CN108676106B - Method for extracting polysaccharide and betaine from seaweed - Google Patents
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Abstract
The invention discloses a method for extracting polysaccharide and betaine from seaweed, which comprises the following steps: s1, weighing the dried and crushed seaweed and water in a glass container, wherein the mass ratio of the seaweed to the water is 1: (30-50); s2, extracting the glass container weighed in the step S1 for 60-90S by microwave with the power of 210-490W to obtain an extracting solution, and filtering to remove solids in the extracting solution to obtain primary clear liquid; s3, concentrating the primary clear liquid to one third of the original volume, adding excessive ethanol, keeping the ethanol concentration more than or equal to 80%, standing for 10h, filtering to obtain polysaccharide and secondary extracting solution, and drying the polysaccharide; s4, adjusting the pH value of the secondary extracting solution to 3-7, and adsorbing the secondary extracting solution by a resin column filled with 732 cation exchange resin until the 732 cation exchange resin is saturated in the adsorption of betaine; s5, eluting 732 cation exchange resin after saturated adsorption by 1% -9% ammonia water to obtain eluent, and concentrating and drying the eluent.
Description
Technical Field
The invention relates to the field of food, beverage and plant stimulant, in particular to a method for extracting polysaccharide and betaine from seaweed.
Background
The "seaweed" is a generic name of marine algae such as kelp, laver, undaria pinnatifida, gelidium amansii, etc., which is an algae growing in the sea. The large-scale seaweed is divided into two categories of large-scale seaweed and microalgae, wherein the large-scale seaweed is various in types and rich in forms, comprises four categories of brown algae, red algae, blue algae, green algae and the like, is a cryptogamic plant in the plant kingdom, and the algae comprises a large number of different types of organisms which generate energy through photosynthesis. They are generally considered to be simple, and are mostly cellular filamentous, membranous, tubular or frond plants, i.e. vascular bundle-free tissues, without differentiation phenomena of true roots, stems, leaves; no flowering, no fruit and no seed; the apomictic embryo has the requirement that chlorophyll can synthesize organic matters through photosynthesis to survive like higher plants.
The large-scale seaweed bioactive substances comprise two major types, one is a small molecular substance which can be digested and absorbed, and specifically comprises halogen compounds, seaweed tannin, terpenoid compounds and the like; the other is viscous polysaccharide which is difficult to digest, such as agar, carrageenan, fucoidan, sulfated polysaccharide, etc. in red algae. Macro-seaweed polysaccharide (polysaccharide for short) is a chain polymer formed by connecting a plurality of same or different monosaccharide groups through glycosidic bonds, exists in seaweed cell walls and interstitium, accounts for more than 50% of the dry weight of seaweed, and has various biological activities and medicinal values of immunoregulation, tumor resistance, virus resistance, oxidation resistance and the like.
The seaweed also contains betaine, which is a general name of a series of substances, and all substances with R- (CH3)3N + CH2 COO-structures belong to betaine. Due to the presence of trimethyl, betaine, a quaternary ammonium salt, has obvious alkali-containing characteristics. Betaine is also a typical surfactant. The betaines found in seaweed include glycine betaine (the simplest betaine in structure, called as "trimethylamine B lactone" or "trimethylglycine"), Y-aminobutyric acid betaine, alanine betaine, homoserine betaine (derived from pure ammonia acid) which is an ether formed from homoserine and glycerol, and homoserine betaine derivatives extracted from seaweed such as 1(3), 2-diacylglycerol-3 (1) -0-4- (N, N, N-trimethyl) homoserine, delta-aminopentanoic acid betaine, N6-trimethyllysine (i.e., laminine which is not only a quaternary amine derivative but also an alpha-amino acid capable of forming a peptide bond), lysine betaine (in which both nitrogen atoms of lysine are completely methylated), and L-lysine betaine, Proline betaine (also known as stachydrine, proline dimethyl inner salt), cis-4-hydroxyproline betaine, trans-4-hydroxy-beta-proline betaine, 1, 3-dimethylhistidine, picolinic acid betaine, trigonelline (N-methylnicotinate), lobster sarcosine, N-dimethyl-1, 2,3, 6-tetrahydropyridine-2-hydroxy salt (piscine betaine).
Betaine is white scaly or prismatic crystalline powder with slight characteristic odor (sweet taste), molecular weight of 117.15, high temperature resistance, melting point of 293 deg.C, and can be decomposed when the temperature reaches 310 deg.C. The solubility (20 ℃) is 160g/100g of water, and the solvent is very easy to dissolve in polar solvents such as water, methanol and the like, is dissolved in ethanol and is slightly soluble in ether. Betaine exists in different forms in solution according to different pH, and generally molecules are in an open ring state when the pH is less than 7 and are in a ring structure when the pH is more than or equal to 7. It is easy to absorb moisture and deliquesce at normal temperature, and has strong moisture retention, stable property and strong oxidation resistance.
There is no prior art method for extracting polysaccharides and betaines from seaweeds.
The invention aims to provide a method for extracting polysaccharide and betaine from seaweed.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for extracting polysaccharides and betaines from seaweeds.
A method for extracting polysaccharide and betaine from seaweed comprises the following steps:
s1, weighing the dried and crushed seaweed and water in a glass container, wherein the mass ratio of the seaweed to the water is 1: (30-50);
s2, extracting the glass container weighed in the step S1 for 60-90S by microwave with the power of 210-490W to obtain an extracting solution, and filtering to remove solids in the extracting solution to obtain primary clear liquid;
s3, concentrating the primary clear liquid to one third of the original volume, adding excessive ethanol, keeping the ethanol concentration more than or equal to 80%, standing for 10h, filtering to obtain polysaccharide and secondary extracting solution, and drying the polysaccharide;
s4, adjusting the pH value of the secondary extracting solution to 3-7, and adsorbing the secondary extracting solution by a resin column filled with 732 cation exchange resin until the 732 cation exchange resin is saturated in the adsorption of betaine;
s5, eluting the 732 cation exchange resin after saturated adsorption by using 1% -9% ammonia water to obtain eluent, and concentrating and drying the eluent to obtain the betaine.
By a microwave water extraction method, polysaccharide and betaine in seaweed are transferred into primary clear liquid by combining the proportion of the seaweed to water, microwave extraction power and microwave extraction time, the primary separation of the polysaccharide and the betaine is realized by utilizing the solubility difference of the betaine and the polysaccharide in water and ethanol respectively, a secondary solution rich in the betaine is adsorbed by 732 cation exchange resin within a specific pH value range, the adsorption rate of the 732 cation exchange resin to the betaine is higher, and finally, ammonia water solution is adopted for elution, so that the further purification of the betaine is completed.
The 732 cation exchange resin is saturated with betaine, which means that the 732 cation exchange resin does not adsorb betaine in the secondary extracting solution any more, namely the concentration of betaine in the secondary extracting solution passing through the resin column filled with 732 cation exchange resin does not change any more.
Preferably, the ratio of the mass of seaweed to the mass of water is 1: 40.
the ratio of the mass of seaweed to the mass of water is 1: at 40 hours, the mass transfer effect in the extraction process is good, and the extraction rate of polysaccharide and betaine by water is high.
Preferably, the microwave extraction power of step S2 is 350W.
Experiments show that after the microwave extraction power exceeds 350W, the polysaccharide content in the primary clear liquid is reduced, and the polysaccharide is decomposed after the microwave extraction power exceeds 350W; therefore, the microwave power is 350W, the higher extraction rate of the polysaccharide by water is ensured, and the loss of the polysaccharide due to decomposition is avoided.
Preferably, step S2 microwave extraction is performed for 75S.
The 350W microwave extraction time is too long, and the polysaccharide is easy to decompose.
Preferably, the pH of step S4 is adjusted to 3.
When the pH of the secondary extract is 3, betaine is in an open-loop state, and a large amount of hydrogen ions are provided in the secondary extract to ionize betaine and exchange the betaine with the 732 cation exchange resin sulfonic acid group for adsorption, at which time the 732 cation exchange resin has an adsorption rate of betaine as high as 94%.
Preferably, step S5 is performed with 5% ammonia water.
5% ammonia water is adopted for elution, the elution rate is up to 75%, under the alkaline condition, hydroxide ions and hydrogen ions on adsorbed betaine cations are neutralized to generate water, so that electrically neutral betaine molecules are eluted from 732 cation exchange resins, meanwhile, the method that acidic solution is adopted for cation exchange elution in the prior art is avoided, and the problem that betaine hydrochloride is generated by the reaction of betaine and Raney's salt in the subsequent detection of the purity of the betaine, so that the accuracy of a test result is influenced is avoided.
Preferably, after the secondary clear liquid is obtained in step S3, impurities are removed by using 717 anion resin.
The experiment finds that the adsorption rate of 717 anions to betaine is lower than 6.5%, the adsorption rate to other substances in the secondary extracting solution is higher, the secondary clear solution is firstly adsorbed by 717 anion resin, the content of impurities in the secondary clear solution is reduced, the impurities are prevented from being influenced in the process of adsorbing and eluting betaine by 732 cation exchange resin, and the final purity of betaine is improved.
Compared with the prior art, the invention has the following advantages and beneficial effects:
polysaccharide and betaine in the seaweed are transferred into primary clear liquid by a microwave water extraction method in combination with proper proportion of the seaweed to water, microwave extraction power and microwave extraction time, primary separation of the polysaccharide and the betaine is realized by utilizing the solubility difference of the betaine and the polysaccharide in water and ethanol respectively, a secondary solution rich in the betaine is adsorbed by 732 cation exchange resin within a specific pH value range, the adsorption rate of the 732 cation exchange resin to the betaine is high, and finally, ammonia water solution is adopted for elution, so that further extraction and purification of the betaine are completed.
Detailed Description
The technical solution of the present invention will be further described with reference to the following examples.
Example 1
This example provides a method for extracting polysaccharides and betaines from seaweeds comprising the steps of:
s1, weighing the dried and crushed seaweed and water in a glass container, wherein the mass ratio of the seaweed to the water is 1: 30, of a nitrogen-containing gas;
s2, placing the glass container weighed in the step S1 in a microwave with the power of 210W for extraction for 60S to obtain an extracting solution, and filtering to remove solids in the extracting solution to obtain primary clear liquid;
s3, concentrating the primary clear liquid to one third of the original volume, adding excessive ethanol, keeping the ethanol concentration more than or equal to 80%, standing for 10h, filtering to obtain polysaccharide and secondary extracting solution, and drying the polysaccharide;
s4, adjusting the pH value of the secondary extracting solution to 5, and adsorbing the secondary extracting solution by using a resin column filled with 732 cation exchange resin until the 732 cation exchange resin is saturated in the adsorption of the betaine;
and S5, eluting the 732 cation exchange resin after saturated adsorption by using 1% ammonia water to obtain an eluent, and concentrating and drying the eluent to obtain the betaine.
Example 2
This example provides a method for extracting polysaccharides and betaines from seaweeds comprising the steps of:
s1, weighing the dried and crushed seaweed and water in a glass container, wherein the mass ratio of the seaweed to the water is 1: 40;
s2, placing the glass container weighed in the step S1 in a microwave with the power of 350W for 75S to obtain an extracting solution, and filtering to remove solids in the extracting solution to obtain primary clear liquid;
s3, concentrating the primary clear liquid to one third of the original volume, adding excessive ethanol, keeping the ethanol concentration more than or equal to 80%, standing for 10h, filtering to obtain polysaccharide and secondary extracting solution, and drying the polysaccharide;
s4, adjusting the pH value of the secondary extracting solution to 3, and adsorbing the secondary extracting solution by using a resin column filled with 732 cation exchange resin until the 732 cation exchange resin is saturated in the adsorption of the betaine;
and S5, eluting the 732 cation exchange resin after saturated adsorption by using 5% ammonia water to obtain an eluent, and concentrating and drying the eluent to obtain the betaine.
Example 3
This example provides a method for extracting polysaccharides and betaines from seaweeds comprising the steps of:
s1, weighing the dried and crushed seaweed and water in a glass container, wherein the mass ratio of the seaweed to the water is 1: 50;
s2, extracting the glass container weighed in the step S1 for 90S by microwave with the power of 490W to obtain an extracting solution, and filtering to remove solids in the extracting solution to obtain primary clear liquid;
s3, concentrating the primary clear liquid to one third of the original volume, adding excessive ethanol, keeping the ethanol concentration more than or equal to 80%, standing for 10h, filtering to obtain polysaccharide and secondary extracting solution, and drying the polysaccharide;
s4, adjusting the pH value of the secondary extracting solution to 7, and adsorbing the secondary extracting solution by using a resin column filled with 732 cation exchange resin until the 732 cation exchange resin is saturated in the adsorption of the betaine;
and S5, eluting the 732 cation exchange resin after saturated adsorption by using 9% ammonia water to obtain an eluent, and concentrating and drying the eluent to obtain the betaine.
Example 4
This example provides a method for extracting polysaccharides and betaines from seaweeds comprising the steps of:
s1, weighing the dried and crushed seaweed and water in a glass container, wherein the mass ratio of the seaweed to the water is 1: 40;
s2, placing the glass container weighed in the step S1 in a microwave with the power of 350W for 75S to obtain an extracting solution, and filtering to remove solids in the extracting solution to obtain primary clear liquid;
s3, concentrating the primary clear liquid to one third of the original volume, adding excessive ethanol, keeping the ethanol concentration more than or equal to 80%, standing for 10h, filtering to obtain polysaccharide and secondary extracting solution, and drying the polysaccharide;
s4, adjusting the pH value of the secondary extracting solution to 3, and adsorbing the secondary extracting solution by using a resin column filled with 732 cation exchange resin until the 732 cation exchange resin is saturated in the adsorption of the betaine;
and S5, eluting the 732 cation exchange resin after saturated adsorption by using 5% ammonia water to obtain an eluent, and concentrating and drying the eluent to obtain the betaine.
After the secondary clear liquid is obtained in step S3, impurities are removed by 717 anion resin.
The polysaccharide content and the betaine content in the extraction processes of examples 1 to 4 were measured, respectively, and the results are shown in table 1.
TABLE 1
Wherein, the content determination of the polysaccharide:
(1) glucose-phenol sulfuric acid standard curve
Taking six test tubes, respectively sucking 0mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL and 1.0mL of prepared 0.1mg/mL standard glucose solution into a 25mL colorimetric tube, supplementing the colorimetric tube with distilled water to 1.0mL, adding 1.0mL of 5% phenol into the test solution, quickly adding 5.0mL of concentrated sulfuric acid (which is added vertically to the liquid level and does not contact with the wall of the test tube so as to be fully mixed with the reaction solution), standing for 10 minutes, shaking uniformly, placing the colorimetric tube in a water bath at 30 ℃ for reaction for 20 minutes, measuring the absorbance (OD value) at 490nm, and establishing a standard curve by taking the mass concentration of glucose as an abscissa and the absorbance as an ordinate.
(2) Determination of polysaccharide content
Weighing 20mg of a sample, dissolving and fixing the volume to a 100mL volumetric flask, sucking 0.5mL of sample liquid, adding distilled water to supplement the distilled water to 1.0mL, then adding 1.0mL of 5% phenol and 5mL of concentrated sulfuric acid, standing for 10 minutes, shaking up, placing a colorimetric tube in a water bath at 30 ℃ for reaction for 20 minutes, measuring an absorbance value (OD value) at 490nm, and calculating the total sugar content according to a standard curve equation.
The polysaccharide content in the sample, expressed in grams per hundred grams (g/100g), as mass fraction ω, is then:
In equation 1: m is1Checking the sugar content of the sample determination solution μ g from the standard curve;
V1- -sample constant volume, ml
m2- -sample mass, g
V2Volume of sample removed for colorimetric determination, ml
0.9 correction factor for glucose to dextran
In equation 2: y-polysaccharide extraction (%)
m-polysaccharide mass (mg)
M-Mass of starting material (mg).
And (3) measuring the content of the betaine: the test adopts a colorimetric method to determine the content of betaine in the solution. Under the condition of the existence of inorganic acid, the betaine reacts with the Rayleigh salt, the reaction has high specificity, the generated silver complex precipitate is dissolved in an acetone solution to form a purple solution, the content of the purple solution is measured under the wavelength of 525nm, and the concentration of the betaine and the absorbance have a certain linear relation under the wavelength.
(1) Solution preparation
Betaine standard solution (1 g/L): accurately weighing 0.1 g of betaine standard substance, dissolving in distilled water, transferring into a 100mL volumetric flask, and fixing the volume, wherein each mL of betaine in the solution is 1mg for drawing a standard curve; betaine standard solution (10 g/L): precisely weighing 1.0g of betaine standard substance, dissolving with appropriate amount of deionized water, fixing the volume in a volumetric flask, and shaking up. Namely 10g/L betaine standard solution; leeb's salt saturated solution (15 g/L): accurately weighing 1.5g of Raney's salt (ammonium tetrasulfacyano diamminechromate), adding 100ml of distilled water, stirring and dissolving for 45min by a stirrer, filtering, and acidifying by concentrated hydrochloric acid until the pH value is 1.0 (the quaternary ammonium Rankine compound is most completely precipitated when the pH value is 1.0) for precipitating the quaternary ammonium compound, wherein the reagent is prepared in situ, and the Raney's salt is only stable in a solid state; concentrated hydrochloric acid: for acidifying a Rayleigh salt solution; acetone solution: uniformly mixing analytically pure acetone and distilled water according to the volume ratio of 7:3 to prepare 70% acetone solution for dissolving the precipitate of the Rankine quaternary ammonium compound; ether wash: taking chromatographic pure ether and distilled water, and mixing uniformly according to a volume ratio of 99:1 to prepare a 99% ether solution for washing and precipitating.
(2) Drawing of betaine standard curve
6 clean centrifuge tubes are numbered. Respectively adding 1mg/mL of standard betaine solution 1, 1.5, 2, 2.5, 3 and 3.5mL, sequentially and respectively adding 2.5, 2, 1.5, 1, 0.5 and 0mL of distilled water, accurately adding 7mL of a Rayleigh saturated solution, placing in a refrigerator, cooling at 4 ℃ for 1h, taking out, centrifuging for 15min (4000 r/min) by using a centrifuge, discarding the supernatant, respectively adding 5mL of 99% diethyl ether washing liquor, uniformly mixing, centrifuging for 15min, discarding the supernatant, after diethyl ether is completely volatilized, dissolving and precipitating by using 70% acetone, transferring the solution to a 10mL bottle, and fixing the volume to be measured. The detection wavelength was fixed at 525nm and the absorbance was measured with reference to an acetone solution. And calculating a regression equation of the standard curve by adopting a linear regression method for the measured data, and drawing the standard curve by taking the absorbance as a vertical coordinate and the content of the betaine as a horizontal coordinate.
Betaine solutions of 10mg/ml having pH values of 3, 5, 7, 9 and 11, respectively, were passed through a chromatography column (10X 300 mm) packed with 2.0g of a cationic resin at a constant flow rate, collected every 5ml, the concentration of betaine in the collected solution was measured, and the adsorption rate was calculated, and the results are shown in Table 2.
TABLE 2
| pH | 3 | 5 | 7 | 9 | 11 |
| Adsorption rate/%) | 94.77 | 91.80 | 90.63 | 88.98 | 86.01 |
The elution rate was calculated by passing 1%, 3%, 5%, 7%, 9% aqueous ammonia solution at a constant flow rate through a chromatography column (10X 300 mm) containing 2.0g of 732 cation exchange resin saturated by adsorption, collecting every 5ml, and measuring the betaine concentration in the collected solution, and the results are shown in Table 3.
TABLE 3
| Concentration of ammonia | 1% | 3% | 5% | 7% | 9% |
| Elution Rate/%) | 68.97 | 72.00 | 75.49 | 77.01 | 78.01 |
The foregoing is a detailed description of the invention, which is described in greater detail and not intended to limit the scope of the invention. It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention, and that such obvious alternatives fall within the scope of the invention.
Claims (6)
1. A method for extracting polysaccharide and betaine from seaweed is characterized by comprising the following steps:
s1, weighing the dried and crushed seaweed and water in a glass container, wherein the mass ratio of the seaweed to the water is 1: (30-50);
s2, extracting the glass container weighed in the step S1 for 60-90S in a microwave with the power of 210-490W to obtain an extracting solution, and filtering to remove solids in the extracting solution to obtain primary clear liquid;
s3, concentrating the primary clear liquid to one third of the original volume, adding excessive ethanol, keeping the ethanol concentration more than or equal to 80%, standing for 10h, filtering to obtain polysaccharide and secondary extracting solution, and drying the polysaccharide;
s4, adjusting the pH value of the secondary extracting solution to 3-7, and adsorbing the secondary extracting solution by a resin column filled with 732 cation exchange resin until the 732 cation exchange resin is saturated in adsorption on the betaine;
s5, eluting the 732 cation exchange resin after saturated adsorption by using 1-9% ammonia water to obtain an eluent, and concentrating and drying the eluent to obtain betaine;
after the secondary clear liquid is obtained in step S3, impurities are removed by using 717 anion exchange resin.
2. The method of extracting polysaccharides and betaines from seaweeds of claim 1 wherein the ratio of the mass of seaweed to the mass of water is 1: 40.
3. the method as claimed in claim 1, wherein the microwave extraction power of step S2 is 350W.
4. The method for extracting polysaccharides and betaines from seaweeds of claim 3 wherein step S2 is microwave extraction for 75S.
5. The method for extracting polysaccharides and betaines from seaweeds of claim 1 wherein the pH of step S4 is adjusted to 3.
6. The method for extracting polysaccharides and betaines from seaweeds of claim 1 wherein step S5 is performed with 5% ammonia water.
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