CN108676106A - A method of extracting polysaccharide and glycine betaine from seaweed - Google Patents

Abstract

The method that the invention discloses a kind of to extract polysaccharide and glycine betaine from seaweed, includes the following steps：In glass container, the quality of seaweed and the mass ratio of water are 1 for S1, the seaweed for weighing drying and crushing and water：（30‑50）；S2, the glass container that step S1 is weighed is obtained into extracting solution in the Microwave Extraction 60s 90s that power is 210W 490W, the solid being filtered to remove in extracting solution obtains primary clear liquid；S3, primary clear liquid is concentrated into the one third for original volume, excessive ethyl alcohol is added, the concentration of ethyl alcohol is greater than or equal to 80%, stands 10h, and polysaccharide and secondary extracting solution is obtained by filtration, polysaccharide is dried；S4, secondary extracting solution pH is adjusted to 37, is adsorbed through the resin column equipped with 732 cation exchange resins, until 732 cation exchange resins are to glycine betaine adsorption saturation；S5,732 cation exchange resins after adsorption saturation are carried out affording eluent using 1% 9% ammonium hydroxide, eluent is concentrated and dried, extracting method of the present invention can extract polysaccharide and glycine betaine from seaweed.

Description

A method for extracting polysaccharide and betaine from seaweed

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 technique

"Seaweed" is a general term for marine algae such as kelp, laver, wakame, and agarose, and is an algae that grows in the sea. It is divided into two categories: macroalgae and microalgae. Macroalgae have many types and rich forms, including brown algae, red algae, cyanobacteria and green algae. They are cryptogamous plants in the plant kingdom. Organisms that produce energy. They are generally considered to be simple, mostly cellular filaments, membranous bodies, tubular bodies or thallus plants that have no vascular tissue, no real differentiation of roots, stems, and leaves; no flowering, no fruit and Seeds; asexual embryos but with chlorophyll can synthesize organic matter through photosynthesis to supply their own survival needs like higher plants.

There are two types of bioactive substances in macroalgae, one is small molecular substances that can be digested and absorbed, specifically halogen compounds, seaweed tannins, terpenoids, etc.; the other is viscous polysaccharides that are difficult to digest, such as red Agar, carrageenan, fucoidan, sulfated polysaccharides in algae, etc. Macroalgae polysaccharides (hereinafter referred to as polysaccharides) are chain polymers formed by multiple identical or different monosaccharide groups linked by glycosidic bonds. They exist in the cell wall and interstitium of seaweed, accounting for more than 50% of the dry weight of seaweed. Immunomodulation, anti-tumor, anti-virus, anti-oxidation and other biological activities and medicinal value.

There is also betaine in seaweed. Betaine is a general term for a series of substances. All substances with the structure of R-(CH3)3N+CH2COO- belong to betaine. Due to the presence of trimethyl, betaine, a quaternary ammonium salt, has obvious alkali-containing properties. Betaine is also a typical surfactant. The betaines found in seaweed are glycine betaine (the simplest betaine, called betaine or trimethylglycine), gamma-aminobutyric acid betaine, alanine betaine, homoserine betaine Alkali (derived from phosphonine), an ether formed from homoserine and glycerol; the other is a derivative of homoserine betaine, 1(3) , 2-diacylglyceryl-3(1)-0-4-(N,N,N-trimethyl)homoserine, δ-aminovaleric acid betaine, N6-trimethyllysine (ie kelp amino acid, which is not only a quaternary amine derivative, but also an α-amino acid that can form peptide bonds), lysine betaine (wherein both nitrogen atoms of lysine are fully methylated), proline Amino acid betaine (also known as stachydrine, that is, proline dimethyl internal salt), cis-4-hydroxyproline betaine, trans-4-hydroxy-β-proline betaine, 1,3 - Dimethylhistidine, Betaine Pyridinecarboxylate, Trigonelline (N-Methylnicotinic Inner Salt), Lobster Creatinine, N,N-Dimethyl-1,2,3,6-tetrahydro Pyridine-2-hydroxyl salt (betaine).

The physical appearance of betaine is white scaly or prismatic crystalline powder, with a slight characteristic odor (sweet taste), molecular weight 117.15, high temperature resistance, melting point 293°C, and will decompose when the temperature reaches 310°C. Solubility (20°C) 160g/100g water, easily soluble in polar solvents such as water and methanol, soluble in ethanol, slightly soluble in ether. According to the different pH in the solution, betaine has different forms of existence. Generally, when the pH is less than 7, the molecule is in an open ring state, and when the pH is greater than or equal to 7, it is in a ring structure. It is easy to absorb moisture and deliquescence at room temperature, has strong moisture retention, relatively stable properties and strong oxidation resistance.

There is no method for extracting polysaccharides and betaine from seaweed in the prior art.

The present invention aims to provide a method for extracting polysaccharide and betaine from seaweed.

Contents of the invention

In view of this, the present invention aims to provide a method for extracting polysaccharide and betaine from seaweed.

A method for extracting polysaccharide and betaine from seaweed, comprising the following steps:

S1. Weigh dried and pulverized seaweed and water in a glass container, the ratio of seaweed mass to water mass is 1: (30-50);

S2. Extract the glass container weighed in step S1 with a microwave with a power of 210W-490W for 60s-90s to obtain an extract, and filter to remove solids in the extract to obtain a primary clear liquid;

S3. Concentrate the primary supernatant to one-third of the original volume, add excess ethanol, the concentration of ethanol is greater than or equal to 80%, let it stand for 10 hours, filter to obtain polysaccharide and secondary extract, and dry the polysaccharide;

S4. Adjust the pH of the secondary extract to 3-7, and absorb it through a resin column equipped with 732 cation exchange resin until the 732 cation exchange resin is saturated with betaine adsorption;

S5. Using 1%-9% ammonia water to elute the saturated 732 cation exchange resin to obtain an eluent, and then concentrate and dry the eluent to obtain betaine.

By microwave water extraction, combined with the ratio of seaweed to water, microwave extraction power and microwave extraction time, the polysaccharides and betaine in seaweed are transferred to the primary clear liquid, and then the betaine and polysaccharides are separated in water and ethanol. The difference in the solubility of the medium, to achieve the initial separation of polysaccharides and betaine, and then the secondary solution rich in betaine in a specific range of pH value through 732 cation exchange resin for adsorption, 732 cation exchange resin for betaine adsorption rate Higher, and finally use ammonia solution to elute, so as to complete the further purification of betaine.

732 cation-exchange resin adsorption saturation of betaine means that 732 cation-exchange resin no longer adsorbs betaine in the secondary extract, that is, the concentration of betaine in the secondary extract by the resin column equipped with 732 cation-exchange resin is not high. Change again.

Preferably, the ratio of the mass of seaweed to the mass of water is 1:40.

When the ratio of the mass of seaweed to the mass of water is 1:40, 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 in step S2 is 350W.

The present invention finds through experiments that after the microwave extraction power exceeds 350W, the polysaccharide content in the preliminary clear liquid decreases, and the polysaccharide decomposes after the microwave extraction power exceeds 350W; , to avoid the loss of polysaccharides due to decomposition.

Preferably, step S2 is microwave extraction for 75s.

350W microwave extraction time is too long, polysaccharides are easy to decompose.

Preferably, the pH of step S4 is adjusted to 3.

When the pH of the secondary extract is 3, the betaine is in an open-ring state, and a large amount of hydrogen ions are provided in the secondary extract to ionize the betaine and then exchange with the sulfonic acid group of the 732 cation exchange resin to be adsorbed. The adsorption rate of 732 cation exchange resin to betaine is as high as 94%.

Preferably, step S5 is eluted with 5% ammonia water.

It is eluted with 5% ammonia water, and the elution rate is as high as 75%. Under alkaline conditions, the hydroxide ions and the hydrogen ions on the adsorbed betaine cations are neutralized to form water, thereby removing the electrically neutral betaine molecules from 732 cation exchange resin for elution, while avoiding the method of cation exchange elution with acidic solution in the prior art, and avoiding the subsequent detection of betaine purity, betaine reacts with Raye’s salt to produce betaine hydrochloride, affect the accuracy of test results.

Preferably, after the secondary clear liquid is obtained in step S3, first use 717 anion resin to remove impurities.

This experiment found that the adsorption rate of 717 anion to betaine is lower than 6.5%, and it has a higher adsorption rate to other substances in the secondary extract. The secondary clear liquid is first adsorbed by 717 anion resin to reduce the secondary The content of impurities in the clear liquid can avoid the impact of impurities in the process of using 732 cation exchange resin to absorb and elute betaine, and improve the final purity of betaine.

Compared with the prior art, the present invention has the following advantages and beneficial effects:

Through the microwave water extraction method, combined with the appropriate ratio of seaweed to water, microwave extraction power and microwave extraction time, the polysaccharides and betaine in the seaweed are transferred to the primary clear liquid, and then the betaine and polysaccharides are separated in water and ethanol. The difference in solubility can realize the preliminary separation of polysaccharide and betaine, and then the secondary solution rich in betaine is adsorbed by 732 cation exchange resin in a specific pH value range, and the adsorption rate of 732 cation exchange resin for betaine is higher High, and finally use ammonia solution for elution, so as to complete the further extraction and purification of betaine.

Detailed ways

The technical solutions of the present invention will be further described below in conjunction with the embodiments.

Example 1

The present embodiment provides a kind of method for extracting polysaccharide and betaine from seaweed, comprises the following steps:

S1. Weigh the dried and pulverized seaweed and water in a glass container, the ratio of seaweed mass to water mass is 1:30;

S2. Extract the glass container weighed in step S1 with a microwave with a power of 210W for 60s to obtain an extract, and filter to remove solids in the extract to obtain a primary clear liquid;

S3. Concentrate the primary supernatant to one-third of the original volume, add excess ethanol, the concentration of ethanol is greater than or equal to 80%, let it stand for 10 hours, filter to obtain polysaccharide and secondary extract, and dry the polysaccharide;

S4, adjusting the pH of the secondary extract to 5, and adsorbing it through a resin column equipped with 732 cation exchange resin until the 732 cation exchange resin is saturated with betaine adsorption;

S5. Using 1% ammonia water to elute the saturated 732 cation exchange resin to obtain an eluent, and then concentrate and dry the eluent to obtain betaine.

Example 2

The present embodiment provides a kind of method for extracting polysaccharide and betaine from seaweed, comprises the following steps:

S1. Weigh the dried and pulverized seaweed and water in a glass container, the ratio of seaweed mass to water mass is 1:40;

S2. Extract the glass container weighed in step S1 with a microwave power of 350W for 75s to obtain an extract, and filter to remove solids in the extract to obtain a primary clear liquid;

S3. Concentrate the primary supernatant to one-third of the original volume, add excess ethanol, the concentration of ethanol is greater than or equal to 80%, let it stand for 10 hours, filter to obtain polysaccharide and secondary extract, and dry the polysaccharide;

S4. Adjust the pH of the secondary extract to 3, and absorb it through a resin column equipped with 732 cation exchange resin until the 732 cation exchange resin is saturated with betaine adsorption;

S5. Using 5% ammonia water to elute the saturated 732 cation exchange resin to obtain an eluent, and then concentrate and dry the eluent to obtain betaine.

Example 3

The present embodiment provides a kind of method for extracting polysaccharide and betaine from seaweed, comprises the following steps:

S1. Weigh the dried and pulverized seaweed and water in a glass container, the ratio of seaweed mass to water mass is 1:50;

S2. Extract the glass container weighed in step S1 with a microwave with a power of 490W for 90s to obtain an extract, and filter to remove solids in the extract to obtain a primary clear liquid;

S3. Concentrate the primary supernatant to one-third of the original volume, add excess ethanol, the concentration of ethanol is greater than or equal to 80%, let it stand for 10 hours, filter to obtain polysaccharide and secondary extract, and dry the polysaccharide;

S4. Adjust the pH of the secondary extract to 7, and absorb it through a resin column equipped with 732 cation exchange resin until the 732 cation exchange resin is saturated with betaine adsorption;

S5, using 9% ammonia water to elute the saturated 732 cation exchange resin to obtain an eluent, and then concentrate and dry the eluent to obtain betaine.

Example 4

The present embodiment provides a kind of method for extracting polysaccharide and betaine from seaweed, comprises the following steps:

S1. Weigh the dried and pulverized seaweed and water in a glass container, the ratio of seaweed mass to water mass is 1:40;

S2. Extract the glass container weighed in step S1 with a microwave power of 350W for 75s to obtain an extract, and filter to remove solids in the extract to obtain a primary clear liquid;

S3. Concentrate the primary supernatant to one-third of the original volume, add excess ethanol, the concentration of ethanol is greater than or equal to 80%, let it stand for 10 hours, filter to obtain polysaccharide and secondary extract, and dry the polysaccharide;

S4. Adjust the pH of the secondary extract to 3, and absorb it through a resin column equipped with 732 cation exchange resin until the 732 cation exchange resin is saturated with betaine adsorption;

S5. Using 5% ammonia water to elute the saturated 732 cation exchange resin to obtain an eluent, and then concentrate and dry the eluent to obtain betaine.

After the secondary clear liquid is obtained in step S3, first use 717 anion resin to remove impurities.

The content of polysaccharide and the content of betaine were tested respectively in the extraction process of Examples 1-4, and the results are shown in Table 1.

Table 1

Wherein the content determination of polysaccharide:

(1) Glucose-phenol sulfate standard curve

Take six test tube numbers, draw 0mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL and 1.0mL of the prepared 0.1mg/ml standard glucose solution into the 25ml colorimetric tube, and then replenish to 1.0mL with distilled water, Add 1.0ml of 5% phenol to the test solution, then quickly add 5.0ml of concentrated sulfuric acid (add it vertically to the liquid surface, do not touch the test tube wall, so as to fully mix with the reaction solution), let stand for 10 minutes, shake well, and then The color tube was placed in a water bath at 30°C for 20 minutes and then the absorbance value (OD value) was measured at 490 nm. The concentration of glucose was taken as the abscissa and the absorbance value as the ordinate to establish a standard curve.

(2) Determination of polysaccharide content

Weigh 20 mg of the sample, dissolve it and set the volume to a 100ml volumetric flask, draw 0.5mL of the sample solution, add distilled water to make up to 1.0mL, then add 1.0mL of 5% phenol and 5mL of concentrated sulfuric acid, let stand for 10 minutes, shake well, and then The color tube was placed in a water bath at 30°C for 20 minutes, and then the absorbance value (OD value) was measured at 490 nm, and the total sugar content was calculated according to the standard curve equation.

The polysaccharide content in the sample is expressed in terms of mass fraction ω, and the unit is expressed in grams per hectogram (g/100g), then:

----- (Formula 1)

In formula 1: m ₁ ---- get the sugar content μg in the sample measurement solution from the standard curve;

V ₁ ---- sample constant volume, ml

m2---sample mass, g

V ₂ ---- the volume of sample measurement solution pipetted during colorimetric determination, ml

0.9----Correction coefficient for converting glucose into dextran

----- (Formula 2)

In formula 2: y—polysaccharide extraction rate (%)

m—polysaccharide mass (mg)

M—mass of raw material (mg).

Determination of betaine content: In this experiment, the colorimetric method was used to determine the content of betaine in the solution. In the presence of inorganic acid, betaine reacts with Rey’s salt, the reaction is highly specific, and the resulting silver complex precipitate is dissolved in acetone solution to form a purple solution, and its content is determined at a wavelength of 525nm. The concentration of betaine has a certain linear relationship with the absorbance.

(1) Solution preparation

Betaine standard solution (1g/L): Accurately weigh 0.1 g of betaine standard, dissolve in distilled water, transfer to a 100mL volumetric flask, and set to volume, 1mg of betaine per mL of this solution is used to draw a standard curve; Betaine standard solution (10g/L): Accurately weigh 1.0g of betaine standard product, dissolve it with an appropriate amount of deionized water and dilute it in a volumetric flask, and shake well. That is, 10g/L betaine standard solution; Reye's salt saturated solution (15g/L): Accurately weigh 1.5g of Reye's salt (tetrathiocyanodiammine ammonium chromate), add 100 ml of distilled water, and use Stir and dissolve with agitator for 45 minutes, filter and acidify with concentrated hydrochloric acid to pH 1.0 (Reynachate quaternary ammonium compound precipitates most completely when pH is 1.0), used for precipitation of quaternary ammonium compound, this reagent must be used and prepared immediately , because Reynolds salt is only stable when it is solid; Concentrated hydrochloric acid: used to acidify Reynolds salt solution; Acetone solution: Mix analytically pure acetone and distilled water in a volume ratio of 7:3 to form a 70% acetone solution, and use Precipitate in dissolving the quaternary ammonium compound of Reynach's acid; diethyl ether washing solution: take chromatographically pure diethyl ether and distilled water at a ratio of 99:1 by volume and mix to form a 99% diethyl ether solution for washing the precipitate.

(2) Drawing of betaine standard curve

Take 6 clean centrifuge tubes and number them. Add 1mg/ml standard betaine solution 1, 1.5, 2, 2.5, 3, 3.5mL respectively, add distilled water 2.5, 2, 1.5, 1, 0.5, 0 mL in turn, and then add 7ml of Raine’s salt saturated solution accurately, Cool in the refrigerator at 4°C for 1 hour, take it out and centrifuge for 15 minutes in a centrifuge (4000r/min), discard the supernatant, add 5ml of 99% ether washing solution, mix well, and centrifuge for 15 minutes, discard the supernatant, After the diethyl ether is volatilized, dissolve the precipitate with 70% acetone, transfer the solution to a 10ml volumetric flask, and set the volume to be tested. The detection wavelength was fixed at 525 nm, and the absorbance was measured with acetone solution as a reference. The linear regression method was used to calculate the regression equation of the standard curve to the measured data, and the absorbance was taken as the ordinate, and the content of betaine was taken as the abscissa, and the standard curve was drawn.

Pass the 10 mg/ml betaine solutions with pHs of 3, 5, 7, 9, and 11 respectively through a chromatographic column (10x300mm) equipped with 2.0 g of cationic resin at a constant flow rate, collect every 5 ml, and measure the concentration of betaine in the collected solution The concentration was calculated to obtain the adsorption rate, 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

Pass 1%, 3%, 5%, 7%, 9% ammonia solution at a constant flow rate through a chromatographic column (10x300mm) equipped with 2.0g of saturated 732 cation exchange resin, collect every 5ml, and measure the concentration of the collected solution. The concentration of betaine was calculated to obtain the elution rate, and the results are shown in Table 3.

table 3

Ammonia concentration 1% 3% 5% 7% 9% Elution rate/% 68.97 72.00 75.49 77.01 78.01

The above is one of the specific implementations of the present invention, and the description thereof is relatively specific and detailed, but it should not be construed as limiting the patent scope of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these obvious replacement forms all belong to the protection scope of the present invention.

Claims (7)

1. a method for extracting polysaccharide and betaine from seaweed, is characterized in that, comprises the following steps: S1. Weigh dried and pulverized seaweed and water in a glass container, the ratio of seaweed mass to water mass is 1: (30-50); S2. Extract the glass container weighed in step S1 with a microwave with a power of 210W-490W for 60s-90s to obtain an extract, and filter to remove solids in the extract to obtain a primary clear liquid; S3. Concentrate the primary supernatant to one-third of the original volume, add excess ethanol, the concentration of ethanol is greater than or equal to 80%, let it stand for 10 hours, filter to obtain polysaccharide and secondary extract, and dry the polysaccharide; S4. Adjust the pH of the secondary extract to 3-7, and absorb it through a resin column equipped with 732 cation exchange resin until the 732 cation exchange resin is saturated with betaine adsorption; S5. Using 1%-9% ammonia water to elute the saturated 732 cation exchange resin to obtain an eluent, and then concentrate and dry the eluent to obtain betaine. 2. the method for extracting polysaccharide and betaine from seaweed according to claim 1, is characterized in that, the ratio of the quality of seaweed and the quality of water is 1:40. 3. the method for extracting polysaccharide and betaine from seaweed according to claim 1, is characterized in that, the microwave extraction power of step S2 is 350W. 4. the method for extracting polysaccharide and betaine from seaweed according to claim 3, is characterized in that, step S2 microwave extraction 75s. 5. the method for extracting polysaccharide and betaine from seaweed according to claim 1, is characterized in that, the pH of step S4 is adjusted to 3. 6. the method for extracting polysaccharide and betaine from seaweed according to claim 1, is characterized in that, step S5 adopts 5% ammonia water to elute. 7. The method for extracting polysaccharides and betaine from seaweed according to claim 1, characterized in that, after step S3 obtains the secondary clear liquid, first use 717 anion exchange resin to remove impurities.