CN100434528C - A method for extracting phycocyanin - Google Patents

Abstract

Translated fromChinese

本发明涉及生物提取藻蓝蛋白的方法，更具体地，本发明涉及一种用微生物的培养液从蓝藻、蓝绿藻中提取藻蓝蛋白的方法。与传统的藻蓝蛋白提取方法相比，本发明的方法具有成本低廉、操作简便，处理量大等优点，并且提取的蛋白纯度高，而且不破坏蛋白的生物活性，是一种非常适合于规模化生产藻蓝蛋白的方法。The invention relates to a method for biologically extracting phycocyanin, more specifically, the invention relates to a method for extracting phycocyanin from blue-green algae and blue-green algae with a culture solution of microorganisms. Compared with the traditional phycocyanin extraction method, the method of the present invention has the advantages of low cost, easy operation, large processing capacity, etc., and the extracted protein has high purity without destroying the biological activity of the protein, and is very suitable for large-scale Method for chemically producing phycocyanin.

Description

Translated fromChinese

一种提取藻蓝蛋白的方法A method for extracting phycocyanin

技术领域technical field

本发明涉及生物提取藻蓝蛋白的方法，更具体地，本发明涉及一种用微生物的培养液从蓝藻、蓝绿藻中提取藻蓝蛋白的方法。The invention relates to a method for biologically extracting phycocyanin, more specifically, the invention relates to a method for extracting phycocyanin from blue-green algae and blue-green algae with a culture solution of microorganisms.

背景技术Background technique

藻蓝蛋白具有很高的开发利用价值^【1】。首先藻蓝蛋白是红藻和蓝藻特有的光合色素，色泽明亮鲜艳，是食品、高级眼影、唇膏的首选纯天然色素，可作为纯天然的色素，用于食品、化妆品和染料等工业^【2】。再则可加工成保健食品和药品^【3】。研究表明，藻蓝蛋白能显著减轻并逐渐消除辐射、化疗对造血功能的损伤^【4】，还能提高淋巴细胞活性，积极清除细胞中氧自由基^【5】，促进伤口愈合，动物实验证明能有效抑制癌细胞生长^【6】，而且还可作为荧光试剂。高纯度的藻蓝蛋白带有很强的荧光，制成的纯天然荧光试剂，广泛应用于医学和生物工程等领域^【7】。Phycocyanin has high development and utilization value^【1】 . First of all, phycocyanin is a photosynthetic pigment unique to red algae and cyanobacteria. It has bright and bright color. It is the first choice of pure natural pigment for food, high-end eye shadow and lipstick. It can be used as a pure natural pigment for food, cosmetics and dye industries^【2】 . Then it can be processed into health food and medicine^[3] . Studies have shown that phycocyanin can significantly reduce and gradually eliminate the damage to hematopoietic function caused by radiation and chemotherapy^[4] , it can also increase the activity of lymphocytes, actively remove oxygen free radicals in cells^[5] , and promote wound healing. Animal experiments have proved that it can It can effectively inhibit the growth of cancer cells^[6] , and it can also be used as a fluorescent reagent. High-purity phycocyanin has strong fluorescence, and the pure natural fluorescent reagent made is widely used in the fields of medicine and bioengineering^[7] .

藻蓝蛋白的提取分为蛋白溶出和蛋白提纯两个过程。蓝藻、蓝绿藻藻细胞外层具鞘，胞壁为革兰氏阴性菌典型构造，由四层组成，每层厚约10nm，要提取出蓝藻、蓝绿藻中的藻蓝蛋白，首先要破碎藻细胞的鞘和细胞壁、细胞膜，使之以水溶状态存在于提取液中，再采用适宜的方法将其进一步纯化，而且还要求全过程都能保持藻蓝蛋白的活性。目前提取方法大致可以总结为机械捣碎、反复冻融、化学处理、超声破碎的蛋白质析出方法，然后进一步采取硫酸铵沉淀、等电点沉淀、柱层析和凝胶层析法等进行纯化处理。往往上述方法都是联合运用，以求达到最好效果。The extraction of phycocyanin is divided into two processes: protein dissolution and protein purification. Cyanobacteria and blue-green algae cells have a sheath on the outer layer, and the cell wall is a typical structure of Gram-negative bacteria. It consists of four layers, each layer is about 10nm thick. To extract phycocyanin from blue-green algae and blue-green algae, the first step is to The sheath, cell wall, and cell membrane of the algal cells are broken to make them exist in the extract in a water-soluble state, and then further purified by an appropriate method, and the activity of phycocyanin is also required to be maintained throughout the process. The current extraction methods can be roughly summarized as mechanical crushing, repeated freezing and thawing, chemical treatment, and ultrasonic crushing for protein precipitation, and then ammonium sulfate precipitation, isoelectric point precipitation, column chromatography, and gel chromatography for purification. . Often the above methods are used in combination to achieve the best results.

我国目前经济微藻，主要是蓝藻和蓝绿藻年生产能力超过1000吨，但大多是微藻初级产品。现在市场上已经有的10多种微藻藻片和胶囊，根据不同的功能通过卫生部批准为保健品，但还没有以微藻深加工产品藻蓝蛋白为主的功能性保健品，药品的研究开发几乎还是空白。这主要是由于蓝藻和蓝绿藻中藻蓝蛋白的提取还处于实验室研究阶段，没有适合于工业化生产的好的工艺方法，使藻蓝蛋白的商品价格昂贵而在应用上受到限制^【8】。my country's current economical microalgae, mainly cyanobacteria and blue-green algae, have an annual production capacity of more than 1,000 tons, but most of them are primary products of microalgae. Now there are more than 10 kinds of microalgae tablets and capsules on the market, which have been approved as health products by the Ministry of Health according to different functions, but there is no research on functional health products and medicines based on microalgae deep-processing products phycocyanin Development is still pretty much blank. This is mainly because the extraction of phycocyanin from blue-green algae and blue-green algae is still in the laboratory research stage, and there is no good process method suitable for industrial production, which makes the commodity price of phycocyanin expensive and limited in application^【8】 .

目前最常运用的蓝藻、蓝绿藻细胞破碎提取方法如下^{【9】【10】【11】}：取新鲜螺旋藻或者藻粉，用0.01M K-磷酸盐缓冲液，pH 6.7，0.15M NaCl的提取缓冲液漂洗干净后，重新悬浮于此缓冲液中，在-15℃状态下冻结，然后解冻并升温到30℃，保持一小时，并持续搅拌，之后置于4℃，过夜，次日18000×g超速离心30分钟，取上清，加入水相萃取液，混合后搅拌再次置于4℃过夜，次日再于8000×g高速离心30分钟后弃沉淀，取上清，加入水萃取液，重复上述离心步骤5次，获得的最后上浮物饱和于40％硫酸铵溶液中，再次离心，沉淀溶解于提取缓冲液中。此时即获得了食品纯和药品纯的藻蓝蛋白。如要获得高纯度的藻蓝蛋白，还需要凝胶层析、柱层析等纯化步骤，得到试剂纯藻蓝蛋白A₆₂₀/A₂₈₀＞4.0。At present, the most commonly used cyanobacteria and blue-green algae cell crushing extraction methods are as follows^{[9] [10] [11]} : take fresh spirulina or algae powder, use 0.01M K-phosphate buffer solution, pH 6.7, 0.15M NaCl After rinsing with the extraction buffer, resuspend in this buffer, freeze at -15°C, then thaw and warm up to 30°C, keep it for one hour, and keep stirring, then place at 4°C overnight, 18000 the next day ×g ultracentrifugation for 30 minutes, take the supernatant, add the aqueous extract, mix and stir again and place it at 4°C overnight, and then centrifuge at 8000×g for 30 minutes the next day, discard the precipitate, take the supernatant, add the water extract , repeat the above centrifugation step 5 times, the final supernatant obtained is saturated in 40% ammonium sulfate solution, centrifuged again, and the precipitate is dissolved in the extraction buffer. At this moment, food pure and pharmaceutical pure phycocyanin are obtained. To obtain high-purity phycocyanin, further purification steps such as gel chromatography and column chromatography are required to obtain reagent pure phycocyanin A₆₂₀ /A₂₈₀ >4.0.

发明内容Contents of the invention

针对上述研究背景，本发明人经过对藻蓝蛋白提取方法的深入研究，非常意外地发现某些种类的微生物的培养液可以使新鲜的蓝藻、蓝绿藻或其制成的藻粉自然破壁，将藻蓝蛋白释放到培养液中，进一步用常规方法纯化可以获得高纯度的藻蓝蛋白。In view of the above-mentioned research background, the present inventor, after in-depth research on the extraction method of phycocyanin, found that the culture solution of certain types of microorganisms can naturally break the wall of fresh blue-green algae, blue-green algae or the algal powder made by them. , release phycocyanin into the culture medium, and further purify by conventional methods to obtain high-purity phycocyanin.

因此，本发明的一个目的是提供一种大量提取藻蓝蛋白的方法，其特征在于将固氮菌中克氏杆菌属和肠细菌属固氮菌的培养液与螺旋藻或藻粉混合。在该过程中，螺旋藻或藻粉被快速降解，藻蓝蛋白被释放到培养液中，用常规方法进一步纯化可以获得高纯度的藻蓝蛋白。Therefore, an object of the present invention is to provide a method for extracting phycocyanin in large quantities, which is characterized in that the culture solution of nitrogen-fixing bacteria of the genus Klebsiella and Enterobacter among nitrogen-fixing bacteria is mixed with spirulina or algae powder. During this process, the spirulina or algae powder is rapidly degraded, and the phycocyanin is released into the culture solution, and further purification by conventional methods can obtain high-purity phycocyanin.

在本发明的一个实施方案中，所述固氮菌是肺炎克雷伯氏菌(Klebsiella pneumoniae)，日勾维肠杆菌(Enterobacter gergoviae)，或产酸克雷伯氏菌(Klebsiella oxytoca)，特别优选肺炎克雷伯氏菌(Klebsiellapneumoniae)IMCAS 1.1734(＝CGCMCC 1.1734)，日勾维肠杆菌CGMCCNo.0510，和产酸克雷伯氏菌(Klebsiella oxytoca)(IBCAS-ZY)，其中日勾维肠杆菌(Enterobacter gergoviae)是日勾维肠杆菌57-7(CGMCC No.0510，其已在中国专利号ZL00133626.6(第一发明人李永兴)中公布授权，保藏在中国微生物菌种保藏管理委员会普通微生物中心)，肺炎克雷伯氏菌(Klebsiella pneumoniae)来自于中国微生物菌种保藏管理委员会普通微生物中心，产酸克雷伯氏菌(Klebsiella oxytoca)(IBCAS-ZY)由中科院植物所保存。In one embodiment of the invention, the nitrogen-fixing bacterium is Klebsiella pneumoniae, Enterobacter gergoviae, or Klebsiella oxytoca, particularly preferably Klebsiella pneumoniae (Klebsiellapneumoniae) IMCAS 1.1734 (= CGCMCC 1.1734), Enterobacter cerevisiae CGMCCNo.0510, and Klebsiella oxytoca (IBCAS-ZY), among which Enterobacter (Enterobacter gergoviae) is Enterobacter gergoviae 57-7 (CGMCC No.0510, which has been published and authorized in Chinese Patent No. ZL00133626.6 (the first inventor Li Yongxing), and is preserved in China Microbiological Culture Collection Management Committee General Microorganisms Center), Klebsiella pneumoniae (Klebsiella pneumoniae) came from the General Microbiology Center of China Committee for Culture Collection of Microorganisms, and Klebsiella oxytoca (IBCAS-ZY) was preserved by the Institute of Botany, Chinese Academy of Sciences.

在本发明的另一个实施方案中，将上述释放到培养液中的藻蓝蛋白进一步用硫酸铵、凝胶层析、羟基磷灰石层析或Sephadex G-100柱层析纯化。In another embodiment of the present invention, the phycocyanin released into the culture medium is further purified by ammonium sulfate, gel chromatography, hydroxyapatite chromatography or Sephadex G-100 column chromatography.

因此，本发明提供了一种生物提取藻蓝蛋白的方法。与传统的藻蓝蛋白提取方法相比，本发明的方法具有成本低廉、操作简便等优点，并且提取的蛋白纯度高，而且不破坏蛋白的生物活性，是一种非常适合大规模生产藻蓝蛋白的方法。本发明避开了传统的物理化学法和溶菌酶法的苛刻条件和繁重操作，利用生物方法自然破壁一步即可提取较纯的藻蓝蛋白，大大简化了提取步骤，使藻蓝蛋白工业化生产成为可能。Therefore, the present invention provides a method for biologically extracting phycocyanin. Compared with the traditional phycocyanin extraction method, the method of the present invention has the advantages of low cost, easy operation, etc., and the extracted protein has high purity without destroying the biological activity of the protein, and is very suitable for large-scale production of phycocyanin. Methods. The invention avoids the harsh conditions and heavy operations of the traditional physical and chemical methods and the lysozyme method, and can extract relatively pure phycocyanin by using biological methods to naturally break the wall in one step, which greatly simplifies the extraction steps and enables the industrial production of phycocyanin become possible.

附图说明Description of drawings

下面结合附图和具体实施方式对本发明作进一步的详细描述，但不应理解为是对本发明进行限定。The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, but it should not be construed as limiting the present invention.

图1.按照本发明方法利用日勾维肠杆菌培养液从螺旋藻中直接提取的提取液的吸收光谱，620nm处的吸收峰是藻蓝蛋白的吸收峰；Fig. 1. utilizes the absorption spectrum of the extract solution that directly extracts from spirulina according to the method of the present invention utilizing Enterobacterium gouvetii culture fluid, and the absorption peak at 620nm place is the absorption peak of phycocyanin;

图2.按照本发明方法利用日勾维肠杆菌培养液从螺旋藻中直接提取的提取液的荧光光谱(室温)，最大发射峰位于650nm(580nm激发)；Fig. 2. utilizes the culture liquid of enterobacterium gouvetii to directly extract the fluorescence spectrum (room temperature) of the extract from spirulina according to the inventive method, and the maximum emission peak is positioned at 650nm (580nm excitation);

图3.利用日勾维肠杆菌培养液最终纯化的藻蓝蛋白的吸收光谱，620nm处的吸收峰是藻蓝蛋白的吸收峰；Fig. 3. Utilize the absorption spectrum of the phycocyanin finally purified from the culture solution of Enterobacter gracilis, the absorption peak at 620nm is the absorption peak of phycocyanin;

图4.利用日勾维肠杆菌培养液最终纯化的藻蓝蛋白的荧光光谱(室温)，最大发射峰位于650nm(580nm激发)；Fig. 4. Utilize the fluorescence spectrum (room temperature) of the finally purified phycocyanin of the culture solution of Enterobacter cerevisiae, the maximum emission peak is located at 650nm (580nm excitation);

图5.利用日勾维肠杆菌培养液最终纯化的藻蓝蛋白的SDS-PAGE电泳图(M，分子量标准；cpc：藻蓝蛋白)。Fig. 5. SDS-PAGE electrophoresis diagram of phycocyanin finally purified by using the culture solution of Enterobacter gracilis (M, molecular weight standard; cpc: phycocyanin).

具体实施方式Detailed ways

实施例1利用细菌培养液提取鲜藻的藻蓝蛋白Embodiment 1 Utilizes bacterial culture solution to extract the phycocyanin of fresh algae

A)螺旋藻培养A) Spirulina culture

将螺旋藻接种于Zarrouk培养基【12】中，该培养基包括(g/l)：NaHCO₃16.8，NaNO₃2.5，K₂HPO₄0.5，K₂SO₄1.0，NaCl 1.0，MgSO₄·7H₂O 0.2，CaCl₂0.04，FeSO₄7H₂O 0.01，EDTA 0.08以及微量元素。130rpm/min振荡培养，30℃恒温，100μmol/m²/s连续光照，培养到藻液的OD值A560达到1.2左右，用0.45μm孔径大小的滤网过滤获得新鲜藻泥，无需清洗而用于以下步骤。Inoculate Spirulina in Zarrouk medium【12】, which contains (g/l): NaHCO₃ 16.8, NaNO₃ 2.5, K₂ HPO₄ 0.5, K₂ SO₄ 1.0, NaCl 1.0, MgSO₄ 7H₂ O 0.2, CaCl₂ 0.04, FeSO₄ 7H₂ O 0.01, EDTA 0.08 and trace elements. Shaking culture at 130rpm/min, constant temperature at 30°C, continuous light at 100μmol/m² /s, cultivate until the OD value A560 of the algae solution reaches about 1.2, filter with a filter with a pore size of 0.45μm to obtain fresh algae mud, which can be used without cleaning The following steps.

B)鱼腥藻、集胞藻培养B) Anabaena and Synechocystis culture

将鱼腥藻sp.595、集胞蓝藻PCC6803(藻种引自于中科院水生生物所)分别接种于BG-11培养基，成分见文献^【13】中，(g/l)：NaNO₃，1.5；K₂HPO₄·3H₂O，0.04；MgSO₄·7H₂O，0.075；CaCl₂·2H₂O，0.036；柠檬酸0.006；柠檬酸铁0.006；Na₂EDTA 0.001；Na₂CO₃，0.02；1ml微量元素(g/l)，包括H₃BO₃，2.86；MnCl₂·4H₂O，1.81；ZnSO₄·7H₂O，0.222；Na₂MoO₄·2H₂O，0.39；CuSO₄·5H₂O，0.079；Co(NO₃)₂·6H₂O，0.0494，调节pH＝7.4。130rpm/min振荡培养，30℃恒温，100μmol/m²/s连续光照培养，培养至对数生长后期，用滤网过滤获得新鲜藻泥，无需清洗。Inoculate Anabaena sp.595 and Synechocystis PCC6803 (algae species from the Institute of Hydrobiology, Chinese Academy of Sciences) on BG-11 medium respectively. The ingredients are listed in^[13] , (g/l): NaNO₃ , 1.5 ; K₂ HPO₄ 3H₂ O, 0.04; MgSO₄ 7H₂ O, 0.075; CaCl₂ 2H₂ O, 0.036; Citric acid 0.006; Ferric citrate 0.006;_{Na 2EDTA0.001} ; ; 1ml trace elements (g/l), including H₃ BO₃ , 2.86; MnCl₂ ·4H₂ O, 1.81; ZnSO₄ ·7H₂ O, 0.222; Na₂ MoO₄ ·2H₂ O, 0.39; CuSO₄ · 5H₂ O, 0.079; Co(NO₃ )₂ 6H₂ O, 0.0494, adjusted to pH=7.4. Shaking culture at 130 rpm/min, constant temperature at 30°C, continuous light culture at 100 μmol/m² /s, culture until late logarithmic growth , use a filter to obtain fresh algae mud without cleaning.

C)微生物的培养C) Cultivation of microorganisms

将肺炎克雷伯氏菌(Klebsiella pneumoniae)IMCAS 1.1734(＝CGCMCC1.1734)，日勾维肠杆菌CGMCC No.0510，和产酸克雷伯氏菌(Klebsiellaoxytoca)(IBCAS-ZY)分别接种于自配简化培养基中，该简化培养基包括(g/l)：蔗糖10，K₂HPO₄10，KH₂PO₄3，MgSO₄0.3，NH₄Cl 0.4，CaCl₂0.02以及常规微量元素，调节PH值到8.0，130rpm/min振荡培养，30℃恒温，培养15小时至OD_600nm值为2.000左右，自然沉淀后，倾倒出上清培养液。Klebsiella pneumoniae (Klebsiella pneumoniae) IMCAS 1.1734 (=CGCMCC1.1734), Enterobacter cerevisiae CGMCC No.0510, and Klebsiella oxytoca (Klebsiellaoxytoca) (IBCAS-ZY) were inoculated in self- In the simplified medium, the simplified medium includes (g/l): sucrose 10, K₂ HPO₄ 10, KH₂ PO₄ 3, MgSO₄ 0.3, NH₄ Cl 0.4, CaCl₂ 0.02 and conventional trace elements, adjusted When the pH value reaches 8.0, shake culture at 130rpm/min, keep the temperature at 30°C, and cultivate for 15 hours until the OD_600nm value is about 2.000. After natural precipitation, pour out the supernatant culture solution.

D)藻蓝蛋白吸收光谱的测定方法D) The assay method of phycocyanin absorption spectrum

吸收光谱使用SHIMSDZU UV-2550紫外可见分光光度计于室温下测定，扫描速率为300nm/min，狭缝宽度为2nm，采用光径为1cm的样品池。620nm处的强吸收峰为藻蓝蛋白的特征峰。Absorption spectra were measured at room temperature using a SHIMSDZU UV-2550 ultraviolet-visible spectrophotometer with a scan rate of 300 nm/min, a slit width of 2 nm, and a sample cell with an optical path of 1 cm. The strong absorption peak at 620nm is the characteristic peak of phycocyanin.

E)藻蓝蛋白荧光光谱的测定方法E) Determination of Phycocyanin Fluorescence Spectrum

室温稳态荧光光谱用HITACHI F-4500荧光分光光度计测量，激发、发射光狭峰为5nm，扫描速率是120nm/min，时间常数为2s，光电倍增管增益为NORMAL。位于650nm(580nm激发)处的最大荧光发射峰是藻蓝蛋白的特征峰。The steady-state fluorescence spectrum at room temperature was measured with a HITACHI F-4500 fluorescence spectrophotometer. The excitation and emission narrow peaks were 5nm, the scan rate was 120nm/min, the time constant was 2s, and the gain of the photomultiplier tube was NORMAL. The maximum fluorescence emission peak located at 650nm (580nm excitation) is a characteristic peak of phycocyanin.

每一步提取纯化步骤之后，均取其澄清藻蓝蛋白液测定其室温荧光、吸收光谱。After each step of extraction and purification, the clarified phycocyanin solution was taken to measure its room temperature fluorescence and absorption spectrum.

F)藻蓝蛋白的纯度检测方法F) The purity detection method of phycocyanin

使用SUNSUNG UV8500II紫外可见分光光度计测定不同波长处的紫外和可见光吸收值，即分别在280nm、615nm、620nm、652nm处的吸光值，A₆₂₀/A₂₈₀的比值代表藻蓝蛋白的纯度：食品级：A₆₂₀/A₂₈₀＞0.7，药品级：A₆₂₀/A₂₈₀＞2.0，试剂级：A₆₂₀/A₂₈₀＞4.0。A为吸收值。藻蓝蛋白的浓度用如下公式计算^【14】Use a SUNSUNG UV8500II UV-Vis spectrophotometer to measure the UV and visible light absorption values at different wavelengths, that is, the absorbance values at 280nm, 615nm, 620nm, and 652nm respectively, and the ratio of A₆₂₀ /A₂₈₀ represents the purity of phycocyanin: food grade : A₆₂₀ /A₂₈₀ ＞0.7, pharmaceutical grade: A₆₂₀ /A₂₈₀ ＞2.0, reagent grade: A₆₂₀ /A₂₈₀ ＞4.0. A is the absorption value. The concentration of phycocyanin was calculated by the following formula^[14]

PC(mg/ml)＝(A₆₁₅-0.474(A₆₅₂))/5.34PC (mg/ml) = (A₆₁₅ -0.474(A₆₅₂ ))/5.34

G)藻蓝蛋白的SDS-PAGE检测方法G) SDS-PAGE detection method of phycocyanin

取50μl提纯后的藻蓝蛋白，加入等体积的增溶液(含5％SDS、2％巯基乙醇和10％甘油的50mM Tris溶液，pH＝6.8)，混匀后静置30分钟，沸水煮5分钟，10000g离心5分钟，取上清上样。电泳使用硼酸缓冲体系，浓缩胶、分离胶浓度分别为6％(pH＝6.1)和13.5％(pH＝9.18)。电泳仪为DYCZ-24D型电泳仪(北京市六一仪器厂)。标准蛋白为SDS-聚丙烯酰胺凝胶电泳低分子量标准蛋白(购自上海西巴斯生物技术开发公司)。Take 50 μl of purified phycocyanin, add an equal volume of enrichment solution (50mM Tris solution containing 5% SDS, 2% mercaptoethanol and 10% glycerol, pH=6.8), mix well, let it stand for 30 minutes, and boil for 5 minutes, centrifuge at 10000g for 5 minutes, and take the supernatant for loading. A boric acid buffer system was used for electrophoresis, and the concentrations of the stacking gel and the separating gel were 6% (pH=6.1) and 13.5% (pH=9.18), respectively. The electrophoresis apparatus is DYCZ-24D electrophoresis apparatus (Beijing Liuyi Instrument Factory). The standard protein is SDS-polyacrylamide gel electrophoresis low molecular weight standard protein (purchased from Shanghai Sebastian Biotechnology Development Company).

H)利用细菌培养液提取鲜藻的藻蓝蛋白H) Utilize bacterial culture solution to extract the phycocyanin of fresh algae

以下以日勾维肠杆菌CGMCC No.0510为例举例说明本发明的藻蓝蛋白提取方法。The phycocyanin extraction method of the present invention will be illustrated below by taking Enterobacter cerevisiae CGMCC No.0510 as an example.

将上述各种菌的上清液分别冲洗藻泥至容器中，并加至菌液和藻泥大约为2∶1的体积比，密封(只为防止水分蒸发，不必很严格)，静置，最好避光，24小时后，藻蓝蛋白析出，细胞壁、细胞膜等非水溶性蛋白沉淀于容器底部，上部即为清亮鲜艳的蓝色藻蓝蛋白粗提物，此时如表1所示，纯度(A_620/280)达到0.9。吸收光谱和荧光发射光谱分别如图1和图2所示，分别显示620nm处的藻蓝蛋白特征吸收峰和650nm(580nm激发)处的藻蓝蛋白最大荧光发射峰。附图所示为用日勾维肠杆菌菌液提取螺旋藻鲜藻泥的藻蓝蛋白的吸收光谱和荧光发射光谱，用上述提到的菌作用于藻后，只是在蛋白析出时间和蛋白析出率上稍有差异，但每一步骤所得样品的光谱特性均是与藻蓝蛋白相同的。所得粗提物用HITACHI 20PR-520离心机5000g离心后，取上清，此时藻蓝蛋白的纯度(A_620/280)可达到1.3-1.5，远高于食品级纯度。Wash the supernatant of the above-mentioned various bacteria into the container respectively, and add the bacterial liquid to the algae mud at a volume ratio of about 2:1, seal it (just to prevent water evaporation, not very strict), let it stand, It is best to avoid light. After 24 hours, phycocyanin precipitates, and water-insoluble proteins such as cell walls and cell membranes precipitate at the bottom of the container. The upper part is the clear and bright blue phycocyanin crude extract. At this time, as shown in Table 1, The purity (A_620/280 ) reaches 0.9. The absorption spectrum and the fluorescence emission spectrum are shown in Figure 1 and Figure 2 respectively, showing the characteristic absorption peak of phycocyanin at 620nm and the maximum fluorescence emission peak of phycocyanin at 650nm (580nm excitation). The accompanying drawing shows the absorption spectrum and fluorescence emission spectrum of phycocyanin extracted from spirulina fresh algae mud with Enterobacter japonicus bacterial liquid. After using the above-mentioned bacteria to act on algae, only the protein precipitation time and protein precipitation There is a slight difference in the rate, but the spectral characteristics of the samples obtained in each step are the same as those of phycocyanin. The obtained crude extract is centrifuged at 5000 g in a HITACHI 20PR-520 centrifuge, and the supernatant is taken. At this time, the purity of phycocyanin (A_620/280 ) can reach 1.3-1.5, which is much higher than the food-grade purity.

之后用60％饱和度的硫酸铵沉淀，余后流程与目前通行方法无太大差别，参照胡一兵^【15】等的方法流程将上清液通过羟基磷灰石层析和Sephadex G-100柱层析，最后可得到纯度4.8-5.2的试剂纯藻蓝蛋白，其吸收光谱和荧光发射光谱分别如图3和图4所示。与图1和图2相同，分别显示620nm处的藻蓝蛋白特征吸收峰和650nm(580nm激发)处的藻蓝蛋白最大荧光发射峰，但是峰形更明显，表明纯度更高。Afterwards, ammonium sulfate with 60% saturation was used for precipitation, and the rest of the process was not much different from the current current method. Referring to the process of Hu Yibing^[15] , the supernatant was passed through hydroxyapatite chromatography and Sephadex G-100 column layer Finally, the reagent pure phycocyanin with a purity of 4.8-5.2 can be obtained, and its absorption spectrum and fluorescence emission spectrum are shown in Figure 3 and Figure 4 respectively. Same as Figure 1 and Figure 2, showing the characteristic absorption peak of phycocyanin at 620nm and the maximum fluorescence emission peak of phycocyanin at 650nm (580nm excitation), respectively, but the peak shape is more obvious, indicating higher purity.

为了检测最终纯化的藻蓝蛋白的纯度，如上使用SDS-PAGE方法检测所得藻蓝蛋白已经达到电泳纯。结果如图5所示，在SDS-PAGE电泳图谱中与标准蛋白相比，显示了藻蓝蛋白的亚单位结构，两条带分别是藻蓝蛋白的α亚基和β亚基，与藻蓝蛋白结构相符合。以上利用光谱特性和电泳结果证明用本发明方法提取的上清的蓝色液体含有高纯度的藻蓝蛋白。In order to detect the purity of the finally purified phycocyanin, the SDS-PAGE method was used to detect that the obtained phycocyanin had reached electrophoretic purity. The results are shown in Figure 5. Compared with the standard protein in the SDS-PAGE electrophoresis pattern, the subunit structure of phycocyanin is shown. The two bands are the α subunit and β subunit of phycocyanin, which are different from the The protein structure matches. The above spectral characteristics and electrophoresis results prove that the blue liquid of the supernatant extracted by the method of the present invention contains high-purity phycocyanin.

实施例2利用细菌培养液提取螺旋藻藻粉中的藻蓝蛋白Embodiment 2 Utilizes bacterial culture fluid to extract the phycocyanin in the spirulina algae powder

以与实施例1中相同的方法进行藻蓝蛋白的提取。不同之处在于，用藻粉代替鲜藻。具体步骤如下：称取云南产香峰牌(北京阳光雨虹峰产品技术开发公司)100％藻粉500g置于容器中，加入实施例1中培养的各种菌液5000ml混合，静置于室温25℃左右，半小时后，藻蓝蛋白析出，上清液清亮湛蓝，倾倒出上清。余下步骤与实施例1相同，得出类似的结果。需要说明的是，利用藻粉获得的藻蓝蛋白粗提液的纯度没有利用鲜藻泥的高，但藻蓝蛋白析出时间藻粉成倍加快，分析可能是藻粉在制备过程中由于处理流程使得螺旋藻的胞壁组构发生变化，导致藻蓝蛋白更易析出。但由于藻粉制备过程中，藻蓝蛋白因为高温干燥等原因，导致藻蓝蛋白部分变性，因此藻蓝蛋白的纯度和浓度都受影响。因此，用鲜藻提取藻蓝蛋白是最佳选择。Extraction of phycocyanin was performed in the same manner as in Example 1. The difference is that algal powder is used instead of fresh algae. The specific steps are as follows: Weigh 500 g of 100% algae powder of Yunnan-produced Xiangfeng brand (Beijing Sunshine Yuhongfeng Product Technology Development Company) and place it in a container, add 5000 ml of various bacterial liquids cultivated in Example 1 to mix, and leave it at room temperature At about 25°C, after half an hour, phycocyanin precipitates, and the supernatant is clear and blue, and the supernatant is poured out. Remaining steps are identical with embodiment 1, obtain similar result. It should be noted that the purity of the phycocyanin crude extract obtained by using algae powder is not as high as that obtained by using fresh algae mud, but the precipitation time of phycocyanin is doubled. The structure of the cell wall of Spirulina changes, resulting in easier precipitation of phycocyanin. However, during the preparation of algae powder, phycocyanin is partially denatured due to high-temperature drying and other reasons, so the purity and concentration of phycocyanin are affected. Therefore, extracting phycocyanin from fresh algae is the best choice.

实施例3藻蓝蛋白的不同粗提取方法的对比The comparison of the different crude extraction methods of embodiment 3 phycocyanin

为了比较本发明的方法和常规的藻蓝蛋白的提取法，在对照条件下按照文献中记载的各种藻蓝蛋白提取方法对鲜藻泥和/或藻粉进行提取，结果如表1所示。In order to compare the extraction method of the method of the present invention and conventional phycocyanin, fresh algal mud and/or algae powder are extracted according to various phycocyanin extraction methods recorded in the literature under control conditions, and the results are shown in Table 1 .

表1  用不同方法粗提鲜藻泥(螺旋藻)中藻蓝蛋白的对照图Table 1 Comparison chart of phycocyanin in fresh algae mud (spirulina) crudely extracted by different methods

提取方法       藻蓝蛋白浓度  藻蓝蛋白纯度  规模和设备Extraction method Phycocyanin concentration Phycocyanin purity Scale and equipment

               (μg/ml)      (A₆₂₀/A₂₈₀)(μg/ml) (A₆₂₀ /A₂₈₀ )

4℃超声破碎    300±12       0.30          设备要求严Ultrasonic crushing at 4°C 300±12 0.30 Strict equipment requirements

                                           格，每次量小grid, small amount

                                           温度难控制Difficult to control temperature

室温25℃压     369±15       0.40          特殊设备，处Room temperature 25 ℃ pressure 369±15 0.40 Special equipment, at

                                           轧破碎理量小，耗力                             , 

室温0℃0.5cm   800±10       0.80          设备温控难，Room temperature 0°C 0.5cm 800±10 0.80 It is difficult to control the temperature of the equipment,

玻璃珠机械破                               时间长，耗能Glass bead mechanical breaking takes a long time and consumes energy

碎broken

-20℃冰冻30℃  950±12       0.66          耗时耗能-20℃ freezing 30℃ 950±12 0.66 Time-consuming and energy-consuming

融化melt

2mg溶菌酶/g    280±10       0.37          耗时，成本高，2mg lysozyme/g 280±10 0.37 time-consuming, high cost,

湿重                                       得率低Wet weight Low yield

本发明的方法   1205±10      0.90          无温度和设备The method of the present invention 1205±10 0.90 without temperature and equipment

(鲜藻泥加入                                限制，省力省(addition of fresh algae mud is limited, saving labor and saving

菌液)                                      能，规模不受Bacterial solution) Capability, the scale is not affected by

                                           限制 limit

本专利发明方    600±15        0.56      同上Invention method of thispatent 600±15 0.56 Same as above

法Law

(藻粉加入菌(algae powder added bacteria

液)liquid)

从表1中可以看出，与各种传统藻蓝蛋白提取方法相比，本发明的微生物培养液提取方法提取的藻蓝蛋白不仅产率高，而且提取的藻蓝蛋白的纯度也优于其他各种方法。本发明的方法不仅具有成本低廉、操作简便等优点，并且提取的蛋白纯度高，而且不破坏蛋白的生物活性，是一种非常适合于规模化生产藻蓝蛋白的方法。由于本发明避开了传统的物理化学法和溶菌酶法的苛刻条件和繁重操作，利用生物方法自然破壁一步即可提取较纯的藻蓝蛋白，大大简化了提取步骤，因而使藻蓝蛋白工业化生产成为可能。As can be seen from Table 1, compared with various traditional phycocyanin extraction methods, the phycocyanin extracted by the microbial culture solution extraction method of the present invention not only has a high yield, but also has a higher purity than other phycocyanin extracts. Various methods. The method of the invention not only has the advantages of low cost, easy operation and the like, but also the extracted protein has high purity and does not destroy the biological activity of the protein, and is very suitable for large-scale production of phycocyanin. Since the present invention avoids the harsh conditions and heavy operations of traditional physical and chemical methods and lysozyme methods, purer phycocyanin can be extracted in one step by natural wall breaking by biological methods, which greatly simplifies the extraction steps, thus making phycocyanin Industrial production is possible.

实施例4不同固氮菌提取藻蓝蛋白的对比Embodiment 4 Comparison of different nitrogen-fixing bacteria extracting phycocyanin

为了比较本发明中不同固氮菌提取藻蓝蛋白的优劣，在对照条件下按照实施例1中的方法对螺旋藻藻粉云南产香峰牌(北京阳光雨虹峰产品技术开发公司)100％藻粉500g进行提取，菌液均培养15小时，OD₆₀₀为2，结果如表2所示。In order to compare the advantages and disadvantages of extracting phycocyanin by different nitrogen-fixing bacteria in the present invention, according to the method in Example 1, 100% of spirulina algae powder Yunnan produced Xiangfeng brand (Beijing Sunshine Yuhongfeng Product Technology Development Company) under control conditions 500g of algae powder was extracted, and the bacterial liquid was cultured for 15 hours, and the_OD600 was 2. The results are shown in Table 2.

表2不同细菌提取藻粉中藻蓝蛋白的对比图Table 2 Comparison chart of phycocyanin in algae powder extracted by different bacteria

                日勾维肠杆菌     肺炎克雷伯氏菌     产酸克雷伯氏菌      Enterobacter gouverii   Klebsiella pneumoniae   Klebsiella oxytoca

破碎时间(分)    30±8            30±540±5Breaking time (min) 30±8 30±540±5

粗提液藻蓝蛋白  0.50±0.20       0.60±0.15         0.55±0.15Crude extract phycocyanin 0.50±0.20 0.60±0.15 0.55±0.15

纯度(A₆₂₀/A₂₈₀)Purity (A₆₂₀ /A₂₈₀ )

粗提液藻蓝蛋白  1300±10         1350±10           1300±15Crude extract phycocyanin 1300±10 1350±10 1300±15

浓度(μg/ml)Concentration (μg/ml)

从表2可以看出，分别使用日勾维肠杆菌CGMCC No.0510，肺炎克雷伯氏菌(Klebsiella pneumoniae)IMCAS 1.1734(＝CGCMCC 1.1734)，和产酸克雷伯氏菌(Klebsiella oxytoca)(IBCAS-ZY)进行实施例1中的实验可以容易地得出类似的结果，都可以在很短时间破碎藻细胞，释放出藻蓝蛋白。藻蓝蛋白纯度和总藻蓝蛋白浓度都很理想。三种菌株都是不错的选择。其中肺炎克雷伯氏菌(Klebsiella pneumoniae)IMCAS1.1734(＝CGCMCC 1.1734)略有优势，说明这些菌属都适用于本发明。As can be seen from Table 2, the use of Enterobacter cerevisiae CGMCC No.0510, Klebsiella pneumoniae (Klebsiella pneumoniae) IMCAS 1.1734 (=CGCMCC 1.1734), and Klebsiella oxytoca (Klebsiella oxytoca) ( IBCAS-ZY) carried out the experiment in embodiment 1 and can draw similar result easily, all can break algae cell in very short time, release phycocyanin. Both phycocyanin purity and total phycocyanin concentration are ideal. All three strains are good choices. Among them, Klebsiella pneumoniae (Klebsiella pneumoniae) IMCAS1.1734 (=CGCMCC 1.1734) has a slight advantage, indicating that these genera are applicable to the present invention.

实施例5按照本发明的方法提取各种蓝藻、蓝绿藻中的藻蓝蛋白Embodiment 5 extracts phycocyanin in various blue-green algae and blue-green algae according to the method of the present invention

为了比较本发明中固氮菌液是否能很好适用于其他蓝藻、蓝绿藻的藻蓝蛋白提取，在对照条件下按照实施例1中的方法，样品为螺旋藻、鱼腥藻、集胞藻的鲜藻泥，湿重为200g，菌液用肺炎克雷伯氏菌(Klebsiellapneumoniae)IMCAS 1.1734(＝CGCMCC 1.1734)，培养15小时，OD₆₀₀为2，结果如表3所示。In order to compare whether the nitrogen-fixing bacteria liquid in the present invention can be well applied to the phycocyanin extraction of other blue-green algae and blue-green algae, according to the method in Example 1 under control conditions, the samples are Spirulina, Anabaena, Synechocystis The fresh algae mud, the wet weight is 200g, the bacterium liquid uses Klebsiella pneumoniae (Klebsiellapneumoniae) IMCAS 1.1734 (=CGCMCC 1.1734), cultivates for 15 hours,_OD600 is 2, and the result is shown in Table 3.

表3按照本发明的方法提取各种蓝藻、蓝绿藻中的藻蓝蛋白Table 3 extracts phycocyanin in various blue-green algae and blue-green algae according to the method of the present invention

                螺旋藻           鱼腥藻            集胞藻                                                 

蛋白析出后显微  未见藻丝，未见   未见藻丝和任何    未见任何完整细After protein precipitation, no algae filaments, no algal filaments and any complete cells were seen under the microscope

镜下观察        任何完整细胞，   完整细胞，细胞    胞，破碎效果很Observing under a microscope any complete cells, complete cells, cells, the broken effect is very

                破碎效果很好     破碎效果很好      好Good breaking effect Good breaking effect Good

藻蓝蛋白析出时  12               15                18When phycocyanin is precipitated 12 15 18

间(小时)time (hour)

粗提液藻蓝蛋白  0.90             0.70              0.68Crude Extract Phycocyanin 0.90 0.70 0.68

纯度(A₆₂₀/A₂₈₀)Purity (A₆₂₀ /A₂₈₀ )

盐析、层析后藻  5.12             4.83              4.15Algae after salting out and chromatography 5.12 4.83 4.15

蓝蛋白纯度Blue protein purity

(A₆₂₀/A₂₈₀)(A₆₂₀ /A₂₈₀ )

粗提液藻蓝蛋白  1350             650               620Crude Extract Phycocyanin 1350 650 620

浓度(μg/ml)Concentration (μg/ml)

从表3可以看出，螺旋藻、鱼腥藻、集胞藻中的藻蓝蛋白均可以用本发明的方法简单提取出来，由于螺旋藻中藻胆蛋白含量高达鲜重的15-20％，且不含藻红蛋白，因此纯度和总蛋白提取都处于明显优势。目前国内最多养殖的也是螺旋藻，因此原材料供应充足，很适宜于提取藻蓝蛋白。本发明同样适用于其他蓝藻，其中集胞藻6803是蓝藻的模式植物，也能在菌液中很快破壁，析出蛋白。但也可以看出，由于鱼腥藻、集胞藻的本身蛋白含量不高，所以藻蓝蛋白提取量不够高，并非因为破壁不完全，而是由于其品系的缘故。所用显微镜是CARL ZEISS JENA，400×。As can be seen from Table 3, the phycocyanin in Spirulina, Anabaena, and Synechocystis can be simply extracted by the method of the present invention, because the phycobiliprotein content in Spirulina is as high as 15-20% of the fresh weight, And it does not contain phycoerythrin, so the purity and total protein extraction are in obvious advantages. At present, spirulina is the most cultivated in China, so the supply of raw materials is sufficient, which is very suitable for extracting phycocyanin. The present invention is also applicable to other cyanobacteria, among which the Synechocystis 6803 is a model plant of cyanobacteria, and it can also quickly break the wall and separate out proteins in the bacterial liquid. However, it can also be seen that since the protein content of Anabaena and Synechocystis is not high, the extraction of phycocyanin is not high enough, not because of incomplete wall breaking, but because of its strain. The microscope used is CARL ZEISS JENA, 400×.

实施例6不同藻和不同菌的反应结果对照The reaction result contrast of embodiment 6 different algae and different bacteria

为了充分了解不同材料之间相互反应是否有特异性差别，设计了如下试验，将不同藻和不同菌分别作用。在对照条件下按照实施例1中的方法，样品为螺旋藻、鱼腥藻、集胞藻的鲜藻泥，湿重为100g，菌液用以上的肺炎克雷伯氏菌、日勾维肠杆菌、产酸克雷伯氏菌，培养15小时，OD₆₀₀为2，结果如表4所示。In order to fully understand whether there is a specific difference in the interaction between different materials, the following experiments were designed, in which different algae and different bacteria were respectively acted on. According to the method in Example 1 under control conditions, the sample is the fresh algae mud of Spirulina, Anabaena, Synechocystis, the wet weight is 100g, and the bacteria liquid uses the above Klebsiella pneumoniae, Glycocystis spp. Bacillus and Klebsiella oxytoca were cultured for 15 hours, and the_OD600 was 2. The results are shown in Table 4.

表4不同菌与不同鲜藻泥的作用对照图Table 4 Effect comparison chart of different bacteria and different fresh algae mud

               螺旋藻           鱼腥藻            集胞藻                                               

日勾维肠杆菌   13小时破碎，粗   17小时破碎，粗    18小时破碎，Enterobacter daily Gouvetii crushed in 13 hours, coarse crushed in 17 hours, coarse crushed in 18 hours,

               提液A₆₂₀/A₂₈₀＝  提液A₆₂₀/A₂₈₀＝   粗提液Extract A₆₂₀ /A₂₈₀ = Extract A₆₂₀ /A₂₈₀ = Crude Extract

               0.9，粗提液蛋白  0.7，粗提液蛋白   A₆₂₀/A₂₈₀＝0.68，粗0.9, crude extract protein 0.7, crude extract protein A₆₂₀ /A₂₈₀ = 0.68, crude extract protein

               浓度(μg/ml)＝   浓度(μg/ml)＝700 提液蛋白浓度Concentration (μg/ml) = Concentration (μg/ml) = 700 Extract protein concentration

               1300                               (μg/ml)＝6901300 (μg/ml)＝690

肺炎克雷伯氏菌 12小时破碎，粗   15小时破碎，粗    18小时破碎，Klebsiella pneumoniae broken in 12 hours, coarse broken in 15 hours, coarse broken in 18 hours,

               提液A₆₂₀/A₂₈₀＝  提液A₆₂₀/A₂₈₀＝   粗提液Extract A₆₂₀ /A₂₈₀ = Extract A₆₂₀ /A₂₈₀ = Crude Extract

               0.9，粗提液藻蓝  0.7，粗提液藻蓝   A₆₂₀/A₂₈₀＝0.68，粗0.9, the crude extract spirulina 0.7, the crude extract spirulina A₆₂₀ /A₂₈₀ = 0.68, the crude

               蛋白浓度         蛋白浓度          提液藻蓝蛋白浓                                                   

               (μg/ml)＝1350   (μg/ml)＝650     度(μg/ml)＝630(μg/ml)＝1350 (μg/ml)＝650 Degree (μg/ml)＝630

产酸克雷伯氏菌 12小时破碎，粗   16小时破碎，粗    17小时破碎，Klebsiella oxytoca breaks in 12 hours, coarse breaks in 16 hours, coarse breaks in 17 hours,

               提液A₆₂₀/A₂₈₀＝  提液A₆₂₀/A₂₈₀＝   粗提液Extract A₆₂₀ /A₂₈₀ = Extract A₆₂₀ /A₂₈₀ = Crude Extract

               0.9，粗提液藻蓝  0.7，粗提液藻蓝   A₆₂₀/A₂₈₀＝0.68，粗0.9, the crude extract spirulina 0.7, the crude extract spirulina A₆₂₀ /A₂₈₀ = 0.68, the crude

               蛋白浓度         蛋白浓度          提液藻蓝蛋白浓                                                   

               (μg/ml)＝1300   (μg/ml)＝670     度(μg/ml)＝650(μg/ml)＝1300 (μg/ml)＝670 Degree (μg/ml)＝650

从表4可以看出，以上各种螺旋藻、鱼腥藻、集胞藻与肺炎克雷伯氏菌、日勾维肠杆菌、产酸克雷伯氏菌两两相互交叉，没有显著性差异，作用基本相同，纯度、浓度和时间主要取决于藻本身。It can be seen from Table 4 that the above-mentioned Spirulina, Anabaena, Synechocystis and Klebsiella pneumoniae, Enterobacter spp. , the role is basically the same, the purity, concentration and time mainly depend on the algae itself.

应当理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，但改动或修改的等价形式同样落在本申请权利要求书所限定的范围内。It should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, but the equivalent forms of changes or modifications also fall within the scope defined by the claims of the present application.

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Claims (3)

Translated fromChinese

1.一种提取藻蓝蛋白的方法，其特征在于将固氮菌的培养液与蓝藻、蓝绿藻的藻泥或藻粉混合，其中所述固氮菌是日勾维肠杆菌(Enterobactergergoviae)或肺炎克雷伯氏菌(Klebsiella pneumoniae)。1. A method for extracting phycocyanin, characterized in that the nutrient solution of nitrogen-fixing bacteria is mixed with the algae mud or algal powder of blue-green algae, blue-green algae, wherein said nitrogen-fixing bacteria is Enterobactergergoviae (Enterobactergergoviae) or pneumoniae Klebsiella pneumoniae.2.按照权利要求1的方法，其中所述固氮菌是日勾维肠杆菌(Enterobacter gergoviae)CGMCC No.0510或肺炎克雷伯氏菌(Klebsiellapneumoniae)IMCAS 1.1734。2. The method according to claim 1, wherein the nitrogen-fixing bacterium is Enterobacter gergoviae CGMCC No. 0510 or Klebsiellapneumoniae IMCAS 1.1734.3.按照权利要求1或2的方法，其另外包含将藻蓝蛋白粗提物进一步用硫酸铵、凝胶层析、羟基磷灰石层析或Sephadex G-200柱层析纯化的步骤。3. The method according to claim 1 or 2, which additionally comprises the step of further purifying the phycocyanin crude extract with ammonium sulfate, gel chromatography, hydroxyapatite chromatography or Sephadex G-200 column chromatography.

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