阅读说明：本技术 一种螺旋藻生物活性物质分级分离方法 (Spirulina bioactive substance grading separation method ) 是由 袁丽 韩丹翔 胡强 余磊 杨小红 于 2020-07-15 设计创作，主要内容包括：本发明涉及一种螺旋藻生物活性物质分级分离方法,其按照顺序依次进行如下步骤：S1:以螺旋藻粉为原料,提取和纯化螺旋藻粉中的藻蓝蛋白,螺旋藻粉变成一级藻渣；S2:以上述一级藻渣为原料,采用超临界二氧化碳萃取法或有机试剂萃取法提取藻渣中的油脂,并进一步纯化得糖脂,所述一级藻渣变成二级藻渣；S3:将二级藻渣置于通风条件下,待有机试剂充分挥发干净后,分装保存,二级藻渣富含其它蛋白和多糖,作为蛋白和多糖的粗产物。本发明不仅可分级分步将不同的活性物质依次提取出来,且保证了提取效率和避免功能性物质失活等问题,该方法藻蓝蛋白和油脂提取率高,具有操作简单、能耗低、易工业化放大等特点,该方案可最大化螺旋藻利用价值,为开发螺旋藻产品提供技术支持。(The invention relates to a spirulina bioactive substance grading separation method, which sequentially comprises the following steps: s1, extracting and purifying phycocyanin in the spirulina powder by taking the spirulina powder as a raw material, and changing the spirulina powder into first-level algae residue; s2, extracting the grease in the primary algae residues as raw materials by a supercritical carbon dioxide extraction method or an organic reagent extraction method, and further purifying to obtain glycolipids, wherein the primary algae residues become secondary algae residues; and S3, placing the secondary algae residue under a ventilation condition, subpackaging and storing after the organic reagent is fully volatilized, wherein the secondary algae residue is rich in other proteins and polysaccharides and is used as a crude product of the proteins and the polysaccharides. The method can sequentially extract different active substances step by step, ensures the extraction efficiency, avoids the problems of inactivation of functional substances and the like, has the characteristics of high extraction rate of phycocyanin and grease, simple operation, low energy consumption, easy industrial amplification and the like, can maximize the utilization value of the spirulina, and provides technical support for developing spirulina products.)

1. A spirulina bioactive substance grading separation method is characterized by sequentially carrying out the following steps:

s1, extracting and purifying phycocyanin in the spirulina powder by taking the spirulina powder as a raw material, and changing the spirulina powder into first-level algae residue;

s2, extracting the grease in the primary algae residues as raw materials by a supercritical carbon dioxide extraction method or an organic reagent extraction method, and further purifying to obtain glycolipids, wherein the primary algae residues become secondary algae residues;

and S3, placing the secondary algae residue under a ventilation condition, subpackaging and storing after the organic reagent is fully volatilized, wherein the secondary algae residue is rich in protein and polysaccharide and is used as a crude product of the spirulina protein and polysaccharide.

2. The method for fractionating biologically active substance according to claim 1, wherein said step S1 comprises:

s11, weighing and mixing the spirulina powder and a potassium phosphate buffer solution, wherein the concentration of potassium phosphate in the potassium phosphate buffer solution is 0.05-0.08M, the spirulina powder and the potassium phosphate buffer solution are uniformly mixed according to the ratio of 1:10-14 in g/ml, and stirring or shaking extraction is carried out at the temperature of 30-42 ℃ for 1-5 hours;

s12, adding 2-4% calcium chloride solution by mass volume percent into the mixed solution, and continuing stirring or extracting for 1-3 hours by oscillation; the mass volume percentage is g/ml percentage;

s13, centrifuging and collecting supernatant;

s14, adding potassium phosphate buffer solution with potassium phosphate concentration of 0.05-0.08M, resuspending the precipitate, and continuing stirring or shaking to extract for 1-3 hours;

s15, centrifuging and collecting supernatant;

s16, repeating the steps S14-S15 at least once to fully extract supernatant rich in phycocyanin, and combining the supernatant of each time; combining the centrifugal precipitates to obtain first-grade algae residues;

s17, performing ultrafiltration purification and concentration on the combined supernatant;

s18, drying to obtain powder with high phycocyanin content, and measuring the content of phycocyanin in the powder.

3. The method for fractionating biologically active substance according to claim 2, wherein said step S1 comprises:

s11, weighing spirulina powder and 0.06M potassium phosphate buffer solution, mixing uniformly according to a ratio of g/ml to 1:11.5, and stirring or shaking for extraction at 40 ℃ for 1-5 hours;

the preparation method of the potassium phosphate buffer solution comprises the following steps: mixing 1M potassium dihydrogen phosphate solution and 1M potassium dihydrogen phosphate solution at a volume ratio of 45:55, diluting by 10 times, and adjusting pH to 7.0;

s12, adding 2.5 percent by mass volume of calcium chloride solution into the spirulina suspension, and continuing stirring or oscillating for 10-30 minutes;

s13, centrifuging for 10 minutes at 4500g, and collecting supernatant;

s14, adding 0.06M potassium phosphate buffer solution, resuspending the precipitate, and continuing stirring or extracting for 1 hour by oscillation;

s15, centrifuging for 10 minutes at 4500g, and collecting supernatant;

s16, repeating the steps S14-S15 once to fully extract the supernatant rich in phycocyanin; combining the supernatants of each time; combining the centrifugal precipitates to obtain first-grade algae residues;

s17, performing ultrafiltration purification and concentration on the combined supernatant through a 50kDa filter membrane;

and S18, drying and carrying out phycocyanin content determination.

4. The method for fractionating biologically active substance according to claim 1 or 2, wherein said step S2 comprises:

s21: taking first-class algae residues as raw materials, and uniformly mixing a porous material which is 0.25-1.0 time of the mass of the raw materials with the first-class algae residues for later use;

s22: subjecting the Spirulina raw material to supercritical CO₂Extraction, wherein the supercritical CO₂In the extraction process, an organic solvent is used as an entrainer, and the volume mass ratio of the entrainer to ml/g of raw materials is 0.5:1-3: 1;

s23, collecting an extract, wherein the extract is grease from first-order algae residue; secondary algae residue is generated after extraction;

s24: further isolated from the extract to obtain glycolipids.

5. The method for fractionating spirulina bioactive substances according to claim 4, wherein in the step S21, the porous material is selected from the group consisting of diatomaceous earth, macroporous resin and molecular sieve, preferably diatomaceous earth: diatomite, macroporous resin and molecular sieve, preferably diatomite.

6. The method for fractionating spirulina bioactive substances according to claim 4, wherein in the step S22, the organic solvent is one or more selected from methanol, ethanol, isopropanol, ethyl acetate, propyl acetate, butyl acetate, acetone and n-hexane.

7. The method for fractionating biologically active substance according to claim 4, wherein said step S22 comprises:

s221: according to the weight percentage of ethanol: adding entrainer into the raw material (0.5: 1-3:1(ml/g volume mass ratio)), mixing with the mixture obtained in step S21, loading into material bag, placing into extraction kettle, and installing the sealing end cap of the extraction kettle;

s222: opening the inlet and outlet valves of the extraction kettle, and using CO₂Purging the whole system pipeline to remove air;

s223: opening a switch of a refrigerator, precooling for 15-25 minutes, and setting the extraction conditions of the system through an equipment control panel, wherein the temperature of the extraction kettle is 30-50 ℃, the temperature of an outlet valve is 40-60 ℃, and the pressure is 4000-;

s224: after the temperature of the extraction kettle is stable, CO₂Inputting the mixture into an extraction kettle through a high-pressure pump;

s225: opening an outlet valve of the extraction kettle after ensuring the extraction retention time of 15-60 minutes, and mixing the entrainer and the supercritical CO₂The mixed fluid is throttled and expanded by a pressure reducing valve, then is discharged, collected and concentrated, and grease extract is obtained.

8. The method for fractionating spirulina bioactive substances according to claim 4, wherein the method for separating glycolipids from said extract in step S24 is: adsorbing with macroporous resin HP20, eluting with ethanol to remove polar ester and phospholipid, and enriching glycolipid; and then obtaining the glycolipid from the grease extract by silica gel column chromatography and an elution system.

Technical Field

The invention relates to the technical field of natural active ingredient extraction, in particular to a spirulina bioactive substance grading separation method.

Background

Spirulina (Spirulina) belongs to the cyanobacteria phylum, is a filamentous prokaryote formed by single cell or multi-cell aggregation, and is also called "Arthrospira". Currently, large-scale artificial culture of spirulina at home and abroad mainly includes spirulina platensis (Arthrospira platensis), spirulina maxima (Arthrospira maxima) and the like. The spirulina is not only rich in nutrition, but also rich in various substances with biological activity, mainly comprising phycocyanin, gamma-linolenic acid, polysaccharide and the like.

Phycocyanin is a scarce natural blue pigment in the market, and is widely used in the food industry in the European and American markets at present. Phycocyanin is also a macromolecule with various biological activities, has strong oxidation resistance, and therefore can effectively prevent and relieve a series of diseases caused by oxidation pressure, such as neurodegenerative diseases (Alzheimer disease, Parkinson disease and the like), diabetic nephropathy, cataract, cardiovascular diseases and the like. In addition, phycocyanin is gradually highlighted in the aspect of anti-tumor in recent years, and is expected to be developed into a natural anti-tumor health-care product and an auxiliary treatment medicine in the process of tumor treatment.

The gamma-linolenic acid in spirulina is combined with lactose to form glycolipid (mainly comprising monogalactosyldiacetyl glyceride and digalactosyldiacetylglyceride), and has the functions of both gamma-linolenic acid and glycolipid, so that the spirulina has wider application range and value. The gamma-linolenic acid is all cis-6, 9, 12-octadecatrienoic acid, and is omega-6 series polyunsaturated fatty acid. An important physiological activity of gamma-linolenic acid is that it is a precursor of the synthesis of prostaglandins by the human body. In addition, the gamma-linolenic acid has the effect of relieving diseases such as rheumatoid arthritis, eczema, colon syndrome, neuralgia and the like.

The content of polysaccharide in spirulina is about 15-20%, and research shows that the spirulina polysaccharide has obvious functions of resisting virus and regulating immunity of organism.

Bioactive peptides are specific protein fragments that have a positive effect on the function or state of the body and ultimately affect the health of the body. The spirulina protein is hydrolyzed, so that the solubility and the in vivo absorption and utilization rate of the spirulina protein can be improved, and the bioactive peptide with special physiological functions can be obtained. At present, various bioactive peptides such as antioxidant peptide, ACE inhibitory peptide, antibacterial peptide and antitumor peptide are successfully extracted from spirulina zymolyte.

Although many studies and reports have been made on the method of extracting a single active substance such as phycocyanin, allophycocyanin, chlorophyll, polysaccharide, etc. from spirulina, there is a lack of research on how to extract different active substances sequentially from spirulina in a stepwise manner, without or with little destruction of other components when extracting one substance, and to ensure extraction efficiency and avoid inactivation of the activity of functional substances as much as possible. The extraction methods are selected for combination, the sequence of extraction, how to ensure the extraction efficiency and the like, and no relevant technical report is disclosed at present.

Disclosure of Invention

Technical problem to be solved

In view of the above problems, it is desirable to provide a method for separating bioactive substances from spirulina, which can separate and extract various bioactive components from spirulina sufficiently, thereby maximizing the utilization value of spirulina and providing technical support for the development of spirulina products.

(II) technical scheme

In order to achieve the purpose, the invention adopts the main technical scheme that:

in a first aspect, an embodiment of the present invention provides a method for separating bioactive substances from spirulina, which sequentially comprises the following steps:

s1, extracting and purifying phycocyanin in the spirulina powder by taking the spirulina powder as a raw material, and changing the spirulina powder into first-level algae residue;

s2, extracting the grease in the primary algae residues as raw materials by a supercritical carbon dioxide extraction method or an organic reagent extraction method, and further purifying to obtain glycolipids, wherein the primary algae residues become secondary algae residues;

and S3, placing the secondary algae residue under a ventilation condition, subpackaging and storing after the organic reagent is fully volatilized, wherein the secondary algae residue contains abundant protein and polysaccharide as crude products of the protein and the polysaccharide.

According to the preferred embodiment of the present invention, step S1 includes:

s11, weighing and mixing the spirulina powder and a potassium phosphate buffer solution, wherein the concentration of potassium phosphate in the potassium phosphate buffer solution is 0.05-0.08M, the spirulina powder and the potassium phosphate buffer solution are uniformly mixed according to the mass gram and the volume milliliter of 1:10-14, and stirring or shaking extraction is carried out at the temperature of 30-42 ℃ for 1-5 hours;

s12, adding 2-4% calcium chloride solution by mass volume percent into the mixed solution, and continuing stirring or extracting for 1-3 hours by oscillation; the mass volume percentage is g/ml percentage;

s13, centrifuging and collecting supernatant;

s14, adding potassium phosphate buffer solution with potassium phosphate concentration of 0.05-0.08M, resuspending the precipitate, and continuing stirring or shaking to extract for 1-3 hours;

s15, centrifuging and collecting supernatant;

s16, repeating the steps S14-S15 at least once to fully extract supernatant rich in phycocyanin, and combining the supernatant of each time; combining the centrifugal precipitates to obtain first-grade algae residues;

s17, performing ultrafiltration purification and concentration on the combined supernatant;

s18, drying to obtain powder with high phycocyanin content, and measuring the content of phycocyanin in the powder.

Further, the S1 includes the following steps:

s11, weighing spirulina powder and 0.06M potassium phosphate buffer solution, mixing uniformly according to a ratio of g/ml to 1:11.5, and stirring or shaking for extraction at 40 ℃ for 1-5 hours;

the preparation method of the potassium phosphate buffer solution comprises the following steps: mixing 1M potassium dihydrogen phosphate solution and 1M potassium dihydrogen phosphate solution at a volume ratio of 45:55, diluting by 10 times, and adjusting pH to 7.0;

s12, adding 2.5 percent by mass volume of calcium chloride solution into the spirulina suspension, and continuing stirring or oscillating for 10-30 minutes;

s13, centrifuging for 10 minutes at 4500g, and collecting supernatant;

s14, adding 0.06M potassium phosphate buffer solution, resuspending the precipitate, and continuing stirring or extracting for 1 hour by oscillation;

s15, centrifuging for 10 minutes at 4500g, and collecting supernatant;

s16, repeating the steps S14-S15 once to fully extract the supernatant rich in phycocyanin; combining the supernatants of each time; combining the centrifugal precipitates to obtain first-grade algae residues;

s17, performing ultrafiltration purification and concentration on the combined supernatant; ultrafiltration is carried out by adopting a 50kDa filter membrane;

and S18, drying and carrying out phycocyanin content determination.

According to the preferred embodiment of the present invention, step S2 includes:

s21: taking first-class algae residues as raw materials, and uniformly mixing a porous material which is 0.25-1.0 time of the mass of the raw materials with the first-class algae residues for later use;

s22: subjecting the Spirulina raw material to supercritical CO₂Extraction, wherein the supercritical CO₂In the extraction process, an organic solvent is used as an entrainer, and the volume mass ratio of the entrainer to ml/g of raw materials is 0.5:1-3: 1;

s23, collecting an extract, wherein the extract is grease from first-order algae residue; secondary algae residue is generated after extraction;

s24: further isolated from the extract to obtain glycolipids.

According to a preferred embodiment of the present invention, in the step S21, the porous material is selected from the group consisting of: diatomite, macroporous resin and molecular sieve, preferably diatomite.

According to a preferred embodiment of the present invention, in the step S22, the organic solvent is selected from the group consisting of: lower alcohols such as methanol, ethanol, isopropanol, etc.; lower esters such as ethyl acetate, propyl acetate, butyl acetate, etc.; acetone; n-hexane; or mixtures of one or more of the foregoing; preferably selected from methanol, ethanol and ethyl acetate or mixtures thereof; more preferably ethanol; more preferably food grade ethanol. Compared with entrainers such as petroleum ether (with high boiling point and flammability and explosiveness) used in some supercritical carbon dioxide extraction processes, the solvent is selected as the entrainer, so that the subsequent extraction and solvent separation is facilitated, the solvent residue is reduced, and the product performance is improved.

According to the preferred embodiment of the present invention, the method of step S22 includes:

s221: according to the weight percentage of ethanol: adding entrainer into the raw material (0.5: 1-3:1(ml/g volume mass ratio)), mixing with the mixture obtained in step S21, loading into material bag, placing into extraction kettle, and installing the sealing end cap of the extraction kettle;

s222: opening the inlet and outlet valves of the extraction kettle, and using CO₂Purging the whole system pipeline to remove air;

s223: opening a switch of a refrigerator, precooling for 15-25 minutes, and setting the extraction conditions of the system through an equipment control panel, wherein the temperature of the extraction kettle is 30-50 ℃, the temperature of an outlet valve is 40-60 ℃, and the pressure is 4000-;

s224: after the temperature of the extraction kettle is stable, CO₂Inputting the mixture into an extraction kettle through a high-pressure pump;

s225: opening an outlet valve of the extraction kettle after ensuring the extraction retention time of 15-60 minutes, and mixing the entrainer and the supercritical CO₂The mixed fluid is throttled and expanded by a pressure reducing valve, then is discharged, collected and concentrated, and grease extract is obtained.

According to a preferred embodiment of the present invention, in the step S24, the method for separating glycolipid from the extract is: adsorbing with macroporous resin HP20, eluting with ethanol to remove polar ester and phospholipid, and enriching glycolipid; and then obtaining the glycolipid from the grease extract by silica gel column chromatography and an elution system.

(III) advantageous effects

The invention relates to a method for preparing spirulina by reacting main active substances in spirulina: the method for extracting phycocyanin, glycolipid and polysaccharide one by one has the advantages of highest extraction efficiency and lower energy consumption, and comprehensively considers the activity, extraction efficiency, difficulty in operation and energy consumption of the spirulina active substances. The invention can not only extract different active substances sequentially by stages and steps, but also ensure the extraction efficiency and avoid the problems of activity inactivation and the like of functional substances.

The method has the characteristics of high extraction rate of phycocyanin and grease, simple operation, low energy consumption, easy industrial amplification and the like, and the biological activity of the extracted substances is not easy to damage due to mild extraction and purification conditions, so the method is a set of technical process easy for industrial amplification. The technical scheme of the invention can maximize the utilization value of the spirulina and provide technical support for developing spirulina products.

Drawings

FIG. 1 shows secondary algal pomace obtained in example 1 of the present invention.

FIG. 2 shows secondary algal residues obtained in example 2 of the present invention.

FIG. 3 shows the relative content and purity of phycocyanin in the samples obtained at each step of example 1 of the present invention, which is the result of SDS-PAGE analysis of proteins (main band of 17-18kDa size is phycocyanin subunit, which shows the relative content and purity of phycocyanin in the samples obtained at each step in the examples).

Detailed Description

For the purpose of better explaining the present invention and to facilitate understanding, the present invention will be described in detail by way of specific embodiments with reference to the accompanying drawings.

The invention provides a spirulina bioactive substance grading separation method, which comprises S1-S3 which are sequentially carried out. Wherein S1, the spirulina powder is used as a raw material, phycocyanin in the spirulina powder is extracted and purified, and the spirulina powder becomes first-level algae residue; s2, extracting the grease in the primary algae residues as raw materials by a supercritical carbon dioxide extraction method or an organic reagent extraction method, and further purifying to obtain glycolipids, wherein the primary algae residues become secondary algae residues; and S3, placing the secondary algae residue under a ventilation condition, subpackaging and storing after the organic reagent is fully volatilized, wherein the secondary algae residue contains abundant protein and polysaccharide as crude products of the protein and the polysaccharide.

One of the main innovations of the invention is an extraction sequence, namely, the phycocyanin in the spirulina is extracted firstly, then the gamma-linolenic acid is extracted, and finally the extraction sequence of the algae residue rich in protein and polysaccharide is obtained; the second innovation lies in the specific technical method for obtaining these substances.

In order to better understand the above technical solutions, exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings.