RU2302429C1 - Method for producing fucoidan from laminaria - Google Patents

Abstract

FIELD: bioengineering.

SUBSTANCE: method involves producing acids polysaccharide fucoidan from laminaria by crushing raw material by treating it with 0.5-1.0% food acid solution during 4-5 h and subjecting acid extract to ultrafiltration on membrane of 100-300 kDa with following end product drying by applying lipophilic or spraying or vacuum drying method.

EFFECT: high purity of end product.

Description

The invention relates to a process for processing brown algae and for the production of a biologically active polysaccharide, in particular fucoidan for use in preventive medicine, microbiology, chemical pharmaceutical, cosmetology, food industry, etc.

An acidic polysaccharide-fucoidan is a water-soluble high molecular weight compound containing many side chains. It has various biological activities, including anticoagulation, cancer prevention, antiviral, lipid-lowering.

Known methods for producing from brown algae acidic polysaccharide - fucoidan:

- RF patent №2247574 dated 12.26.2002, IPC А61К 35/80 "Means with anticoagulant and immunotropic effects", including the treatment of brown alga Fucus evanescens with ethanol, then its drying and grinding, treatment with water first at room temperature and then at temperature 50-60 ° C. The concentration of the extract, separation of the precipitate, obtaining the sodium salt of fucoidan by planting it with a standard, washing with water and drying with ethanol;

- RF patent No. 2135518 dated 06/17/1998 "Method for producing water-soluble brown algae polysaccharides." The method involves the processing of fresh or frozen, or dry algae with solvents (ethanol, acetone, chloroform), sequential extraction of polysaccharides of 0.1 N hydrochloric acid at room temperature and water at 50-60 ° C. The preparation of fucoidan is carried out using hydrophobic chromatography;

- RF patent No. 2028153 dated 02/09/1995 "Method for the production of biologically active substances from kelp." The method involves treating fresh, frozen or dry brown algae with an alcohol solution, followed by precipitation from an alcohol solution of a complex of laminaran with fucoidan. To separate fucoidan, the precipitate is treated with a 0.4% hydrochloric acid solution at room temperature and water at a temperature of 50-60 ° C for 2 hours; then, a 0.5 M solution of cetavlon is added to the resulting solution to convert acidic polysaccharides to the precipitate. The resulting precipitate is dissolved in a 0.4% hydrochloric acid solution, and then treated with an ethanol solution to obtain fucoidan;

- RF patent No. 2240816 dated 07/28/03, wherein brown algae is treated with ethanol, the treated algae is extracted with hydrochloric acid solution, the extract is concentrated to 1/5 of the original volume in an ultrafiltration unit with a membrane having pores from 5 to 100 kDa, neutralized to pH 6.0 sodium hydroxide solution and evaporated on a rotary evaporator to 1 liter The resulting concentrate is neutralized, and then fucoidan is precipitated with ethanol and dried;

     - RF patent No. 2233104, 09.12.02, "A method for the complex processing of brown algae to obtain iodine-containing and polysaccharide products", while the brown algae is extracted with a water-alcohol solution, the solid residue is treated with 1% hydrochloric acid solution for 6-10 hours at room temperature . The resulting mass is heated to a temperature of 45-55 ° C for 4-6 hours. The resulting acid extract is first neutralized to pH 7, evaporated in vacuo at a temperature of 50-55 ° C, and then fucoidan is isolated by precipitation with ethanol and dried.

The disadvantages of these methods is that the raw materials are first treated with an alcohol-containing solvent, and either expensive chemicals (alcohol or cetavlon) or expensive equipment, namely hydrophobic chromatography, rotary evaporators, are used to obtain the acid polysaccharide-fucoidan. Drying is also carried out mainly with alcohol.

The closest in technical essence the method of producing acidic polysaccharides is the method according to patent No. 2041656 of 23.10 1991, IPC 6 A23L 1/337 "Method for the preparation of food semi-finished products from laminaria algae", including grinding brown algae, processing them with food acid at pH 1-3 in for 2-3 hours at room temperature, to obtain a gel-like product, which also includes acid polysaccharide - fucoidan.

The disadvantage of this method is to obtain only a complex acid polysaccharide, which includes both low molecular weight products and high molecular weight polysaccharides, in particular fucoidan.

The objective of the invention is to obtain a pure acidic polysaccharide - fucoidan while maintaining its native properties, as well as increasing the purity of the final product.

The problem is solved by grinding raw materials, processing brown algae with an acid solution with a pH of 1-3, separating the acid extract and obtaining the final product from it, according to the invention, the processing of brown algae is carried out with a 0.5-1% acid solution for 4-5 hours at room temperature, the final product is obtained by one-time ultrafiltration on a membrane from 100 to 300 kDa, and then dried. Moreover, the drying is carried out using freeze drying, or spray, or vacuum.

The proposed technical solution meets the criteria of "novelty", "inventive step" and "industrial application".

The essence of the method is as follows.

Raw algae (or dry, after preliminary soaking in water at 80 ° C for 2-3 hours) is ground on a minced top and an acid treatment is carried out with a 0.5-1.0% solution of food acid (pH 1-3) for 4- 5 hours at room temperature. The acid extract is separated from the solid fraction by filtration through a sieve and a fabric filter and sent to ultrafiltration with a membrane from 100 to 300 kDa, the resulting final product is dried.

New in the claimed technical solution is that the source of raw materials - brown algae is treated with a 0.5-1.0% acid solution for 4-5 hours at room temperature, and the final product is obtained by one-time ultrafiltration on a membrane from 100 to 300 kDa of an acid extract with subsequent drying.

Processing of raw materials with a 0.5-1.0% acid solution with a pH of 1-3 for 4-5 hours at room temperature allows you to maximize the preservation of the native properties of the final.

The use of one-time ultrafiltration on a membrane from 100 to 300 kDa allows you to get a high molecular weight acid polysaccharide - fucoidan with a molecular weight of 100,000 to 300,000 daltons, to completely get rid of low molecular weight polysaccharides that reduce the native activity of the drug and cause side effects, in particular allergies, and get a polysaccharide possessing high biological activity, namely anti-inflammatory, antioxidant and anticoagulation, antiviral. The yield of the final product is 6.0-8.0% of dry raw materials.

The drying of the final product is carried out without the use of alcohol, which significantly reduces the drying time and labor costs.

The inventive method is characterized by simplicity and manufacturability, because It mainly includes extraction and ultrafiltration. Using the last dose, native fucoidan is obtained with a purity of 92-96%.

Quantification of fucoidan was carried out spectrophotometrically by the reaction of fucoidan with thioglycolic acid in sulfuric acid and an analysis of a fucoidan sample by NMR on C ¹³ and H ¹ nuclei.

The resulting fucoidan is a white powder, highly soluble in water and well absorbed in the gastrointestinal tract. This is due to the fact that the molecular weight of fucoidan is 100,000-300,000 daltons. The molecular weight and quantity of sulfate groups ensures its dissolution in water and acidic solutions, which makes it possible to use fucoidan therapeutic using the claimed technology.

The acute toxicity of the drug was studied in non-inbred mice by adding it to food and introducing it under the skin. The resulting fucoidan is non-toxic when taken in doses up to 500 mg / kg.

Concrete example

Example 1

10 kg of raw kelp are ground in a minced meat top and placed in an acidic solution (12 l of water and 1 l of 80% acetic acid at pH 3 for 5 hours at room temperature). The resulting mixture was filtered through a sieve and a fabric filter. The resulting acidic extract is passed through an ultrafiltration unit with a membrane having pores of 200 kDa. In the ultrafiltration process, fucoidan with a molecular weight of 200,000 daltons is obtained, which is dried by freeze drying. The resulting final product, fucoidan, is a white powder, readily soluble in water.

Its output is 70 g Product purity 94%.

Example 2

10 kg of frozen raw kelp are thawed, ground in a minced top and placed in a 1% hydrochloric acid solution for 4 hours at room temperature. The resulting mixture was filtered through a sieve and a fabric filter, then the acid extract was passed through an ultrafiltration unit with a membrane having pores of 100 kDa. In the ultrafiltration process, fucoidan with a molecular weight of 100,000 daltons is obtained, which is then dried by spray drying.

The product is a white powder, soluble in water.

Its output is 60 g Product purity - 96%.

Example 3

1.3 kg of dry kelp is soaked in 15 l of water at 80 ° C for 2-3 hours, ground in a minced top and placed in a 0.8% hydrochloric acid solution for 5 hours at room temperature. Then the resulting mixture is filtered through a sieve and a fabric filter, the acidic extract is separated and passed through an ultrafiltration unit with a membrane having pores of 300 kDa. After ultrafiltration, fucoidan with a molecular weight of 300,000 daltons is obtained, which is then dried by drum drying.

The product is a white powder, soluble in water.

Its output is 80 g Product purity 92%.

Currently, an experimental batch of fucoidan in the amount of 150 g has been received at the experimental base of the Federal State Unitary Enterprise TINRO-Center.

Claims (2)

1. A method of producing fucoidan from kelp, including grinding the raw material, processing with an acid solution, separating the acid extract and obtaining the final product from it, characterized in that the processing of kelp is carried out with a 0.5-1.0% solution of food acid for 4- 5 hours, and the final product is obtained by ultrafiltration of the acid extract on a membrane with a pore size of from 100 to 300 kDa, and then dried. 2. The method according to claim 1, characterized in that the drying of the final product is carried out either by lyophilic, or spray, or vacuum drying.