CN104672325A - Method for preparing phycocyanin from fresh spirulina - Google Patents
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- 108010053210 Phycocyanin Proteins 0.000 title claims abstract description 69
- 235000016425 Arthrospira platensis Nutrition 0.000 title claims abstract description 50
- 240000002900 Arthrospira platensis Species 0.000 title claims abstract description 50
- 229940082787 spirulina Drugs 0.000 title claims abstract description 50
- 238000000034 method Methods 0.000 title claims abstract description 26
- 239000002202 Polyethylene glycol Substances 0.000 claims abstract description 42
- 229920001223 polyethylene glycol Polymers 0.000 claims abstract description 42
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 36
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims abstract description 36
- 235000011130 ammonium sulphate Nutrition 0.000 claims abstract description 36
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 27
- 241000195493 Cryptophyta Species 0.000 claims abstract description 20
- 239000002994 raw material Substances 0.000 claims abstract description 16
- 238000000605 extraction Methods 0.000 claims abstract description 12
- 238000012545 processing Methods 0.000 claims abstract description 12
- 150000004676 glycans Chemical class 0.000 claims abstract description 9
- 229920001282 polysaccharide Polymers 0.000 claims abstract description 9
- 239000005017 polysaccharide Substances 0.000 claims abstract description 9
- 238000009777 vacuum freeze-drying Methods 0.000 claims abstract description 9
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 7
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 7
- 238000005185 salting out Methods 0.000 claims abstract description 7
- 229930002875 chlorophyll Natural products 0.000 claims abstract description 4
- 235000019804 chlorophyll Nutrition 0.000 claims abstract description 4
- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 claims abstract description 4
- 239000007788 liquid Substances 0.000 claims description 50
- 239000002244 precipitate Substances 0.000 claims description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- 239000012141 concentrate Substances 0.000 claims description 16
- 238000003756 stirring Methods 0.000 claims description 12
- 239000007787 solid Substances 0.000 claims description 10
- 150000003863 ammonium salts Chemical class 0.000 claims description 6
- 239000000084 colloidal system Substances 0.000 claims description 6
- 239000012528 membrane Substances 0.000 claims description 6
- 238000000926 separation method Methods 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 2
- 230000008569 process Effects 0.000 abstract description 11
- 238000002360 preparation method Methods 0.000 abstract description 10
- 230000009286 beneficial effect Effects 0.000 abstract description 4
- 230000004071 biological effect Effects 0.000 abstract 1
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- 239000000047 product Substances 0.000 description 10
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- 238000004925 denaturation Methods 0.000 description 2
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- 238000011160 research Methods 0.000 description 2
- 230000002411 adverse Effects 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
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- 239000007864 aqueous solution Substances 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
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- 239000003814 drug Substances 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
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- 239000007850 fluorescent dye Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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Abstract
本发明以简便、高效、有利于不同组分综合利用为工艺设计的基础，以新鲜螺旋藻为加工原料，通过低温破壁、硫酸铵分级盐析、聚乙二醇2000/(NH4)2SO4双水相萃取、超滤、真空冷冻干燥的步骤，制得螺旋藻藻蓝蛋白。本发明能实现较高的螺旋藻藻蓝蛋白得率，所得螺旋藻藻蓝蛋白多糖含量低，纯度A620/A280约3.7，并适用于以其他藻类为原料制备藻蓝蛋白，且制备过程中能很好的保持螺旋藻各组分的生物活性，得到的其余分离部位可经进一步加工得到活性多糖、叶绿素、藻蛋白等系列产品，具有良好的推广价值及应用前景。The present invention is based on simple, efficient, and beneficial to the comprehensive utilization of different components as the basis of process design, uses fresh spirulina as processing raw material, through low-temperature wall breaking, ammonium sulfate graded salting out, polyethylene glycol 2000/(NH 4 ) 2 Spirulina phycocyanin is obtained through the steps of SO 4 aqueous two-phase extraction, ultrafiltration, and vacuum freeze-drying. The invention can realize higher yield of spirulina phycocyanin, the obtained spirulina phycocyanin polysaccharide content is low, the purity A 620 /A 280 is about 3.7, and it is suitable for preparing phycocyanin with other algae as raw materials, and the preparation process It can well maintain the biological activity of each component of Spirulina, and the remaining isolated parts can be further processed to obtain a series of products such as active polysaccharides, chlorophyll, and algae protein, which have good promotion value and application prospects.
Description
技术领域 technical field
本发明属于藻类功能组分深加工技术领域，具体涉及一种用新鲜螺旋藻高效制备藻蓝蛋白的方法。 The invention belongs to the technical field of deep processing of functional components of algae, and in particular relates to a method for efficiently preparing phycocyanin with fresh spirulina.
背景技术 Background technique
目前，市场上以螺旋藻粉为基础原料生产的螺旋藻片、胶囊、复合饮料等品种繁多，但由于行业内质量评价体系的缺失，使一些生产企业忽视产品生产过程中的质量控制，造成同类产品生产工艺、产品质量差异较大，同时，对产品功效的夸大宣传，导致人们对螺旋藻产品营养、安全、功效等产生怀疑，严重影响了螺旋藻产品的开发应用和螺旋藻产业的健康发展。 At present, there are many varieties of spirulina tablets, capsules, and compound beverages produced on the market based on spirulina powder. However, due to the lack of a quality evaluation system in the industry, some manufacturers ignore the quality control during the production process, resulting in similar The production process and product quality of the products are quite different. At the same time, the exaggerated publicity of the efficacy of the products has caused people to doubt the nutrition, safety, and efficacy of spirulina products, which has seriously affected the development and application of spirulina products and the healthy development of the spirulina industry. .
为保障螺旋藻产业的持续健康发展，应注重加强螺旋藻养殖加工系统集成技术的研究。其中，螺旋藻多糖、藻蓝蛋白等精深加工产品的开发，加工工艺对螺旋藻组分结构、特性的影响，及提高螺旋藻组分综合利用率等方面，是产业发展需要重点解决的关键问题。 In order to ensure the sustainable and healthy development of the spirulina industry, attention should be paid to strengthening the research on the integration technology of the spirulina cultivation and processing system. Among them, the development of deep-processing products such as spirulina polysaccharides and phycocyanin, the influence of processing technology on the structure and characteristics of spirulina components, and the improvement of the comprehensive utilization rate of spirulina components are key issues that need to be solved in industrial development. .
藻蓝蛋白是一种光合色素，其水溶液为亮蓝色，可作为天然色素应用于食品、化妆品等行业；藻蓝蛋白还具有诸多生理活性功能，如提高免疫力、抗癌、抗氧化等，在医药保健领域也有良好的应用前景；此外，由于藻蓝蛋白具有强烈荧光，高纯度的藻蓝蛋白可制成荧光试剂、荧光探针、荧光示踪物质等，用于临床医学诊断、免疫化学及生物工程等研究领域中，具有很高的经济价值。而目前市场上尚未有功效明确、可靠的藻蓝蛋白精深加工产品，这是由于现有报道大多采用盐析、离心、透析和色谱柱层析等工艺从螺旋藻及其他藻类原料中分离纯化藻蓝蛋白，但其在实际生产过程中存在步骤繁琐、能耗高、产率低、操作周期长、综合利用率低等缺点，导致高纯度藻蓝蛋白的价格极其昂贵，制约了藻蓝蛋白的应用。因此，探索一种工艺简化、条件温和、便于工业放大、有利于共存组分综合利用、加工成本低、经济效益高的藻蓝蛋白制备方法，对于藻蓝蛋白的开发应用具有重要的意义。 Phycocyanin is a kind of photosynthetic pigment, and its aqueous solution is bright blue, which can be used as a natural pigment in food, cosmetics and other industries; phycocyanin also has many physiologically active functions, such as improving immunity, anti-cancer, anti-oxidation, etc. It also has good application prospects in the field of medicine and health care; in addition, due to the strong fluorescence of phycocyanin, high-purity phycocyanin can be made into fluorescent reagents, fluorescent probes, fluorescent tracers, etc., for clinical medical diagnosis, immunochemistry, etc. It has high economic value in research fields such as bioengineering and bioengineering. At present, there is no clear and reliable phycocyanin deep-processing product on the market. This is because most of the existing reports use processes such as salting out, centrifugation, dialysis and column chromatography to separate and purify algae from spirulina and other algae raw materials. cyanocyanin, but in the actual production process, there are disadvantages such as cumbersome steps, high energy consumption, low yield, long operation cycle, and low comprehensive utilization rate, which lead to the extremely expensive price of high-purity phycocyanin, which restricts the production of phycocyanin. application. Therefore, it is of great significance for the development and application of phycocyanin to explore a phycocyanin preparation method with simplified process, mild conditions, easy industrial scale-up, comprehensive utilization of coexisting components, low processing cost and high economic benefit.
发明内容 Contents of the invention
本发明的目的在于提供一种用新鲜螺旋藻制备藻蓝蛋白的方法，经本发明方法制备藻蓝蛋白具有得率高、糖含量低的特点，且其制备工艺简单、制备成本低、经济效益高，又有利于提高共存组分的综合利用率，具有良好的推广价值及应用前景。 The purpose of the present invention is to provide a method for preparing phycocyanin with fresh spirulina, the preparation of phycocyanin by the method of the present invention has the characteristics of high yield and low sugar content, and its preparation process is simple, the preparation cost is low, and the economic benefit It is also beneficial to improve the comprehensive utilization rate of coexisting components, and has good promotion value and application prospect.
为实现上述目的，本发明采用如下技术方案： To achieve the above object, the present invention adopts the following technical solutions:
一种用新鲜螺旋藻制备藻蓝蛋白的方法，是以新鲜螺旋藻为加工原料，通过低温破壁、硫酸铵分级盐析、聚乙二醇2000/(NH4)2SO4双水相萃取、超滤、真空冷冻干燥的步骤，制得螺旋藻藻蓝蛋白。 A method for preparing phycocyanin from fresh spirulina, using fresh spirulina as processing raw material, through low-temperature wall breaking, ammonium sulfate graded salting out, polyethylene glycol 2000/(NH 4 ) 2 SO 4 two-phase extraction , ultrafiltration, and vacuum freeze-drying steps to prepare spirulina phycocyanin.
其具体包括以下步骤： It specifically includes the following steps:
1）将新鲜螺旋藻于-20℃下冷冻4-24h，解冻，胶体磨预处理后，25-35MPa压力下均质破壁，1300Orpm离心15min，除去沉淀，得一次离心分离液； 1) Freeze fresh spirulina at -20°C for 4-24 hours, thaw, pretreat with a colloid mill, homogeneously break the wall under a pressure of 25-35MPa, centrifuge at 1300Orpm for 15min, remove the precipitate, and obtain a centrifuge;
2）取步骤1）所得离心分离液，快速搅拌下缓慢加入粉末状固体硫酸铵，使混合液中硫酸铵的饱和度达25%，静置2h，1300Orpm离心15-25min，除去沉淀，得二次离心分离液； 2) Take the centrifuged liquid obtained in step 1), slowly add powdered solid ammonium sulfate under rapid stirring, so that the saturation of ammonium sulfate in the mixed liquid reaches 25%, let it stand for 2 hours, and centrifuge at 1300Orpm for 15-25min to remove the precipitate and obtain two secondary centrifuge;
3）取步骤2）所得离心分离液，快速搅拌下缓慢加入粉末状固体硫酸铵，使混合液中硫酸铵的饱和度达55%，静置2h，1300Orpm离心15min，除去离心分离液，取沉淀； 3) Take the centrifuged liquid obtained in step 2), slowly add powdered solid ammonium sulfate under rapid stirring, so that the saturation of ammonium sulfate in the mixed liquid reaches 55%, let it stand for 2 hours, centrifuge at 1300Orpm for 15min, remove the centrifuged liquid, and take the precipitate ;
4）取步骤3）所得沉淀加水溶解，得待处理液；分别将质量浓度为22%的聚乙二醇2000溶液和质量浓度为44%的硫酸铵溶液缓慢加入到所配制的待处理液中，形成聚乙二醇2000总浓度为10%、硫酸铵总浓度为20%的聚乙二醇2000/(NH4)2SO4双水相萃取体系，充分搅拌后静置2h，分液收集聚乙二醇相； 4) Take the precipitate obtained in step 3) and dissolve it with water to obtain the liquid to be treated; slowly add the polyethylene glycol 2000 solution with a mass concentration of 22% and the ammonium sulfate solution with a mass concentration of 44% to the prepared liquid to be treated , to form a polyethylene glycol 2000/(NH 4 ) 2 SO 4 two-phase extraction system with a total concentration of polyethylene glycol 2000 of 10% and a total concentration of ammonium sulfate of 20%, stir well, let stand for 2 hours, and collect by liquid separation polyethylene glycol phase;
5）采用分子量为3500kDa的超滤膜对步骤4）所收集的聚乙二醇相进行超滤，超滤时采用聚乙二醇相、水交替进入超滤器的供液模式，聚乙二醇相每次进料后的间隔时间逐渐增长，水每次进料后的间隔时间逐渐缩短，以脱除聚乙二醇、铵盐；超滤后段停止进水，以体系循环超滤，以浓缩得藻蓝蛋白浓缩液； 5) Use an ultrafiltration membrane with a molecular weight of 3500kDa to perform ultrafiltration on the polyethylene glycol phase collected in step 4). During ultrafiltration, the polyethylene glycol phase and water enter the ultrafilter alternately. The interval time after each feed of the alcohol phase is gradually increased, and the interval time after each feed of water is gradually shortened to remove polyethylene glycol and ammonium salts; the water feed is stopped at the post-ultrafiltration stage, and the system circulates the ultrafiltration, Concentrate to obtain phycocyanin concentrate;
6）将步骤5）所得藻蓝蛋白浓缩液真空冷冻干燥，制备得螺旋藻藻蓝蛋白。 6) Vacuum freeze-drying the phycocyanin concentrate obtained in step 5) to prepare spirulina phycocyanin.
步骤1）-步骤4）中分离除去的部分经进一步合并、加工，能制备出活性多糖、叶绿素、藻蛋白等系列产品。 The parts separated and removed in step 1)-step 4) can be further combined and processed to prepare a series of products such as active polysaccharides, chlorophyll, and algae protein.
本发明的显著优点在于：Significant advantage of the present invention is:
（1）本发明以新鲜螺旋藻为加工制备藻蓝蛋白的基础原料，不仅免去了藻粉制备时高温干燥等原料加工的工序，达到节约能耗、降低生产成本的目的，而且新鲜螺旋藻便于实现藻细胞的破壁和细胞液的释放、分离；同时，采用新鲜螺旋藻为加工原料可有效保持各组分的结构特征，避免藻粉高温干燥造成的藻蓝蛋白变性，及其他功效组分的活性丧失、组成结构发生变化。 (1) The present invention uses fresh spirulina as the basic raw material for processing and preparing phycocyanin, which not only eliminates the process of raw material processing such as high-temperature drying during the preparation of algae powder, and achieves the purpose of saving energy and reducing production costs, but also fresh spirulina It is convenient to break the wall of algae cells and release and separate the cell fluid; at the same time, using fresh spirulina as the processing raw material can effectively maintain the structural characteristics of each component, avoid the denaturation of phycocyanin caused by high-temperature drying of algae powder, and other functional groups The activity of the component is lost and the composition structure changes.
（2）本发明采用低温冷冻，避免了高温或长时间放置对藻蓝蛋白及其他功效组分的不利影响，并为后续破壁加工过程提供了必要的基础条件；采用胶体磨对冷冻鲜藻原料进行预处理，使原料形态呈液态，并能够使原料保持低温状态，有利于保护螺旋藻中各组分的活性；采用均质破壁，严格控制破壁进程，可使藻细胞液充分释放，细胞碎片粒度适宜，为充分离心分离提供了有利条件，保证了细胞液中藻蓝蛋白的提取效率。 (2) The present invention adopts low-temperature freezing, which avoids the adverse effects of high temperature or long-term storage on phycocyanin and other functional components, and provides the necessary basic conditions for the subsequent wall-breaking process; the use of colloid mills for frozen fresh algae The raw material is pretreated to make the raw material in a liquid state and keep the raw material at a low temperature, which is beneficial to protect the activity of each component in the spirulina; the homogeneous wall breaking is adopted, and the breaking process is strictly controlled, so that the algae cell fluid can be fully released , the particle size of the cell fragments is suitable, which provides favorable conditions for sufficient centrifugation and ensures the extraction efficiency of phycocyanin in the cell fluid.
（3）本发明采用硫酸铵分级盐析工艺分离藻蓝蛋白，即以饱和度为25%的硫酸铵盐析，分离除去藻多糖及杂蛋白，以减轻后处理的压力；再以饱和度为55%的硫酸铵盐析，可使绝大多数藻蓝蛋白沉淀，并有效的将剩余藻多糖和杂蛋白分离除去，获得藻蓝蛋白的粗沉淀。其中，分离得到的藻多糖和杂蛋白可回收用于制备饲料添加剂。 (3) The present invention adopts ammonium sulfate graded salting-out process to separate phycocyanin, that is, salting out with ammonium sulfate with a saturation of 25%, separating and removing algal polysaccharides and miscellaneous proteins, so as to reduce the pressure of post-processing; 55% ammonium sulfate salting out can precipitate most of the phycocyanin, and effectively separate and remove the remaining algal polysaccharides and miscellaneous proteins to obtain a coarse precipitate of phycocyanin. Among them, the separated algae polysaccharides and miscellaneous proteins can be recovered for the preparation of feed additives.
（4）本发明采用聚乙二醇2000/(NH4)2SO4双水相萃取分离技术，能够实现高效分离纯化藻蓝蛋白的目的，且有利于稳定藻蓝蛋白的结构。 (4) The present invention adopts polyethylene glycol 2000/(NH 4 ) 2 SO 4 two-phase extraction and separation technology, which can achieve the purpose of efficient separation and purification of phycocyanin, and is beneficial to stabilize the structure of phycocyanin.
（5）本发明在双水相萃取分离藻蓝蛋白后，所得聚乙二醇相与水采用交替进入超滤器的供液模式进行超滤，以脱聚乙二醇和脱盐，可有效避免堵膜，提高超滤效率，并具良好的浓缩效果。 (5) After the separation of phycocyanin by two-phase extraction in the present invention, the obtained polyethylene glycol phase and water are alternately fed into the ultrafilter for ultrafiltration to remove polyethylene glycol and desalination, which can effectively avoid clogging Membrane, improve ultrafiltration efficiency, and has a good concentration effect.
（6）本发明采用真空冷冻干燥工艺对所得藻蓝蛋白浓缩液进行干燥，制备得到螺旋藻藻蓝蛋白，可有效避免藻蓝蛋白失活，提高产品的质量。 (6) The present invention uses a vacuum freeze-drying process to dry the obtained phycocyanin concentrate to prepare spirulina phycocyanin, which can effectively avoid the inactivation of phycocyanin and improve the quality of the product.
（7）本发明制备的螺旋藻藻蓝蛋白多糖含量低，纯度A620/A280约3.7。此外，本发明在制备螺旋藻藻蓝蛋白的同时，对藻蓝蛋白以外的其他组分的活性进行了有效保护，其余分离部位可经进一步加工制备出活性多糖、叶绿素、藻蛋白等系列产品，提高了鲜藻各组分的综合利用度。 (7) The spirulina phycocyanin prepared by the present invention has a low content of phycocyanin, and the purity A 620/ A 280 is about 3.7. In addition, while preparing spirulina phycocyanin, the present invention effectively protects the activity of other components other than phycocyanin, and the remaining separated parts can be further processed to prepare a series of products such as active polysaccharides, chlorophyll, and algae protein. The comprehensive utilization of each component of fresh algae is improved.
（8）本发明提出的螺旋藻藻蓝蛋白的制备方法，不仅适用于以螺旋藻制备藻蓝蛋白，而且同样适用于以其他藻类为原料制备藻蓝蛋白，具有良好的推广价值。 (8) The method for preparing phycocyanin from spirulina proposed by the present invention is not only applicable to the preparation of phycocyanin from spirulina, but also applicable to the preparation of phycocyanin from other algae as raw materials, and has good promotion value.
具体实施方式 Detailed ways
为了使本发明所述的内容更加便于理解，下面结合具体实施方式对本发明所述的技术方案做进一步的说明，但是本发明不仅限于此。 In order to make the content of the present invention easier to understand, the technical solutions of the present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited thereto.
实施例1 Example 1
一种用新鲜螺旋藻制备藻蓝蛋白的方法，其具体包括以下步骤： A method for preparing phycocyanin with fresh spirulina, which specifically comprises the following steps:
1）将新鲜螺旋藻500g于-20℃下冷冻24h，解冻，胶体磨预处理后，25MPa压力下均质破壁，1300Orpm离心15min，除去沉淀，得一次离心分离液； 1) Freeze 500g of fresh spirulina at -20°C for 24 hours, thaw, pretreat with a colloid mill, homogeneously break the wall under a pressure of 25MPa, centrifuge at 1300Orpm for 15min, remove the precipitate, and obtain a centrifugal liquid;
2）取步骤1）所得离心分离液，快速搅拌下缓慢加入粉末状固体硫酸铵，使混合液中硫酸铵的饱和度达25%，静置2h，1300Orpm离心15min，除去沉淀，得二次离心分离液； 2) Take the centrifuged liquid obtained in step 1), slowly add powdered solid ammonium sulfate under rapid stirring, so that the saturation of ammonium sulfate in the mixed liquid reaches 25%, let it stand for 2 hours, and centrifuge at 1300Orpm for 15 minutes to remove the precipitate and obtain a second centrifugation separating liquid;
3）取步骤2）所得离心分离液，快速搅拌下缓慢加入粉末状固体硫酸铵，使混合液中硫酸铵的饱和度达55%，静置2h，1300Orpm离心15min，除去离心分离液，取沉淀； 3) Take the centrifuged liquid obtained in step 2), slowly add powdered solid ammonium sulfate under rapid stirring, so that the saturation of ammonium sulfate in the mixed liquid reaches 55%, let it stand for 2 hours, centrifuge at 1300Orpm for 15min, remove the centrifuged liquid, and take the precipitate ;
4）取步骤3）所得沉淀加水配制成2 L待处理液；将12.5L质量浓度为22%的聚乙二醇2000溶液和12.5L质量浓度为44%的硫酸铵溶液缓慢加入到所配制的待处理液中，形成聚乙二醇2000总浓度为10%、硫酸铵总浓度为20%的聚乙二醇2000/(NH4)2SO4双水相萃取体系，充分搅拌后静置2h，分液收集聚乙二醇相； 4) Take the precipitate obtained in step 3) and add water to prepare 2 L of liquid to be treated; slowly add 12.5 L of polyethylene glycol 2000 solution with a mass concentration of 22% and 12.5 L of ammonium sulfate solution with a mass concentration of 44% to the prepared In the liquid to be treated, a polyethylene glycol 2000/(NH 4 ) 2 SO 4 two-phase extraction system with a total concentration of polyethylene glycol 2000 of 10% and a total concentration of ammonium sulfate of 20% was formed, which was fully stirred and allowed to stand for 2 hours. , collecting the polyethylene glycol phase by liquid separation;
5）采用分子量为3500kDa的超滤膜对步骤4）所收集的聚乙二醇相进行超滤，采用聚乙二醇相、水交替进入超滤器的供液模式，聚乙二醇相每次进料后的间隔时间逐渐增长，水每次进料后的间隔时间逐渐缩短，以脱除聚乙二醇、铵盐；超滤后段停止进水，以体系循环超滤浓缩至总体积0.2L，得藻蓝蛋白浓缩液； 5) Use an ultrafiltration membrane with a molecular weight of 3500kDa to perform ultrafiltration on the polyethylene glycol phase collected in step 4), and use the polyethylene glycol phase and water to alternately enter the liquid supply mode of the ultrafilter. The interval time after each feed is gradually increased, and the interval after each feed of water is gradually shortened to remove polyethylene glycol and ammonium salts; the water feed is stopped at the end of the ultrafiltration, and the system circulates the ultrafiltration to concentrate to the total volume 0.2L to obtain phycocyanin concentrate;
6）将步骤5）所得藻蓝蛋白浓缩液真空冷冻干燥，制备得螺旋藻藻蓝蛋白。 6) Vacuum freeze-drying the phycocyanin concentrate obtained in step 5) to prepare spirulina phycocyanin.
实施例2 Example 2
一种用新鲜螺旋藻制备藻蓝蛋白的方法，其具体包括以下步骤： A method for preparing phycocyanin with fresh spirulina, which specifically comprises the following steps:
1）将新鲜螺旋藻1000g于-20℃下冷冻8h，解冻，胶体磨预处理后，30MPa压力下均质破壁，1300Orpm离心15min，除去沉淀，得一次离心分离液； 1) Freeze 1000g of fresh spirulina at -20°C for 8h, thaw, pretreat with colloid mill, homogeneously break the wall under 30MPa pressure, centrifuge at 1300Orpm for 15min, remove the precipitate, and obtain a centrifuge liquid;
2）取步骤1）所得离心分离液，快速搅拌下缓慢加入粉末状固体硫酸铵，使混合液中硫酸铵的饱和度达25%，静置2h，1300Orpm离心20min，除去沉淀，得二次离心分离液； 2) Take the centrifuged liquid obtained in step 1), slowly add powdered solid ammonium sulfate under rapid stirring, so that the saturation of ammonium sulfate in the mixed liquid reaches 25%, let it stand for 2 hours, and centrifuge at 1300Orpm for 20min to remove the precipitate and obtain a second centrifugation separating liquid;
3）取步骤2）所得离心分离液，快速搅拌下缓慢加入粉末状固体硫酸铵，使混合液中硫酸铵的饱和度达55%，静置2h，1300Orpm离心15min，除去离心分离液，取沉淀； 3) Take the centrifuged liquid obtained in step 2), slowly add powdered solid ammonium sulfate under rapid stirring, so that the saturation of ammonium sulfate in the mixed liquid reaches 55%, let it stand for 2 hours, centrifuge at 1300Orpm for 15min, remove the centrifuged liquid, and take the precipitate ;
4）取步骤3）所得沉淀加水配制成4 L待处理液；将23L质量浓度为22%的聚乙二醇2000溶液和23L质量浓度为44%的硫酸铵溶液缓慢加入到所配制的待处理液中，形成聚乙二醇2000总浓度为10%、硫酸铵总浓度为20%的聚乙二醇2000/(NH4)2SO4双水相萃取体系，充分搅拌后静置2h，分液收集聚乙二醇相； 4) Take the precipitate obtained in step 3) and add water to prepare 4 L of liquid to be treated; slowly add 23 L of polyethylene glycol 2000 solution with a mass concentration of 22% and 23 L of ammonium sulfate solution with a mass concentration of 44% to the prepared liquid to be treated In the solution, a polyethylene glycol 2000/(NH 4 ) 2 SO 4 two-phase extraction system with a total concentration of polyethylene glycol 2000 of 10% and a total concentration of ammonium sulfate of 20% was formed, which was fully stirred and allowed to stand for 2 hours, and divided into Liquid collection polyethylene glycol phase;
5）采用分子量为3500kDa的超滤膜对步骤4）所收集的聚乙二醇相进行超滤，采用聚乙二醇相、水交替进入超滤器的供液模式，聚乙二醇相每次进料后的间隔时间逐渐增长，水每次进料后的间隔时间逐渐缩短，以脱除聚乙二醇、铵盐；超滤后段停止进水，以体系循环超滤浓缩至总体积0.5L，得藻蓝蛋白浓缩液； 5) Use an ultrafiltration membrane with a molecular weight of 3500kDa to perform ultrafiltration on the polyethylene glycol phase collected in step 4), and use the polyethylene glycol phase and water to alternately enter the liquid supply mode of the ultrafilter. The interval time after each feed is gradually increased, and the interval after each feed of water is gradually shortened to remove polyethylene glycol and ammonium salts; the water feed is stopped at the end of the ultrafiltration, and the system circulates the ultrafiltration to concentrate to the total volume 0.5L to obtain phycocyanin concentrate;
6）将步骤5）所得藻蓝蛋白浓缩液真空冷冻干燥，制备得螺旋藻藻蓝蛋白。 6) Vacuum freeze-drying the phycocyanin concentrate obtained in step 5) to prepare spirulina phycocyanin.
实施例3 Example 3
一种用新鲜螺旋藻制备藻蓝蛋白的方法，其具体包括以下步骤： A method for preparing phycocyanin with fresh spirulina, which specifically comprises the following steps:
1）将新鲜螺旋藻2000g于-20℃下冷冻4h，解冻，胶体磨预处理后，35MPa压力下均质破壁，1300Orpm离心15min，除去沉淀，得一次离心分离液； 1) Freeze 2000g of fresh spirulina at -20°C for 4h, thaw, pretreat with a colloid mill, homogeneously break the wall under a pressure of 35MPa, centrifuge at 1300Orpm for 15min, remove the precipitate, and obtain a centrifuge;
2）取步骤1）所得离心分离液，快速搅拌下缓慢加入粉末状固体硫酸铵，使混合液中硫酸铵的饱和度达25%，静置2h，1300Orpm离心25min，除去沉淀，得二次离心分离液； 2) Take the centrifuged liquid obtained in step 1), slowly add powdered solid ammonium sulfate under rapid stirring, so that the saturation of ammonium sulfate in the mixed liquid reaches 25%, let it stand for 2 hours, and centrifuge at 1300Orpm for 25min to remove the precipitate and obtain the second centrifugation separating liquid;
3）取步骤2）所得离心分离液，快速搅拌下缓慢加入粉末状固体硫酸铵，使混合液中硫酸铵的饱和度达55%，静置2h，1300Orpm离心15min，除去离心分离液，取沉淀； 3) Take the centrifuged liquid obtained in step 2), slowly add powdered solid ammonium sulfate under rapid stirring, so that the saturation of ammonium sulfate in the mixed liquid reaches 55%, let it stand for 2 hours, centrifuge at 1300Orpm for 15min, remove the centrifuged liquid, and take the precipitate ;
4）取步骤3）所得沉淀加水配制成8 L待处理液；将46L质量浓度为22%的聚乙二醇2000溶液和46L质量浓度为44%的硫酸铵溶液缓慢加入到所配制的待处理液中，形成聚乙二醇2000总浓度为10%、硫酸铵总浓度为20%的聚乙二醇2000/(NH4)2SO4双水相萃取体系，充分搅拌后静置2h，分液收集聚乙二醇相； 4) Take the precipitate obtained in step 3) and add water to prepare 8 L of liquid to be treated; slowly add 46 L of polyethylene glycol 2000 solution with a mass concentration of 22% and 46 L of ammonium sulfate solution with a mass concentration of 44% to the prepared liquid to be treated In the solution, a polyethylene glycol 2000/(NH 4 ) 2 SO 4 two-phase extraction system with a total concentration of polyethylene glycol 2000 of 10% and a total concentration of ammonium sulfate of 20% was formed, which was fully stirred and allowed to stand for 2 hours, and divided into Liquid collection polyethylene glycol phase;
5）采用分子量为3500kDa的超滤膜对步骤4）所收集的聚乙二醇相进行超滤，采用聚乙二醇相、水交替进入超滤器的供液模式，聚乙二醇相每次进料后的间隔时间逐渐增长，水每次进料后的间隔时间逐渐缩短，以脱除聚乙二醇、铵盐；超滤后段停止进水，以体系循环超滤浓缩至总体积1.0L，得藻蓝蛋白浓缩液； 5) Use an ultrafiltration membrane with a molecular weight of 3500kDa to perform ultrafiltration on the polyethylene glycol phase collected in step 4), and use the polyethylene glycol phase and water to alternately enter the liquid supply mode of the ultrafilter. The interval time after each feed is gradually increased, and the interval after each feed of water is gradually shortened to remove polyethylene glycol and ammonium salts; the water feed is stopped at the end of the ultrafiltration, and the system circulates the ultrafiltration to concentrate to the total volume 1.0L, to obtain phycocyanin concentrate;
6）将步骤5）所得藻蓝蛋白浓缩液真空冷冻干燥，制备得螺旋藻藻蓝蛋白。 6) Vacuum freeze-drying the phycocyanin concentrate obtained in step 5) to prepare spirulina phycocyanin.
现有技术采用藻粉为原料制备藻蓝蛋白，但由于藻粉的制备过程经历高温干燥处理，大量藻蓝蛋白在高温下易发生变性，同时也使破壁难度加大，藻细胞内的藻蓝蛋白释放率降低，所得藻蓝蛋白的得率低、纯度差（A620/A280<3）。与传统采用藻粉为原料制备藻蓝蛋白的方法进行对比，本发明以鲜藻为原料，经破壁、分级铵盐沉降前处理，再经双水相萃取制备藻蓝蛋白，所得藻蓝蛋白得率高、纯度高（A620/A280约3.7）、糖脱除率高（96%），且本发明工艺过程不涉及层析等处理工艺，工艺简单、省时省力、成本低、原料综合利用率高。 The existing technology uses algae powder as raw material to prepare phycocyanin, but because the preparation process of algae powder is subjected to high-temperature drying treatment, a large amount of phycocyanin is prone to denaturation at high temperature, and it also makes it more difficult to break the wall. The release rate of cyanocyanin is reduced, and the yield and purity of the obtained phycocyanin are low (A 620/ A 280 <3). Compared with the traditional method of preparing phycocyanin by using algae powder as raw material, the present invention uses fresh algae as raw material, undergoes wall breaking, graded ammonium salt sedimentation pretreatment, and then prepares phycocyanin by aqueous two-phase extraction, and the obtained phycocyanin High yield, high purity (A 620/ A 280 about 3.7), high sugar removal rate (96%), and the process of the present invention does not involve chromatography and other treatment processes, simple process, time-saving and labor-saving, low cost, and raw materials Comprehensive utilization rate is high.
以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所做的均等变化与修饰，皆应属本发明的涵盖范围。 The above descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.
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