技术领域Technical Field
本发明涉及藻类提取技术领域，具体为一种用于提取丝状蓝藻生物活性成分的方法及其应用。The invention relates to the technical field of algae extraction, in particular to a method for extracting biologically active components of filamentous cyanobacteria and application thereof.
背景技术Background Art
丝状蓝藻是一类在水体中常见的蓝藻，它们以丝状体的形式存在，具有重要的生态功能和环境意义；丝状蓝藻在水体中可以形成水华，影响水质和水生生态系统的健康；它们的生长和功能特性受到多种环境因素的影响，其中温度是一个关键因素；根据研究，丝状蓝藻的生长速率和光合活性会随着温度的变化而变化；例如，拟柱孢藻在温度从15℃增加至30℃时，其比生长速率会显著增加，同时光合活性也会在一定范围内波动；此外，丝状蓝藻的氮和磷消耗量也会随着温度的升高而增加，这表明它们在不同温度条件下能够通过调整生理特性来维持生长优势。Filamentous cyanobacteria are a type of cyanobacteria commonly found in water bodies. They exist in the form of filaments and have important ecological functions and environmental significance. Filamentous cyanobacteria can form algal blooms in water bodies, affecting water quality and the health of aquatic ecosystems. Their growth and functional characteristics are affected by a variety of environmental factors, among which temperature is a key factor. According to research, the growth rate and photosynthetic activity of filamentous cyanobacteria change with changes in temperature. For example, when the temperature increases from 15°C to 30°C, the specific growth rate of Pseudo-cylindrical spores increases significantly, and the photosynthetic activity also fluctuates within a certain range. In addition, the nitrogen and phosphorus consumption of filamentous cyanobacteria also increases with increasing temperature, indicating that they can maintain their growth advantage by adjusting their physiological characteristics under different temperature conditions.
丝状蓝藻的原始特征包括它们的单细胞或群体形态，无鞭毛，细胞壁由黏肽组成，无真正的细胞核，细胞内含有叶绿素、类胡萝卜素以及藻蓝素等色素，且主要通过分裂生殖进行繁殖在农业应用方面，丝状蓝藻可以作为天然的微生物肥料，固定大气中的氮，促进农作物生长，并且死亡的藻细胞可以转化为有机肥料，改善土壤结构，减少化肥的使用，对发展低碳农业具有重要意义在水华防治方面，丝状蓝藻的控制和管理是水体环境保护的重要内容。通过研究和实践，人们已经开发出多种方法来控制蓝藻水华，包括物理、化学和生物方法，以减少蓝藻对水体环境的影响。The original characteristics of filamentous cyanobacteria include their single-cell or colony morphology, no flagella, cell walls composed of mucopeptides, no true nucleus, cells containing chlorophyll, carotenoids, and pigments such as phycocyanin, and mainly reproduce by fission. In agricultural applications, filamentous cyanobacteria can be used as natural microbial fertilizers to fix nitrogen in the atmosphere and promote crop growth. Dead algal cells can be converted into organic fertilizers, improve soil structure, and reduce the use of chemical fertilizers. It is of great significance to the development of low-carbon agriculture. In terms of water bloom prevention and control, the control and management of filamentous cyanobacteria is an important part of water environment protection. Through research and practice, people have developed a variety of methods to control cyanobacterial blooms, including physical, chemical, and biological methods to reduce the impact of cyanobacteria on the water environment.
然而，丝状蓝藻含有丰富的生物活性成分，如多糖、蛋白质、维生素、矿物质等，具有重要的药用价值和保健功能；然而，传统提取方法存在提取效率低、成本高、纯度不高等问题，限制了其在实际应用中的推广，因此，本领域的技术人员提供了一种用于提取丝状蓝藻生物活性成分的方法及其应用，以解决上述背景技术中提出的问题。However, filamentous cyanobacteria contain rich bioactive ingredients, such as polysaccharides, proteins, vitamins, minerals, etc., and have important medicinal value and health functions; however, traditional extraction methods have problems such as low extraction efficiency, high cost, and low purity, which limits their promotion in practical applications. Therefore, technicians in this field provide a method for extracting bioactive ingredients from filamentous cyanobacteria and its application to solve the problems raised in the above background technology.
发明内容Summary of the invention
(一)解决的技术问题1. Technical issues to be solved
针对现有技术的不足，本发明提供了一种用于提取丝状蓝藻生物活性成分的方法及其应用，解决了传统提取方法存在提取效率低、成本高和纯度不高的问题。In view of the deficiencies in the prior art, the present invention provides a method for extracting bioactive components of filamentous cyanobacteria and its application, which solves the problems of low extraction efficiency, high cost and low purity in traditional extraction methods.
(二)技术方案(II) Technical solution
为实现以上目的，本发明通过以下技术方案予以实现：一种用于提取丝状蓝藻生物活性成分的方法，包括：To achieve the above objectives, the present invention is implemented by the following technical scheme: A method for extracting biologically active components from filamentous cyanobacteria, comprising:
培养条件优化，选择螺旋藻作为目标丝状蓝藻，通过优化培养基配方的培养条件，其中包括氮源、磷源和光照强度，提高螺旋藻的生物活性成分产量；Optimization of culture conditions: Spirulina was selected as the target filamentous cyanobacteria, and the production of biologically active components of Spirulina was increased by optimizing the culture conditions of the medium formulation, including nitrogen source, phosphorus source and light intensity;
细胞破碎，使用超声波破碎技术破碎螺旋藻细胞壁，通过优化超声波功率和处理时间，提高细胞破碎效率；Cell disruption: using ultrasonic disruption technology to disrupt the cell wall of Spirulina, and improving the efficiency of cell disruption by optimizing ultrasonic power and processing time;
提取方法，选择乙醇作为提取溶剂，通过优化提取温度、时间和乙醇浓度相关的参数，提高螺旋藻生物活性成分的提取效率；The extraction method selects ethanol as the extraction solvent and improves the extraction efficiency of the biologically active components of Spirulina by optimizing the parameters related to extraction temperature, time and ethanol concentration;
纯化步骤，采用离子交换层析和凝胶过滤层析相结合的方法，对提取液进行纯化，获得高纯度的螺旋藻生物活性成分。In the purification step, the extract is purified by combining ion exchange chromatography and gel filtration chromatography to obtain high-purity biologically active components of Spirulina.
优选的，所述培养条件优化包括以下步骤：Preferably, the culture condition optimization comprises the following steps:
S1.菌株选择与培养基配方，选择具有高生物活性成分含量的丝状蓝藻菌株，如螺旋藻，根据螺旋藻菌株的生长需求，设计含有适宜营养成分的培养基，通常包括硝酸钠、磷酸二氢钾、锰、维生素B12和碳酸氢钠；S1. Strain selection and culture medium formulation: select filamentous cyanobacteria strains with high content of bioactive ingredients, such as Spirulina, and design a culture medium containing appropriate nutrients according to the growth requirements of the Spirulina strain, which usually includes sodium nitrate, potassium dihydrogen phosphate, manganese, vitamin B12 and sodium bicarbonate;
S2.光照条件，确定适宜的光照强度，通常为2000-5000lux，以模拟蓝藻在自然环境中的光照条件，采用12小时光照/12小时黑暗，以模拟自然界的日夜循环；S2. Light conditions: determine the appropriate light intensity, usually 2000-5000 lux, to simulate the light conditions of cyanobacteria in the natural environment, using 12 hours of light/12 hours of darkness to simulate the day and night cycle in nature;
S3.温度控制，将温度调整在25-30摄氏度之间，以促进蓝藻的生长和生物活性成分的积累，避免温度波动对蓝藻生长造成影响；S3. Temperature control, adjusting the temperature between 25-30 degrees Celsius to promote the growth of cyanobacteria and the accumulation of bioactive components, and avoiding the impact of temperature fluctuations on the growth of cyanobacteria;
S4.pH值控制，调整培养基的初始pH值至适宜范围，通常为8.0-9.0，以利于蓝藻的生长，并在培养过程中定期监测pH值，并通过添加酸碱溶液进行调整，以维持pH值的稳定。S4. pH control: adjust the initial pH of the culture medium to an appropriate range, usually 8.0-9.0, to facilitate the growth of cyanobacteria. Monitor the pH value regularly during the culture process and adjust it by adding acid and alkali solutions to maintain a stable pH value.
优选的，所述细胞破碎包括以下步骤：Preferably, the cell disruption comprises the following steps:
S1.细胞收集，将采集到的蓝藻样品放入清水中，用轻柔的搅拌或冲洗，去除附着在外表面的泥土和沉淀物，将培养好的丝状蓝藻细胞通过离心或过滤的方式收集，离心时，转速为1000-3000rpm，时间为10-20分钟，以确保细胞沉淀完全；S1. Cell collection: put the collected cyanobacteria sample into clean water, gently stir or rinse to remove the dirt and sediment attached to the outer surface, and collect the cultured filamentous cyanobacteria cells by centrifugation or filtration. During centrifugation, the speed is 1000-3000 rpm and the time is 10-20 minutes to ensure that the cells are completely precipitated;
S2.细胞洗涤，使用去离子或缓冲液洗涤收集的细胞，以去除残留的培养基和杂质，重复洗涤三遍以上，以确保细胞干净；S2. Wash the cells: Use deionized water or buffer to wash the collected cells to remove residual culture medium and impurities. Repeat the washing three times or more to ensure that the cells are clean.
S3.细胞悬浮，将洗涤后的细胞重新悬浮于适量的缓冲液中，缓冲液的pH值和离子强度应根据目标成分的性质进行选择，以保持细胞的稳定性和活性；S3. Cell suspension: resuspend the washed cells in an appropriate amount of buffer. The pH value and ionic strength of the buffer should be selected according to the properties of the target components to maintain the stability and activity of the cells.
S4.破碎处理，将细胞悬浮液置于超声波破碎仪中，通过超声波产生的空化效应破碎细胞壁，控制超声波功率在200-500W，处理时间控制在5-15min，以确保细胞破碎完全且生物活性成分不被破坏；S4. Disruption treatment: placing the cell suspension in an ultrasonic disruptor to disrupt the cell wall through the cavitation effect generated by ultrasound. The ultrasonic power is controlled at 200-500W and the treatment time is controlled at 5-15min to ensure that the cells are completely disrupted and the bioactive components are not destroyed.
S5.破碎后处理，破碎完成后，再次进行离心处理，离心的转速为10000g，处理时间为10-20min去除细胞碎片和未破碎的细胞，收集上清液，上清液中应包含释放出的生物活性成分；S5. After the crushing is completed, centrifugation is performed again, the centrifugal speed is 10000g, the processing time is 10-20min to remove cell debris and unbroken cells, and the supernatant is collected, and the supernatant should contain the released bioactive components;
S6.通过显微镜观察细胞破碎的结果，确保大部分细胞已被破碎，且目标成分已释放到上清液中；S6. Observe the results of cell disruption under a microscope to ensure that most cells have been disrupted and the target components have been released into the supernatant;
S7.后续处理，对收集的上清液进行进一步的处理，其中包括再次进行过滤、离心和透析处理，以去除残留的细胞碎片和杂质。S7. Subsequent processing: the collected supernatant is further processed, including filtering, centrifuging and dialysis again to remove residual cell debris and impurities.
优选的，所述提取方法包括以下步骤：Preferably, the extraction method comprises the following steps:
S1.提取溶剂的选择，根据目标生物活性成分的化学性质选择合适的提取溶剂，目标成分是水溶性的，选择水或者含有一定比例水的混合溶剂，目标成分是脂溶性的，可以选择乙醇、甲醇或丙酮有机溶剂；S1. Selection of extraction solvent: Select a suitable extraction solvent according to the chemical properties of the target bioactive component. If the target component is water-soluble, select water or a mixed solvent containing a certain proportion of water. If the target component is fat-soluble, select ethanol, methanol or acetone organic solvents;
S2.提取条件的确定，确定提取溶剂的浓度、温度和时间参数，对水溶性成分，使用热水提取，提取温度控制在65-80摄氏度；对于脂溶性成分，可以使用有机溶剂在室温或加热条件下提取；S2. Determination of extraction conditions, including the concentration, temperature and time parameters of the extraction solvent. For water-soluble components, hot water is used for extraction, and the extraction temperature is controlled at 65-80 degrees Celsius. For fat-soluble components, organic solvents can be used for extraction at room temperature or under heating conditions;
S3.提取操作，将破碎后的细胞悬浮液与提取溶剂混合，进行提取，通过采用超声波辅助提取，对于热敏感的成分，可以采用室温或低温提取，以减少热降解；S3. Extraction operation, mixing the broken cell suspension with an extraction solvent, extracting by using ultrasonic assisted extraction, and for heat-sensitive components, room temperature or low temperature extraction can be used to reduce thermal degradation;
S4.固液分离，提取完成后，通过离心的方式将提取液与细胞残渣分离，转速为10000rpm，时间为30min，以确保充分分离，分离完成后通过采用聚醚砜滤膜去除细胞碎片和不溶性杂质；S4. Solid-liquid separation. After the extraction is completed, the extract is separated from the cell residue by centrifugation at a speed of 10,000 rpm for 30 minutes to ensure sufficient separation. After the separation is completed, the cell debris and insoluble impurities are removed by using a polyethersulfone filter membrane;
S5.提取液的浓缩，通过使用旋转蒸发器将提取液进行浓缩，以提高目标成分的浓度，浓缩过程中，应将温度控制在40-60摄氏度之间，压力控制在10-100mbar之间，避免高温导致成分的损失和变性；S5. Concentration of the extract: The extract is concentrated by using a rotary evaporator to increase the concentration of the target component. During the concentration process, the temperature should be controlled between 40-60 degrees Celsius and the pressure should be controlled between 10-100 mbar to avoid loss and denaturation of the components caused by high temperature;
S6.提取液的纯化，通过采用凝胶过滤层析、萃取和结晶的方式对浓缩后的提取液进行纯化处理，以去除杂质，提高目标成分的纯度；S6. Purification of the extract, by purifying the concentrated extract by gel filtration chromatography, extraction and crystallization to remove impurities and improve the purity of the target component;
S7.纯化后处理，通过使用冷冻干燥法对纯化后的提取物进行干燥处理，得到最终的生物活性成分产品，干燥后的产品应进行质量检测，其中包括成分含量、纯度和活性指标的测定。S7. After purification, the purified extract is dried using freeze-drying to obtain the final bioactive ingredient product. The dried product should be quality tested, including the determination of ingredient content, purity and activity indicators.
优选的，所述纯化步骤包括以下步骤：Preferably, the purification step comprises the following steps:
S1.初步纯化，首先通过过滤的方式去除提取液中的细胞碎片、不溶性杂质的大颗粒物质，同时通过加入硫酸铵调节pH值来实现初步纯化；S1. Preliminary purification, firstly removing cell debris and large particles of insoluble impurities in the extract by filtering, and adjusting the pH value by adding ammonium sulfate to achieve preliminary purification;
S2.层析技术，通过采用凝胶过滤层析技术进行过滤和分离处理，利用凝胶介质的孔径大小，根据分子大小进行分离，主要用于去除小分子杂质和分离不同分子量的蛋白质；S2. Chromatography technology, through the use of gel filtration chromatography technology for filtration and separation, using the pore size of the gel medium to separate according to the molecular size, mainly used to remove small molecule impurities and separate proteins of different molecular weights;
S3.萃取法，采用液-液萃取，利用不同溶剂对目标成分和杂质的溶解度差异，通过多次萃取来分离和纯化目标成分，溶剂包括非极性溶剂、中等极性溶剂和极性溶剂，其中非极性溶剂可采用乙烷、二氯甲烷、氯仿或乙醚中的一种，中等极性溶剂可采用乙酸乙酯、丙酮或乙醇中的一种，极性溶剂可采用甲醇、乙醇、丙酮或乙腈中的一种；S3. Extraction method, using liquid-liquid extraction, using the difference in solubility of different solvents for target components and impurities, separating and purifying the target components through multiple extractions, the solvents include non-polar solvents, medium-polar solvents and polar solvents, wherein the non-polar solvent can be one of ethane, dichloromethane, chloroform or ether, the medium-polar solvent can be one of ethyl acetate, acetone or ethanol, and the polar solvent can be one of methanol, ethanol, acetone or acetonitrile;
S4.结晶法，可以通过改变溶剂的饱和度或温度，诱导目标成分结晶，从而实现纯化；S4. Crystallization method can induce crystallization of target components by changing the saturation or temperature of the solvent, thereby achieving purification;
S5.干燥与收集，通过使用冷冻干燥来去除水分，得到干燥的纯化产品；S5. Drying and collection, by removing water using freeze drying to obtain a dried purified product;
S6.纯度检测与分析，可通过采用高效液相色谱、质谱或紫外-可见光谱中的一种检测方式进行纯度检测和分析；S6. Purity detection and analysis can be performed by using one of the detection methods of high performance liquid chromatography, mass spectrometry or ultraviolet-visible spectroscopy;
S7.质量控制，对纯化后的样品进行质量控制，确保其符合ISO9001质量管理体系和ISO22000食品安全管理体系。S7. Quality control: perform quality control on the purified samples to ensure that they comply with the ISO9001 quality management system and ISO22000 food safety management system.
根据权利要求1至5所述的一种用于提取丝状蓝藻生物活性成分的方法，在日化产品和医药产品中应用，丝状蓝藻中含有丰富的抗氧化剂，有助于清除自由基，减少氧化应激，从而降低慢性疾病的风险，同时丝状蓝藻中的维生素和矿物质对皮肤健康有益，可以帮助改善皮肤状况。According to claims 1 to 5, a method for extracting bioactive ingredients from filamentous cyanobacteria is used in daily chemical products and pharmaceutical products. The filamentous cyanobacteria are rich in antioxidants, which help to scavenge free radicals and reduce oxidative stress, thereby reducing the risk of chronic diseases. At the same time, the vitamins and minerals in the filamentous cyanobacteria are beneficial to skin health and can help improve skin conditions.
(三)有益效果(III) Beneficial effects
本发明提供了一种用于提取丝状蓝藻生物活性成分的方法及其应用。具备以下有益效果：The present invention provides a method for extracting biologically active components from filamentous cyanobacteria and its application, which has the following beneficial effects:
1、本发明中，通过选择合适的提取溶剂和优化提取条件，可以提高提取物的纯度，减少杂质的含量，从而提高产品的质量。1. In the present invention, by selecting a suitable extraction solvent and optimizing the extraction conditions, the purity of the extract can be improved, the content of impurities can be reduced, and thus the quality of the product can be improved.
2、本发明中，通过采用温和的细胞破碎方法，可以减少对生物活性成分的破坏，保持其活性和稳定性，同时采用层析技术进行纯化，可以有效地去除提取物中的杂质，简化纯化步骤，提高纯化效率。2. In the present invention, by adopting a mild cell disruption method, the damage to the biologically active components can be reduced, and their activity and stability can be maintained. At the same time, the use of chromatography technology for purification can effectively remove impurities in the extract, simplify the purification steps, and improve the purification efficiency.
3、本发明中，通过优化提取和纯化过程，可以减少溶剂的使用和废弃物的产生，降低对环境的影响，促进可持续发展。3. In the present invention, by optimizing the extraction and purification process, the use of solvents and the generation of waste can be reduced, the impact on the environment can be reduced, and sustainable development can be promoted.
具体实施方式DETAILED DESCRIPTION
下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整的描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
实施例：Example:
本发明实施例提供一种用于提取丝状蓝藻生物活性成分的方法，包括：The embodiment of the present invention provides a method for extracting biologically active components from filamentous cyanobacteria, comprising:
培养条件优化，选择螺旋藻作为目标丝状蓝藻，通过优化培养基配方的培养条件，其中包括氮源、磷源和光照强度，提高螺旋藻的生物活性成分产量；Optimization of culture conditions: Spirulina was selected as the target filamentous cyanobacteria, and the production of biologically active components of Spirulina was increased by optimizing the culture conditions of the medium formulation, including nitrogen source, phosphorus source and light intensity;
细胞破碎，使用超声波破碎技术破碎螺旋藻细胞壁，通过优化超声波功率和处理时间，提高细胞破碎效率；Cell disruption: using ultrasonic disruption technology to disrupt the cell wall of Spirulina, and improving the efficiency of cell disruption by optimizing ultrasonic power and processing time;
提取方法，选择乙醇作为提取溶剂，通过优化提取温度、时间和乙醇浓度相关的参数，提高螺旋藻生物活性成分的提取效率；The extraction method selects ethanol as the extraction solvent and improves the extraction efficiency of the biologically active components of Spirulina by optimizing the parameters related to extraction temperature, time and ethanol concentration;
纯化步骤，采用离子交换层析和凝胶过滤层析相结合的方法，对提取液进行纯化，获得高纯度的螺旋藻生物活性成分。In the purification step, the extract is purified by combining ion exchange chromatography and gel filtration chromatography to obtain high-purity biologically active components of Spirulina.
培养条件优化包括以下步骤：Optimization of culture conditions includes the following steps:
S1.菌株选择与培养基配方，选择具有高生物活性成分含量的丝状蓝藻菌株，如螺旋藻，根据螺旋藻菌株的生长需求，设计含有适宜营养成分的培养基，通常包括硝酸钠、磷酸二氢钾、锰、维生素B12和碳酸氢钠；S1. Strain selection and culture medium formulation: select filamentous cyanobacteria strains with high content of bioactive ingredients, such as Spirulina, and design a culture medium containing appropriate nutrients according to the growth requirements of the Spirulina strain, which usually includes sodium nitrate, potassium dihydrogen phosphate, manganese, vitamin B12 and sodium bicarbonate;
S2.光照条件，确定适宜的光照强度，通常为2000-5000lux，以模拟蓝藻在自然环境中的光照条件，采用12小时光照/12小时黑暗，以模拟自然界的日夜循环；S2. Light conditions: determine the appropriate light intensity, usually 2000-5000 lux, to simulate the light conditions of cyanobacteria in the natural environment, using 12 hours of light/12 hours of darkness to simulate the day and night cycle in nature;
S3.温度控制，将温度调整在25-30摄氏度之间，以促进蓝藻的生长和生物活性成分的积累，避免温度波动对蓝藻生长造成影响；S3. Temperature control, adjusting the temperature between 25-30 degrees Celsius to promote the growth of cyanobacteria and the accumulation of bioactive components, and avoiding the impact of temperature fluctuations on the growth of cyanobacteria;
S4.pH值控制，调整培养基的初始pH值至适宜范围，通常为8.0-9.0，以利于蓝藻的生长，并在培养过程中定期监测pH值，并通过添加酸碱溶液进行调整，以维持pH值的稳定。S4. pH control: adjust the initial pH of the culture medium to an appropriate range, usually 8.0-9.0, to facilitate the growth of cyanobacteria. Monitor the pH value regularly during the culture process and adjust it by adding acid and alkali solutions to maintain a stable pH value.
细胞破碎包括以下步骤：Cell disruption involves the following steps:
S1.细胞收集，将采集到的蓝藻样品放入清水中，用轻柔的搅拌或冲洗，去除附着在外表面的泥土和沉淀物，将培养好的丝状蓝藻细胞通过离心或过滤的方式收集，离心时，转速为1000-3000rpm，时间为10-20分钟，以确保细胞沉淀完全；S1. Cell collection: put the collected cyanobacteria sample into clean water, gently stir or rinse to remove the dirt and sediment attached to the outer surface, and collect the cultured filamentous cyanobacteria cells by centrifugation or filtration. During centrifugation, the speed is 1000-3000 rpm and the time is 10-20 minutes to ensure that the cells are completely precipitated;
S2.细胞洗涤，使用去离子或缓冲液洗涤收集的细胞，以去除残留的培养基和杂质，重复洗涤三遍以上，以确保细胞干净；S2. Wash the cells: Use deionized water or buffer to wash the collected cells to remove residual culture medium and impurities. Repeat the washing three times or more to ensure that the cells are clean.
S3.细胞悬浮，将洗涤后的细胞重新悬浮于适量的缓冲液中，缓冲液的pH值和离子强度应根据目标成分的性质进行选择，以保持细胞的稳定性和活性；S3. Cell suspension: resuspend the washed cells in an appropriate amount of buffer. The pH value and ionic strength of the buffer should be selected according to the properties of the target components to maintain the stability and activity of the cells.
S4.破碎处理，将细胞悬浮液置于超声波破碎仪中，通过超声波产生的空化效应破碎细胞壁，控制超声波功率在200-500W，处理时间控制在5-15min，以确保细胞破碎完全且生物活性成分不被破坏；S4. Disruption treatment: placing the cell suspension in an ultrasonic disruptor to disrupt the cell wall through the cavitation effect generated by ultrasound. The ultrasonic power is controlled at 200-500W and the treatment time is controlled at 5-15min to ensure that the cells are completely disrupted and the bioactive components are not destroyed.
S5.破碎后处理，破碎完成后，再次进行离心处理，离心的转速为10000g，处理时间为10-20min去除细胞碎片和未破碎的细胞，收集上清液，上清液中应包含释放出的生物活性成分；S5. After the crushing is completed, centrifugation is performed again, the centrifugal speed is 10000g, the processing time is 10-20min to remove cell debris and unbroken cells, and the supernatant is collected, and the supernatant should contain the released bioactive components;
S6.通过显微镜观察细胞破碎的结果，确保大部分细胞已被破碎，且目标成分已释放到上清液中；S6. Observe the results of cell disruption under a microscope to ensure that most cells have been disrupted and the target components have been released into the supernatant;
S7.后续处理，对收集的上清液进行进一步的处理，其中包括再次进行过滤、离心和透析处理，以去除残留的细胞碎片和杂质。S7. Subsequent processing: the collected supernatant is further processed, including filtering, centrifuging and dialysis again to remove residual cell debris and impurities.
提取方法包括以下步骤：The extraction method comprises the following steps:
S1.提取溶剂的选择，根据目标生物活性成分的化学性质选择合适的提取溶剂，目标成分是水溶性的，选择水或者含有一定比例水的混合溶剂，目标成分是脂溶性的，可以选择乙醇、甲醇或丙酮有机溶剂；S1. Selection of extraction solvent: Select a suitable extraction solvent according to the chemical properties of the target bioactive component. If the target component is water-soluble, select water or a mixed solvent containing a certain proportion of water. If the target component is fat-soluble, select ethanol, methanol or acetone organic solvents;
S2.提取条件的确定，确定提取溶剂的浓度、温度和时间参数，对水溶性成分，使用热水提取，提取温度控制在65-80摄氏度；对于脂溶性成分，可以使用有机溶剂在室温或加热条件下提取；S2. Determination of extraction conditions, including the concentration, temperature and time parameters of the extraction solvent. For water-soluble components, hot water is used for extraction, and the extraction temperature is controlled at 65-80 degrees Celsius. For fat-soluble components, organic solvents can be used for extraction at room temperature or under heating conditions;
S3.提取操作，将破碎后的细胞悬浮液与提取溶剂混合，进行提取，通过采用超声波辅助提取，对于热敏感的成分，可以采用室温或低温提取，以减少热降解；S3. Extraction operation, mixing the broken cell suspension with an extraction solvent, extracting by using ultrasonic assisted extraction, and for heat-sensitive components, room temperature or low temperature extraction can be used to reduce thermal degradation;
S4.固液分离，提取完成后，通过离心的方式将提取液与细胞残渣分离，转速为10000rpm，时间为30min，以确保充分分离，分离完成后通过采用聚醚砜滤膜去除细胞碎片和不溶性杂质；S4. Solid-liquid separation. After the extraction is completed, the extract is separated from the cell residue by centrifugation at a speed of 10,000 rpm for 30 minutes to ensure sufficient separation. After the separation is completed, the cell debris and insoluble impurities are removed by using a polyethersulfone filter membrane;
S5.提取液的浓缩，通过使用旋转蒸发器将提取液进行浓缩，以提高目标成分的浓度，浓缩过程中，应将温度控制在40-60摄氏度之间，压力控制在10-100mbar之间，避免高温导致成分的损失和变性；S5. Concentration of the extract: The extract is concentrated by using a rotary evaporator to increase the concentration of the target component. During the concentration process, the temperature should be controlled between 40-60 degrees Celsius and the pressure should be controlled between 10-100 mbar to avoid loss and denaturation of the components caused by high temperature;
S6.提取液的纯化，通过采用凝胶过滤层析、萃取和结晶的方式对浓缩后的提取液进行纯化处理，以去除杂质，提高目标成分的纯度；S6. Purification of the extract, by purifying the concentrated extract by gel filtration chromatography, extraction and crystallization to remove impurities and improve the purity of the target component;
S7.纯化后处理，通过使用冷冻干燥法对纯化后的提取物进行干燥处理，得到最终的生物活性成分产品，干燥后的产品应进行质量检测，其中包括成分含量、纯度和活性指标的测定。S7. After purification, the purified extract is dried using freeze-drying to obtain the final bioactive ingredient product. The dried product should be quality tested, including the determination of ingredient content, purity and activity indicators.
纯化步骤包括以下步骤：The purification step includes the following steps:
S1.初步纯化，首先通过过滤的方式去除提取液中的细胞碎片、不溶性杂质的大颗粒物质，同时通过加入硫酸铵调节pH值来实现初步纯化；S1. Preliminary purification, firstly removing cell debris and large particles of insoluble impurities in the extract by filtering, and adjusting the pH value by adding ammonium sulfate to achieve preliminary purification;
S2.层析技术，通过采用凝胶过滤层析技术进行过滤和分离处理，利用凝胶介质的孔径大小，根据分子大小进行分离，主要用于去除小分子杂质和分离不同分子量的蛋白质；S2. Chromatography technology, through the use of gel filtration chromatography technology for filtration and separation, using the pore size of the gel medium to separate according to the molecular size, mainly used to remove small molecule impurities and separate proteins of different molecular weights;
S3.萃取法，采用液-液萃取，利用不同溶剂对目标成分和杂质的溶解度差异，通过多次萃取来分离和纯化目标成分，溶剂包括非极性溶剂、中等极性溶剂和极性溶剂，其中非极性溶剂可采用乙烷、二氯甲烷、氯仿或乙醚中的一种，中等极性溶剂可采用乙酸乙酯、丙酮或乙醇中的一种，极性溶剂可采用甲醇、乙醇、丙酮或乙腈中的一种；S3. Extraction method, using liquid-liquid extraction, using the difference in solubility of different solvents for target components and impurities, separating and purifying the target components through multiple extractions, the solvents include non-polar solvents, medium-polar solvents and polar solvents, wherein the non-polar solvent can be one of ethane, dichloromethane, chloroform or ether, the medium-polar solvent can be one of ethyl acetate, acetone or ethanol, and the polar solvent can be one of methanol, ethanol, acetone or acetonitrile;
S4.结晶法，可以通过改变溶剂的饱和度或温度，诱导目标成分结晶，从而实现纯化；S4. Crystallization method can induce crystallization of target components by changing the saturation or temperature of the solvent, thereby achieving purification;
S5.干燥与收集，通过使用冷冻干燥来去除水分，得到干燥的纯化产品；S5. Drying and collection, by removing water using freeze drying to obtain a dried purified product;
S6.纯度检测与分析，可通过采用高效液相色谱、质谱或紫外-可见光谱中的一种检测方式进行纯度检测和分析；S6. Purity detection and analysis can be performed by using one of the detection methods of high performance liquid chromatography, mass spectrometry or ultraviolet-visible spectroscopy;
S7.质量控制，对纯化后的样品进行质量控制，确保其符合ISO9001质量管理体系和ISO22000食品安全管理体系。S7. Quality control: perform quality control on the purified samples to ensure that they comply with the ISO9001 quality management system and ISO22000 food safety management system.
根据权利要求1至5所述的一种用于提取丝状蓝藻生物活性成分的方法，在日化产品和医药产品中应用，丝状蓝藻中含有丰富的抗氧化剂，有助于清除自由基，减少氧化应激，从而降低慢性疾病的风险，同时丝状蓝藻中的维生素和矿物质对皮肤健康有益，可以帮助改善皮肤状况。According to claims 1 to 5, a method for extracting bioactive ingredients from filamentous cyanobacteria is used in daily chemical products and pharmaceutical products. The filamentous cyanobacteria are rich in antioxidants, which help to scavenge free radicals and reduce oxidative stress, thereby reducing the risk of chronic diseases. At the same time, the vitamins and minerals in the filamentous cyanobacteria are beneficial to skin health and can help improve skin conditions.
实验例一：抗氧化活性评估；Experimental Example 1: Evaluation of antioxidant activity;
样品准备：从丝状蓝藻中提取活性成分，如多糖、蛋白质、类胡萝卜素；Sample preparation: Extraction of active ingredients such as polysaccharides, proteins, and carotenoids from filamentous cyanobacteria;
DPPH实验：将活性成分与DPPH溶液混合，测定其在一定时间内的吸光度变化，计算自由基清除率；DPPH experiment: The active ingredient is mixed with DPPH solution, the absorbance change over a certain period of time is measured, and the free radical scavenging rate is calculated;
ABTS实验：制备ABTS自由基溶液，与活性成分反应后测定吸光度，计算自由基清除率；ABTS experiment: prepare ABTS free radical solution, measure the absorbance after reaction with active ingredients, and calculate the free radical scavenging rate;
实验结果：活性成分显示出显著的自由基清除能力，与阳性对照维生素C相比，具有相似的抗氧化活性。Experimental results: The active ingredients showed significant free radical scavenging ability and had similar antioxidant activity compared to the positive control vitamin C.
实验例二：抗炎活性评估；Experimental Example 2: Evaluation of anti-inflammatory activity;
细胞培养：培养RAW264.7巨噬细胞系，用LPS诱导炎症反应；Cell culture: RAW264.7 macrophage cell line was cultured and inflammatory response was induced with LPS;
细胞处理：将丝状蓝藻活性成分加入培养基中，设置不同浓度梯度；Cell treatment: Add the active ingredients of filamentous cyanobacteria into the culture medium and set different concentration gradients;
细胞因子测定：通过ELISA测定培养上清液中炎症因子的浓度；Cytokine determination: The concentration of inflammatory factors in the culture supernatant was determined by ELISA;
实验结果：活性成分显著降低炎症因子的表达，表明具有抗炎作用。Experimental results: The active ingredients significantly reduced the expression of inflammatory factors, indicating an anti-inflammatory effect.
实验例三：免疫调节活性评估；Experimental Example 3: Evaluation of immunomodulatory activity;
细胞培养：培养脾细胞或树突细胞；Cell culture: culture splenocytes or dendritic cells;
细胞处理：将丝状蓝藻活性成分加入培养基中，设置不同浓度梯度；Cell treatment: Add the active ingredients of filamentous cyanobacteria into the culture medium and set different concentration gradients;
细胞因子测定：通过ELISA测定培养上清液中免疫调节因子的浓度；Cytokine determination: The concentration of immunomodulatory factors in the culture supernatant was determined by ELISA;
细胞增殖测定：使用MTT或CCK-8试验评估活性成分对细胞增殖的影响；Cell proliferation assay: The effect of active ingredients on cell proliferation was evaluated using MTT or CCK-8 assay;
实验结果：活性成分能促进免疫细胞增殖和免疫因子的分泌，表明其具有免疫调节作用。Experimental results: The active ingredients can promote the proliferation of immune cells and the secretion of immune factors, indicating that it has an immunomodulatory effect.
实验例四：皮肤健康评估；Experimental Example 4: Skin Health Assessment;
体外实验：使用皮肤细胞模型，如角质形成细胞或成纤维细胞，评估活性成分对细胞增殖、胶原合成的影响；In vitro experiments: Use skin cell models, such as keratinocytes or fibroblasts, to evaluate the effects of active ingredients on cell proliferation and collagen synthesis;
体内实验：使用动物模型，小白鼠，评估活性成分对皮肤保湿和抗衰老的影响；In vivo experiments: using animal models, mice, to evaluate the effects of active ingredients on skin moisturizing and anti-aging;
实验结果：活性成分能促进皮肤细胞的增殖和胶原合成，改善皮肤保湿和抗衰老效果。Experimental results: The active ingredients can promote the proliferation of skin cells and collagen synthesis, and improve skin moisturizing and anti-aging effects.
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.
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