CN111732671A - Preparation method of spirulina polysaccharide - Google Patents
Preparation method of spirulina polysaccharide Download PDFInfo
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- CN111732671A CN111732671A CN202010693023.0A CN202010693023A CN111732671A CN 111732671 A CN111732671 A CN 111732671A CN 202010693023 A CN202010693023 A CN 202010693023A CN 111732671 A CN111732671 A CN 111732671A
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- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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Abstract
The invention discloses a preparation method of spirulina polysaccharide, which comprises the following specific steps: the preparation of raw materials, the extraction of mixture, the separation of extract, cooling, purification and precipitation, the washing of precipitate, solid-liquid separation, crushing and sieving, the invention avoids the damage of spirulina to the polysaccharide structure in the high-temperature drying process, improves the yield and quality of polysaccharide, reduces the alcohol dosage required by decolorization, saves cost, has higher practicability, increases the extraction amount of spirulina polysaccharide, has the functions of resisting tumor and radiation, promoting DNA synthesis and enhancing immunity when being used as auxiliary treatment drugs on the market, improves the yield and quality of polysaccharide, reduces the alcohol dosage required by decolorization, the production cost can be greatly reduced, the quality is improved, the fresh spirulina is used as the raw material to prepare the polysaccharide, any chemical reagent is not required to be added for processing, the chemical residue is avoided, and the experimental result is closer to the required result.
Description
Technical Field
The invention relates to the technical field of spirulina polysaccharide, in particular to a preparation method of spirulina polysaccharide.
Background
Spirulina belongs to Cyanophyta, Cyanophyceae, Oscillatoriaceae, Spirulina, is an ancient low-grade prokaryotic unicellular or multicellular aquatic plant shaped like clockwork spring, and is in spiral blue-green color, so the algae is also called blue-green algae, the surface of the algae body does not have a colloid sheath, the algae body is not easy to be attached by microorganism, air bubbles exist in cells, the floating property is good, the suitable growth temperature is generally 28-35 ℃, the minimum and maximum growth temperatures are respectively at 15 ℃ and 40 ℃, the thermophilic and heat-resistant strain can be cultured at 35-40 ℃, the spirulina has positive significance for preventing and treating anemia, one gram of the spirulina is equal to the sum of 1 kilogram of vitamins and mineral substances in various vegetables and fruits, the spirulina contains a large amount of linolenic acid, it is an unsaturated fatty acid essential for human body, and is an ideal substance for nourishing brain, improving intelligence, removing blood lipid, regulating blood pressure, and reducing cholesterol.
The spirulina polysaccharide in the spirulina has the functions of resisting radiation damage and improving side effects caused by radiotherapy and chemotherapy, so that the spirulina polysaccharide is a good food therapy product for tumor patients, the content of chlorophyll in the spirulina is very rich and is more than 10 times of that of common vegetables, the spirulina polysaccharide has positive effects of promoting human digestion, neutralizing toxin in blood, improving allergic constitution, eliminating visceral inflammation and the like, the content of fat in the spirulina is only 5%, cholesterol is not contained, and excessive heat can be prevented from being taken by a human body when necessary protein is supplemented.
The existing preparation method of spirulina polysaccharide has the defects that the spirulina polysaccharide cannot be really applied and produced industrially because the extraction of the spirulina polysaccharide is long in time consumption, high in cost, easy to damage the polysaccharide activity and the like at present, the spirulina cannot be self-sufficient during the experiment due to the technology of reversely culturing the spirulina in laboratories of most scientific research and teaching units, the large area and the places and researches of culture equipment, the further research of the spirulina is limited, and the experiment result of extracting the polysaccharide by using spirulina powder as a raw material on the market mainly reflects the defects in the aspects of the wall breaking, the solid-liquid ratio, the extraction temperature, the extraction time, the alcohol precipitation decoloration and the like of spirulina cells.
Therefore, it is necessary to develop a method for preparing spirulina polysaccharides to solve the above problems.
Disclosure of Invention
In order to overcome the above defects in the prior art, the embodiment of the present invention provides a preparation method of spirulina polysaccharide, which comprises the following specific steps:
s1: preparation of raw materials: 100-300 parts of spirulina, 10-20 parts of water, 100-300 parts of dimethyl sulfoxide, 50-150 parts of papain, 80-100 parts of trypsin, 0.2-0.3 part of powdery active polypeptide, 10 parts of TCA solution and 50-200 parts of activated carbon, wherein the required auxiliary materials comprise the following components in percentage by weight: 8-10 parts of 95% ethanol, 10-20 parts of 95% phenolic aldehyde and 5-20 parts of 95% acetone.
S2: extraction of the mixture: placing the processed and prepared spirulina mud into a stainless steel heating pot, injecting 100 portions of dimethyl sulfoxide and heating, dissolving spirulina polysaccharide in the dimethyl sulfoxide, heating to boil, and obtaining a pre-immersion liquid, wherein the dosage of the extraction liquid is three times that of spirulina;
s3: and (3) separating the extract: injecting enzyme such as papain and trypsin into the extracted mixture for treatment, further purifying by chromatography, concentrating the mixed solution as much as possible to reduce the volume, introducing one end of a stationary phase, eluting by a solvent, adsorbing different solutes in a mobile phase by using active molecules or active groups on the surface of an adsorption chromatography medium, and separating by using the strength of the adsorption capacity of the active molecules or active groups on the surface of the adsorption chromatography medium;
s4: and (3) cooling: putting the liquid after further purification by chromatography into a refrigerating chamber for cooling treatment;
s5: purifying and precipitating: putting the cooled concentrated solution into a stainless steel barrel, precipitating with an organic solvent, adding 8-10 parts of ethanol, 5-20 parts of acetone and 10-20 parts of phenolic aldehyde into the stainless steel barrel, precipitating polysaccharide in the stainless steel barrel when the pH value of the internal liquid is 7.0, and taking out the precipitate after precipitation is finished;
s6: washing the precipitate: washing the precipitate with ethanol;
s7: solid-liquid separation: adding powder active polypeptide, dispersing water in the washed precipitate into fog drops by using an atomizer, drying the fog drops by using hot air, and evaporating the water to obtain granular polysaccharide substances;
s8: crushing and sieving: and finally, crushing and sieving the dried polysaccharide.
Preferably, the leaching process in S2 is as follows: adding 100 parts of dimethyl sulfoxide 300 parts into the extract, and leaching at 67-74 deg.C for 3-4h to obtain extractive solution.
Preferably, the stationary phase in S3 is a solid adsorbent with adsorption activity, and is made of silica gel and alumina, so that the concentrated solvent contracts or expands.
Preferably, the solvent in S3 elutes: the elution solvent is ethanol and acetone.
Preferably, the enzyme injection treatment of S3 can remove proteins, and the ratio of enzyme additive to raw material is set to 3: 2.
Preferably, the amount of ethanol added in S5 is four times the volume of the solution after concentration.
Preferably, the number of washing in S6 is 2 to 3.
The invention has the technical effects and advantages that:
through the experimental process flow, the energy consumption is reduced, the cost is saved, the practicability is higher, the extraction amount of the spirulina polysaccharide is increased, the polysaccharide is extracted to be used as an auxiliary treatment medicine on the market, the polysaccharide has the functions of resisting tumor and radiation, promoting DNA synthesis and enhancing immunity, compared with the method for extracting the polysaccharide by using the spirulina powder as a raw material on the market, the method avoids the damage of the spirulina powder to the polysaccharide structure in the high-temperature drying process and avoids the phenomenon of scorching, the common vacuum drying method has the advantages of low heat transfer speed, large energy consumption, low efficiency and long drying time, the principle of the spray drying method is that the dried liquid material is dispersed into a plurality of fine liquid drops through an atomizer and enters flowing hot air flow, the drying speed is extremely high due to the extremely large total surface area, the moisture evaporation is completed within a few seconds, and the method has the characteristic of instant drying, the method has the advantages that the yield and the quality of the polysaccharide are improved, the alcohol consumption required by decolorization is reduced, the production cost can be greatly reduced, and the quality can be improved.
Detailed Description
The following will clearly and completely describe the technical solutions in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
the invention provides a preparation method of spirulina polysaccharide, which comprises the following steps:
s1: preparation of raw materials: 100 parts of spirulina, 10 parts of water, 100 parts of dimethyl sulfoxide, 50 parts of papain, 80 parts of trypsin and 0.2 part of powder active polypeptide, wherein the required auxiliary materials comprise the following components in percentage by weight: 8 parts of 95% ethanol, 10 parts of 95% phenolic aldehyde and 5 parts of 95% acetone.
S2: extraction of the mixture: placing the processed and prepared spirulina mud into a stainless steel heating pot, injecting 100 parts of dimethyl sulfoxide, heating, dissolving spirulina polysaccharide in dimethyl sulfoxide, heating to boil, and extracting for 3-4h, wherein the amount of the extracting solution is three times that of spirulina to obtain a pre-leaching solution;
s3: and (3) separating the extract: injecting 50 parts of papain, 80 parts of trypsin and other enzymes into the extracted mixture for treatment, further purifying by chromatography, concentrating the mixed solution as much as possible to reduce the volume, introducing one end of a stationary phase, eluting by 2 parts of ethanol solvent, generating adsorption action on different solutes in a mobile phase by utilizing active molecules or active groups on the surface of an adsorption chromatography medium, and separating by utilizing the strength of the adsorption capacity of the adsorption chromatography medium on the different solutes;
s4: and (3) cooling: putting the liquid after further purification by chromatography into a refrigerating chamber for cooling treatment;
s5: purifying and precipitating: putting the cooled concentrated solution into a stainless steel barrel, precipitating with an organic solvent, adding 4 parts of ethanol, 5 parts of acetone and 10 parts of phenolic aldehyde into the stainless steel barrel, precipitating the polysaccharide in the stainless steel barrel when the pH value of the internal liquid is 7.0, and taking out the precipitate after precipitation is finished;
s6: washing the precipitate: washing the precipitate with 2 parts of ethanol;
s7: solid-liquid separation: adding 0.2 part of powder active polypeptide, dispersing water in the washed precipitate into fog drops by using an atomizer, drying the fog drops by using hot air, and evaporating the water to obtain a granular polysaccharide substance;
s8: crushing and sieving: and finally, crushing and sieving the dried polysaccharide.
The spirulina polysaccharide prepared in the embodiment has uniform output particles, so that the extraction amount of the spirulina polysaccharide is increased, in addition, the spirulina polysaccharide prepared in the embodiment is used for the auxiliary treatment medicines on the market, and has the effects of resisting tumors and radiation, promoting DNA synthesis and enhancing immunity, in the experimental case, the extracting solution in the spirulina polysaccharide is 75kg, the supernatant is 35.6kg, the concentrated solution is 7.1kg, the using amount of ethanol is 8 parts, and the yield of the polysaccharide is 236 g, so that the production cost is reduced.
Example 2:
the invention provides a preparation method of spirulina polysaccharide, which comprises the following steps:
s1: preparation of raw materials: 200 parts of spirulina, 15 parts of water, 200 parts of dimethyl sulfoxide, 100 parts of papain, 90 parts of trypsin and 0.25 part of powder active polypeptide, wherein the required auxiliary materials comprise the following components in percentage by weight: 9 parts of 95% ethanol, 15 parts of 95% phenolic aldehyde and 10 parts of 95% acetone.
S2: extraction of the mixture: placing the processed and prepared spirulina mud into a stainless steel heating pot, injecting 200 parts of dimethyl sulfoxide, heating, dissolving spirulina polysaccharide in dimethyl sulfoxide, heating to boil, and extracting for 3-4h, wherein the amount of the extracting solution is three times that of spirulina to obtain a pre-leaching solution;
s3: and (3) separating the extract: injecting the extracted mixture into enzymes such as 100 parts of papain, 90 parts of trypsin and the like for treatment, further purifying by chromatography, concentrating the mixed solution as much as possible to reduce the volume, introducing one end of a stationary phase, then eluting by 2 parts of ethanol solvent, generating adsorption action on different solutes in a mobile phase by utilizing active molecules or active groups on the surface of an adsorption chromatography medium, and separating by utilizing the strength of the adsorption capacity of the adsorption chromatography medium on the different solutes;
s4: and (3) cooling: putting the liquid after further purification by chromatography into a refrigerating chamber for cooling treatment;
s5: purifying and precipitating: putting the cooled concentrated solution into a stainless steel barrel, precipitating with an organic solvent, adding 5 parts of ethanol, 10 parts of acetone and 15 parts of phenol formaldehyde into the stainless steel barrel, precipitating the polysaccharide in the stainless steel barrel when the pH value of the internal liquid is 7.0, and taking out the precipitate after precipitation is finished;
s6: washing the precipitate: washing the precipitate with 2 parts of ethanol;
s7: solid-liquid separation: adding 0.25 part of powder active polypeptide, dispersing water in the washed precipitate into fog drops by using an atomizer, drying the fog drops by using hot air, and evaporating the water to obtain a granular polysaccharide substance;
s8: crushing and sieving: and finally, crushing and sieving the dried polysaccharide.
The spirulina polysaccharide prepared in the embodiment has uniform output particles, so that the extraction amount of the spirulina polysaccharide is increased, in addition, the spirulina polysaccharide prepared in the embodiment is used for the auxiliary treatment medicines on the market, and has the effects of resisting tumors and radiation, promoting DNA synthesis and enhancing immunity, in the experimental case, the extracting solution in the spirulina polysaccharide is 90kg, the supernatant is 50kg, the concentrated solution is 8.1kg, the using amount of ethanol is 10 parts, and the gram of polysaccharide is 300 g, so that the output amount and the quality of the polysaccharide are improved, and the production cost is reduced.
Comparative example 1, this implementation is through with taking the algae mud to compare as raw and other materials, and this implementation case obviously knows, needs to consume a large amount of energy, and the content that the polysaccharide obtained is less, needs to increase the stoving process of algae powder, can't practice thrift the cost, and spirulina algae powder can cause the destruction to the polysaccharide structure at high temperature stoving in-process, takes place the phenomenon of scorching easily.
Experimental example 3:
the invention provides a preparation method of spirulina polysaccharide, which comprises the following steps:
s1: preparation of raw materials: 300 parts of spirulina, 20 parts of water, 300 parts of dimethyl sulfoxide, 150 parts of papain, 100 parts of trypsin and 0.3 part of powder active polypeptide, wherein the required auxiliary materials comprise the following components in percentage by weight: 10 parts of 95% ethanol, 20 parts of 95% phenolic aldehyde and 20 parts of 95% acetone;
s2: extraction of the mixture: placing the processed and prepared spirulina mud into a stainless steel heating pot, injecting 300 parts of dimethyl sulfoxide, heating, dissolving spirulina polysaccharide in dimethyl sulfoxide, heating to boil, and extracting for 3-4h, wherein the amount of the extracting solution is three times that of spirulina to obtain a pre-leaching solution;
s3: and (3) separating the extract: injecting the extracted mixture into enzyme such as 150 parts of papain, 100 parts of trypsin and the like for treatment, further purifying by chromatography, concentrating the mixed solution as much as possible to reduce the volume, introducing one end of a stationary phase, then eluting by 3 parts of ethanol solvent, generating adsorption action on different solutes in a mobile phase by utilizing active molecules or active groups on the surface of an adsorption chromatography medium, and separating by utilizing the strength of the adsorption capacity of the adsorption chromatography medium on the different solutes;
s4: and (3) cooling: putting the liquid after further purification by chromatography into a refrigerating chamber for cooling treatment;
s5: purifying and precipitating: putting the cooled concentrated solution into a stainless steel barrel, precipitating with an organic solvent, adding 3 parts of ethanol, 20 parts of acetone and 20 parts of phenol formaldehyde into the stainless steel barrel, precipitating the polysaccharide when the pH value of the internal liquid is 7.0, and taking out the precipitate after precipitation;
s6: washing the precipitate: washing the precipitate with 4 parts of ethanol;
s7: solid-liquid separation: adding 0.3 part of powder active polypeptide, dispersing water in the washed precipitate into fog drops by using an atomizer, drying the fog drops by using hot air, and evaporating the water to obtain a granular polysaccharide substance;
s8: crushing and sieving: and finally, crushing and sieving the dried polysaccharide.
The spirulina polysaccharide prepared in the embodiment has uniform output particles, so that the extraction amount of the spirulina polysaccharide is increased, in addition, the spirulina polysaccharide prepared in the embodiment is used for the auxiliary treatment medicines on the market, and has the effects of resisting tumors and radiation, promoting DNA synthesis and enhancing immunity, the experimental case is that the extracting solution in the spirulina polysaccharide is 80kg, the supernatant is 40kg, the concentrated solution is 7.5kg, the using amount of ethanol is 9 parts, and the yield of the polysaccharide is 250 g, so that the output and the quality of the polysaccharide are improved, and the production cost is reduced.
The following table is obtained according to examples 1 to 3:
as can be seen from the above table, the raw material proportion of the example 2 is moderate, the processing temperature is moderate, the spirulina polysaccharide content prepared by the process is the highest, the experimental process can reduce the equipment addition in the spirulina drying process, reduce the energy consumption, save the cost and have higher practicability, compared with the method for extracting polysaccharide from spirulina powder in the market, the method avoids the damage of the spirulina powder to the polysaccharide structure in the high-temperature drying process, avoids the phenomenon of scorching, improves the yield and quality of the polysaccharide, reduces the alcohol dosage required by decoloration, the invention can greatly reduce the production cost and improve the quality, and the process of preparing the polysaccharide by taking the fresh spirulina as the raw material does not need to add any chemical reagent for treatment, thereby avoiding chemical residue and leading the experimental result to be closer to the required result.
Finally, it should be noted that: the above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that are within the spirit and principle of the present invention are intended to be included in the scope of the present invention.
Claims (7)
1. A preparation method of spirulina polysaccharide is characterized in that: the preparation steps are as follows:
s1: preparation of raw materials: 100-300 parts of spirulina, 10-20 parts of water, 100-300 parts of dimethyl sulfoxide, 50-150 parts of papain, 80-100 parts of trypsin, 0.2-0.3 part of powdery active polypeptide, 10 parts of TCA solution and 50-200 parts of activated carbon, wherein the required auxiliary materials comprise the following components in percentage by weight: 8-10 parts of 95% ethanol, 10-20 parts of 95% phenolic aldehyde and 5-20 parts of 95% acetone.
S2: extraction of the mixture: placing the processed and prepared spirulina mud into a stainless steel heating pot, injecting 100 portions of dimethyl sulfoxide and heating, dissolving spirulina polysaccharide in the dimethyl sulfoxide, heating to boil, and obtaining a pre-immersion liquid, wherein the dosage of the extraction liquid is three times that of spirulina;
s3: and (3) separating the extract: injecting enzyme such as papain and trypsin into the extracted mixture for treatment, further purifying by chromatography, concentrating the mixed solution as much as possible to reduce the volume, introducing one end of a stationary phase, eluting by a solvent, adsorbing different solutes in a mobile phase by using active molecules or active groups on the surface of an adsorption chromatography medium, and separating by using the strength of the adsorption capacity of the active molecules or active groups on the surface of the adsorption chromatography medium;
s4: and (3) cooling: putting the liquid after further purification by chromatography into a refrigerating chamber for cooling treatment;
s5: purifying and precipitating: putting the cooled concentrated solution into a stainless steel barrel, precipitating with an organic solvent, adding 8-10 parts of ethanol, 5-20 parts of acetone and 10-20 parts of phenolic aldehyde into the stainless steel barrel, precipitating polysaccharide in the stainless steel barrel when the pH value of the internal liquid is 7.0, and taking out the precipitate after precipitation is finished;
s6: washing the precipitate: washing the precipitate with ethanol;
s7: solid-liquid separation: adding powder active polypeptide, dispersing water in the washed precipitate into fog drops by using an atomizer, drying the fog drops by using hot air, and evaporating the water to obtain granular polysaccharide substances;
s8: crushing and sieving: and finally, crushing and sieving the dried polysaccharide.
2. The method for preparing spirulina polysaccharide as claimed in claim 1, wherein: the leaching process in the S2 comprises the following steps: adding 100 parts of dimethyl sulfoxide 300 parts into the extract, and leaching at 67-74 deg.C for 3-4h to obtain extractive solution.
3. The method for preparing spirulina polysaccharide as claimed in claim 1, wherein: the stationary phase in the S3 is a solid adsorbent with adsorption activity, is made of silica gel and alumina, and enables the concentrated solvent to shrink or expand.
4. The method for preparing spirulina polysaccharide as claimed in claim 1, wherein: solvent elution in S3: the elution solvent is ethanol and acetone.
5. The method for preparing spirulina polysaccharide as claimed in claim 1, wherein: the enzyme injection treatment of S3 removed protein, and the ratio of enzyme additive to raw material was set at 3: 2.
6. The method for preparing spirulina polysaccharide as claimed in claim 1, wherein: the amount of ethanol added in S5 was four times the volume of the solution after concentration.
7. The method for preparing spirulina polysaccharide as claimed in claim 1, wherein: the number of washing in S6 was 2 to 3.
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2020
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