阅读说明：本技术 一种高品质琼脂糖的制备方法 (Preparation method of high-quality agarose ) 是由 张全斌 鞠豪 耿丽华 岳洋 王晶 于 2019-11-19 设计创作，主要内容包括：本发明公开了一种联合絮凝法从琼脂中分离制备高品质琼脂糖的方法。该方法包括以下步骤：配制一定浓度的琼胶溶液、加热溶解；配制适宜浓度的聚合氯化铝和聚丙烯酰胺溶液；在40～100℃的水浴锅中保温一段时间；在搅拌的琼胶溶液中加入一定体积的聚合氯化铝溶液并搅拌一定时间；在上述反应的溶液中,加入一定体积的聚丙烯酰胺溶液并搅拌一定时间；过滤,去掉滤渣,收集滤液；滤液降温成为凝胶、凝胶冻结脱水、洗涤、晾干,粉碎得到琼脂糖。本发明使用的制备方法是通过聚合氯化铝无机阳离子絮凝剂和聚丙烯酰胺阳离子絮凝剂的联合使用去除琼脂糖溶液中的硫琼胶和色素,凝胶性好,能够达到分子生物学等领域对高品质琼脂糖的要求。(The invention discloses a method for separating and preparing high-quality agarose from agar by a combined flocculation method. The method comprises the following steps: preparing agar solution with a certain concentration, and heating for dissolving; preparing polyaluminium chloride and polyacrylamide solution with proper concentration; keeping the temperature in a water bath kettle at 40-100 ℃ for a period of time; adding a certain volume of polyaluminum chloride solution into the stirred agar solution and stirring for a certain time; adding a certain volume of polyacrylamide solution into the solution obtained by the reaction, and stirring for a certain time; filtering, removing filter residue, and collecting filtrate; cooling the filtrate to gel, freezing and dehydrating the gel, washing, airing and crushing to obtain the agarose. The preparation method provided by the invention removes the sulfur agar and the pigment in the agarose solution by the combined use of the polyaluminium chloride inorganic cationic flocculant and the polyacrylamide cationic flocculant, has good gelling property, and can meet the requirements of fields such as molecular biology and the like on high-quality agarose.)

1. A preparation method of high-quality agarose is characterized by comprising the following steps:

(1) adding water to swell the agar, heating to 90-100 ℃, stirring and dissolving to prepare 0.5-5 mass percent of agar solution, and standing at a constant temperature of 40-90 ℃;

(2) preparing 0.2-0.6% of polyaluminium chloride inorganic flocculant, and standing at a constant temperature of 40-90 ℃;

(3) preparing 0.2-1.2% of polyacrylamide organic flocculant, and placing at a constant temperature of 40-70 ℃;

(4) adding the agar solution obtained in the step (1) into the polyaluminum chloride solution prepared in the step (2) under stirring, wherein the material-liquid ratio of the two solutions is 1: 1-10: 1, and stirring for 10-360 min;

(5) stirring the solution obtained in the step (4), adding the polyacrylamide solution prepared in the step (3) into the solution, wherein the material-liquid ratio of the two solutions is 1: 1-10: 1, and mixing and stirring for 10-360 min;

(6) immediately filtering or centrifuging the solution obtained in the step (5), and collecting filtrate or supernatant;

(7) standing the filtrate at a temperature lower than 40 ℃ to form gel;

(8) cutting the gel into strips, freezing at low temperature to freeze water in the gel into ice; unfreezing and dehydrating the frozen gel strip, washing with water and drying in the air, drying the dried agar strip, and crushing to obtain agarose; or squeezing the gel for dewatering, and drying to obtain agarose.

2. The method for producing high-quality agarose according to claim 1, wherein: the agar used in the step (1) is prepared from marine red algae of Gelidium amansii, Gracilaria, Porphyra and the like.

3. The method for producing high-quality agarose according to claim 1, wherein: the polyaluminium chloride inorganic flocculant used in the step (2) is a polyaluminium chloride aqueous solution, the polyacrylamide organic flocculant used in the step (3) is a polyacrylamide aqueous solution, and the polyacrylamide is cationic polyacrylamide.

4. The method for producing high-quality agarose according to claim 1, wherein: the high-quality agarose has a sulfate radical content of not more than 0.13%, an electro-endosmosis of not more than 0.13, and a gel strength of not less than 900g/cm²The whiteness is more than or equal to 83.

Technical Field

The invention relates to the field of biochemistry, in particular to a production process for extracting and preparing high-quality agarose from agar by a flocculant combination method, namely a preparation method of the high-quality agarose.

Background

Agar, jelly, agar jelly, and agar, is a substance extracted from red algae of Enteromorpha, Gelidium, Gracilaria, Pterocephalus, and Porphyra, and is widely used in many fields such as food, medicine, daily chemical industry, and biological engineering. Agar is a mixture of agarose derivatives which are formed by transiting neutral sugar (containing different methyl groups) consisting of an agarose skeleton into agarose derivatives with high-charge acidic substituents through a series of sulfate groups and pyruvic acid groups with low charges, and mainly consists of agarose and sulfur agar.

Agarose, also known as agar, is a natural polysaccharide with high gel property, widely exists in agar, is a sulfate-free nonionic polysaccharide, and is a linear macromolecular polysaccharide which is composed of disaccharide units of 1, 3-linked beta-D-galactose and 1, 4-linked alpha-3, 6-lactonic galactose; the sulfur agar is agarose used as a basic skeleton, is combined with polar groups such as sulfate, carboxylic acid, pyruvic acid, uronic acid and the like, and is combined with inorganic ions such as calcium, magnesium and the like. Because of the influence of sulfate and carboxyl, agarose gel containing sulfur agar is used as an electrophoresis support, and has the defects of large electroosmosis, strong capability of adsorbing basic dye and protein, non-specific precipitation of some proteins caused in an acid buffer solution, poor transparency and the like. Agarose is ideally a neutral polysaccharide free of polar groups, but it is practically difficult to obtain a neutral polysaccharide completely free of polar groups. The size of electro-endosmosis is one of the main differences between agar and agarose, and is also a main index for measuring the quality of agarose. When agarose gel electrophoresis is carried out, excessive sulfur agar in agarose enables the gel to generate convection under a direct current electric field, so that the migration and separation of a sample are influenced, and the electrophoresis is directly expressed as electro-endosmosis and protein adsorption. The size of the electro-endosmosis is mainly related to sulfate groups and carboxyl groups contained in agarose, so that the polysaccharide containing sulfate groups and carboxyl groups is mainly removed in the agarose preparation process, the electro-endosmosis is reduced, and a small amount of algae protein, insoluble precipitate, soluble salts and pigments doped in the agar production process are removed, so that the agarose purity and the product stability are improved.

The agarose has good physical, chemical and thermal stability, is the best quality of agar series products, and has wide application in medical science and scientific research. Because of the low chemical complexity, agarose rarely causes denaturation and adsorption to sensitive biological macromolecules, and is an ideal inert carrier which can be used as a support for gel electrophoresis and an affinity chromatography carrier; in the fields of biochemistry and medicine, agarose is an important reagent for researching virus phage, bacteria, clinical examination, biochemical analysis, protein, nucleic acid, antigen and antibody, and can be used for separating and purifying polysaccharide and preparing medicines; the agarose can also be used as a solid culture medium, connected with hyaluronic acid and the like to be used as a polypeptide carrier, used as a tissue engineering scaffold, prepared into a microcapsule artificial membrane and the like. Agarose has become an indispensable substance in the course of scientific research.

The purification technology of the agar sugar begins in the last century, and agarose is firstly separated from the agar in 1937 when the composition and the structure of the agar are researched by barren woods. In 1961, Swedish scientists S.Hjerten firstly utilized agar to purify agarose, the agarose is increasingly researched after the agarose is found to have excellent service performance for the first time, and is started to be used for industrial production, in 1971, Duckworth and Yaphe establish a method for further grading the agar to obtain agarose, and in 1974, Arnott proposes a gel mechanism of the agarose and indicates that agarose molecules exist in a double helix structure form in a left-handed form. M. djibourov in 1989 gave a model of agarose gel processing by low angle light scattering SAXS. Watase also gives the physicochemical structure of agarose gel at a certain concentration by methods such as DSC, XRD, NMR, etc. The research on agarose is far earlier than that in China, at present, the countries for the industrial production of agarose mainly comprise Spain, America, Switzerland, British and the like, and the agarose does not form large-scale production in China. The agarose extraction technology reported in China mainly comprises a dimethyl sulfoxide (DMSO) method, a cetylpyridinium chloride (CPC) method, a sodium iodide method, an ammonium sulfate method, a chitin method, a Ravannao method, a polyethylene glycol method, a DEAE-cellulose method, an acetylation method, an EDTA sodium salt method, a chelation method, a urea method and the like, and the methods except the DEAE-cellulose method commonly used at present have the problems of low yield, poor product color, low gel strength, high production cost and the like. Although the DEAE cellulose method is a relatively mature process, the DEAE cellulose has poor applicability to agars from different sources and is expensive, so that the application range and the large-scale production of the agaroses are limited. Therefore, the search for new agarose production technology is of great significance.

The key of the technology for producing agarose by using agar is to separate agarose from sulfur agar (agarose), wherein a flocculating agent mainly comprises groups with positive (negative) electricity and particles which are difficult to separate and have negative (positive) electricity in water or particles which are difficult to separate are close to each other, the potential of the particles is reduced, the particles are in an unstable state, and the particles are concentrated by utilizing the polymerization property of the particles and are separated by a physical or chemical method. Sulphur agar is negatively charged due to linkage of sulphate and carboxyl groups and we bind it by a flocculating agent, producing a flocculent precipitate which is separated from the agar solution, thus obtaining an agarose solution.

Disclosure of Invention

The invention aims to provide a flocculant-flocculation combined method for separating and preparing low-sulfate-radical agarose from agar, the method is simple, efficient, economical, environment-friendly and easy to operate and implement, the sulfate radical content of the prepared agarose is less than 0.13%, the gel property is good, and the requirements of fields of molecular biology and the like on agarose purity can be met.

A method for separating and preparing high-quality agarose from agar by a flocculant combination method, wherein the electro-endosmosis of the high-quality agarose is less than 0.13, the sulfate radical content is less than 0.13%, the whiteness of a 1.5% solution is more than 83, and the gel strength is more than 900g/cm²The preparation method comprises the following steps:

(1) adding water to swell the agar, heating to 90-100 ℃, stirring and dissolving to prepare 0.5-5 mass percent of agar solution, and standing at a constant temperature of 40-90 ℃;

(2) preparing 0.2-0.6% of polyaluminium chloride inorganic flocculant, and standing at a constant temperature of 40-90 ℃;

(3) preparing 0.2-1.2% of polyacrylamide organic flocculant, and placing at a constant temperature of 40-70 ℃;

(4) adding the agar solution obtained in the step (1) into the polyaluminum chloride solution prepared in the step (2) under stirring, wherein the material-liquid ratio of the two solutions is 1: 1-10: 1, and stirring for 10-360 min;

(5) stirring the solution obtained in the step (4), adding the polyacrylamide solution prepared in the step (3) into the solution, wherein the material-liquid ratio of the two solutions is 1: 1-10: 1, and mixing and stirring for 10-360 min;

(6) immediately filtering or centrifuging the solution obtained in the step (5), and collecting filtrate or supernatant;

(7) standing the filtrate at a temperature lower than 40 ℃ to form gel;

(8) cutting the gel into strips, freezing at low temperature to freeze water in the gel into ice; unfreezing and dehydrating the frozen gel strip, then washing with water for 3-6 times and drying in the air, drying the dried agar strip, and crushing to obtain agarose; or squeezing the gel for dewatering, and drying to obtain agarose.

The agar used in the step (1) is prepared from marine red algae of Gelidium amansii, Gracilaria, Porphyra and the like. The polyaluminium chloride inorganic flocculant used in the step (2) is a polyaluminium chloride aqueous solution, the polyacrylamide organic flocculant used in the step (3) is a polyacrylamide aqueous solution, and the polyacrylamide is cationic polyacrylamide.

The method removes sulfur agar and pigment in the agar by combining two flocculation methods, thereby preparing the agarose with low sulfate radical content and low electroosmosis and realizing agarose decoloration at the same time.

THE ADVANTAGES OF THE PRESENT INVENTION

The preparation method used by the invention is to remove the sulfur agar and the pigment in the agarose solution by the combined use of the polyaluminium chloride inorganic cationic flocculant and the polyacrylamide cationic flocculant, is simple, efficient, economic and easy to operate and implement, and the prepared agarose has the sulfate radical content of less than 0.13 percent, the electro-endosmosis of less than 0.13 percent, the whiteness of more than 83 and good gel property, and can meet the requirements of the fields of molecular biology and the like on high-quality agarose.

The recovery rate of the agarose to the agar is more than 60 percent, and the produced product has stable property at normal temperature.

The advantages of the invention will become clearer and the effect of the method can be visually seen after reading the specific embodiment of the invention with the attached drawings.

Drawings

FIG. 1 is a diagram showing the state of dissolved agar used in the present invention,

FIG. 2 is a view showing the state of an agarose solution obtained by the present invention.

Detailed Description

The invention discloses a method for separating and preparing high-quality agarose from agar by a flocculant combination method, which comprises the following steps:

(1) preparing 0.5-5 mass percent of agar solution, adding water at room temperature for swelling, heating to 90-100 ℃, stirring for dissolving, and standing at a constant temperature of 40-90 ℃;

(2) preparing 0.2-0.6% of polyaluminium chloride aqueous solution, stirring and dissolving, and standing at the constant temperature of 40-90 ℃;

(3) preparing 0.2-1.2% polyacrylamide aqueous solution, stirring for dissolving, and standing at a constant temperature of 40-90 ℃;

(4) stirring the agar solution obtained in the step (1), adding the polyaluminum chloride solution prepared in the step (2) into the agar solution, wherein the material-liquid ratio of the two solutions is 1: 1-10: 1, and stirring for 10-360 min;

(5) stirring the solution obtained in the step (4), adding the polyacrylamide solution prepared in the step (3) into the solution, wherein the material-liquid ratio of the two solutions is 1: 1-10: 1, and mixing and stirring for 10-360 min;

(6) filtering or centrifuging the solution obtained in the step (5) while the solution is hot, and collecting filtrate (or supernatant);

(7) standing the filtrate at 40 deg.C to form gel, cutting the gel into strips, and freezing at low temperature to freeze water in the gel. Thawing and dehydrating the frozen gel strips, washing with water for 3-6 times, and air drying;

(8) and drying the aired agar strip, and crushing to obtain the agarose.