RU2240329C2 - Method for preparing biologically active acid sulfated polysaccharide fucoidan from marine brown algae - Google Patents

Abstract

FIELD: food, perfume-cosmetic industry, medicine.

SUBSTANCE: milled marine brown algae are extracted 3-4-fold with water by countercurrent method at temperature 20-60^oC. Extract is separation by filtration and the end product is precipitated with ethyl alcohol in consumption of absolute ethyl alcohol 0.7-1.1 ml per 1 ml of extract. Invention provides significant shortening the technological process, to reduce consumption of reagents and to enhance the yield of ready product by 40%.

EFFECT: improved preparing method.

3 tbl, 9 ex

Description

The invention relates to the integrated processing of marine brown algae, and in particular to the preparation of a biologically active acidic water-soluble sulfated polysaccharide from marine brown algae - fucoidan, and can be used in food, perfume and cosmetic industries and in medicine.

A known method of producing water-soluble polysaccharides of brown algae (RF Patent No. 2135518. MKI C 08 V 37/00), which includes a number of stages:

1. Subsequent treatment with dry (50% aqueous ethanol) or preferably with fresh or freshly frozen brown algae with ethanol (1: 1 by weight), acetone and chloroform, where salts, partially polyphenols, low molecular weight substances (including mannitol, mono- , oligosaccharides, amino acids, peptides, pigments, lipids, iodine-containing substances, vitamins A, B, etc.). About 30-40% of the dry weight of the algae is extracted into solvents. Further, an alcoholic extract of brown algae can be directly used in the cosmetic industry and medicine as an iodine-containing preparation, a treatment for tonsillitis, and mucous diseases. Substances extracted with acetone and chloroform can also be used in the cosmetics industry.

2. Sequential extraction of polysaccharides from algae residues 0.1 N Hcl with the addition of 40% formaldehyde (1 ml / l) at room temperature and water at elevated temperature (60-70 ° C). Sequential extraction at various temperatures allows the separation of the main carbohydrate components according to molecular weights, degree of branching, and fucoidans according to the monosaccharide composition already at the isolation stage.

3. The obtained extracts are separately applied to columns with a hydrophobic carrier (polytetrafluoroethylene). Lamaran is adsorbed by a hydrophobic carrier. Highly sulfated fucoidans easily elute with water. A stepwise gradient of alcohol in water is eluted from the laminaran carrier in a mixture with low sulfated fucoidan (5% aqueous ethanol) and the laminaran fraction (10-, 15% aqueous ethanol).

4. Fucoidans from the solution are precipitated completely with 80% aqueous ethanol or sequentially with 60 and 80% aqueous ethanol. Fractions eluted from polychrome with 1-, 5-, 10-, 15% aqueous ethanol are freeze-dried.

5. The hydrophobic carrier is regenerated with ethanol, which is more hydrophobic than laminaran, eluting from the column. If necessary (for example, in the case of strong pigmentation of the carrier after passing the extract), additional regeneration is carried out with 0.5-1.0 M solutions of alkali and / or hydrochloric acid, depending on the nature of the pigment.

This method is focused on the fractionation of fucoidans and laminaranes in order to obtain standard fractions of these biologically active, heterogeneous polysaccharides.

The complexity, multi-stage process, the use of a significant amount of various chemicals (ethanol, acetone, chloroform, hydrochloric acid, formaldehyde) and materials (polytetrafluoroethylene) indicate the possibility of using this method mainly in laboratory conditions.

A known method of producing biologically active substances from kelp by extraction of crushed raw materials, separation of the extract, its evaporation and isolation of the target product (RF Patent No. 2028153, MKI A 61 K 35/80 - prototype). According to this method, the raw materials are first dried at 50 ° C, after which they are freed from mechanical impurities. Then the raw material is ground and extracted with 90-96% ethanol in the ratio (raw material: ethanol) 1: 6 in a Soxhlet apparatus at the boiling point of the extractant for 3 hours. The filtered alcoholic extract is used to obtain mannitol. Then, after removing the residual alcohol from the algae, the latter are extracted twice with water in a ratio of 1:10 at a temperature of 80 ° C for 30 minutes, the aqueous extracts are combined, filtered, evaporated under vacuum at a residual pressure of 0.3 atm and a temperature of 70 ° C to a density of 1 05-1.06 g / cm ³ ; the water-soluble polysaccharide complex is precipitated with 96% ethanol from the cooled extract in the ratio (extract: ethanol) 1: 2, the complex is filtered, purified three times with 96% ethanol, and dried. The alcoholic mother liquor remaining after cooling the water-soluble polysaccharide complex is evaporated to 1/10 of the original volume, after which the condensed extract is treated with 80% ethanol in the ratio (extract: ethanol) 1: 3. The resulting precipitate (laminaran and fucoidan) is centrifuged. To separate laminaran and fucoidan, a centrifuged precipitate is treated with a 0.4% hydrochloric acid solution at a ratio (precipitate: hydrochloric acid solution) of 1: 5 at room temperature for 2 hours; then, a 0.5 M solution of cetavlon is added to the resulting solution to convert the acid polysaccharides into a precipitate. The precipitate was separated, and the supernatant was treated with 80% ethanol (to precipitate laminaran) in the ratio of supernatant: ethanol 1: 3. The resulting laminaran is filtered, purified with 96% ethanol, and dried. The precipitate formed after the addition of cetavlon was dissolved in a 0.4% hydrochloric acid solution in the ratio (precipitate: hydrochloric acid solution) 1:10 and treated with 70% ethanol in the ratio (solution: ethanol) 1: 3, a precipitate formed fucoidan, which is filtered, purified, dried. After that, the remaining pulp of algae is sent to obtain alginate.

The disadvantages of this method are:

1. Alcoholic extraction of the feedstock for 3 hours at the boiling point and distillation of alcohol residues from the extracted algae, which does not allow alginates with high molecular weight to be obtained from algae. Elevated temperatures during the above processes reduce the molecular weight of fucoidan, which leads to an increase in its losses during isolation from extracts at the filtration stage.

2. The processes for producing mannitol, laminaran, and fucoidan are multistage, energy-intensive, and time-consuming.

3. The method requires the use of hydrochloric acid and cetavlon.

In view of the foregoing, the described method can only be used in laboratory conditions when performing technological studies.

The invention is aimed at solving the problem of reducing the material and energy intensity of the process, its duration and increasing the yield of the target product.

This is achieved by the fact that the extraction of algae is carried out 3-4 times with water at a temperature of 20-60 ° C, and the precipitation is carried out with ethyl alcohol at a flow rate of absolute alcohol of 0.7-1.1 ml per 1 ml of extract. The extracted algae are used to produce high molecular weight alginates, and laminaran, mannitol, mineral concentrates and algae extracts are obtained from a water-alcohol mixture.

The method is as follows.

The process begins with the extraction of the first portion of the raw material with water at a ratio by weight of algae and water of 1:12 for 20-30 minutes at a temperature of 45-50 ° C. Then the extract is separated, and the algae is extracted one more time under the same conditions, and after separation of the second extract is directed to obtain alginates. Next, the process is carried out in a predetermined mode as follows.

A portion (example 1) of air-dried crushed algae weighing 2 kg (mass fraction of water 7.9%) is poured with the first extract from the previous portion (t = 45-50 ° C) V = 23 L. The extraction is carried out for 30 minutes with stirring. The resulting extract with a volume of 11 l is poured into a container for collecting the finished extracts.

Once the extracted portion of the algae is poured into the second extract from the previous portion of the algae. The extraction is carried out for 30 minutes with stirring at a temperature of 45-50 ° C.

The resulting extract is served on a fresh portion of algae, which is extracted as described above. The finished extract with a volume of 11 l is sent to a container for collecting extracts.

A twice-extracted portion of algae is poured into 24 liters of water with a temperature of 45-50 ° C. Extraction is carried out for 30 minutes with stirring. The resulting extract is sent to a once-extracted subsequent portion of algae and the process is carried out until the finished extract is obtained, as described above. The resulting extract with a volume of 11 l is sent to a container for collecting extracts. The triple-extracted portion of the algae is sent to produce alyninate.

Thus, after extraction in the operating mode of three servings of raw materials, 2 kg each, the total volume of extracts is 33 liters. The combined extracts are separated from solids by passing through a filter material. Then, 28.00 l of 96% ethanol is added to the filtrate with stirring, which in terms of absolute ethanol is 26.88 l. Alcohol consumption of 0.8 ml per 1 ml of extract.

The sulfated polysaccharide released in the form of fibers is separated after 20 minutes from the liquid phase by filtration through a filter material. The liquid phase is directed to obtain mannitol.

The selected target product is treated with 96% ethyl alcohol to remove moisture, dried in air and crushed. The yield of 188 g with a moisture content of 9.41%, i.e. the absolutely dry mass of the target product is 170.3 g or 3.08% of the absolutely dry mass of the extracted algae.

The resulting product is a white powder with a grayish or cream tint, when dissolved in water forms viscous solutions, so the viscosity of a 0.25% solution is 2.5 ° Oe. The mineral part is 36.1-37.2%, SO $\genfrac{}{}{0ex}{}{\text{-2}}{\text{4}}$ - 15.1-16.5%.

Examples 2-4 are carried out under the conditions of example 1, but at different temperatures of extraction. The data are presented in Table 1. The increased viscosity of the filtrate with a decrease in the yield of the finished product at an extraction temperature below 20 ° C makes it inappropriate to use temperatures up to 20 ° C. An increase in extraction temperatures of more than 60 ° C causes a decrease in the molecular weight of both alginic acids in algae and fucoidan, which leads to an increase in the duration of the filtration process during the separation of fucoidan from the water-alcohol mixture and to its losses.

Examples 5-9 are carried out under the conditions of example 1, but with a different ratio of extract and absolute ethyl alcohol. The data are presented in table 2

The consumption of absolute ethyl alcohol of less than 0.7 ml per 1 ml of extract does not allow to isolate the polysaccharide, the consumption of more than 1.1 ml / ml is impractical due to the unjustified increase in reagent consumption without increasing the yield of the target product.

Comparative characteristics of the known method and the claimed are presented in table.3

The positive effect of the proposed method for producing fucoidan is to reduce the duration of the process by more than six times in the absence of technological operations performed at a temperature of more than 60 ° C; replacing non-regenerated reagents (hydrochloric acid and cetavlon) with regenerated (ethyl alcohol) while increasing the yield of the finished product by 40%. All this allowed to introduce the proposed method in a production environment.

Claims (1)

A method of obtaining a biologically active acidic sulfated polysaccharide from marine brown algae - fucoidan - by extraction of crushed raw materials, separation of the extract and isolation of the target product, characterized in that the extraction of algae is carried out 3-4 times with water at a temperature of 20-60 ° C, and the deposition is carried out ethyl alcohol at a flow rate of absolute alcohol of 0.7-1.1 ml per 1 ml of extract.