CN110156797B - Process for extracting chlorophyll from spirulina - Google Patents
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Abstract
The invention discloses a process for extracting chlorophyll from spirulina, which comprises the steps of raw material pretreatment, soaking, enzymolysis, filtering and drying. When chlorophyll in spirulina is extracted, the chlorophyll is obtained by adopting the modes of soaking, enzymolysis, microwave and ultrasonic treatment, so that the use of an organic solvent is avoided, and the edible safety of the chlorophyll is improved; the spirulina is subjected to enzymolysis treatment by using protease, cellulase, pectinase, ligninase, amylase, esterase and other enzymes, so that other substances except chlorophyll in the spirulina are degraded, chlorophyll in the spirulina cannot be damaged, the chlorophyll contained in the spirulina is extracted as far as possible, products do not contain other impurities, the yield and the purity of the chlorophyll are improved, the yield of the chlorophyll is higher than 99.5%, and is higher than that of a traditional solvent extraction method by more than 8.8%, and the purity of the chlorophyll is higher than 99.96%.
Description
Technical Field
The invention belongs to the technical field, and particularly relates to a process for extracting chlorophyll from spirulina.
Background
Spirulina (the scientific name: Spirulina) is a kind of lower organisms, and has effects of reducing cholesterol, regulating blood sugar, enhancing immune system, resisting oxidation, relieving fatigue, treating anemia, resisting tumor, preventing cancer, suppressing cancer, relieving toxic and side effects of cancer radiotherapy and chemotherapy, improving immunity, and reducing blood lipid. The spirulina contains high content of protein, low content of fat and cellulose, and a great variety of vitamins, and is a food with the highest content of vitamin B12 and beta-carotene. In addition, the iron content in all foods is the highest, and the algae protein with the functions of preventing and treating cancers and other mineral elements and bioactive substances for improving the immunity of the organism are also found to be contained. The spirulina is also a natural treasure house of chlorophyll, is abundant and excellent in quality, accounts for 1.1% of the algae, is 2-3 times of most terrestrial plants, and is 10 times of common vegetables. The type of chlorophyll contained in spirulina is mainly chlorophyll a, the molecular structure of which is very similar to human heme, and the spirulina is a direct raw material for human hemoglobin synthesis, which is called green blood and contains up to 7600mg/kg of spirulina powder. The spirulina is a novel medicine and food dual-purpose resource, the active ingredients of the spirulina comprise phycobiliprotein, chlorophyll, carotenoid, water-soluble polysaccharide and various trace elements, and the spirulina has extremely important development potentials of nutrition, health care, medicine and the like and becomes a healthy food recommended by grain and agriculture organizations in the United nations.
Chlorophyll is a safe and nontoxic natural fat-soluble pigment and is a general name of a green pigment which has a structure that magnesium is combined with nitrogen on four pyrrole rings and porphyrin is used as a framework. Chlorophyll is not very stable and can be decomposed by light, acid, alkali, oxygen, oxidizing agents, etc. Under acidic conditions, chlorophyll molecules easily lose magnesium in the porphyrin ring to become pheophytin. Chlorophyll has many uses such as hemopoiesis, vitamin supply, detoxification, disease resistance, etc. Chlorophyll has a structure similar to heme, is easily absorbed by human body, has good physiological effect on human body, and has wide application in medicine, food and cosmetic industries.
The extraction method of chlorophyll and carotenoid in spirulina is roughly divided into 3 types, namely: organic solvent extraction method, ultrasonic extraction method, and supercritical CO2An extraction method. The common organic solvent extraction method is time-consuming in operation, large in solvent consumption, high in toxicity, high in cost and easy to decompose chlorophyll at high temperature, and nutritional ingredients are damaged, so that the yield and the biological activity are reduced. At present, although there are reports on relevant documents for extracting chlorophyll from spirulina, the prior art is still immature in comprehensive development and utilization technology of biomass such as spirulina, most of the biomass such as spirulina cannot be fully utilized, and huge waste of biomass energy is caused; in addition, the chlorophyll in the spirulina can be dissolved in organic solvents such as acetone, methanol, ethanol, ethyl acetate and the like and is not dissolved in water, so that organic solvents are used for dehydrating spirulina mud during extraction and processing, chlorophyll is extracted, precipitation, filtration and other operations are carried out after alcohol extraction, the solubility of carotene and lutein in the organic solvents is high, the chlorophyll prepared by the method contains carotene and lutein, the prepared chlorophyll contains a certain amount of organic solvents, and the health of people can be affected after eating the chlorophyll. For example:
1. patent application CN201710169066.7 discloses an extraction process of phycocyanin, chlorophyll and spirulina polysaccharide in fresh spirulina, and relates to the technical field of extraction processes of active substances in spirulina. Weighing fresh Spirulina, centrifuging and washing for 1-3 times, weighing Spirulina mud, and adding 0. 5-4 times of water by weight of the algae mud, uniformly stirring, freezing and thawing twice, freezing overnight at minus 20 ℃, dissolving at room temperature, adding 2-8 times of water by weight of the algae mud after the second dissolution, standing for 2-8 hours, centrifuging to obtain a supernatant a and a solid b, extracting the solid b in an ethanol soxhlet extraction mode to obtain an extracting solution and a solid c, wherein the supernatant a is used for extracting phycocyanin; extracting the solid b from the extracting solution to obtain sodium zinc chlorophyllin; the solid c is used for extracting spirulina polysaccharide; the process is simple, can fully and effectively utilize the existing spirulina resources, realizes the comprehensive utilization and the deep development of the spirulina, and has positive significance for the development of the spirulina industry.
2. Patent application CN201510528132.6 discloses a method for preparing high-purity chlorophyll a, which comprises the following steps: (1) taking spirulina platensis cultured to the end of logarithmic growth phase as a raw material, and centrifuging to collect algal bodies; (2) extracting algae with ethanol for three times, and mixing extractive solutions; (3) adding a proper amount of normal hexane or petroleum ether into the extract, adding water until phase separation is carried out for back extraction, and washing the solution containing the pigment twice by using water; (4) performing chromatographic separation and purification on chlorophyll a by using a 100-200-mesh neutral alumina column, firstly rinsing the alumina column by using a normal hexane or petroleum ether solution containing 0.3% of ethanol, and performing gradient washing by using an ethanol-normal hexane or ethanol-petroleum ether mixed solvent after sampling; (5) and (3) carrying out reduced pressure rotary evaporation on the solution containing the chlorophyll a to recover the solvent, thus obtaining the high-purity chlorophyll a. The chlorophyll a prepared by the invention does not contain chlorophyll b, and the HPLC purity is more than 99.5%.
3. Patent application CN200910016683.9 discloses a process for extracting phycocyanin, chlorophyll and spirulina polysaccharide from spirulina, comprising the following steps: weighing fresh spirulina, centrifugally washing, weighing spirulina mud, adding water into supernatant, uniformly stirring, freezing and thawing twice (to crack cell walls), dissolving for the second time, adding water, standing for 2-8h, centrifuging to obtain supernatant and precipitate A, and subjecting the precipitate A to soxhlet extraction with ethanol to obtain extract and precipitate B, wherein the supernatant is used for extracting phycocyanin; the sediment A is used for extracting sodium zinc chlorophyllin; the precipitate B is used for extracting spirulina polysaccharide. The extraction process of the invention integrates the extraction of active substances such as phycocyanin, chlorophyll, spirulina polysaccharide and the like, fully and effectively utilizes the existing spirulina resources, and avoids the waste of resources.
Therefore, how to fully utilize the biomass energy of spirulina, reduce the resource waste, improve the chlorophyll extraction rate and purity, and ensure the production of healthy, safe, qualified and stable pure natural chlorophyll products is a technical problem which needs to be solved urgently in the prior art.
Disclosure of Invention
The invention provides a process for extracting chlorophyll from spirulina, which aims to solve the technical problems.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a process for extracting chlorophyll from spirulina comprises the following steps:
(1) pretreatment of raw materials: cleaning fresh spirulina, carrying out shade treatment at 1-9 ℃ for 80-90 min, then crushing at 1-8 ℃ until the spirulina is sieved by a sieve of 50-100 meshes, conveying the spirulina into a centrifugal tube filled with steel balls, pouring liquid nitrogen, covering a centrifugal tube cover when the liquid nitrogen is just volatilized, placing the centrifugal tube cover on a vortex instrument, and carrying out vortex until the spirulina powder is sieved by a sieve of 250-350 meshes;
(2) soaking: under the dark and the protection of argon or nitrogen, adding deionized water into the spirulina powder for soaking treatment, and performing microwave and ultrasonic treatment assisted soaking to obtain spirulina aqueous solution;
(3) enzymolysis: adding mixed enzyme A into the spirulina water solution for enzymolysis under the dark and argon or nitrogen protection conditions, performing microwave treatment-assisted enzymolysis, adding mixed enzyme B, and further performing ultrasonic treatment-assisted enzymolysis to obtain an enzymolysis mixture;
(4) and (3) filtering: filtering the enzymolysis mixture by using an ultrafiltration membrane under the protection of darkness and argon or nitrogen, and removing a filtering mother solution to obtain a chlorophyll semi-finished product;
(5) drying: and under the protection of dark and argon or nitrogen, sending the prepared chlorophyll semi-finished product into a dryer for drying, sealing and packaging, and storing in a dark place to obtain the chlorophyll.
Further, in the step (1), the centrifugal tube is repeatedly inverted for 3-5 times in the vortex process.
Further, in step (2), the microwave and ultrasonic treatment assisted soaking mode is as follows: firstly, performing microwave treatment for 45-65 s at a microwave temperature of 2-6 ℃ and a microwave power of 50-60W, then performing ultrasonic treatment for 3-7 min at an ultrasonic power of 105-120W and an ultrasonic temperature of 18-23 ℃, and alternately performing microwave treatment and ultrasonic treatment for 2-3 times according to the above modes.
Further, in step (2), the soaking parameters are controlled as follows: the soaking pressure is 0.05-0.09 MPa, the soaking temperature is 1-8 ℃, and the soaking time is 2.5-3 h.
Further, in step (3), the microwave treatment: the microwave power is 85-95W, the microwave time is 60-120 s, and the microwave temperature is 2-7 ℃.
Further, in step (3), the ultrasonic treatment: the ultrasonic power is 105-120W, the ultrasonic time is 3-9 min, and the ultrasonic temperature is 18-25 ℃.
Further, in the step (3), the mixed enzyme a is protease, cellulase, pectinase, hemicellulase, and peroxidase in a mass ratio of 3:1 to 2:1:0.3:0.1 to 0.2.
Further, before adding the mixed enzyme B in the step (3), heating to 40-50 ℃, adding an enzyme inhibitor to adjust the pH value for inactivating the enzyme, cooling to 1-10 ℃, and then adding the mixed enzyme B; after the ultrasonic treatment is finished to assist enzymolysis, heating the material to 42-50 ℃, adding an enzyme inhibitor to adjust PH for enzyme deactivation, and cooling to 1-8 ℃ to obtain an enzymolysis mixture. The enzyme inhibitor is one or more of protease inhibitor, cellulase inhibitor, pectinase inhibitor, hemicellulase inhibitor, peroxidase inhibitor, ligninase inhibitor, amylase inhibitor, and lipoxygenase inhibitor.
Further, in the step (3), the mixed enzyme B is ligninase, cellulase, amylase, esterase, lipoxygenase and pectinase in a mass ratio of 1-2: 2:1:0.5:0.5: 1-1.5.
Further, in the step (5), the temperature of reduced pressure drying is 26-30 ℃, the relative humidity of air is less than or equal to 65%, the drying time is 22-30 min, and the drying is carried out until the water content of chlorophyll is less than 20%.
Due to the adoption of the technical scheme, the invention has the following beneficial effects:
(1) when the chlorophyll is extracted from the spirulina, the chlorophyll is obtained by adopting the modes of soaking, enzymolysis, microwave and ultrasonic treatment, the use of an organic solvent is avoided, the edible safety of the chlorophyll is improved, the cellulose and pectin frameworks forming the spirulina cell wall can be degraded by the cellulase and the pectinase in the used enzyme, the cell wall framework structure of the spirulina is further destroyed, the dissolution of the chlorophyll in cells is increased, the chlorophyll extraction yield is higher than 99.5 percent and higher than 8.8 percent compared with the traditional solvent extraction method, and the chlorophyll purity is higher than 99.96 percent.
(2) This application adopts enzymes such as protease, cellulase, pectinase, ligninase, amylase, esterase to carry out enzymolysis to spirulina, degrades except that other material of chlorophyll in the spirulina, and chlorophyll in the spirulina can not destroyed, makes the chlorophyll that contains in the spirulina draw out as far as possible, and the product does not contain other impurity, has improved the yield and the purity of chlorophyll.
(3) Because spirulina contains more protein, and chlorophyll combines very tightly with protein, soak earlier in this application, then carry out enzyme treatment, help going on smoothly of enzymolysis, and degrade spirulina's protein with protease earlier, help cellulase etc. to degrading the cellulose and the pectin skeleton of spirulina cell wall, be favorable to improving chlorophyll extraction rate.
(4) According to the method, the protease and the esterase are used for decomposing proteins outside spirulina chlorophyll, and the peroxidase and the lipoxygenase are used for catalyzing the oxidation of substrates of the protease and the esterase for degrading the proteins, so that the reaction is promoted, the reaction speed is increased, and the chlorophyll extraction time is shortened.
(5) Each step of this application all goes on under complete light-resistant and gas protection, isolated chlorophyll and oxygen and light contact, avoids chlorophyll content to be degraded at the extraction in-process in the spirulina, shortens chlorophyll extraction time, increases chlorophyll's extraction efficiency.
(6) The ultrasonic extraction technology adopted by the application has good cavitation effect and mechanical effect, the microwave can generate good heat effect, so that polar substances in cells generate a large amount of heat, the operation is simple, the extraction time is shortened, the energy consumption is reduced, the cost is saved, and the extraction rate can be effectively improved.
(7) Since spirulina cells are easily swollen, organic solvent extraction is considered whether to destroy spirulina phycobiliprotein, but the application is provided.
(8) This application carries out shredding to spirulina and has added the liquid nitrogen in raw materials preliminary treatment, and the gasification process can condense in the twinkling of an eye and reduce spirulina's temperature when the liquid nitrogen is vortex, is favorable to abundant grinding and improves grinding efficiency, and can keep the grinding process to go on at low temperature, can effectively prevent and treat chlorophyll's degradation.
(9) When this application carries out the preliminary treatment to spirulina, carry out shading before smashing and handle, reduced spirulina's water content, convenient follow-up to chlorophyll draws, improves chlorophyll and draws the yield.
Drawings
In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the drawings needed in the description of the embodiments or the prior art will be briefly introduced below, it is obvious that the drawings in the following description are only some examples of the present invention, and for a person skilled in the art, without inventive step, other drawings can be obtained according to these drawings:
FIG. 1 is a flow chart of a process for extracting chlorophyll from Spirulina.
Detailed Description
The following is a detailed description of the embodiments of the present invention, but the present invention is not limited to these embodiments, and any modifications or substitutions in the basic spirit of the embodiments are included in the scope of the present invention as claimed in the claims.
Example 1
A process for extracting chlorophyll from spirulina comprises the following steps:
(1) pretreatment of raw materials: cleaning fresh spirulina, treating at 1 deg.C in the shade for 80min, pulverizing at 8 deg.C to 50 mesh, feeding into a centrifuge tube containing steel balls, pouring liquid nitrogen, covering the centrifuge tube with a centrifugal tube cover when liquid nitrogen is volatilized completely, and vortexing to obtain 250 mesh spirulina powder; the centrifuge tube is repeatedly inverted for 3 times in the vortex process;
(2) soaking: under the dark and the protection of argon or nitrogen, adding deionized water into the spirulina powder for soaking treatment, and performing microwave and ultrasonic treatment assisted soaking to obtain spirulina aqueous solution;
the microwave and ultrasonic treatment auxiliary soaking mode is as follows: firstly, performing microwave treatment for 45s at a microwave power of 50W and a microwave temperature of 2 ℃, then performing ultrasonic treatment for 3min at an ultrasonic power of 105W and an ultrasonic temperature of 18 ℃, and alternately performing microwave treatment and ultrasonic treatment for 2 times according to the above mode; the soaking parameters are controlled as follows: the soaking pressure is 0.05MPa, the soaking temperature is 1 ℃, and the soaking time is 2.5 h;
(3) enzymolysis: adding mixed enzyme A into the spirulina water solution for enzymolysis under the dark and argon or nitrogen protection conditions, performing microwave treatment-assisted enzymolysis, adding mixed enzyme B, and further performing ultrasonic treatment-assisted enzymolysis to obtain an enzymolysis mixture;
the microwave treatment comprises the following steps: the microwave power is 85W, the microwave time is 60s, and the microwave temperature is 2 ℃; the ultrasonic treatment comprises the following steps: the ultrasonic power is 105W, the ultrasonic time is 3min, and the ultrasonic temperature is 18 ℃; the mixed enzyme A is protease, cellulase, pectinase, hemicellulase and peroxidase according to a mass ratio of 3:1:1:0.3: 0.1; before adding the mixed enzyme B, heating to 40 ℃, adding an enzyme inhibitor to adjust the pH value for inactivating the enzyme, cooling to 1 ℃, and then adding the mixed enzyme B; after the ultrasonic treatment is finished to assist enzymolysis, heating the material to 42 ℃, adding an enzyme inhibitor to adjust the pH value for enzyme deactivation, and cooling to 8 ℃ to obtain an enzymolysis mixture; the mixed enzyme B is ligninase, cellulase, amylase, esterase, lipoxygenase and pectinase according to the mass ratio of 1:2:1:0.5:0.5: 1;
(4) and (3) filtering: filtering the enzymolysis mixture by using an ultrafiltration membrane under the protection of darkness and argon or nitrogen, and removing a filtering mother solution to obtain a chlorophyll semi-finished product;
(5) drying: sending the prepared chlorophyll semi-finished product into a dryer for drying under the protection of darkness and argon or nitrogen, sealing and packaging, and storing in dark place to obtain the chlorophyll; the temperature of reduced pressure drying is 26 ℃, the relative humidity of air is less than or equal to 65%, the drying time is 22min, and the drying is carried out until the water content of chlorophyll is less than 20%.
Example 2
A process for extracting chlorophyll from spirulina comprises the following steps:
(1) pretreatment of raw materials: cleaning fresh spirulina, treating at 9 deg.C in the shade for 90min, pulverizing at 1 deg.C to 100 mesh, feeding into a centrifuge tube containing steel balls, pouring liquid nitrogen, covering the centrifuge tube with a centrifugal tube cover when liquid nitrogen is volatilized completely, and vortexing to obtain 350 mesh spirulina powder; the centrifuge tube is repeatedly inverted for 5 times in the vortex process;
(2) soaking: under the dark and the protection of argon or nitrogen, adding deionized water into the spirulina powder for soaking treatment, and performing microwave and ultrasonic treatment assisted soaking to obtain spirulina aqueous solution;
the microwave and ultrasonic treatment auxiliary soaking mode is as follows: firstly, carrying out microwave treatment for 65s at the microwave power of 60W and the microwave temperature of 6 ℃, then carrying out ultrasonic treatment for 7min at the ultrasonic power of 120W and the ultrasonic temperature of 18 ℃, and alternately carrying out microwave treatment and ultrasonic treatment for 3 times according to the above mode; the soaking parameters are controlled as follows: the soaking pressure is 0.09MPa, the soaking temperature is 8 ℃, and the soaking time is 3 h;
(3) enzymolysis: adding mixed enzyme A into the spirulina water solution for enzymolysis under the dark and argon or nitrogen protection conditions, performing microwave treatment-assisted enzymolysis, adding mixed enzyme B, and further performing ultrasonic treatment-assisted enzymolysis to obtain an enzymolysis mixture;
the microwave treatment comprises the following steps: the microwave power is 95W, the microwave time is 120s, and the microwave temperature is 7 ℃; the ultrasonic treatment comprises the following steps: the ultrasonic power is 120W, the ultrasonic time is 9min, and the ultrasonic temperature is 25 ℃; the mixed enzyme A is protease, cellulase, pectinase, hemicellulase and peroxidase according to a mass ratio of 3:2:1:0.3: 0.2; before adding the mixed enzyme B, heating to 50 ℃, adding an enzyme inhibitor to adjust the pH value for inactivating enzyme, cooling to 10 ℃, and then adding the mixed enzyme B; after the ultrasonic treatment is finished to assist enzymolysis, heating the material to 50 ℃, adding an enzyme inhibitor to adjust the pH value for enzyme deactivation, and cooling to 1 ℃ to obtain an enzymolysis mixture; the mixed enzyme B is ligninase, cellulase, amylase, esterase, lipoxygenase and pectinase according to the mass ratio of 2:2:1:0.5:0.5: 1.5;
(4) and (3) filtering: filtering the enzymolysis mixture by using an ultrafiltration membrane under the protection of darkness and argon or nitrogen, and removing a filtering mother solution to obtain a chlorophyll semi-finished product;
(5) drying: sending the prepared chlorophyll semi-finished product into a dryer for drying under the protection of darkness and argon or nitrogen, sealing and packaging, and storing in dark place to obtain the chlorophyll; the temperature of reduced pressure drying is 30 ℃, the relative humidity of air is less than or equal to 65%, the drying time is 30min, and the drying is carried out until the water content of chlorophyll is less than 20%.
Example 3
A process for extracting chlorophyll from spirulina comprises the following steps:
(1) pretreatment of raw materials: cleaning fresh spirulina, treating at 2 deg.C in the shade for 82min, pulverizing at 7 deg.C to 60 mesh, feeding into a centrifuge tube containing steel balls, pouring liquid nitrogen, covering the centrifuge tube with a centrifugal tube cover when liquid nitrogen is volatilized completely, and vortexing to obtain spirulina powder passing through 270 mesh; the centrifuge tube is repeatedly inverted for 5 times in the vortex process;
(2) soaking: under the dark and the protection of argon or nitrogen, adding deionized water into the spirulina powder for soaking treatment, and performing microwave and ultrasonic treatment assisted soaking to obtain spirulina aqueous solution;
the microwave and ultrasonic treatment auxiliary soaking mode is as follows: firstly, performing microwave treatment for 50s at a microwave power of 52W and a microwave temperature of 3 ℃, then performing ultrasonic treatment for 4min at an ultrasonic power of 110W and an ultrasonic temperature of 19 ℃, and alternately performing microwave treatment and ultrasonic treatment for 2 times according to the above mode; the soaking parameters are controlled as follows: the soaking pressure is 0.06MPa, the soaking temperature is 2 ℃, and the soaking time is 2.6 h;
(3) enzymolysis: adding mixed enzyme A into the spirulina water solution for enzymolysis under the dark and argon or nitrogen protection conditions, performing microwave treatment-assisted enzymolysis, adding mixed enzyme B, and further performing ultrasonic treatment-assisted enzymolysis to obtain an enzymolysis mixture;
the microwave treatment comprises the following steps: the microwave power is 87W, the microwave time is 80s, and the microwave temperature is 3 ℃; the ultrasonic treatment comprises the following steps: the ultrasonic power is 110W, the ultrasonic time is 4min, and the ultrasonic temperature is 19 ℃; the mixed enzyme A is protease, cellulase, pectinase, hemicellulase and peroxidase according to a mass ratio of 3:1.2:1:0.3: 0.12; before adding the mixed enzyme B, heating to 42 ℃, adding an enzyme inhibitor to adjust the pH value for inactivating enzyme, cooling to 2 ℃, and then adding the mixed enzyme B; after the ultrasonic treatment is finished to assist enzymolysis, heating the material to 43 ℃, adding an enzyme inhibitor to adjust the pH value for enzyme deactivation, and cooling to 3 ℃ to obtain an enzymolysis mixture; the mixed enzyme B is ligninase, cellulase, amylase, esterase, lipoxygenase and pectinase according to the mass ratio of 1.2:2:1:0.5:0.5: 1.2;
(4) and (3) filtering: filtering the enzymolysis mixture by using an ultrafiltration membrane under the protection of darkness and argon or nitrogen, and removing a filtering mother solution to obtain a chlorophyll semi-finished product;
(5) drying: sending the prepared chlorophyll semi-finished product into a dryer for drying under the protection of darkness and argon or nitrogen, sealing and packaging, and storing in dark place to obtain the chlorophyll; the temperature of reduced pressure drying is 27 ℃, the relative humidity of air is less than or equal to 65%, the drying time is 25min, and the drying is carried out until the water content of chlorophyll is less than 20%.
Example 4
A process for extracting chlorophyll from spirulina comprises the following steps:
(1) pretreatment of raw materials: cleaning fresh spirulina, treating at 8 deg.C in the dark for 88min, pulverizing at 3 deg.C to 90 mesh, feeding into a centrifuge tube containing steel balls, pouring liquid nitrogen, covering the centrifuge tube with a centrifugal tube cover when liquid nitrogen is volatilized completely, and vortexing to obtain 330 mesh spirulina powder; the centrifuge tube is repeatedly inverted for 5 times in the vortex process;
(2) soaking: under the dark and the protection of argon or nitrogen, adding deionized water into the spirulina powder for soaking treatment, and performing microwave and ultrasonic treatment assisted soaking to obtain spirulina aqueous solution;
the microwave and ultrasonic treatment auxiliary soaking mode is as follows: firstly, performing microwave treatment for 60s at a microwave temperature of 5 ℃ and a microwave power of 58W, then performing ultrasonic treatment for 6min at an ultrasonic power of 1115W and an ultrasonic temperature of 22 ℃, and alternately performing microwave treatment and ultrasonic treatment for 3 times according to the above manner; the soaking parameters are controlled as follows: the soaking pressure is 0.08MPa, the soaking temperature is 6 ℃, and the soaking time is 2.9 h;
(3) enzymolysis: adding mixed enzyme A into the spirulina water solution for enzymolysis under the dark and argon or nitrogen protection conditions, performing microwave treatment-assisted enzymolysis, adding mixed enzyme B, and further performing ultrasonic treatment-assisted enzymolysis to obtain an enzymolysis mixture;
the microwave treatment comprises the following steps: the microwave power is 93W, the microwave time is 100s, and the microwave temperature is 6 ℃; the ultrasonic treatment comprises the following steps: the ultrasonic power is 115W, the ultrasonic time is 8min, and the ultrasonic temperature is 24 ℃; the mixed enzyme A is protease, cellulase, pectinase, hemicellulase and peroxidase according to a mass ratio of 3:1.8:1:0.3: 0.18; before adding the mixed enzyme B, heating to 48 ℃, adding an enzyme inhibitor to adjust the pH value for inactivating the enzyme, cooling to 8 ℃, and then adding the mixed enzyme B; after the ultrasonic treatment is finished to assist enzymolysis, heating the material to 49 ℃, adding an enzyme inhibitor to adjust the pH value for enzyme deactivation, and cooling to 7 ℃ to obtain an enzymolysis mixture; the mixed enzyme B is ligninase, cellulase, amylase, esterase, lipoxygenase and pectinase according to the mass ratio of 1.8:2:1:0.5:0.5: 1.4;
(4) and (3) filtering: filtering the enzymolysis mixture by using an ultrafiltration membrane under the protection of darkness and argon or nitrogen, and removing a filtering mother solution to obtain a chlorophyll semi-finished product;
(5) drying: sending the prepared chlorophyll semi-finished product into a dryer for drying under the protection of darkness and argon or nitrogen, sealing and packaging, and storing in dark place to obtain the chlorophyll; the temperature of reduced pressure drying is 29 ℃, the relative humidity of air is less than or equal to 65%, the drying time is 28min, and the drying is carried out until the water content of chlorophyll is less than 20%.
Example 5
A process for extracting chlorophyll from spirulina comprises the following steps:
(1) pretreatment of raw materials: cleaning fresh spirulina, treating at 5 deg.C in the shade for 85min, pulverizing at 4 deg.C to 80 mesh, feeding into a centrifuge tube containing steel balls, pouring liquid nitrogen, covering the centrifuge tube with a centrifugal tube cover when liquid nitrogen is volatilized completely, and vortexing to obtain 300 mesh spirulina powder; the centrifuge tube is repeatedly inverted for 4 times in the vortex process;
(2) soaking: under the dark and the protection of argon or nitrogen, adding deionized water into the spirulina powder for soaking treatment, and performing microwave and ultrasonic treatment assisted soaking to obtain spirulina aqueous solution;
the microwave and ultrasonic treatment auxiliary soaking mode is as follows: firstly, performing microwave treatment for 55s at a microwave power of 55W and a microwave temperature of 4 ℃, then performing ultrasonic treatment for 5min at an ultrasonic power of 112W and an ultrasonic temperature of 20 ℃, and alternately performing microwave treatment and ultrasonic treatment for 3 times according to the above mode; the soaking parameters are controlled as follows: the soaking pressure is 0.07MPa, the soaking temperature is 5 ℃, and the soaking time is 2.8 h;
(3) enzymolysis: adding mixed enzyme A into the spirulina water solution for enzymolysis under the dark and argon or nitrogen protection conditions, performing microwave treatment-assisted enzymolysis, adding mixed enzyme B, and further performing ultrasonic treatment-assisted enzymolysis to obtain an enzymolysis mixture;
the microwave treatment comprises the following steps: the microwave power is 90W, the microwave time is 90s, and the microwave temperature is 5 ℃; the ultrasonic treatment comprises the following steps: the ultrasonic power is 113W, the ultrasonic time is 6min, and the ultrasonic temperature is 21 ℃; the mixed enzyme A is protease, cellulase, pectinase, hemicellulase and peroxidase according to a mass ratio of 3:1.5:1:0.3: 0.15; before adding the mixed enzyme B, heating to 45 ℃, adding an enzyme inhibitor to adjust the pH value for inactivating enzyme, cooling to 5 ℃, and then adding the mixed enzyme B; after the ultrasonic treatment is finished to assist enzymolysis, heating the material to 46 ℃, adding an enzyme inhibitor to adjust the pH value for enzyme deactivation, and cooling to 4 ℃ to obtain an enzymolysis mixture; the mixed enzyme B is ligninase, cellulase, amylase, esterase, lipoxygenase and pectinase according to the mass ratio of 1.5:2:1:0.5:0.5: 1.3;
(4) and (3) filtering: filtering the enzymolysis mixture by using an ultrafiltration membrane under the protection of darkness and argon or nitrogen, and removing a filtering mother solution to obtain a chlorophyll semi-finished product;
(5) drying: sending the prepared chlorophyll semi-finished product into a dryer for drying under the protection of darkness and argon or nitrogen, sealing and packaging, and storing in dark place to obtain the chlorophyll; the temperature of reduced pressure drying is 28 ℃, the relative humidity of air is less than or equal to 65%, the drying time is 26min, and the drying is carried out until the water content of chlorophyll is less than 20%.
Comparative example 1
The examples in patent application CN201710169066.7 were subjected to chlorophyll extraction.
Comparative example 2
The examples in patent application CN201510528132.6 were subjected to chlorophyll extraction.
Comparative example 3
The examples in patent application CN200910016683.9 were subjected to chlorophyll extraction.
The method for extracting chlorophyll according to the embodiments 1-5 and the comparative examples 1-3 is adopted, the chlorophyll extraction yield is checked, the purity and the organic solvent content of the prepared chlorophyll are shown in the following table 1.
TABLE 1
| Group of | Extraction yield | Purity of | Organic solvent content |
| Example 1 | 99.7％ | 99.98％ | 0％ |
| Example 2 | 99.5％ | 99.96％ | 0％ |
| Example 3 | 99.7％ | 99.98％ | 0％ |
| Example 4 | 99.6％ | 99.97％ | 0％ |
| Example 5 | 99.8％ | 99.99％ | 0％ |
| Comparative example 1 | 90.5％ | 97.03％ | 2.7％ |
| Comparative example 2 | 90.7％ | 99.52％ | 0.4％ |
| Comparative example 3 | 87.6％ | 96.15％ | 3.5％ |
As can be seen from the experimental data in Table 1, the chlorophyll prepared by the method does not contain organic solvent, and the chlorophyll extraction yield is higher than 99.5%, and is higher than that of the chlorophyll extraction yield by more than 8.8% and the chlorophyll purity is higher than 99.96% by adopting the traditional solvent method.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (8)
1. A process for extracting chlorophyll from spirulina is characterized by comprising the following steps:
(1) pretreatment of raw materials: cleaning fresh spirulina, carrying out shade treatment at 1-9 ℃ for 80-90 min, then crushing at 1-8 ℃ until the spirulina is sieved by a sieve of 50-100 meshes, conveying the spirulina into a centrifugal tube filled with steel balls, pouring liquid nitrogen, covering a centrifugal tube cover when the liquid nitrogen is just volatilized, placing the centrifugal tube cover on a vortex instrument, and carrying out vortex until the spirulina powder is sieved by a sieve of 250-350 meshes;
(2) soaking: under the dark and the protection of argon or nitrogen, adding deionized water into the spirulina powder for soaking treatment, and performing microwave and ultrasonic treatment assisted soaking to obtain spirulina aqueous solution;
(3) enzymolysis: adding mixed enzyme A into the spirulina water solution for enzymolysis under the dark and argon or nitrogen protection conditions, performing microwave treatment-assisted enzymolysis, adding mixed enzyme B, and further performing ultrasonic treatment-assisted enzymolysis to obtain an enzymolysis mixture;
(4) and (3) filtering: filtering the enzymolysis mixture by using an ultrafiltration membrane under the protection of darkness and argon or nitrogen, and removing a filtering mother solution to obtain a chlorophyll semi-finished product;
(5) drying: sending the prepared chlorophyll semi-finished product into a dryer for drying under the protection of darkness and argon or nitrogen, sealing and packaging, and storing in dark place to obtain the chlorophyll;
in the step (3), the mixed enzyme A is protease, cellulase, pectinase, hemicellulase and peroxidase in a mass ratio of 3: 1-2: 1:0.3: 0.1-0.2;
in the step (3), the mixed enzyme B is ligninase, cellulase, amylase, esterase, lipoxygenase and pectinase according to a mass ratio of 1-2: 2:1:0.5:0.5: 1-1.5.
2. The process of claim 1, wherein the chlorophyll is extracted from spirulina by the following steps: in the step (1), the centrifugal tube is repeatedly inverted for 3-5 times in the vortex process.
3. The process of claim 1, wherein the chlorophyll is extracted from spirulina by the following steps: in step (2), the microwave and ultrasonic treatment assisted soaking mode is as follows: firstly, performing microwave treatment for 45-65 s at a microwave temperature of 2-6 ℃ and a microwave power of 50-60W, then performing ultrasonic treatment for 3-7 min at an ultrasonic power of 105-120W and an ultrasonic temperature of 18-23 ℃, and alternately performing microwave treatment and ultrasonic treatment for 2-3 times according to the above modes.
4. The process of claim 1, wherein the chlorophyll is extracted from spirulina by the following steps: in step (2), the soaking parameters are controlled as follows: the soaking pressure is 0.05-0.09 MPa, the soaking temperature is 1-8 ℃, and the soaking time is 2.5-3 h.
5. The process of claim 1, wherein the chlorophyll is extracted from spirulina by the following steps: in step (3), the microwave treatment: the microwave power is 85-95W, the microwave time is 60-120 s, and the microwave temperature is 2-7 ℃.
6. The process of claim 1, wherein the chlorophyll is extracted from spirulina by the following steps: in step (3), the ultrasonic treatment: the ultrasonic power is 105-120W, the ultrasonic time is 3-9 min, and the ultrasonic temperature is 18-25 ℃.
7. The process of claim 1, wherein the chlorophyll is extracted from spirulina by the following steps: before adding the mixed enzyme B in the step (3), firstly heating to 40-50 ℃, adding an enzyme inhibitor to adjust the pH value for inactivating enzyme, cooling to 1-10 ℃, and then adding the mixed enzyme B; after the ultrasonic treatment is finished to assist enzymolysis, heating the material to 42-50 ℃, adding an enzyme inhibitor to adjust PH for enzyme deactivation, and cooling to 1-8 ℃ to obtain an enzymolysis mixture.
8. The process of claim 1, wherein the chlorophyll is extracted from spirulina by the following steps: in the step (5), the drying is reduced-pressure drying, the temperature of the reduced-pressure drying is 26-30 ℃, the relative humidity of air is less than or equal to 65%, the drying time is 22-30 min, and the drying is carried out until the water content of chlorophyll is less than 20%.
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