重的0.218％(John ABet al.1997)。 (3) the use of fungi produce: fungi such as Phaffia yeast (Phaffia rhodozyma) wild strains containing 0.05% astaxanthin; some mutant strains of astaxanthin content of up to 0.218% dry cell weight ( John ABet al.1997). 真菌中虾青素的含量普遍很低。 Fungal astaxanthin content is generally low. 利用酵母菌生产虾青素除产量低外，发酵成本高也是不利于大规模生产的原因。 The use of reason yeast production of astaxanthin addition to low yields, the high cost is not conducive to large-scale fermentation production.
(4)利用微藻生产：雨生红球藻(Haematococcus pluvialis)细胞中虾青素含量很高，一般达干重的1.5-3.0％；根据最新报道虾青素含量可占生物量的6-8％左右(Tsavalos et al.，1992)。 (4) the use of microalgae production: pluvialis algae (Haematococcus pluvialis) cells in high astaxanthin content, usually up to 1.5-3.0% of the dry weight; according to the latest reports astaxanthin content can be accounted for biomass 6- about 8% (Tsavalos et al., 1992). 雨生红球藻细胞内天然虾青素的含量相对较高，而且与天然生长的三文鱼体内的类胡萝卜素结构完全相同(GoodwinT.W.1984；Steven DM1948；Fex DL1957；Khare A.et al 1973)，因此，利用红球藻生产虾青素具有巨大的商业及经济价值。 Content within pluvialis algal cells natural astaxanthin is relatively high, and the carotenoid structure identical to the natural growth of salmon in vivo (GoodwinT.W.1984; Steven DM1948; Fex DL1957; Khare A.et al 1973 ), therefore, the use of Haematococcus astaxanthin production has great commercial and economic value.
目前国外大规模培养都采用二步法。 At present, foreign cultures have adopted the two-step scale. 所谓的二步法是红球藻的营养生长和虾青素累积人为地分为两个阶段，这两个阶段分别在不同的光生物反应器中，不同的培养条件(包括培养基)下进行的，维持营养生长的条件和诱导虾青素累积的条件有很大的不同。 The so-called two-step is Haematococcus vegetative growth and astaxanthin accumulation artificially divided into two stages, the two phases were in a different light bioreactor under different culture conditions (including the media) were maintain vegetative growth conditions and induction of astaxanthin cumulative conditions are very different. 二步法的优点是解决了红球藻细胞生长繁殖与孢子形成、积累虾青素所需条件不同的矛盾，其不足之处在于(1)工艺复杂；(2)诱导虾青素累积的措施会导致采收后的培养液不能重复使用，致使生产过程中大量排出废水，对水环境有污染作用。 The advantage of the two-step solution to pluvialis cell growth and reproduction and spore formation, accumulation of astaxanthin contradictory elements required for different conditions, its shortcomings in that (1) the complexity of the process; (2) induction of astaxanthin accumulation of measures will lead to the culture medium after harvest can not be reused, resulting in the production process of a large number of discharged waste water, water environment pollution effect.
发明内容 SUMMARY
本发明的目的在于提供一种培养雨红球藻生产虾青素的方法，培养基配制合理，方法简便，生产周期短，培养基循环使用，解决了培养过程中废水排放的问题。 The purpose of the present invention is to provide a rain pluvialis astaxanthin production methods culture media preparation and reasonable method is simple, short production cycle, medium recycling, solve the problem of the training process wastewater discharges.
为了达到上述目的，本发明采用如下技术措施。 In order to achieve the above object, the present invention adopts the following technical measures.
1.配制培养基(MHM)培养基配方如下：硝酸钾(KNO3) 0.4-0.7g/L磷酸氢二钾(K2HPO4) 0.08-0.12g/L硫酸镁(MgSO4·7H2O) 0.08-0.12g/L硫酸钙(CaSO4·2H2O) 0.04-0.06g/L三氯化铁(FeCL3·6H2O) 2.38-2.50×10-4g/L乙二胺四乙酸二钠(EDTA·Na2) 1.85-3.7×10-4g/L氯化锌(ZnCL2) 3.7-5.0×10-6g/L硼酸(H3BO3) 5.5-8.0×10-5g/L氯化钴(CoCL2·6H2O) 4.8-9.6×10-6g/L硫酸铜(CuSO4·5H2O) 6.4-8.5×10-6g/L氯化锰(MnCL2·4H2O) 3.8-7.6×10-6g/L钼酸铵((NH4)6Mo7O24·4H2O) 3.0-5.0×10-5g/L维生素B12(Vitamin B12) 1.0-3.0×10-6g/L生物素(Biotin) 1.0-3.0×10-6g/L2.一步法培养利用雨生红球藻生产虾青素，采用一步法。 1. Preparation of culture medium (MHM) medium formulation as follows: potassium nitrate (KNO3) 0.4-0.7g / L dipotassium hydrogen phosphate (K2HPO4) 0.08-0.12g / L magnesium sulfate (MgSO4 · 7H2O) 0.08-0.12g / L calcium sulfate (CaSO4 · 2H2O) 0.04-0.06g / L ferric chloride (FeCL3 · 6H2O) 2.38-2.50 × 10-4g / L disodium ethylenediamine tetraacetate (EDTA · Na2) 1.85-3.7 × 10-4g / L zinc chloride (ZnCL2) 3.7-5.0 × 10-6g / L boric acid (H3BO3) 5.5-8.0 × 10-5g / L cobalt chloride (CoCL2 · 6H2O) 4.8-9.6 × 10-6g / L copper sulfate ( CuSO4 · 5H2O) 6.4-8.5 × 10-6g / L manganese chloride (MnCL2 · 4H2O) 3.8-7.6 × 10-6g / L ammonium molybdate ((NH4) 6Mo7O24 · 4H2O) 3.0-5.0 × 10-5g / L Vitamin B12 (Vitamin B12) 1.0-3.0 × 10-6g / L biotin (Biotin) 1.0-3.0 × 10-6g / L2. One-step training use pluvialis algae produce astaxanthin, one-step. 即雨生红球藻的营养细胞的生长、孢子转化和虾青素累积是在同一个光生物反应器、同一培养基中完成，通过调控培养液的pH值促进孢子转化和虾青素累积。 That pluvialis algae growing vegetative cells, spores transformation and astaxanthin accumulation is in the same light bioreactor, the same medium is completed, the pH of the culture to promote spore transformation and astaxanthin accumulation by regulation. 培养周期均为12-15天，前5-6天是营养生长期，藻细胞持续进行细胞分裂，以指数形式增殖，达到最大密度。 Culture period were 12-15 days, 5-6 days before a vegetative growth, algal cells continued cell division, proliferation exponentially, reaching maximum density. 后7-9天为孢子转化与虾青素积累期。 After 7-9 days for spore transformation and astaxanthin accumulation period. 两个阶段自然连贯。 Two stages of natural continuity. 红球藻在12-15天内完成营养生长、孢子转化和虾青素积累的全过程。 Haematococcus completed in 12-15 days, vegetative growth, spore transformation and astaxanthin accumulation process.
3.循环使用培养基3.1回收培养基的处理回收上一次采收后的培养基，用活性碳处理回收的培养基，活性碳的用量为0.2-1.0g/L，活性碳处理时间为2-5小时，过滤得到澄清的水液。 3. recycling medium medium 3.1 medium was recovered on treating the recovered after the first harvest, the culture medium was recovered by treatment with activated carbon, activated carbon in an amount of 0.2-1.0g / L, the activated carbon treatment time is 2 5 hours, filtered water to give a clear solution. 活性炭处理和过滤的作用有2个：(1)吸附培养基中的有机物。 The role of activated carbon treatment and filtration have two: (1) adsorption of organic medium. (2)去除杂质，包括剩余的藻细胞、少量原生动物和菌类；3.2重新配制培养基向过滤得到的澄清水液中加入培养基配方中的各种化学物质，每种的用量为原配方的80％，得到新的培养基，用于下一次培养。 (2) to remove impurities, including the remaining algal cells, a small amount of protozoa and fungi; 3.2 reconstituted medium was filtered to clarify aqueous medium formulation was added various chemicals, each in an amount of the original formula 80% to obtain a new medium for the next culture.
4.分阶段调控培养液的pH值在培养的前期(5-6天)，将培养液的pH值调节在7-8.5的范围内，提供有利于细胞快速生长繁殖的条件，使红球藻的生物量快速增长。 4. The pH regulation stages in the culture broth of the early (5-6 days), the culture medium pH is adjusted in the range of 7-8.5, and provide beneficial conditions for rapid cell growth and reproduction, so that Haematococcus The rapid growth of the biomass. 在培养的后期(7-9天)，将培养液的pH值提高到8.5-10.0的范围内，促进孢子的形成和虾青素积累。 In the late culture (7-9 days), the pH of the culture is increased to the range of 8.5 to 10.0, and promote the formation and accumulation of astaxanthin spores.
本发明与现有技术相比，具有以下优点和效果：1.培养基重复使用，减少生产过程的废水排放，保护环境；2.工艺简便，整个培养过程在一个光生物反应器和一种培养基中完成；3.生产周期短，12-15天内内完成营养生长、孢子转化和虾青素积累的全过程。 Compared with the prior art, the present invention has the following advantages and effects: a medium reuse, reduce wastewater discharge production process, to protect the environment; two simple process, the whole training process in a photobioreactor and a culture. group completed; 3 short production cycle, completed within 12-15 days of vegetative growth, spore transformation and astaxanthin accumulation process.
4.产量高，质量好，红球藻孢子产量大于1克(干重)/升，孢子中虾青素含量2-3％(孢子产量和虾青素含量随不同的藻种(品系)变化)。 4. high yield, good quality, Haematococcus spore production is greater than 1 g (dry weight) / liter, spores astaxanthin content of 2-3% (spore production and astaxanthin content (strains) varies with different algae species ).
5.用二氧化碳(CO2)调节培养液pH值控制红球藻孢子的形成和虾青素积累，简单易行，经济高效。 5. Carbon dioxide (CO2) to adjust the pH of the culture medium Haematococcus control spore formation and accumulation of astaxanthin, simple, economical and efficient.
具体实施方式 DETAILED DESCRIPTION
1.配制培养基：环形培养池中放入20cm水，体积为600升，向其中加入各种营养成分及其浓度如下：硝酸钾(KNO3) 0.5g/L磷酸氢二钾(K2HPO4) 0.1g/L硫酸镁(MgSO4·7H2O) 0.1g/L硫酸钙(CaSO4·2H2O) 0.05g/L三氯化铁(FeCL36H2O) 2.43×10-4g/L乙二胺四乙酸二钠(EDTA·Na2) 1.88×10-4g/L氯化锌(ZnCL2) 4.1×10-6g/L硼酸(H3BO3) 6.1×10-5g/L氯化钴(CoCL2·6H2O) 5.1×10-6g/L硫酸铜(CuSO4·5H2O) 6.6×10-6g/L氯化锰(MnCL2·4H2O) 4.1×10-6g/L钼酸铵((NH4)6Mo7O24·4H2O) 3.8×10-5g/L 1. Preparation of Medium: 20cm ring culture into the pool water volume of 600 liters, to which a variety of nutrients and their concentration as follows: potassium nitrate (KNO3) 0.5g / L potassium phosphate dibasic (K2HPO4) 0.1g / L magnesium sulfate (MgSO4 · 7H2O) 0.1g / L calcium sulfate (CaSO4 · 2H2O) 0.05g / L ferric chloride (FeCL36H2O) 2.43 × 10-4g / L disodium ethylenediamine tetraacetate (EDTA · Na2) 1.88 × 10-4g / L zinc chloride (ZnCL2) 4.1 × 10-6g / L boric acid (H3BO3) 6.1 × 10-5g / L cobalt chloride (CoCL2 · 6H2O) 5.1 × 10-6g / L copper sulfate (CuSO4 · 5H2O) 6.6 × 10-6g / L manganese chloride (MnCL2 · 4H2O) 4.1 × 10-6g / L ammonium molybdate ((NH4) 6Mo7O24 · 4H2O) 3.8 × 10-5g / L
维生素B12(Vitamin B12) 2.0×10-6g/L生物素(Biotin) 2.5×10-6g/L2.接种：将旺盛生长的红球藻绿色游动细胞接种到配制好的培养基中，使细胞密度达到5×104cells/ml左右；3.培养培养过程光照采用自然光；利用机械搅拌使培养液保持均匀的流动；温度调控在15-28℃范围内；在培养的前6天，通过向培养液中通二氧二碳(CO2)，将培养液的pH值调节在7-8.5的范围内。 Vitamin B12 (Vitamin B12) 2.0 × 10-6g / L biotin (Biotin) 2.5 × 10-6g / L2 vaccination: The strong growth of Haematococcus green motile cells were inoculated into the prepared culture medium, the cells were density of 5 × 104cells / ml or so; 3. Training training process uses natural light illumination; broth using mechanical agitation to maintain a uniform flow; temperature control in the range of 15-28 ℃; 6 days in culture before, through the medium Carbon dioxide was passed through two (CO2), the pH of the culture was adjusted in the range of 7-8.5. 在这一阶段内，红球藻细胞快速生长繁殖，生物量快速增长，藻液呈绿色。 During this stage, Haematococcus rapid cell growth and reproduction, rapid growth in biomass, algae green liquid.
4.诱导虾青素的积累从培养的第7天开始，控制通入二氧化碳(CO2)的频率和数量将培养液的pH值调节在8.5-10.0的范围内。 4. induced accumulation of astaxanthin from the 7th day of culture began, control leads to carbon dioxide (CO2) in the frequency and amount of the culture solution to adjust the pH in the range of 8.5-10.0. 在这一阶段内，红球藻游动细胞逐渐转化为孢子，虾青素大量积累。 During this stage, Haematococcus motile cells gradually transformed into spores, astaxanthin accumulation. 藻液颜色由绿色变为黄绿色，进一步变为桔红色，再变为红色。 Algae solution color from green to yellow-green, and further changes to orange and then to red.
5.采收红球藻培养进行到第12天，培养液中95％的细胞转化为孢子，孢子内充满了红色的虾青素，藻液呈深红色，即可采收。 5. Harvest pluvialis culture to the first 12 days, the culture medium for 95% of the cells into spores, the spores inside is filled with red astaxanthin, algae solution was dark red, can be harvested. 将藻液静置，红球藻孢子很快沉淀下来，收集上清液备用，孢子在低于100℃的温度下干燥。 The solution was allowed to stand algae, Haematococcus spores quickly precipitated, the supernatant was collected standby, spores dried at a temperature of less than 100 ℃.
6.培养基的重新配制收集的上清液加入0.5g/L活性碳处理3小时、过滤，得到澄清的液体600升，再向其中加入各种营养成分配制成新的培养基，以备下一次培养使用。 6. The reconstituted supernatant medium collected was added 0.5g / L activated charcoal for 3 hours, filtered to give a clear liquid 600 liters, to which was added various nutritional prepared to have a new medium to prepare The next train to use. 加入的营养成份及其浓度如下硝酸钾(KNO3) 0.4g/L磷酸氢二钾(K2HPO4) 0.08g/L硫酸镁(MgSO4·7H2O) 0.08g/L硫酸钙(CaSO4·2H2O) 0.04g/L三氯化铁(FeCL36H2O) 1.94×10-4g/L乙二胺四乙酸二钠(EDTA·Na2) 1.5×10-4g/L氯化锌(ZnCL2) 3.28×10-6g/L硼酸(H3BO3) 4.88×10-5g/L氯化钴(CoCL2·6H2O) 4.08×10-6g/L硫酸铜(CuSO4·5H2O) 5.28×10-6g/L氯化锰(MnCL2·4H2O) 3.28×10-6g/L钼酸铵((NH4)6Mo7O24·4H2O) 3.04×10-5g/L维生素B12(Vitamin B12) 1.6×10-6g/L Added nutrients and the following concentrations of potassium nitrate (KNO3) 0.4g / L dipotassium hydrogen phosphate (K2HPO4) 0.08g / L magnesium sulfate (MgSO4 · 7H2O) 0.08g / L of calcium sulfate (CaSO4 · 2H2O) 0.04g / L ferric chloride (FeCL36H2O) 1.94 × 10-4g / L disodium ethylenediamine tetraacetate (EDTA · Na2) 1.5 × 10-4g / L zinc chloride (ZnCL2) 3.28 × 10-6g / L boric acid (H3BO3) 4.88 × 10-5g / L cobalt chloride (CoCL2 · 6H2O) 4.08 × 10-6g / L copper sulfate (CuSO4 · 5H2O) 5.28 × 10-6g / L manganese chloride (MnCL2 · 4H2O) 3.28 × 10-6g / L ammonium molybdate ((NH4) 6Mo7O24 · 4H2O) 3.04 × 10-5g / L of vitamin B12 (Vitamin B12) 1.6 × 10-6g / L
生物素(Biotin) 2.0×10-6g/L7.培养基的循环使用配制好的培养基的使用方法与第一次的培养基相同。 Biotin (Biotin) 2.0 × 10-6g / L7. Recycled medium medium formulated using the same method with the first medium. 经过接种—培养—诱导虾青素的积累—采收—培养基回收—处理—重新配制，又成为新的培养基，如此循环，培养基至少可以循环使用6次。 After inoculation - Training - induced accumulation of astaxanthin - Harvest - medium Recycling - Processing - reconstituted, has become a new medium, so the cycle, the medium can be recycled at least six times.
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| Date | Code | Event | Description |
|---|---|---|---|
| Jan 22, 2003 | C06 | Publication | |
| Apr 16, 2003 | C10 | Request of examination as to substance | |
| Dec 22, 2004 | C14 | Granted |
