CN113388026A - Method for synchronously extracting phycocyanin and algae oil from spirulina - Google Patents
Method for synchronously extracting phycocyanin and algae oil from spirulina Download PDFInfo
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Abstract
The invention provides a method for synchronously extracting phycocyanin and algae oil from spirulina, which comprises the following steps: s1: preparing a block polymer aqueous solution; s2: adding spirulina powder into the block polymer solution to obtain an spirulina powder soaking solution; s3: crushing spirulina cells by an ultrasonic method; s4: taking the solution after ultrasonic treatment, and performing low-temperature extraction; s5: raising the temperature of the solution after low-temperature extraction, and centrifuging to separate the phases; s6: transferring the upper polymer phase to a separating funnel, adding sodium chloride, raising the temperature, standing, and collecting an upper oil phase to obtain purified and separated spirulina oil; transferring the middle layer water phase into a dialysis bag, dialyzing, freezing at ultralow temperature, and performing vacuum freeze drying to obtain the spirulina phycocyanin. The invention belongs to the technical field of food biology, realizes the synchronous extraction of spirulina phycocyanin and algae oil, and has high efficiency, low energy consumption and good environmental benefit.
Description
Technical Field
The invention belongs to the technical field of food biology, and particularly relates to a method for synchronously extracting phycocyanin and algae oil from spirulina.
Background
The spirulina is a photoautotrophic aquatic plant utilizing solar energy to fix carbon dioxide and has high nutritive value. Spirulina cells are rich in proteins, amino acids, vitamins, fatty acids, etc., and have been developed as various health foods. Among them, phycocyanin and algal oil have higher economic value and have been widely studied. Phycocyanin is an intracellular protein, has good water solubility, presents brilliant blue color, has the effects of resisting oxidation, eliminating free radicals and the like, and is widely applied to food, cosmetics and pharmaceutical industries; algal oil, broadly referred to as hydrophobic lipids formed within algal cells, is an important green energy source for biomass.
There are many reports on methods for extracting and separating phycocyanin or algae oil, such as techniques of salting out, chromatographic separation, and aqueous two-phase extraction for phycocyanin extraction (shuli yan. nature of phycocyanin and progress of research on extraction technique. Fujian light textile, 2020(05)), and techniques of organic solvent extraction, sulfuric acid hydrolysis, and supercritical extraction for algae oil extraction (Liu Yi Lin, Chen hong cultivate, Gong Shi Yu, Pan rain Yang, Zeng Feng, Liu. progress on extraction and function research of microalgae oil, science and technology in food industry, 2019,40 (05)). However, no technical report of synchronously extracting phycocyanin and algae oil is found in the prior art, which is mainly because phycocyanin is water-soluble and algae oil is hydrophobic, and the existing technologies such as extraction and the like have incompatibility for synchronously extracting phycocyanin and algae oil. For example, phycocyanin is easily denatured by organic solvent or sulfuric acid extraction of algal oil, resulting in loss of biological activity; when the phycocyanin is extracted by the two-aqueous phase extraction technology, the phycoerythrin cannot be dissolved in the phycocyanin aqueous solution due to strong hydrophobicity and is abandoned in cell fragments.
The aqueous solution of the nonionic surfactant can form two phases with different polarities at a certain temperature, and can be used for extracting and separating hydrophobic compounds. The invention patent application CN 101269275A reports that a cloud point system formed by polyethylene glycol (PEG) induction can be used for separating nonvolatile organic solvent in aqueous solution, and the invention patent application CN 103190522A reports a method for extracting protein from sesame, and the cloud point system is used for removing hydrophobic impurities to obtain high-purity protein. The cloud point system formed by the surfactant is widely applied to the separation of target products, however, technical reports of synchronously extracting hydrophilic phycocyanin and hydrophobic algae oil from a spirulina sample by using the cloud point system are not found.
Therefore, the method for synchronously extracting the phycocyanin and the algae oil from the spirulina has important significance.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a method for synchronously extracting spirulina phycocyanin and algae oil, which realizes the synchronous extraction of the spirulina phycocyanin and the algae oil, and has the advantages of high efficiency, low energy consumption and good environmental benefit.
The objects of the invention will be further illustrated by the following detailed description.
The invention provides a method for synchronously extracting phycocyanin and algae oil from spirulina, which comprises the following steps:
s1: preparing a block polymer aqueous solution with the mass concentration of 5-20% by taking a polyethylene glycol-polypropylene glycol-polyethylene glycol block polymer for later use;
s2: adding spirulina powder into the block polymer solution, and oscillating or stirring uniformly to obtain an spirulina powder impregnation solution;
s3: crushing spirulina cells by using the algae powder infiltration solution by using an ultrasonic method;
s4: taking the solution after ultrasonic treatment, and oscillating at constant temperature of 5-20 ℃ for low-temperature extraction;
s5: raising the temperature of the solution after low-temperature extraction to 25-30 ℃, centrifuging and carrying out phase separation, wherein water-soluble phycocyanin is in the middle-layer water phase, and hydrophobic algae oil is in the upper-layer polymer phase;
s6: transferring the upper polymer phase into a separating funnel, adding sodium chloride, raising the temperature to 55-65 ℃, standing, and layering a solution: an upper oil phase, a middle sodium chloride salt solution phase and a lower polymer concentrated phase; collecting the upper oil phase to obtain purified and separated spirulina oil;
transferring the middle-layer water phase to a dialysis bag with the molecular weight cutoff of 12-15 ku, dialyzing the purified concentrated solution, freezing at ultralow temperature, and performing vacuum freeze drying to obtain the spirulina phycocyanin.
The composition of the block polymer and its concentration are preferred embodiments determined by a number of experimental screens. The polyethylene glycol-polypropylene glycol-polyethylene glycol block polymer (PEG-PPG-PEG) is used, a cloud point system can be formed under the conditions of specific concentration and temperature, the synchronous extraction and separation of spirulina phycocyanin and algae oil are realized, and the yield is high; the concentration of the block polymer aqueous solution also has an important influence on the extraction and separation of the algae oil, when the mass concentration of the PEG-PPG-PEG is lower than 5%, the extraction efficiency of the algae oil is obviously reduced, and when the mass concentration of the PEG-PPG-PEG is higher than 20%, the phase separation volume is too large, so that the separation of the algae oil is not facilitated. Extracting at low temperature of 5-20 ℃ is beneficial to forming a cloud point system, then raising the temperature to 25-30 ℃ to facilitate cloud point phase separation, and if the temperature is lower than 25 ℃, phase separation is difficult to perform. In step S5, the algae oil is extracted into the PEG-PPG-PEG coacervate phase, and the overall density is relatively low, so that the algae oil is on the upper layer; after sodium chloride is added into S6, water in the PEG-PPG-PEG phase is taken away by the sodium chloride to form a salting-out effect, the temperature is increased to 55-65 ℃ to improve the phase separation efficiency, the algae oil is separated out and floats on the upper layer after the water content of the PEG-PPG-PEG is reduced, the PEG-PPG-PEG is settled on the lower layer, and the sodium chloride solution is medium in density and is positioned in the middle layer.
Preferably, the polyethylene glycol-polypropylene glycol-polyethylene glycol block polymer has an average molecular weight of 2700 to 2900.
Preferably, the addition amount of the sodium chloride is 5 to 10 percent of the mass of the upper polymer phase.
Preferably, the conditions of the ultrasonic method are: carrying out ice bath and ultrasonic power of 80-1000 w, and carrying out continuous ultrasonic for 3-30 min.
Preferably, the constant temperature oscillation time is 30-60 min.
Preferably, the conditions of the centrifugation are: centrifuging at 3000-4000 rpm for 8-10 min.
Preferably, the temperature of the ultra-low temperature freezing is-85 to-60 ℃.
Compared with the prior art, the invention has the beneficial effects that:
(1) before extraction, the algae powder does not need pretreatment, so that the operation steps are saved, and the operation period is shortened.
(2) The method realizes the synchronous separation of the phycocyanin and the algae oil of the spirulina, and has high efficiency, low energy consumption and good environmental benefit.
(3) Compared with organic solvent extraction, the method does not use organic solvent, and is environment-friendly; compared with a double aqueous phase extraction method, the phycocyanin in the method does not use salt in the aqueous phase, so that protein denaturation is avoided; compared with supercritical fluid extraction, the method has the advantages of no high-pressure process, mild conditions, low equipment requirement and low energy consumption cost.
(4) The PEG-PPG-PEG block polymer can be repeatedly used, reduces waste, has low raw material cost and is suitable for industrial popularization.
Drawings
FIG. 1 is a schematic process diagram of the process of the present invention.
FIG. 2 is a UV-visible full-wavelength scanning spectrum of phycocyanin in the method of the present invention; the non-purified phycocyanin in the middle layer water phase in step S5, and the purified phycocyanin in the concentrated solution after dialysis purification.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples.
In the present invention, PEG-PPG-PEG is commercially available as polyether, and is available from Shanghai Allan Biotechnology Ltd, with the following product number: p131344. The experimental procedures, in which specific conditions are not indicated in the examples, are generally carried out according to the conditions conventional in the art or according to the conditions recommended by the manufacturers.
Example a method for synchronously extracting phycocyanin and algae oil from spirulina
A method for synchronously extracting phycocyanin and algae oil from spirulina comprises the following steps:
s1: preparing a block polymer aqueous solution with the mass concentration of 10% by taking a polyethylene glycol-polypropylene glycol-polyethylene glycol (PEG-PPG-PEG) block polymer for later use;
s2: adding 5g of spirulina powder into 1L of the block polymer solution, and oscillating at a constant speed for 10min to obtain an spirulina powder impregnating solution;
s3: crushing spirulina cells by using the algae powder infiltration solution by using an ultrasonic method; the conditions of the ultrasonic method were: carrying out ice bath and ultrasonic power of 800w, and carrying out ultrasonic treatment for 10 min;
s4: taking the solution after ultrasonic treatment, and oscillating at constant temperature of 10 ℃ for 45min for low-temperature extraction;
s5: increasing the temperature of the solution after low-temperature extraction to 25 ℃, centrifuging at 4000rpm for 8min for phase separation, wherein the water-soluble phycocyanin is in the middle-layer water phase, and the hydrophobic algae oil is in the upper-layer polymer phase; the spirulina cell debris is deposited at the bottom of the centrifugal tube and can be directly discarded;
s6: transferring the upper polymer phase into a separating funnel, adding sodium chloride accounting for 8% of the mass of the upper polymer phase, raising the temperature to 60 ℃, standing for 20min, and layering the solution: an upper oil phase, a middle sodium chloride salt solution phase and a lower polymer concentrated phase; collecting the upper oil phase to obtain purified and separated spirulina oil; the lower polymer concentrated phase can be recovered and reused; transferring the middle layer water phase to a dialysis bag with the molecular weight cutoff of 13ku, dialyzing the purified concentrated solution, freezing at the ultralow temperature of-65 ℃, and performing vacuum freeze drying to obtain the spirulina phycocyanin. The process schematic is shown in fig. 1. The results of full-wavelength scanning of phycocyanin with the intermediate aqueous phase of step S5 and the dialyzed and purified concentrate using an ultraviolet-visible spectrophotometer are shown in fig. 2.
The extraction rate of the spirulina oil is 98.2 percent; phycocyanin has characteristic absorption peak at 620nm, while 280nm is the absorption peak of common protein, the ratio of the characteristic absorption peak to the absorption peak is in direct proportion to the content of phycocyanin, the purity A620/A280 of phycocyanin is 2.05, and the extraction rate is 94.5%.
Example synchronous extraction method of phycocyanin and algae oil from Spirulina
A method for synchronously extracting phycocyanin and algae oil from spirulina comprises the following steps:
s1: preparing a block polymer aqueous solution with the mass concentration of 15% by taking a polyethylene glycol-polypropylene glycol-polyethylene glycol block polymer for later use;
s2: adding 8g of spirulina powder into 1L of the block polymer solution, and oscillating at a constant speed for 12min to obtain an spirulina powder soaking solution;
s3: crushing spirulina cells by using the algae powder infiltration solution by using an ultrasonic method; the conditions of the ultrasonic method were: carrying out ice bath and ultrasonic power of 1000w, and carrying out ultrasonic treatment for 15 min;
s4: taking the solution after ultrasonic treatment, and oscillating at constant temperature of 15 ℃ for 50min for low-temperature extraction;
s5: the solution after low-temperature extraction is heated to 28 ℃, centrifuged at 3800rpm for 9min for phase separation, the water-soluble phycocyanin is in the middle layer water phase, and the hydrophobic algae oil is in the upper layer polymer phase;
s6: transferring the upper polymer phase into a separating funnel, adding sodium chloride accounting for 6% of the mass of the upper polymer phase, raising the temperature to 58 ℃, standing for 18min, and layering the solution: an upper oil phase, a middle sodium chloride salt solution phase and a lower polymer concentrated phase; collecting the upper oil phase to obtain purified and separated spirulina oil;
transferring the middle layer water phase to a dialysis bag with the molecular weight cutoff of 14ku, dialyzing the purified concentrated solution, freezing at the ultralow temperature of-70 ℃, and performing vacuum freeze drying to obtain the spirulina phycocyanin.
The extraction rate of algae oil is 93.76%; the purity of phycocyanin is 2.18, and the extraction rate is 96.27%.
Example influence of TriPEG-PPG-PEG Recycling on phycocyanin and algae oil extraction yield
S1: phycocyanin and algae oil were extracted according to the method described in example 1, and the extraction rates of phycocyanin and algae oil are shown in table 1;
s2: after recovering the PEG-PPG-PEG block polymer in S1, the PEG-PPG-PEG block polymer in S1 was replaced with the first recovered PEG-PPG-PEG polymer according to the method described in example 1, and used for extraction of phycocyanin and algae oil. After weighing the phycocyanin and the algae oil, the influence of the first recovered polymer on the extraction rate of the phycocyanin and the algae oil is calculated according to the formula 1.
S3: after recovering the PEG-PPG-PEG block polymer in S2, the first recovered PEG-PPG-PEG polymer was replaced with the second recovered PEG-PPG-PEG block polymer according to the method described in example 1, and used for extraction of phycocyanin and algae oil. After weighing the phycocyanin and the algae oil, calculating the influence of the secondary recovered polymer on the extraction rate of the phycocyanin and the algae oil according to a formula 1;
s4: after recovering the PEG-PPG-PEG block polymer in S3, the second recovered PEG-PPG-PEG block polymer was replaced with the third recovered PEG-PPG-PEG block polymer according to the method described in example 1, and then used for extraction of phycocyanin and algae oil. Weighing phycocyanin and algae oil, and calculating the extraction rate of the recovered polymer for three times on the phycocyanin and the algae oil according to a formula 1;
the extraction rates of phycocyanin and algae oil of the fresh PEG-PPG-PEG block polymer and the recovered PEG-PPG-PEG block polymer were compared, and the results are shown in Table 1.
TABLE 1 extraction rates of phycocyanin and algae oil from completely new and recovered PEG-PPG-PEG polymers
With the increase of the recovery times of the PEG-PPG-PEG block polymer, the extraction rates of phycocyanin and algae oil are slightly reduced, but are not obvious. The polymer recovered for the third time can also maintain the extraction rates of phycocyanin and algae oil to be respectively more than 95% and 90%. The reduction in extraction yield may be associated with a small loss of PEG-PPG-PEG block polymer in the aqueous phase during the extraction process. Therefore, in the process of popularization and application, the fresh block polymer is supplemented to the recovered PEG-PPG-PEG block polymer in a proper amount, which is beneficial to maintaining high extraction rate of phycocyanin and algae oil, and can greatly reduce extraction cost while reducing waste by recycling for multiple times.
Example four different extraction Agents for the extraction of Spirulina oil and phycocyanin
To demonstrate the extraction effect of the method of the invention, the inventors designed comparative experiments of the extraction method of example 1 with other polymer-based extraction methods.
Experimental groups: the extraction agent adopts the PEG-PPG-PEG block polymer of the invention, and the experimental steps refer to example 1;
comparative group 1: the extractant adopts polyethylene oxide-block-polypropylene oxide-block-polyethylene oxide (PEO-PPO-PEO) block polymer L62, belongs to poloxamer block polymers, and is a conventional commercially available product. Experimental procedures reference example 1;
comparative group 2: the extractant adopts Triton X-114, belongs to polyoxyethylene mono-tert-octyl phenyl ether, and is a conventional commercial product. Experimental procedure reference was made to example 1.
The comparison group 2 is different from the experimental group and the comparison group 1 in that when the extraction agent extracts and separates algal oil and phycocyanin, PEG-PPG-PEG and PEO-PPO-PEO phases are in the upper phase, and Triton X-114 phase is in the lower phase due to the larger specific gravity.
The results of the experiment are shown in table 2, as compared with the comparative groups 1 and 2:
TABLE 2 extraction rates of different extractants for spirulina phycocyanin and algae oil
Compared with PEG-PPG-PEG block polymer, the extraction rate of phycocyanin and algae oil from PEO-PPO-PEO and Triton X-114 is obviously reduced. Wherein, the extraction efficiency of the PEO-PPO-PEO on the algae oil is slightly good, but the extraction rate of the phycocyanin is obviously lower; triton X-114 is suitable for extracting phycocyanin, but the extraction rate of algae oil is low. Therefore, the PEG-PPG-PEG block polymer is used as the extracting agent of the invention, and the method of the invention is matched to realize the synchronous extraction of the spirulina phycocyanin and the spirulina oil, and the method has the advantages of simple and safe operation, high extraction rate, low cost and easy popularization.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
Claims (7)
1. A method for synchronously extracting phycocyanin and algae oil from spirulina is characterized by comprising the following steps: the method comprises the following steps:
s1: preparing a block polymer aqueous solution with the mass concentration of 5-20% by taking a polyethylene glycol-polypropylene glycol-polyethylene glycol block polymer for later use;
s2: adding spirulina powder into the block polymer solution, and oscillating or stirring uniformly to obtain an spirulina powder impregnation solution;
s3: crushing spirulina cells by using the algae powder infiltration solution by using an ultrasonic method;
s4: taking the solution after ultrasonic treatment, and oscillating at constant temperature of 5-20 ℃ for low-temperature extraction;
s5: raising the temperature of the solution after low-temperature extraction to 25-30 ℃, centrifuging and carrying out phase separation, wherein water-soluble phycocyanin is in the middle-layer water phase, and hydrophobic algae oil is in the upper-layer polymer phase;
s6: transferring the upper polymer phase into a separating funnel, adding sodium chloride, raising the temperature to 55-65 ℃, standing, and layering a solution: an upper oil phase, a middle sodium chloride salt solution phase and a lower polymer concentrated phase; collecting the upper oil phase to obtain purified and separated spirulina oil;
transferring the middle-layer water phase to a dialysis bag with the molecular weight cutoff of 12-15 ku, dialyzing the purified concentrated solution, freezing at ultralow temperature, and performing vacuum freeze drying to obtain the spirulina phycocyanin.
2. The method for synchronously extracting phycocyanin and algae oil in spirulina according to claim 1, wherein the method comprises the following steps: the average molecular weight of the polyethylene glycol-polypropylene glycol-polyethylene glycol block polymer is 2700-2900.
3. The method for synchronously extracting phycocyanin and algae oil in spirulina according to claim 1, wherein the method comprises the following steps: the addition amount of the sodium chloride is 5-10% of the mass of the upper polymer phase.
4. The method for synchronously extracting phycocyanin and algae oil in spirulina according to claim 1, wherein the method comprises the following steps: the conditions of the ultrasonic method are as follows: carrying out ice bath and ultrasonic power of 80-1000 w, and carrying out continuous ultrasonic for 3-30 min.
5. The method for synchronously extracting phycocyanin and algae oil in spirulina according to claim 1, wherein the method comprises the following steps: the constant-temperature oscillation time is 30-60 min.
6. The method for synchronously extracting phycocyanin and algae oil in spirulina according to claim 1, wherein the method comprises the following steps: the centrifugation conditions were: centrifuging at 3000-4000 rpm for 8-10 min.
7. The method for synchronously extracting phycocyanin and algae oil in spirulina according to claim 1, wherein the method comprises the following steps: the temperature of the ultra-low temperature freezing is-85 to-60 ℃.
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