RU2793805C1 - Method for obtaining polysaccharides from meal (processing waste) of brown algae - Google Patents

Abstract

FIELD: chemical; pharmaceutical; food industries.

SUBSTANCE: invention relates to the methods for obtaining polysaccharides from carbohydrate-containing seaweed processing waste. A method for obtaining polysaccharides from brown algae meal (processing waste) is disclosed, it consists in extracting algae thalli, division of reaction mixture into fractions, concentration, drying of the liquid extract that is characterized by the fact that algae meal (processing waste) is formed in the process of obtaining polyphenolic extracts using natural deep eutectic solvents (DES) based on lactic acid, after separating the liquid PGES extract by filtration or centrifugation, the algae meal is loaded into the extractor and extracted at pH=2.0–5.0 at a temperature of 20–60°C for 1–24 hours. The pH is adjusted by adding a 10–80% solution of lactic acid, an ultrasonic device of the "horn" type is used to intensify the process of extraction and mixing of the medium, then the reaction mixture is neutralized by adding a solution of 0.1 N NaOH, kept at a temperature extraction 1–24 h, separated from the solid residue by filtration or centrifugation, liquid extract is filtered through a membrane with a pore diameter of 300–500 kDa to separate high molecular weight polysaccharides, the filtrate is passed through a 50–100 kDa membrane to obtain medium molecular weight polysaccharides, then through a 5–30 kDa membrane to remove low molecular weight impurities, concentrated liquid extracts are freeze-dried.

EFFECT: claimed invention provides a simple and economical method for obtaining polysaccharides while maintaining their native properties, with a high yield of both polysaccharide fractions and the main active substance with the rational use of waste from the processing of aquatic organisms (brown algae).

4 cl, 4 tbl, 4 ex

Description

The invention relates to medical and preventive practice, to the chemical-pharmaceutical, food, cosmetic industries, and in particular to methods for obtaining polysaccharides from carbohydrate-containing seaweed processing waste.

State of the art

Natural deep eutectic solvents (DGE) are an environmentally friendly and safe alternative to organic solvents such as ethanol, acetone, chloroform for the extraction of biologically active substances from plant materials, including brown algae [Obluchinskaya, E.D., Daurtseva, A.V., Pozharitskaya , O.N., Flisyuk, E.V., Shikov, A.N. (2019). Natural deep eutectic solvents as alternative extractants for the extraction of brown algae phlorotannins. Chemical Pharmaceutical Journal, 53(3), 45-49.; Obluchinskaya, E.D., Pozharitskaya, O.N., Zakharova, L.V., Daurtseva, A.V., Flisyuk, E.V., Shikov, A.N. (2021). Efficacy of natural deep eutectic solvents for extraction of hydrophilic and lipophilic compounds from Fucus vesiculosus. Molecules, 26(14), 4198]. Organic solvents have a negative effect on the environment and the work area, require the use of special equipment, complex cleaning systems, and are not suitable for the implementation of environmentally friendly technologies. PGER are mainly used to extract phenolic compounds, alkaloids, glycosides, pigments from natural raw materials. Their action is based on the donor-acceptor interaction of the solvent components, where the role of donors is played by substances such as choline chloride, betaine, the role of acceptors is performed by organic acids (lactic, malic and others), monosaccharides, amino acids and other compounds. PGER have a wide variety of compositions. So, at least 10-15 solvents have been created based on lactic acid, and PGER based on malic acid is also often used (for examples, see Table 1). To prepare eutectic solvents for PGER, L-lactic acid, betaine, choline chloride, malic acid, glucose, and other components are used in certain molar ratios (see Table 1). The components of the mixture are heated to 50-80°C with stirring until a transparent homogeneous liquid is formed. PGER prepared in this way are used to obtain extracts, in particular those containing polyphenols, pigments and other biologically active substances from plants and algae.

At the same time, the problem of utilization of meal (waste from processing of raw materials) after PGE treatment has not yet been solved, since in most cases it is a thick mass that is difficult to dry and has a strongly acidic reaction of the medium. In a series of preliminary experiments, we found that the meal after obtaining PGER-extracts from brown algae contains a sufficient amount of carbohydrates - polysaccharides of fucoidan, laminaran, alginates, taking into account their different content in the feedstock (table 2).

The scientific and technical literature practically does not describe methods for obtaining polysaccharides - alginates, laminarans and fucoidans from algae processing waste using a common simple technology. Usually, a separate technology or a separate stage of multi-stage complex processing of fresh, frozen or dry thalli is provided for each polysaccharide. These technologies or steps are often individual, depending on the type of algae used. It is important to note that the polysaccharide composition and content in brown algae, in addition to species specificity, changes under the influence of various factors, such as the collection season, salinity, temperature, the phase of reproduction and growth of algae. Therefore, the same thallus of algae may contain too low amounts of polysaccharides and their production may not be economically feasible. For practical production purposes, it is essential to develop a simple flexible technology for the production of polysaccharides, suitable for any type of brown algae, with different content, which is especially important for algae processing waste.

A known method for producing sulfated brown algae fucoidan polysaccharide with anticoagulant and immunotropic action (RF patent for the invention No. 2247574). It is a fucoidan obtained by water extraction of Fucus evanescens brown algae at 20-60°C followed by ethanol precipitation. Fucoidan is obtained in the following way. Fresh or frozen (after defrosting) algae is treated with ethanol to degrease it and remove the pigment. The algae is then air-dried, coarsely ground and treated with water (1:20 w/v) first at room temperature (5 hours) and then at 50-60° C. with stirring for 5 hours. The extract is concentrated under reduced pressure to 1/4-1/5 of the original volume and the pH of the suspension is adjusted to a value of 3.0. Then the precipitate formed is separated by centrifugation, the pH of the supernatant is adjusted to neutral by titration with sodium hydroxide, and dialyzed against water. The fucoidan sodium salt is then precipitated with two volumes of 96% ethanol. The precipitate is washed three times with 50% aqueous ethanol and dried with 96% ethanol. Get the target product fucoidan.

A known method of obtaining water-soluble polysaccharides of brown algae (RF patent for invention No. 2135518). The method involves the treatment of fresh, or freshly frozen, or dry algae with organic solvents to remove low molecular weight substances, extraction of polysaccharides with 0.1 N hydrochloric acid at room temperature and water at 50-60°C. The separation of laminarans and fucoidans and their subsequent fractionation is carried out using hydrophobic chromatography. Sequentially obtained extracts of polysaccharides are applied to the columns separately. Polychrome-1 is used as a hydrophobic sorbent.

A known method of complex processing of brown algae to obtain drugs for medical and cosmetic purposes (RF patent for the invention No. 2240816). The method consists in the fact that the plant material is treated with ethanol, the extract is separated, the ethanol is distilled off, a concentrate of biologically active and low molecular weight compounds is obtained, then the treated algae is extracted, the extract is concentrated, neutralized and a concentrate containing polysaccharide-1 is obtained, which is a mixture of laminaran and fucoidan, a mixture neutralize, sequentially precipitate fucoidan (F1) and laminaran (L1), the algae residue is extracted twice, the extracts are combined, concentrated, dried, polysaccharide-2 is obtained, which is a mixture of laminaran, fucoidan and polymannuronic acid, the pH is adjusted to a certain value, the precipitate of polymannuronic acid is separated , the precipitate is dissolved, neutralized and precipitated with a salt of polymannuronic acid (M), the supernatant is neutralized, fucoidan (F2) and laminaran (L2) are precipitated successively, then the algae residue is subjected to alkaline treatment, the extract is concentrated, neutralized and precipitated polysaccharide-3, which is a salt of alginic acid (A), the precipitate is washed and dried. The method allows complex processing of brown algae and at the same time obtaining individual preparations of acidic and neutral polysaccharides and a concentrate of low molecular weight biologically active substances.

A known method of processing algae to obtain a product containing sodium alginate (RF patent for invention No. 2019981). The essence of the invention: the grinding of algae to a particle size of 5-15 mm is carried out by pre-treatment in an acidic environment, acid treatment is carried out for 0.5-1.5 h at pH 2.0-4.0, after which they are washed in the sea, and then in fresh water to pH 6.0 - 6.6, before treatment with carbonate salt, the washed mass is heated to a temperature of 60 - 70 ° C, treatment with carbonate salt is carried out with vigorous stirring for 10-15 minutes, sodium bicarbonate is used as carbonate salt , and the grinding of the resulting mass is carried out by homogenization, after which the mass is frozen.

The disadvantages of these methods are: the use of toxic flammable solvents - ethanol 96% and other organic solvents, the use of a high-cost hydrophobic sorbent, the use of hydrochloric acid, which is a toxic and potent substance, the concentration of aqueous extracts under reduced pressure - vacuum evaporation - energy-intensive and in an uneconomical way. Precipitation with alcohol is also accompanied by large losses of end products. In the process of obtaining fucoidan or alginate, other valuable polysaccharides are separately lost, the yield of the target product is low when using raw materials with a low content of target polysaccharides. The disadvantages of the known methods, in addition to the above, is the multi-stage and wide variability of the techniques used, which entails the use of a variety of equipment, often expensive.

The closest technical solution is described in the technology for obtaining an extract of fucus with anticoagulant action (RF patent for the invention No. 2506089). A method for obtaining a dry extract of fucus by complex processing of fucus vesiculosus Fucus vesiculosus, which consists in the fact that the dry thalli of algae are crushed, extracted with a mixture of methylene chloride with ethyl alcohol in a ratio of 94.2:5.86% in a Soxhlet apparatus, concentrated in a vacuum on a rotary evaporator, dried in a vacuum on a rotary evaporator, dried in a vacuum oven to obtain a lipid-pigment complex - a thick extract, then the meal is extracted by percolation with 85-90% ethanol with forced circulation of the extractant every hour of infusion for 3-5 hours, filtered, concentrated in vacuum on a rotary evaporator, crystallized, filtered on a suction filter, dried and purified by column chromatography using aluminum oxide and purified mannitol is obtained, then the remaining meal is extracted by percolation with 5-15% aqueous ethanol for 4-6 hours at a temperature of 30-40 ° C and pH = 3-4 by percolation with ultrasonic treatment, filtered, concentrated on an ultrafiltration unit and dried in a spray dryer and a polysaccharide complex is obtained - a dry extract of fucus, then the meal is extracted with a 1.5% carbonate solution sodium, treated with sulfuric acid, concentrated on a rotary evaporator, purified by dialysis through a cellophane membrane, dried in a vacuum oven or freeze-dried to obtain the polysaccharide sodium alginate, filtered, concentrated in an ultrafiltration unit and dried in a spray dryer.

The disadvantage of the method can also be attributed to the use of organic solvents and multi-stage production of biologically active substances.

The method for obtaining polysaccharides from brown algae meal (processing waste), as well as known ones, consists in extracting the algae thalli, the reaction mixture is divided into fractions, concentrated, the liquid extract is dried.

The technical problem of the invention is the development of a simple, environmentally friendly and economical technology for the rational use of brown algae processing waste.

The technical result of the invention is the development of a single-stage method for obtaining polysaccharides from brown algae meal (processing waste) while maintaining their native properties, with a high yield, with a high content of the main active ingredient, taking into account the characteristics of each type of raw material.

The technical result is achieved by the fact that algae processing wastes generated in the process of obtaining polyphenol extracts using natural deep eutectic solvents (DGE) based on lactic or malic acid are used as raw materials. After separating the liquid PGER extract by filtration or centrifugation, the algae meal is loaded into the extractor and extracted at pH=2.0-5.0 at a temperature of 20-60°C for 1-24 hours. The pH is adjusted by adding a 10-80% solution of milk or malic acid, respectively. To intensify the process of extraction and mixing of the medium, an ultrasonic apparatus of the "horn" type is used. Then the reaction mixture is neutralized by adding a solution of 0.1 N NaOH or KOH, maintained at an extraction temperature of 1-24 h, separated from the solid residue by filtration or centrifugation. The liquid extract is filtered through a membrane with a pore diameter of 300-500 kDa to separate high molecular weight polysaccharides. The filtrate is passed through a 50-100 kDa membrane to obtain medium molecular weight polysaccharides, then through a 5-30 kDa membrane to remove low molecular weight impurities. The concentrated liquid extracts are freeze-dried or under vacuum or in a spray dryer.

Meal (processing waste) of any kind of brown algae, which were originally in a fresh, frozen or dry state (lat. Phaeophyceae), is used as a raw material, having previously assessed the polysaccharide composition of the meal according to known methods [Quality, safety and methods for analyzing products from aquatic organisms. Issue. 3. - M.: Publishing house of VNIRO, 2009. - 108 p.].

The proposed technology provides a solution to the problem of rational use of brown algae processing waste and helps to reduce environmental pollution due to the absence of organic solvents, and the fact that brown algae processing waste is used as a raw material, which is formed, for example, after the extraction of polyphenolic extracts obtained on the basis of deep eutectic solvents based on lactic or malic acid. With this processing of brown algae, the waste consists of meal, which is a dense crushed wet mass containing a large amount of carbohydrates (polysaccharides). An important feature is the simplicity of the instrumentation of the process, since for its implementation it is necessary and sufficient to have one batch extractor, a Nutsch or Druk filter, a device for membrane filtration or ultrafiltration, a lyophilic, spray or vacuum dryer. Such a set of equipment exists in many food or pharmaceutical industries.

Confirmation of the possibility of obtaining the claimed technical result by this method - the development of a simple, environmentally friendly, economical and one-stage method for obtaining polysaccharides from meal (processing waste) of brown algae, while maintaining their native properties, with a high yield of both polysaccharide fractions and the main active substance, is given in the following specific examples.

Example 1. Obtaining polysaccharides from the meal of fresh fucus algae from the Barents Sea.

1 kg of brown algae fucus vesiculosus (Fucus vesiculosus L.) meal, after separation by filtration of the liquid PGER-extract based on lactic acid (PGER 1, see Table 1), is loaded into the extractor and extracted with 50 l of an aqueous solution of lactic acid at pH = 2.0 at a temperature of 60°C for 1 hour pH is adjusted by adding 80% lactic acid solution. To intensify the process of extraction and mixing of the medium, an ultrasonic apparatus of the "Volna" series of the "horn" type is used, the intensity of ultrasonic treatment was 600 W/dm3. Then the reaction mixture is neutralized by adding a solution of 0.1 N NaOH to pH=7.0. The reaction mixture is kept at the extraction temperature for 1 h, and hot is separated from the solid residue by filtration on a suction filter. The liquid extract is filtered through a membrane with a pore diameter of 300 kDa to separate high molecular weight polysaccharides (VMP Fraction 1-1). The filtrate is passed through a 50 kDa membrane to obtain medium molecular weight polysaccharides (VMP 2-1 Fraction), then through a 5 kDa membrane (VMP 3-1 Fraction) to remove low molecular weight impurities. Concentrated liquid extracts of fractions VMP 1-3 freeze-dried.

The chemical composition of the obtained products is shown in table 3. The composition of monosaccharides is presented in table 4.

Example 2. Obtaining polysaccharides from the meal of dry fucus algae from the Barents Sea.

1 kg of brown algae meal double-ended Fucus (Fucus distichus L.) after separation by filtration of the liquid PGER-extract based on malic acid (PGER 8 see Table 1) is loaded into the extractor and extracted with 50 l of an aqueous solution of malic acid at pH=5.0 at 20° C. for 24 hours. The pH is adjusted by adding a 20% malic acid solution. To intensify the process of extraction and mixing of the medium, an ultrasonic apparatus of the "Volna" series of the "horn" type is used, the intensity of ultrasonic treatment was 600 W/dm3. Then the reaction mixture is neutralized by adding a solution of 0.1 n KOH to pH=7.0. The reaction mixture is kept at the extraction temperature for 24 hours and separated from the solid residue by centrifugation at 6000 rpm. The liquid extract is filtered through a membrane with a pore diameter of 500 kDa to separate high molecular weight polysaccharides (VMP fraction 1-2). The filtrate is passed through a 100 kDa membrane to obtain medium molecular weight polysaccharides (VMP 2-2 Fraction), then through a 30 kDa membrane (VMP 3-2 Fraction) to remove low molecular weight impurities. Concentrated liquid extracts of VMP 1 -3 fractions are dried in a vacuum oven.

The chemical composition of the obtained products is shown in table 3. The composition of monosaccharides is presented in table 4.

Example 3. Obtaining polysaccharides from the meal of frozen kelp algae in the Barents Sea.

1 kg of brown algae meal, sugary kelp (Saccharina latissima L.), after separation by filtration of the liquid PGER-extract based on malic acid (PGER 5, see Table 1), is loaded into the extractor and extracted with 50 l of an aqueous solution of malic acid at pH = 5.0 at a temperature of 50°C for 3 hours, the pH is adjusted by adding a 10% solution of malic acid. To intensify the process of extraction and mixing of the medium, an ultrasonic apparatus of the "Volna" series of the "horn" type is used, the intensity of ultrasonic treatment was 600 W/dm3. Then the reaction mixture is neutralized by adding a solution of 0.1 N NaOH to pH=7.0. The reaction mixture is kept at the extraction temperature for 3 hours, separated from the solid residue by filtration on a suction filter. The liquid extract is filtered through a membrane with a pore diameter of 500 kDa to separate high-molecular polysaccharides (VMP fraction 1-3). The filtrate is passed through a 50 kDa membrane to obtain medium molecular weight polysaccharides (VMP fraction 2-3), then through a 5 kDa membrane (VMP fraction 3-3) to remove low molecular weight impurities. Concentrated liquid extracts of fractions VMP 1-3 are dried in a spray dryer.

The chemical composition of the obtained products is shown in table 3. The composition of monosaccharides is presented in table 4.

Example 4. Obtaining polysaccharides from the meal of the frozen ascophyllum of the Barents Sea.

1 kg of meal of brown alga Ascophyllum knotty (Ascophyllum nodosum L. le Jolis) after separation by filtration of the liquid PGER-extract based on lactic acid (PGER 4 see Table 1) is loaded into the extractor and extracted with 50 l of an aqueous solution of lactic acid at pH=3 0 at 40° C. for 12 hours. The pH is adjusted by adding a 60% malic acid solution. To intensify the process of extraction and mixing of the medium, an ultrasonic apparatus of the "Volna" series of the "horn" type is used, the intensity of ultrasonic treatment was 600 W/dm3. Then the reaction mixture is neutralized by adding a solution of 0.1 N NaOH to pH=7.0. The reaction mixture is kept at the extraction temperature for 12 hours and is separated from the solid residue by filtration on a Druk filter. The liquid extract is filtered through a membrane with a pore diameter of 500 kDa to separate high molecular weight polysaccharides (VMP fraction 1-4). The filtrate is passed through a 100 kDa membrane to obtain medium molecular weight polysaccharides (VMP fraction 2-4), then through a 30 kDa membrane (VMP fraction 3-4) to remove low molecular weight impurities. Concentrated liquid extracts of fractions VMP 1-3 freeze-dried.

The proposed method is simple, fast and environmentally friendly, since it excludes the use of toxic and flammable explosive reagents, provides for the rational use of seaweed processing waste using malic or lactic acids and water, does not require the use of complex expensive apparatus, and does not pollute the environment. Polysaccharides are obtained with a high technological yield, as well as with a high content of the main active ingredient (fucoidan, laminaran, alginate).

Polysaccharides obtained by this method, taking into account the characteristics of each type of raw material, can be used in the medical, pharmaceutical, cosmetic and food industries.

Claims (5)

1. A method for obtaining polysaccharides from brown algae meal (processing waste), which consists in extracting algae thalli, the reaction mixture is divided into fractions, concentrated, the liquid extract is dried, characterized in that algae meal (processing waste) is used as a raw material, formed in the process of obtaining polyphenolic extracts using natural deep eutectic solvents (DDE) based on lactic acid, after separating the liquid DDE extract by filtration or centrifugation, the algae meal is loaded into the extractor and extracted at pH = 2.0-5.0 at a temperature of 20- 60°C for 1-24 h, pH is adjusted by adding a 10-80% solution of lactic acid, an ultrasonic device of the "horn" type is used to intensify the extraction process and mixing of the medium, then the reaction mixture is neutralized by adding a solution of 0.1 N NaOH, kept at an extraction temperature of 1-24 h, separated from the solid residue by filtration or centrifugation, the liquid extract is filtered through a membrane with a pore diameter of 300-500 kDa to separate high molecular weight polysaccharides, the filtrate is passed through a 50-100 kDa membrane to obtain medium molecular weight polysaccharides, then through membrane 5-30 kDa to remove low molecular weight impurities, concentrated liquid extracts are freeze-dried. 2. The method according to p. 1, characterized in that the raw material used is waste from the processing of algae formed in the process of obtaining polyphenol extracts using natural deep eutectic solvents (PGER) based on malic acid, the pH is adjusted by adding a 10-80% solution of malic acid 3. The method according to p. 1, characterized in that the reaction mixture is neutralized by adding a solution of 0.1 n KOH. 4. The method according to p. 1, characterized in that the concentrated liquid extracts are dried under vacuum. 5. The method according to p. 1, characterized in that the concentrated liquid extracts are dried in a spray dryer.