Low electric endosmosis agarose and a method for preparing the same

Abstract

The application belongs to the technical field of biochemistry and relates to a low electric endosmosis agarose and a preparation method thereof. The method comprises the following steps: 1. preparing agar solution and heating and dissolving; 2. preparing inorganic flocculating agent solution and dimethyl diallyl ammonium chloride polymer solution and keeping in a water bath pot at 40-100 DEG C; 3. under stirring, adding the inorganic flocculating agent solution into the agar solution and stirring; 4. adding the dimethyl diallyl ammonium chloride polymer solution into the solution of the above reaction and stirring; 5. filtering, removing the filter residue and collecting the filtrate; 5. adding ethanol into the filtrate for alcohol precipitation; 6. centrifuging, washing with water and vacuum freeze-drying; and 7. crushing to obtain the low electric endosmosis agarose. The application is simple, efficient, economical, environment-friendly and easy to operate and implement, the prepared agarose gel has good performance and high gel strength, and can meet the requirements of low electric endosmosis agarose in the field of molecular biology.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry and relates to a low-electro-endoosmotic agarose and its preparation method. Background Technology

[0002] Agar, also known as agar, gelatin, or agar powder, is a polysaccharide extracted from red algae such as those in the genera *Gnaphalium*, *Gracilaria*, *Igoromo*, *Porphyra*, and *Porphyra*. It is widely used in food, medicine, daily chemicals, and bioengineering. Agar is primarily a mixture of neutral sugars (containing different methyl groups) with an agarbiose backbone, transitioning through a series of low-charge sulfate and pyruvate groups to high-charge, acidic substituents. It mainly consists of agarose and sulfur agar. The melting temperature, solidification temperature, gel strength, polysaccharide composition, and yield of agar depend on the type of seaweed, harvest season, growth environment, and extraction method. Agar, alginate, and carrageenan are considered the three most widely used seaweed gum industrial products in the world.

[0003] Agarose is a non-ionic polysaccharide without sulfate groups. It is a copolymer formed by the linear alternation of agarobiose, consisting of 1,3-linked β-D-galactose and 1,4-linked α-3,6-galactose, and possesses gelling properties. Sulfate agar, also known as agar pectin, is a complex polysaccharide in agar and does not possess gelling properties. Sulfate agar has a backbone linkage similar to agarose, but some of the 3,6-galactose is replaced by galactose-6-sulfate groups and binds inorganic ions such as calcium and magnesium. Due to the influence of sulfate and carboxyl groups, agarose gel containing sulfur agar, when used as an electrophoretic support, suffers from drawbacks such as high electroendoosmosis, strong adsorption of basic dyes and proteins, potential for non-specific protein precipitation in acidic buffers, and poor transparency.

[0004] Ideally, agarose should be a neutral polysaccharide free of polar groups, but in reality, it is difficult to obtain a completely neutral polysaccharide free of polar groups. Currently, agarose prepared using existing technologies all contain trace amounts of polar groups such as sulfate and pyruvate, possibly due to residual trace amounts of sulfur agar. Electroendoosmosis is one of the main differences between agar and agarose, and a key indicator of agarose quality. During agarose gel electrophoresis, excessive sulfur agar in agarose causes convection in the gel under a DC electric field, affecting sample migration and separation, directly manifesting as electroendoosmosis and protein adsorption. The magnitude of electroendoosmosis is mainly related to the sulfate and carboxyl groups contained in the agarose. Therefore, the main focus of agarose preparation is to remove polysaccharides containing sulfate and carboxyl groups to reduce electroendoosmosis, while also removing small amounts of algal proteins, insoluble precipitates, soluble salts, and pigments introduced during agar production, thereby improving agarose purity and product stability.

[0005] The purification technology of agarose began in the last century. In 1937, Araki, while studying the composition and structure of agar, first prepared agarose by removing sulfur agar from agar using the acetylation method. In 1961, Swedish scientist S. Hjerten first purified agarose using agar, and after discovering the excellent performance of agarose, research on agarose increased and its application in industrial production began. In 1969, Barteling et al. prepared agarose by adsorbing charged sulfur agar from hot agar solution using an insoluble adsorbent. In 1971, Duckworth and Yaphe established a method for further fractionation of agar to obtain agarose. Also in 1971, Allan et al. proposed a method for preparing agarose using a mixture of chitin and chitosan with agar as a co-precipitation agent. In recent years, ionic liquids (ILs) have begun to be used as a novel solvent in the preparation of agarose. The biodegradable and non-toxic properties of bio-ionic liquids have enhanced their function as a solvent for agarose separation. There are many agarose extraction technologies, which can be broadly categorized based on their preparation principles into sulfur agar precipitation, agarose precipitation, ion exchange, ionic liquid methods, and others. Except for the DEAE cellulose method, other methods suffer from low yields, poor product color, low gel strength, and high production costs. While the DEAE cellulose method is a relatively mature process, its poor applicability to different agar sources and high price limit its application and the large-scale production of agarose. Therefore, finding new agarose production technologies is of great significance. Summary of the Invention

[0006] The purpose of this invention is to provide a flocculant-based method for separating and preparing low-electroendoosmotic agarose from agar. This method is simple, efficient, economical, environmentally friendly, and easy to operate. The resulting agarose has a sulfate content of less than 0.1%, an electroendoosmotic content of less than 0.1%, and good gelation properties, meeting the purity requirements of agarose in fields such as molecular biology.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is: a method for preparing low-electro-endoosmotic agarose, comprising the following steps: (1) Prepare an agar solution with a mass fraction of 0.5% to 5%, swell at room temperature, heat to 90 to 100 °C and stir to dissolve, and place in a water bath at 40 to 90 °C for constant temperature; (2) Prepare an inorganic flocculant aqueous solution with a mass fraction of 0.1% to 2%, stir and dissolve it at room temperature, and place it in a water bath at 40 to 90°C for constant temperature; (3) Prepare an aqueous solution of dimethyl diallyl ammonium chloride (DMDAAC) polymer with a mass fraction of 0.1% to 4%, stir to dissolve at room temperature and place in a water bath at 40 to 70°C for constant temperature; (4) Add the agar solution obtained in step (1) to the inorganic flocculant solution prepared in step (2) under stirring. The volume ratio (W / W) of the two solutions is 1:1 to 10:1. Stir for 10 to 420 min. (5) Add the solution obtained in step (4) to the organic flocculant solution prepared in step (3) under stirring. The volume ratio (W / W) of the two solutions is 1:1 to 10:1. Mix and stir for 10 to 420 min. (6) Filter or centrifuge the solution obtained in step (5) while it is still hot, and collect the filtrate or supernatant; (7) Add anhydrous ethanol to the filtrate obtained in step (6), let it stand at 4 °C for 6 h to 24 h, centrifuge, then wash with water several times and freeze dry under vacuum; (8) Pulverize the dried agarose to obtain low electro-endoosmotic agarose.

[0008] Preferably, the agar raw material used in step (1) is derived from marine red algae.

[0009] Preferably, the marine red algae is any one or more of the following: Gracilaria, Lithops, Gracilaria, Isophyllaria, Porphyra, Laminaria japonica, and Laminaria spp.

[0010] Preferably, the inorganic flocculant used in step (2) is one of polyferric chloride, polyferric aluminum sulfate, polyphosphoric ferric chloride, and polyferric sulfate.

[0011] Preferably, in step (7), after the agarose filtrate and anhydrous ethanol solution are mixed, the volume fraction of ethanol in the solution is 50% to 80%.

[0012] The present invention also provides a low electroendoosmotic agarose prepared by the above method, having a sulfate content ≤0.1%, electroendoosmosis ≤0.1%, and gel strength ≥1200 g / cm³. 2 Whiteness ≥ 90.

[0013] The key to producing agarose from agar is separating agarose from sulfur agar. Flocculants primarily work by bringing positively (negatively) charged groups and negatively (positively) charged, difficult-to-separate particles in water closer together, lowering their potential and making them unstable. Their polymerization properties then cause these particles to aggregate, allowing for separation through physical or chemical methods. Sulfur agar carries a negative charge due to the linking of sulfate and carboxyl groups. This invention uses a flocculant to bind with sulfur agar, producing a flocculent precipitate that separates from the agar solution, thus obtaining an agarose solution.

[0014] The method of this invention is simple, efficient, economical, environmentally friendly, easy to operate and implement, and the prepared agarose gel has good performance, high gel strength, sulfate content of less than 0.1%, electroendoosmosis of less than 0.1%, and whiteness of more than 90, which can meet the requirements of low electroendoosmosis agarose in fields such as molecular biology. Attached Figure Description

[0015] Figure 1 This refers to the state of the agarose solution obtained using the method of this invention; Figure 2 It is an agarose product obtained by the method of the present invention; Figure 3 The images shown are gel electrophoresis images of agarose obtained using the method of this invention. All experimental samples are markers. Among them, A is the sample prepared in Example 1; B is the sample prepared in Example 2; and C is commercially available agarose. Detailed Implementation

[0016] The present invention will be further explained and described below with reference to the accompanying drawings and embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the contents of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by this invention.

[0017] Example 1: This example provides a method for preparing low-electro-endoosmotic agarose, comprising the following steps: (1) Take 2g of agar, add 200ml of ultrapure water, let it swell for 2 hours, put it into boiling water and stir until dissolved, and put it in a 60°C water bath for more than 30 minutes; (2) Add 50ml of ultrapure water to a beaker and turn on the magnetic stirrer. Then add 0.4g of polyferric chloride and continue stirring for more than 30 minutes. After dissolution, place the beaker in a 60°C water bath and keep it warm for 30 minutes. (3) Add 50ml of ultrapure water to a beaker and turn on the magnetic stirrer. Then add 0.9g of dimethyl diallyl ammonium chloride (DMDAAC) polymer and continue stirring for more than 30min. After dissolution, place it in a 60°C water bath and keep it warm for 30min. (4) In a 60°C water bath, add 50 ml of polyferric chloride solution to 200 ml of agar sample solution and stir for 60 min; (5) In a water bath at 60°C, add 50 ml of dimethyl diallyl ammonium chloride (DMDAAC) polymer solution to the solution that has been flocculated with polyferric chloride, stir for 120 min until flocculent matter settles, and filter while hot.

[0018] (6) Add 600 ml of anhydrous ethanol to the filtrate, let it stand at 4°C for 6 h, centrifuge, then wash with water several times and freeze-dry under vacuum. After pulverization, agarose was obtained, and its sulfate content was determined to be 0.085%, electroosmosis 0.10, and gel strength 1220 g / cm³. 2 The whiteness is 91.72, such as... Figure 1-3 As shown.

[0019] Example 2: This example provides a method for preparing low-electro-endoosmotic agarose, comprising the following steps: (1) Take 1g of agar, add 200ml of ultrapure water, let it swell for 2 hours, put it into boiling water and stir until dissolved, and put it into an 80°C water bath and keep it warm for more than 30 minutes. (2) Add 50ml of ultrapure water to a beaker and turn on the magnetic stirrer. Then add 0.1g of polyphosphoric ferric chloride and continue stirring for more than 30 minutes. After dissolution, place the beaker in a 60°C water bath and keep it warm for 30 minutes. (3) Add 50ml of ultrapure water to a beaker and turn on the magnetic stirrer. Then add 0.1g of dimethyl diallyl ammonium chloride (DMDAAC) polymer and continue stirring for more than 30min. After dissolution, place it in a 60°C water bath and keep it warm for 30min. (4) In a water bath at 60°C, add 50 ml of polyphosphoric ferric chloride solution to 200 ml of agar sample solution and stir for 120 min; (5) In a water bath at 60°C, add 50 ml of dimethyl diallyl ammonium chloride (DMDAAC) polymer solution to the solution that has been flocculated with polyphosphoric ferric chloride, stir for 120 min until flocculent matter settles, and filter while hot.

[0020] (6) Add 300 ml of anhydrous ethanol to the filtrate, let it stand at 4°C for 12 h, centrifuge, then wash with water several times and freeze-dry under vacuum. After pulverization, agarose was obtained, and its sulfate content was determined to be 0.065%, electroosmosis 0.09, and gel strength 1100 g / cm³. 2 The whiteness is 92.87, such as Figure 1-3 As shown.

[0021] Example 3: This example provides a method for preparing low-electro-endoosmotic agarose, comprising the following steps: (1) Take 4g of agar, add 100ml of ultrapure water, let it swell for 2 hours, put it into boiling water and stir until dissolved, and put it in a 70°C water bath for more than 30 minutes. (2) Add 50ml of ultrapure water to a beaker and turn on the magnetic stirrer. Then add 2g of polyferric sulfate and continue stirring for more than 30 minutes. After dissolution, place the beaker in a 70°C water bath and keep it warm for 30 minutes. (3) Add 50ml of ultrapure water to a beaker and turn on the magnetic stirrer. Then add 2g of dimethyl diallyl ammonium chloride (DMDAAC) polymer and continue stirring for more than 30min. After dissolution, place it in a 70°C water bath and keep it warm for 30min. (4) In a water bath at 70°C, add 50 ml of polyferric sulfate solution to 100 ml of agar sample solution and stir for 120 min; (5) In a water bath at 70°C, add 50 ml of dimethyl diallyl ammonium chloride (DMDAAC) polymer to the solution that has been flocculated with polyferric sulfate, stir for 120 min until flocculent material settles, and filter while hot.

[0022] (6) Add 400 ml of anhydrous ethanol to the filtrate, let it stand at 4°C for 12 h, centrifuge, then wash with water several times and freeze-dry under vacuum. After pulverization, agarose was obtained, and its sulfate content was determined to be 0.062%, electroosmosis 0.08, and gel strength 1150 g / cm³. 2 The whiteness is 92.21, such as Figure 1-3 As shown.

[0023] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the technical scope of the present invention. For those skilled in the art, minor modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A method for preparing low-electro-endoosmotic agarose, characterized in that: The steps include the following: (1) Prepare an agar solution with a mass fraction of 0.5% to 5%, swell at room temperature, heat to 90 to 100°C and stir to dissolve, and place in a water bath at 40 to 90°C for constant temperature; (2) Prepare an inorganic flocculant aqueous solution with a mass fraction of 0.1% to 2%, stir and dissolve it at room temperature, and place it in a water bath at 40 to 90°C for constant temperature; the inorganic flocculant used is one of polyferric chloride, polyferric aluminum sulfate, polyphosphoric ferric chloride, and polyferric sulfate; (3) Prepare an aqueous solution of dimethyl diallyl ammonium chloride polymer with a mass fraction of 0.1% to 4%, stir and dissolve at room temperature, and place in a water bath at 40 to 70°C for constant temperature; (4) Add the agar solution obtained in step (1) to the inorganic flocculant solution prepared in step (2) under stirring. The volume ratio (W / W) of the two solutions is 1:1 to 10:

1. Stir for 10 to 420 min. (5) Add the solution obtained in step (4) to the organic flocculant solution prepared in step (3) under stirring. The volume ratio (W / W) of the two solutions is 1:1 to 10:

1. Mix and stir for 10 to 420 min. (6) Filter or centrifuge the solution obtained in step (5) while it is still hot, and collect the filtrate or supernatant; (7) Add anhydrous ethanol to the filtrate obtained in step (6), let it stand at 4°C for 6 to 24 hours, centrifuge, then wash with water several times and freeze dry under vacuum. (8) Pulverize the dried agarose to obtain low electro-endoosmotic agarose.

2. The method for preparing low-electro-endoosmotic agarose according to claim 1, characterized in that: The agar raw material used in step (1) is derived from marine red algae.

3. The method for preparing low-electro-endoosmotic agarose according to claim 2, characterized in that: The marine red algae mentioned are any one or more of the following: Gracilaria, Lithops, Gracilaria, Isophyllaria, Porphyra, and Laminaria japonica.

4. The method for preparing low-electro-endoosmotic agarose according to claim 1, characterized in that: In step (7), after the agarose filtrate and anhydrous ethanol solution are mixed, the volume fraction of ethanol in the solution is 50% to 80%.

Images