Method for preparing agarose and agarose prepared thereby

Abstract

The invention relates to a method for preparing agarose, comprising the following steps: preparing agar into an acidic suspension, adding a sulfate radical precursor substance, and carrying out desulfurization at at least 30 DEG C to obtain agarose. The invention also relates to agarose prepared by the method of the invention.

Description

Technical Field

[0001] The present invention relates to a method for preparing agarose, comprising the following steps: preparing agar into an acidic suspension, adding a sulfate radical precursor, and desulfurizing at at least 30°C to obtain agarose. The present invention also relates to agarose prepared by the method of the present invention. Background Art

[0002] At present, the commonly used techniques for preparing agarose mainly include polyethylene glycol method, acetylation method, CPC method, DMSO method, sodium iodide method, ammonium sulfate method, chitosan method, ion exchange method, chelating agent method, enzyme method, etc. The principle of these methods is mainly to separate sulfur agar in agar. Sulfur agar and agarose are the two main components of agar. The precipitation method mainly uses the difference in the solubility of sulfur agar and agarose in salt and alcohol solutions to achieve the purpose of separating agarose. The representative one is EDTA-Na 2 Method, polyethylene glycol method. Ion chromatography mainly uses anion exchange resin to absorb acidic polysaccharides and does not absorb neutral polysaccharide agarose, thereby achieving the purpose of separating agarose. However, the above method has certain drawbacks in the process of producing agarose. Its raw material agar often requires seaweed to be further processed after the agar solution is produced by traditional alkali treatment process. There are problems such as long production cycle, low production yield, complicated production process and high cost.

[0003] In recent years, a new hydrogen peroxide method for preparing agarose has emerged, which mainly uses the hydroxyl radicals produced by hydrogen peroxide to desulfurize agar powder. However, hydrogen peroxide has defects such as easy decomposition when exposed to light, easy deterioration during storage, easy volatility or generation of oxygen during the reaction process, and easy explosion in the ethanol reaction system, making it difficult to achieve large-scale production.

[0004] Since the traditional method is a homogeneous treatment process, seaweed is treated with traditional alkali to produce an agar solution with a concentration of 1.0 to 1.5%. 2 The yield of agarose obtained after treatment with , polyethylene glycol or anion exchange resin is low.

[0005] The hydrogen peroxide method is to desulfurize agar in a heterogeneous ethanol-water system. Since agar easily absorbs water and swells, it cannot be stirred, which affects mass transfer and heat transfer. Therefore, it is necessary to desulfurize agar in an ethanol-water system. Since hydrogen peroxide easily releases oxygen during the reaction, ethanol is easy to volatilize under high temperature conditions, and there is a risk of explosion under the above conditions.

[0006] The use of hydroxyl radical desulfurization requires overcoming the problem of excessive decomposition of agar, and currently there is no technology in the prior art that can solve this problem.

[0007] In addition, the use of hydrogen peroxide can reduce the sulfate content to 0.34%. If hydroxyl radicals are used for multiple cycles of desulfurization, the final sulfate content can be reduced to 0.1%, but the gel strength will be lower than 200g / cm 2 (1.5% w / v). Using traditional methods to separate sulfur agar, even with a combination of multiple separation processes, it is difficult to reduce the sulfate content to 0.15%, and a large amount of ion exchange media is required, the process is cumbersome, the cost is high, and the yield is low. Summary of the invention

[0008] The purpose of the present invention is to solve the above-mentioned problems in the prior art.

[0009] The present invention provides a method for preparing agarose, the method comprising the following steps:

[0010] Agar is prepared into an acidic suspension, a sulfate radical precursor is added, and desulfurization is carried out at at least 30° C. to obtain agarose.

[0011] In a preferred embodiment of the method of the present invention, the agarose obtained by the first desulfurization is prepared into a neutral to alkaline suspension, a sulfate radical precursor is added, and a second desulfurization is performed at at least 30° C. to obtain agarose. Specifically, the difference between the pH value of the second desulfurization and the pH value of the first desulfurization can be at least 2, preferably 3 to 8, more preferably 4 to 7, and particularly preferably 5 to 6.

[0012] The present invention also provides agarose prepared by the method of the present invention.

[0013] Various aspects of the present invention are explained in more detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Shown is the electrophoresis effect diagram of agarose prepared in Comparative Example 1;

[0015] Figure 2 Shown is the electrophoresis effect diagram of agarose prepared in Example 1;

[0016] Figure 3 Shown is the electrophoresis effect diagram of agarose prepared in Example 2;

[0017] Figure 4 Shown is the electrophoresis effect diagram of agarose prepared in Example 3;

[0018] Figure 5 Shown is the electrophoresis effect diagram of agarose prepared in Example 4;

[0019] Figure 6 Shown is the electrophoresis effect diagram of agarose prepared in Example 5;

[0020] Figure 7 Shown is the electrophoresis effect diagram of agarose prepared in Example 6; and

[0021] Figure 8 Shown is the electrophoresis effect diagram of agarose prepared in Example 7. DETAILED DESCRIPTION

[0022] Unless otherwise indicated, all publications, patent applications, patents, and other references mentioned in this application are incorporated by reference in their entirety as if fully set forth herein.

[0023] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of conflict, the present specification, including definitions, will control.

[0024] When a certain amount, concentration or other value or parameter is expressed in the form of a range, a preferred range, or a preferred upper numerical limit and a preferred lower numerical limit, it should be understood that it is equivalent to specifically disclosing any range by combining any pair of upper range limits or preferred numerical values ​​with any lower range limit or preferred numerical value, regardless of whether the range is specifically disclosed. Unless otherwise indicated, the numerical ranges listed herein are intended to include the endpoints of the range, and all integers and fractions within the range.

[0025] The present invention relates to a method for preparing agarose, the method comprising the following steps:

[0026] i) preparing agar into an acidic suspension, adding a sulfate radical precursor, and performing a first desulfurization at at least 30° C.; and

[0027] ii) the agarose obtained by the first desulfurization is prepared into a neutral to alkaline suspension, a sulfate radical precursor is added, and a second desulfurization is carried out at at least 30° C. to obtain agarose.

[0028] In one embodiment of the method of the present invention, the difference between the pH value of the second desulfurization and the pH value of the first desulfurization may be at least 2, preferably 3 to 8, more preferably 4 to 7, and particularly preferably 5 to 6.

[0029] First desulfurization

[0030] In one embodiment of the method of the present invention, the pH value of the first desulfurization may be 2 to 6, preferably 3 to 5.

[0031] In one embodiment of the method of the present invention, the temperature of the first desulfurization may be 35 to 80°C, preferably 40 to 60°C.

[0032] In one embodiment of the method of the present invention, the duration of the first desulfurization may be at least 30 minutes, preferably 40 to 120 minutes, more preferably 60 to 90 minutes.

[0033] In one embodiment of the method of the present invention, the sulfate radical precursor substance added in the first desulfurization can be selected from: peroxymonosulfate and peroxydisulfate. Specifically, the sulfate radical precursor substance added in the first desulfurization can be selected from: sodium persulfate, potassium persulfate, ammonium persulfate, potassium hydrogen persulfate and sodium hydrogen persulfate.

[0034] Second desulfurization

[0035] In one embodiment of the method of the present invention, the pH value of the second desulfurization may be 7 to 10, preferably 8 to 9.

[0036] In one embodiment of the method of the present invention, the temperature of the second desulfurization is 35 to 80°C, preferably 40 to 60°C.

[0037] In one embodiment of the method of the present invention, the duration of the second desulfurization is at least 30 minutes, preferably 40 to 120 minutes, more preferably 60 to 90 minutes.

[0038] In one embodiment of the method of the present invention, the sulfate radical precursor substance added in the second desulfurization can be selected from: peroxymonosulfate and peroxydisulfate. Specifically, the sulfate radical precursor substance added in the second desulfurization can be selected from: sodium persulfate, potassium persulfate, ammonium persulfate, potassium hydrogen persulfate and sodium hydrogen persulfate.

[0039] The present invention also relates to agarose obtained by the method of the present invention, the sulfate content of which can be reduced to less than 2.5%, preferably less than 2.0%, more preferably less than 1.5%, particularly preferably less than 1.0%, in particular as low as 0.05%, without substantially impairing the gel strength.

[0040] Studies have shown that the oxidation mechanisms of hydroxyl radicals and sulfate radicals are different. OH· is prone to addition and hydrogen abstraction reactions, while SO 4 – ·It is more likely to undergo electron transfer reaction, but the mechanism of both in agar desulfurization has not yet been elucidated.

[0041] According to the present invention, agarose is prepared by removing sulfate groups in agar structure by using sulfate radicals in a heterogeneous room temperature system. Based on the phenomena and reaction conditions during the treatment, the characteristics of sulfate radicals in agarose preparation can be summarized as follows:

[0042] Sulfate free radicals and various persulfates that can generate sulfate free radicals are both extremely strong oxidizing agents. Under normal circumstances, they can decompose the pigment proteins in agar, and the effect is quite significant.

[0043] Persulfate is more soluble in aqueous solution, so the generated sulfate radicals can quickly contact the agar in the water and produce oxidation, and the miscibility is good, which makes the decomposition rate higher. Then, the oxidant properties of the generated sulfate radicals are stable. Compared with hydrogen peroxide, which is also an oxidant, various persulfates are easier to store, and are not easy to change their properties during storage, and can maintain their properties for a long time.

[0044] Sulfate radicals can adapt to a wide pH range (pH = 2-10), which can increase the pH range for treating agar. Regardless of acidic or alkaline conditions, the reaction activity of sulfate radicals is not significantly affected.

[0045] Persulfate does not volatilize or produce gas, compared to O 3 and H 2 O 2 , persulfate does not generate volatile gases during the activation step, causing explosion risks, and does not cause the concentration to decrease the production of sulfate radicals due to volatilization.

[0046] Sulfate radicals can exist in aqueous solution for a longer time, and generally their half-life can reach about 4 seconds. The longer duration increases their efficiency in removing sulfate groups from agar.

[0047] Example

[0048] The agar source applicable to the present invention is not limited to the imported Gracilaria from Indonesia, the Philippines, Malaysia, Chile, Argentina and other countries, and is also applicable to the domestic Gracilaria from Fujian, Guangdong, Shandong, Hainan and other provinces. In general, the present invention has a good desulfurization effect on agar raw materials from different sources, and agar raw materials from different sources can be used to prepare high-quality agarose using the technology of the present invention.

[0049] The source of agar used in the following examples: imported Gracilaria, produced in Java, Indonesia; basic properties: sulfate content 0.56%, electro-endosmotic pressure 0.365, gel strength 1154 g / cm 2 , molecular weight Mw = 384kDa.

[0050] Comparative Example 1 (desulfurization with hydrogen peroxide)

[0051] (1) Prepare agar with a mass concentration of 20% (w / v) (sulfate content of 0.56%, electro-endosmotic pressure of 0.365, gel strength of 1154 g / cm 2 , molecular weight Mw = 384 kDa) suspension;

[0052] (2) adding 2% (v / v) hydrogen peroxide to the agar suspension, adjusting the system pH to 9, and performing a desulfurization reaction at 40° C. for 120 min;

[0053] (3) After the desulfurization reaction is completed, filter and wash until no hydrogen peroxide remains, and collect the precipitate sample;

[0054] (4) The precipitate was dried and crushed to obtain desulfurized agar, the sulfate content of the agar was 0.34%, the electro-endosmotic pressure was 0.315, and the gel strength was 1052 g / cm 2 , molecular weight Mw = 304kDa. Figure 1 Shown is the electrophoresis effect diagram of agarose prepared in Comparative Example 1, and the bands cannot be effectively separated.

[0055] Example 1

[0056] (1) preparing an agar suspension with a mass concentration of 20% (w / v), adjusting the solution pH to 3 and the temperature to 60° C.;

[0057] (2) adding sodium persulfate to the agar suspension at a sodium persulfate concentration of 2 mM and performing the desulfurization reaction for 60 min;

[0058] (3) After the reaction is completed, the precipitate is filtered and washed immediately, and then collected. The precipitate is dried and crushed to obtain desulfurized agar. The sulfate content of the agar is 0.21%, the electro-endosmotic pressure is 0.18, and the gel strength is 1170 g / cm 2 , molecular weight Mw = 372kDa. Figure 2 The figure shows the electrophoresis effect of the agarose prepared in Example 1, and the bands can be effectively separated.

[0059] Example 2

[0060] (1) preparing an agar suspension with a mass concentration of 20% (w / v), adjusting the solution pH to 3 and the temperature to 60° C.;

[0061] (2) adding sodium persulfate to the agar suspension at a sodium persulfate concentration of 2 mM and performing the desulfurization reaction for 120 min;

[0062] (3) After the reaction is completed, the precipitate is filtered and washed immediately, and then collected. The precipitate is dried and crushed to obtain desulfurized agar. The sulfate content of the agar is 0.20%, the electro-endosmotic pressure is 0.18, and the gel strength is 1153 g / cm 2 , molecular weight Mw = 359 kDa. Figure 3 The figure shows the electrophoresis effect of the agarose prepared in Example 2, and the bands can be effectively separated.

[0063] Example 3

[0064] (1) preparing an agar suspension with a mass concentration of 20% (w / v), adjusting the solution pH to 3 and the temperature to 60° C.;

[0065] (2) adding sodium persulfate to the agar suspension at a sodium persulfate concentration of 2 mM and performing the desulfurization reaction for 60 min;

[0066] (3) After the desulfurization reaction is completed, filter and wash until no sodium persulfate remains;

[0067] (4) adding deionized water to prepare an agar suspension with a mass concentration of 20% (w / v), adjusting the solution pH to 9 and the temperature to 40° C.;

[0068] (5) adding sodium persulfate to the agar suspension at a sodium persulfate concentration of 2 mM and performing the desulfurization reaction for 60 min;

[0069] (6) After the reaction, the mixture was filtered and washed immediately, and the precipitate was collected. The precipitate was dried and crushed to obtain high-quality agarose. The sulfate content of the agarose was 0.05%, the electro-endosmotic pressure was 0.05, and the gel strength was 1230 g / cm 2 , molecular weight Mw = 368kDa. Figure 4 The figure shows the electrophoresis effect of the agarose prepared in Example 3, and the bands can be effectively separated.

[0070] Example 4

[0071] (1) preparing an agar suspension with a mass concentration of 20% (w / v), adjusting the solution pH to 3 and the temperature to 40° C.;

[0072] (2) adding sodium persulfate to the agar suspension at a sodium persulfate concentration of 2 mM and performing the desulfurization reaction for 60 min;

[0073] (3) After the desulfurization reaction is completed, filter and wash until no sodium persulfate remains;

[0074] (4) adding deionized water to prepare an agar suspension with a mass concentration of 20% (w / v), adjusting the solution pH to 9 and the temperature to 40° C.;

[0075] (5) adding sodium persulfate to the agar suspension at a sodium persulfate concentration of 2 mM and performing the desulfurization reaction for 60 min;

[0076] (6) After the reaction, the precipitate was filtered and washed immediately, and then dried and crushed to obtain high-quality agarose. The sulfate content of the agarose was 0.11%, the electro-endosmotic pressure was 0.10, and the gel strength was 1158 g / cm 2 , molecular weight Mw = 382kDa. Figure 5 The figure shows the electrophoresis effect of the agarose prepared in Example 4, and the bands can be effectively separated.

[0077] Example 5

[0078] (1) preparing an agar suspension with a mass concentration of 20% (w / v), adjusting the solution pH to 6 and the temperature to 60° C.;

[0079] (2) adding sodium persulfate to the agar suspension at a sodium persulfate concentration of 2 mM and performing the desulfurization reaction for 60 min;

[0080] (3) After the desulfurization reaction is completed, filter and wash until no sodium persulfate remains;

[0081] (4) adding deionized water to prepare an agar suspension with a mass concentration of 20% (w / v), adjusting the solution pH to 9 and the temperature to 40° C.;

[0082] (5) adding sodium persulfate to the agar suspension at a sodium persulfate concentration of 2 mM and performing the desulfurization reaction for 60 min;

[0083] (6) After the reaction, the precipitate was filtered and washed immediately, and then dried and crushed to obtain high-quality agarose. The sulfate content of agarose was 0.18%, the electro-endosmotic pressure was 0.18, and the gel strength was 1050 g / cm 2 , molecular weight Mw = 390kDa. Figure 6 The figure shows the electrophoresis effect of the agarose prepared in Example 5, and the bands can be effectively separated.

[0084] Example 6

[0085] (1) preparing an agar suspension with a mass concentration of 20% (w / v), adjusting the solution pH to 3 and the temperature to 60° C.;

[0086] (2) adding sodium persulfate to the agar suspension at a sodium persulfate concentration of 2 mM and performing the desulfurization reaction for 90 min;

[0087] (3) After the desulfurization reaction is completed, filter and wash until no sodium persulfate remains;

[0088] (4) adding deionized water to prepare an agar suspension with a mass concentration of 20% (w / v), adjusting the solution pH to 9 and the temperature to 40° C.;

[0089] (5) adding sodium persulfate to the agar suspension at a sodium persulfate concentration of 2 mM and performing the desulfurization reaction for 60 min;

[0090] (6) After the reaction is completed, the precipitate is immediately filtered and washed, and the precipitate is collected. The precipitate is dried and crushed to obtain high-quality agarose. The sulfate content of the agarose is 0.05%, the electro-endosmotic pressure is 0.05, and the gel strength is 1200 g / cm 2 , molecular weight Mw = 382kDa. Figure 7The figure shows the electrophoresis effect of the agarose prepared in Example 6, and the bands can be effectively separated.

[0091] Example 7

[0092] (1) preparing an agar suspension with a mass concentration of 20% (w / v), adjusting the solution pH to 3 and the temperature to 60° C.;

[0093] (2) adding sodium persulfate to the agar suspension at a sodium persulfate concentration of 2 mM and performing the desulfurization reaction for 60 min;

[0094] (3) After the desulfurization reaction is completed, filter and wash until no sodium persulfate remains;

[0095] (4) adding deionized water to prepare an agar suspension with a mass concentration of 20% (w / v), adjusting the solution pH to 7 and the temperature to 40° C.;

[0096] (5) adding sodium persulfate to the agar suspension at a sodium persulfate concentration of 2 mM and performing the desulfurization reaction for 60 min;

[0097] (6) After the reaction, the mixture was filtered and washed immediately, and the precipitate was collected. The precipitate was dried and crushed to obtain high-quality agarose. The sulfate content of the agarose was 0.14%, the electro-endosmotic pressure was 0.13, and the gel strength was 1235 g / cm 2 , molecular weight Mw = 366kDa. Figure 8 The figure shows the electrophoresis effect of the agarose prepared in Example 7, and the bands can be effectively separated.

[0098] The specific embodiments described above are only used to illustrate the concept of the present application and should not be construed as limiting the scope of the present invention in any way. On the contrary, it should be clearly understood that after reading the description herein, those of ordinary skill in the art can implement other technical solutions, modifications, etc. without departing from the spirit of the present invention.

Claims

1. A method for preparing agarose, the method comprising the following steps: Agar is prepared into an acidic suspension, a sulfate radical precursor is added, and desulfurization is carried out at at least 30° C. to obtain agarose.

2. The method according to claim 1, characterized in that The agarose obtained by the first desulfurization is prepared into a neutral to alkaline suspension, a sulfate radical precursor is added, and the second desulfurization is carried out at at least 30° C. to obtain agarose.

3. The method according to claim 1 or 2, characterized in that: The difference between the pH value of the second desulfurization and the pH value of the first desulfurization is at least 2, preferably 3 to 8, more preferably 4 to 7, and particularly preferably 5 to 6.

4. The method according to any one of claims 1 to 3, characterized in that The pH value of the first desulfurization is 2 to 6, preferably 3 to 5.

5. The method according to any one of claims 1 to 4, characterized in that The pH value of the second desulfurization is 7 to 10, preferably 8 to 9.

6. The method according to any one of claims 1 to 5, characterized in that The temperatures of the first desulfurization and the second desulfurization are each independently 35 to 80°C, preferably 40 to 60°C.

7. The method according to any one of claims 1 to 6, characterized in that The duration of the first desulfurization and the second desulfurization is each independently at least 30 minutes, preferably 40 to 120 minutes, more preferably 60 to 90 minutes.

8. The method according to any one of claims 1 to 7, characterized in that The sulfate radical precursor substances added in the first desulfurization and the second desulfurization are each independently selected from: peroxymonosulfate and peroxydisulfate.

9. The method according to any one of claims 1 to 7, characterized in that The sulfate radical precursor substances added in the first desulfurization and the second desulfurization are each independently selected from the group consisting of sodium persulfate, potassium persulfate, ammonium persulfate, potassium hydrogen persulfate and sodium hydrogen persulfate.

10. Agarose obtained by the method according to any one of claims 1 to 9.

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