PROCESS OF EXTRACTION OF POLISACARIDES FROM BROWN ALGAE

专利摘要:
extraction process of brown algae polysaccharides, brown algae polysaccharide and sodium alginate with fragments rich in manuronic acid reveals a process of extraction of active polysaccharides from brown algae through a chemical process with microwave and use of polysaccharides of brown algae obtained by the process. the process comprises: placing raw material of pulverized brown algae in a microwave reaction chamber to react it with an added acid solution, optionally concentrating, then washing with the addition of an organic solvent to remove excess acid; then carry out gradual alcohol precipitation after aqueous extraction to obtain algin, fucoidan and / or laminaran respectively with fragments rich in manuronic acid; add an alkaline solution to the brown algae residue to perform an alkaline digestion, filter and adjust the pH until neutral, and then perform precipitation with alcohol to obtain precipitates of algin with fragments rich in guluronic acid.
公开号:BR112014010922B1
申请号:R112014010922-2
申请日:2012-11-01
公开日:2020-12-15
发明作者:Jinsong Zhang；Mingtian Li；Zhiyu Liu；Lei Xu
申请人:Shenyang Kesi High-Technology Co. Ltd；
IPC主号:

专利说明:

Technical Field
[001] This invention refers to the field of pharmaceutical chemistry, it refers to a brown algae polysaccharide extraction process and it is, in particular, a brown algae polysaccharide extraction process based on a chemical method with micro- waves. Description of the Prior Art
[002] Algae, a collective term for marine algae, usually attach themselves to structures on the seabed or to some solid structures, are a single or a series of simple plants that comprise basic cells. Nutrients in algae include polysaccharides, proteins, lipids, pigments and low molecular weight substances. Proven by traditional Chinese medicine and modern scientific research, the main component of substances with immunity-enhancing and anti-cancer activity in algae comprises polysaccharides.
[003] Algae includes red algae, green algae, brown algae, etc., and current research and application of algae polysaccharides is mainly focused on brown algae polysaccharides. Brown algae comprise a superior class of algae, with about 250 genera and more than 1,500 species. The body of brown algae is yellowish brown or dark brown and contains substances such as polysaccharides, proteins, lipids, mannitol, etc. Some members of the class, like Japan, also contain large amounts of iodine in their cells.
[004] Brown algae polysaccharides are important components of them, including algin, fucoidan, laminarane, etc. Algin, generally referring to sodium alginate, is a linear copolymer with homopolymeric polysaccharide blocks of β-D-manuronate (M) and α-L-guluronate (G) (1-4) -related residues, which presents a high content in brown algae. There are three types of structure of segment chains of the same: M blocks of continuous M units, G blocks of continuous G units and an MG block formed with G and M units alternately connected. Algina is also the most representative class of algae chemicals. Data show that annual algin production in China is the highest in the world. For ease of use and storage, algin is generally converted to sodium alginate as the final product. As a sodium salt of the polyanionic polysaccharide (alginic acid), sodium alginate has a very wide variety of applications in the industry for its inherent physical and chemical properties.
[005] In the food industry, sodium alginate is an excellent food additive, due to its low calorie, non-toxicity, easy expansion and high flexibility, and when it is added to food, it performs many functions such as coagulation, thickening, emulsion, suspension, stabilization and prevention of dehydration of food, etc.
[006] In the textile industry, sodium alginate has characteristics such as it colors easily, has high color yield, color shine and softening of printed fabrics, etc., and is the most commonly used paste in printing with reactive dye of fabrics from cotton. However, it can also be used as a soluble fiber in industrial processes, such as warp slurry, waterproof processing and lace making.
[007] In the cosmetic industry, sodium alginate is used as a base for toothpaste, shampoos, hair styling agent, etc. In the paper industry, it is used as a scaler. In the rubber industry, it can be used as a latex concentrate and can also be made as water-based coatings and water-resistant coating.
[008] Sodium alginate is also an important biomedical material, being widely used as a drug release agent, polymeric film, in cell encapsulation, in wound dressings, surgical sponge, embolic agent, etc. He is attracting more and more attention in biomedical materials science, clinical medicine, tissue engineering, pharmaceutical science and other fields. So far, algin production basically still uses alkaline digestion, the basic principle of which is the use of sodium carbonate (Na2CO3) to convert various water-insoluble alginate salts into sodium alginate, to dissolve sodium alginate in water and obtaining the dried sodium alginate powder through filtration, acid / calcium precipitation and conversion of sodium salt.
[009] Fucoidan is a heteropolysaccharide, which comprises fucose and sulfate, as well as monosaccharides including galactose, xylose, mannose, uronic acid, etc. Due to its unique structure and excellent physiological activity, such as the regulation of blood lipids, blood sugar reduction, blood pressure reduction, anticoagulation, anti-tumor, anti-mutagenic and anti-radiation and antiviral function, of enhancing the immune system, etc., fucoidan has become one of the points of excellence for marine pharmaceutical research in this century. Laminaran is also a polysaccharide with a variety of physiological activities. The preparation of fucoidan and laminarane mainly uses residues from the production of algin, iodine and mannitol as main raw materials, which are subjected to classification-precipitation with alcohol, after being immersed in water.
[010] The current algin production process has the following main disadvantages:
[011] It consumes a lot of water and energy and is very polluting. This is also one of the main factors that restrict the development of the algae industry. Alkaline digestion is simple in principle, but it often requires dozens of processes in actual industrial production, some of which are very difficult. For example, in the concentrated sodium alginate slurry formed from japonica through the treatment of sodium carbonate, a lot of water-insoluble cellulose and other ingredients need to be filtered. Due to the high viscosity of the concentrated slurry, in addition to the addition of a filter aid such as diatomite, it is also necessary to consume a large amount of water for dilution and has great demands in terms of the quality of the water used. According to statistics, the production of one ton of finished sodium alginate needs about 700 to 1000 tons of water. In addition, there are mainly two approaches, namely, addition of acid and the addition of calcium salt in the chemical process of conventional alkaline digestion: first, the water-insoluble alginate salts in brown algae are converted into water-soluble sodium alginate , the addition of acid or calcium ions causes sodium alginate to form precipitates of alginic acid or calcium alginate which, after washing, is converted back to sodium alginate and, finally, sodium alginate is subsequently processed into various products. Regardless of the method used, it will produce a large amount of industrial waste water, which poses a serious threat to the ecological environment.
[012] Meanwhile, the current sodium alginate product is a single product variety, it does not have excellent quality and has low added value. Sodium alginates, in terms of structure, can be divided into three categories: high G / M ratio, medium G / M ratio and low G / M ratio, in terms of viscosity, they can be divided into ultra-low viscosity sodium alginate , low viscosity, medium viscosity, high viscosity and ultra-high viscosity, in terms of purity, they can be divided into three levels: industrial, food and clinical. The current domestic production of sodium alginate mainly comprises products of medium viscosity. As health or pharmaceutical products, their application is limited due to their strong gelling properties, low solubility, etc., and their activity cannot be fully achieved.
[013] To overcome these difficulties, domestic and overseas researchers have made long-term efforts on these processes, to address the above disadvantages and have achieved many important achievements and progress. These studies include improving the conventional process and developing a new process. For example, a newly reported process of reactive alginous extrusion by foreign students has advantages such as saving water, saving time, using less base, etc. and the product's viscosity and yield reached some degree of improvement, but the process cannot directly produce water-soluble polysaccharide, with good biological activities. The modification for conventional algae polysaccharide products includes the use of biological, chemical or physical methods to break down sodium alginate and fucoidan into low molecular weight alginate, oligosaccharides and fucose oligosaccharides, to adjust the sulfur content of fucoidan, etc.
[014] The researchers also confirmed that the activities of sodium alginate and fucoidan after degradation have been effectively improved, some low molecular weight sodium alginates already have physiological anti-tumor and antiviral activities like heparin and can be used in medical research for diseases cardiovascular diseases and viruses. Some of them have properties of regulation of the intestine and detoxification, reduction of glucose and lipids in the blood, anticoagulant, anti-inflammatory and immunomodulatory effects and can be used as dietary foods for patients with diabetes, obesity, colorectal cancer and with frequent constipation.
[015] Especially in recent years, the unique physiological functions of modified low molecular weight algin or fucoidan continue to be discovered, and their activity and medicinal value has become one of the points of excellence of new research.
[016] In addition, polymanuronic acid (M) and polyguluronic acid (G) are unique components in the algin molecule and have not been found to exist independently in nature. When the proportion of the two alduronic acid polysaccharides (M / G), or the structure and disposition of such a block in the algin vary, the algin's performance will show a significant difference. Therefore, there are many researchers obtaining different fragments of their polysaccharides and oligosaccharides with unique structures through different methods of degradation and separation, to study their exclusive biological activity and to develop drugs with special effectiveness.
[017] All of these series of oligosaccharides or oligosis with different structures greatly enrich the diversity of algal polysaccharide products. However, all of these studies use medium viscosity algin as raw materials and fundamentally do not solve the problems in the conventional production process of sodium alginates, such as high water and energy consumption, high pollution generation, low yield / content ratio , etc.
[018] A rapid microwave assisted method for the hydrolysis of sodium alginate for the determination of the M / G ratio (Mahesh Chhatbar, CarbohydratePolymers Vol 7 6 (2009) 650-656) discloses a microwave oven for use domestic use, the use of a solution of oxalic acid or a solution of sulfuric acid diluted as a solvent and partial degradation of sodium alginate, and its main purpose is to find a simple, fast and mild method to determine the M / G ratio (manuronic acid / guluronic acid) in sodium alginate. However, the sodium alginate material used in this method is still prepared by conventional extraction methods and does not solve the problem of consuming large amounts of water and energy and other defects in the conventional process.
[019] Microwave-assisted desulfation of sulfated polysaccharides (Diego A, Navaroo, CarbohydratePolymers Vol 69 (2007) 742-747) discloses, in a microwave oven for home use, the use of a micro-assisted method -waves that remove sulfur from red algae polysaccharide carrageenan, agar and fucoidan in brown algae and chondroitin sulfate from animal polysaccharides, in order to overcome the deficiencies of the commonly used hydrochloric acid desulfurization method. The method still uses polysaccharides as a starting material and has not overcome defects such as the large consumption of water and energy, etc. In addition, the purpose of seeking a high rate of desulfurization in the article is intended to improve the convenience of sample analysis and does not take into account the activity of polysaccharides.
[020] It is therefore necessary to continue to carry out research and development on new technologies, new technologies, new products in marine science and in the algae industry. Summary of the Invention
[021] To overcome the above technical flaws, the present invention provides a new process for extracting brown algae polysaccharides, that is, a process for extracting active polysaccharides from brown algae by means of a chemical microwave method. The process of extracting active polysaccharides from brown algae using the microwave chemical method of the invention comprises the following steps: 1) placing powdered brown algae powder in a microwave reaction chamber, adding acid solution of a concentration in mass from 5 to 99%, carry out the mixer reaction for 5 to 120 min at a microwave power with a mass power density of 1 kW per kilogram of material to 10 kilowatts per kilogram of material, under a working pressure of 20 mmHg to 760 mmHg; optionally, concentrate the mixer and then wash with organic solvent to remove excess acid. 2) add the aqueous solution to the product obtained from step 1) for aqueous extraction, concentrate the extraction solution, adjust the pH to neutrality with a base, carry out gradual alcohol precipitation to obtain fragments rich in manuronic acid (rich in M) algin, fucoidan and / or laminarane, respectively; and the remaining brown algae residue. 3) add an alkaline solution to the brown algae residue from step 2), carry out the alkaline digestion reaction at a temperature of 35 to 60 ° C for 20 to 80 minutes, remove the residues by filtration, adjust the pH of the filtrate until neutrality, perform the precipitation with alcohol after concentration to obtain precipitates of algin with fragments rich in guluronic acid (rich in G).
[022] In the process of the present invention, powdered brown algae powder is brown algae powder obtained using conventional spray methods in the area, or commercially available powdered brown algae powder is purchased and used directly in the process of the invention, in that the granulometry of the powdered brown algae powder is normal in the area, or is determined by a person skilled in the art in the combination of the present invention and common knowledge.
[023] In the process of the present invention, as one of the modalities, where in the acid solution added in step 1) an organic acid is a non-volatile acid, there is no need to remove the acid by concentration after the microwave reaction; where an added organic acid is volatile acid, after the microwave reaction is complete, the concentration is performed to remove the acid, preferably by microwave heating under reduced pressure, and then washing with organic solvent is performed to remove a small amount of residual acid.
[024] In the process of the present invention, as one of the modalities, the method of applying microwave power in said step 1) is a continuous microwave mode or a combination of continuous microwave mode and pulsed microwave; in which, in the modality of using the combination of continuous microwave and pulsed microwave, continuous microwave irradiation is first used until the acid solution refluxes, then the irradiation is converted into pulsed microwave during 5 min to 120 min; the continuous microwave is also maintained for 5 min to 120 min after refluxing the solution.
[025] In the process of the present invention, as one of the additional preferred modalities, in said step 1), in the case of continuous microwave, the mass power density is from 1 kW per kilogram of material to 5 kilowatts per kilogram of material; in the case of pulsed microwaves, the mass power density is from 2 kilowatts per kilogram of material to 10 kilowatts per kilogram of material, with the service ratio being A / B, where A = 1 s to 100 s and B = 1 to 100 s.
[026] In the process of the present invention, the selection of the microwave reaction chamber can be determined by a person skilled in the art in combination with the present invention and common knowledge in the art, which is a microwave reaction chamber. propagation wave or a reaction chamber of microwave resonance.
[027] In the process of the present invention, as one of the modalities, in said step 1), the weight ratio range of the raw material of brown algae and the acid solution is equal to 5/1 to 1/5.
[028] In the process of the present invention, as an embodiment, the acid solution in step 1) is selected from an organic acid or a mixed solution of an organic acid and an inorganic acid.
[029] In the process of the present invention, as one of the additional embodiments, organic acid is selected from oxalic acid, from a concentration in weight of 5% to 50%, preferably oxalic acid from 10% to 35%; formic acid from 10% to 99%, preferably formic acid from 30 to 85%; acetic acid from 10% to 99%, preferably acetic acid from 60 to 95%; or propionic acid from 10% to 99%, preferably aqueous solution of propionic acid from 70 to 95%.
[030] The acid solutions of concentration mentioned above can be purchased directly from the market, or be prepared using conventional methods in the field, for example, by adding an appropriate amount of water in an acid of a concentration of 100% (for example, formic acid pure) to dilute said acid to the corresponding concentration.
[031] In the process of the present invention, as one of the modalities, when a mixed solution of organic and inorganic acids is used, in a mixed solution of organic and inorganic acid, the concentration of organic acid in the mixed solution is above the defined concentration of the organic acid; and the mass percentage concentration of inorganic acid is 0.1% to 15%; as one of the additional preferred embodiments, inorganic acid is selected from hydrochloric acid, sulfuric acid, nitric acid or phosphoric acid.
[032] The inorganic acid solution mentioned above can be commercially available and then, using conventional methods, prepare the corresponding concentration of the mixed organic-inorganic acid solution, for example, by adding hydrochloric acid of a concentration of 36% to the acid organic to achieve the corresponding concentration.
[033] In the process of the present invention, controllable degradation of brown algae polysaccharides can be achieved by controlling reaction conditions, using cooperative effects between microwaves, organic acid molecules and brown algae polysaccharides, and selectively cleaving bonds glycosides of brown algae polysaccharides.
[034] In the process of the present invention, as one of the modalities, in said step 1), the organic solvent used to wash the residual acid is selected from methanol, ethanol, propanol or acetone, or from a combination of two or more of the same . There is no special requirement in terms of the concentration of the organic solvent, which can be determined by a person skilled in the art with a combination of the prior art or common knowledge in the field.
[035] In the process of the present invention, as one of the modalities, in step 2), the amount of water is 5 to 8 times the volume of the product obtained in step 1). A person skilled in the art, with the combination of the present invention and common knowledge, can adjust the amount by addition or reduction; when the extraction solution is concentrated, the extraction solution is preferably concentrated to 1/5 to 1/8 of its original volume.
[0036] In the process of the present invention, as one of the modalities, in step 2), the base used for pH adjustment is selected from sodium carbonate or sodium hydroxide; wherein sodium carbonate or sodium hydroxide are solutions of sodium carbonate or sodium hydroxide in a concentration commonly used in the art; its main purpose is to complete the conversion of alginic acid to sodium alginates.
[037] In the process of the present invention, as one of the modalities, the gradual alcohol precipitation in said step 2) comprises: adding ethanol up to 20% by weight to 40% by weight of alcohol content, performing the centrifugation or filtration to obtain oligoprecipitates of algin with fragments rich in manuronic acid; then, add ethanol to an alcohol content of 60% by weight to 70% by weight, perform filtration or centrifugation to obtain precipitates of fucoidan; and finally, add ethanol to an alcohol content of 80% by weight to 85% by weight, and perform filtration or centrifugation to obtain laminarane precipitates.
[038] In the process of the present invention, as one of the modalities, the base of said step 3) is selected from sodium carbonate or sodium hydroxide; wherein sodium carbonate or sodium hydroxide are solutions of sodium carbonate or sodium hydroxide in a concentration commonly used in the art.
[039] In the process of the present invention, as one of the modalities, the acid used to adjust the pH value in step 3) is selected from hydrochloric acid, where hydrochloric acid is a solution of hydrochloric acid in a concentration commonly used in technical.
[040] In the process of the present invention, as one of the modalities, the alcohol used in the precipitation with alcohol in said step 3) is selected from ethanol or methanol. When ethanol or methanol is used in precipitation with alcohol, in principle, alcohol is added to the solution until there is no more precipitation, and preferably until the alcohol content in the solution reaches 80% to 85%.
[041] In the process of the present invention, brown algae used to extract polysaccharides from brown algae using the process of the present invention include, but are not limited to, Japanese Laminaria (Laminaria), Sargassum (Sargassum) sargassum, sea millet, fusiform , seaweed, creeping seaweed sticks, Fucus (Fucus) fucus, fucus, Pelvetia carragena (Pelvetia), Undaria (Undaria) wakame or Maerocystis (Maerocystis) kelp.
[042] As one of the preferred embodiments, the present invention also provides a process for preparing active polysaccharides using Japanese laminate. The process is as follows: put dry Japanese powder in a microwave extraction chamber, add 60% to 85% formic acid from 0.5 to 1.5 times its weight, at a power density microwaves of 1 to 2 kW / Kg under pressure of 500 mmHg to 760 mmHg, maintaining the reflux of the formic acid solution for 15 to 30 min and then evaporating under reduced pressure the formic acid solution to dryness . An ethanol solution of 3 to 5 times the weight of japonica powder is added to the reaction chamber. The mixer is stirred and washed for 40 to 60 minutes and filtered. The filter residue, after drying, is extracted twice with water, in which each time the amount of water is 4 to 6 times the weight of the residue, the extraction temperature is 60 to 80 ° C and the time of extraction is about 40 minutes. The filtration is carried out and the two filtrate solutions are combined, neutralized with a sodium hydroxide solution until neutral and concentrated to 1/5 of the extract volume; then, the ethanol is added to the solution to an ethanol content equal to 35% and the solution is left to stand for 4 to 8 hours and is filtered; and the filtered cake is washed with anhydrous ethanol and ether, and is dried to obtain polysaccharide A; ethanol continues to be added to the filtrate until it reaches 65% ethanol in the solution, and the solution is left to stand for 4 to 8 hours and is filtered; and the filtered cake is washed with anhydrous ethanol and ether, and is dried to obtain polysaccharide B. Ethanol continues to be added to the filtrate to an 85% ethanol content of the solution and it is left to stand for 4 to 8 hours and is filtered; and the filtered cake is washed with anhydrous ethanol and ether, and is dried to obtain polysaccharide C.
[043] The japonica residue, after the aqueous extraction above, is added with a solution of sodium carbonate at a temperature of 35 to 60 ° C for an alkaline digestion reaction for about 40 minutes, and filtered, the filtrate has the pH adjusted to neutrality with hydrochloric acid, concentrated and subjected to precipitation with alcohol to obtain polysaccharide D.
[044] Where polysaccharide A is sodium alginate rich in manuronic acid (rich in M), polysaccharide B is fucoidan, polysaccharide C is laminarane and polysaccharide D is sodium alginate rich in guluronic acid (rich in G) .
[045] As one of the preferred embodiments, the present invention also provides a process for preparing active polysaccharides using Sargassum fusiforme. The process is as follows: place dry fusiform powder in a microwave extraction chamber, add 10% to 20% oxalic acid solution from 0.5 to 1.5 times its weight, in a density microwave power from 1 to 2 kW / Kg under pressure of 500 mmHg to 760 mmHg, maintaining the reflux of the oxalic acid solution for 15 to 25 min and then evaporating under reduced pressure to dryness. An ethanol solution of 4 to 6 times the weight of the fusiform powder is added to the reaction chamber. The mixer is stirred and washed for 40 to 60 minutes and filtered, and the filter residue is dried. The filtrate is distilled to recycle ethanol and oxalic acid. The filter residue, after drying, is extracted twice with water, where each time the amount of water is 4 to 6 times the weight of the residue, the extraction temperature is 70 ° C and the extraction time is about 40 minutes; the above two filtrate solutions, after filtration, are combined, neutralized with a sodium hydroxide solution to neutrality and concentrated to 1/5 of the extract volume; then, ethanol is added to the solution to a content of 30% ethanol and the solution remains for 4 to 8 hours and is filtered; and the filtered cake is washed with anhydrous ethanol and ether, and is dried to obtain polysaccharide A; ethanol continues to be added to the filtrate to a 60% ethanol content in the solution, which remains in place for 4 to 8 hours and is filtered; and the filtered cake is washed with anhydrous ethanol and ether, and is dried to obtain polysaccharide B. Ethanol continues to be added to the filtrate to an 80% ethanol content of the solution and it is left to stand for 4 to 8 hours and is filtered; and the filtered cake is washed with anhydrous ethanol and ether, and is dried to obtain polysaccharide C. The fusiform residue, after the aqueous extraction above, is added with a solution of sodium carbonate at a temperature of 35 to 60 ° C for alkaline digestion reaction for 40 to 60 minutes, and filtered, the filtrate has the pH adjusted to neutrality with concentrated hydrochloric acid and is subjected to precipitation with alcohol to obtain polysaccharide D.
[046] Where polysaccharide A is sodium alginate rich in manuronic acid (rich in M), polysaccharide B is fucoidan, polysaccharide C is laminarane and polysaccharide D is sodium alginate rich in guluronic acid (rich in G) .
[047] As one of the preferred embodiments, the present invention also provides a process for preparing active polysaccharides using Fucus fucus. The process is as described below: place the dried fucus powder in a microwave extraction chamber, add 80% to 95% propionic acid solution 1 to 2 times the same, in a micro power density -waves of 3 to 5 kW / Kg, after refluxing the propionic acid solution, maintaining for 40 to 60 minutes under pressure from 500 mmHg to 760 mmHg and then evaporating under reduced pressure to the propionic acid solution until dryness. An ethanol solution of 3 to 5 times the weight of fucus powder is added to the reaction chamber. The mixer is stirred and washed for 30 to 60 minutes and filtered, and the filter residue is dried. The filter residue, after drying, is extracted with water of 4 to 6 times the weight of the residue, is extracted at 70 ° C for 40 minutes and is filtered; the above extraction process is repeated again. The two solutions of the filtrate, after filtration, are combined, neutralized with a sodium hydroxide solution until neutral and concentrated to 1/5 of the volume of the extraction solution; then, the ethanol is added to the solution until the ethanol content of 35% and the solution remains for 4 to 8 hours and is filtered; and the filtered cake is washed with anhydrous ethanol and ether, and is dried to obtain polysaccharide A; ethanol continues to be added to the filtrate until it reaches 65% ethanol content in the solution, which remains in place for 4 to 8 hours and is filtered; and the filtered cake is washed with anhydrous ethanol and ether, and is dried to obtain polysaccharide B. Ethanol continues to be added to the filtrate to an 85% ethanol content of the solution and it is left to stand for 4 to 8 hours and is filtered; and the filtered cake is washed with anhydrous ethanol and ether, and is dried to obtain the polysaccharide C. The fucus residue, after the aqueous extraction above, is added with a solution of sodium carbonate at a temperature of 35 to 60 ° C for alkaline digestion reaction for 40 to 60 minutes and filtered, and the filtrate is pH adjusted to neutrality with concentrated hydrochloric acid and subjected to precipitation with alcohol to obtain polysaccharide D.
[048] Where polysaccharide A is sodium alginate rich in manuronic acid (rich in M), laminarane and polysaccharide D is sodium alginate rich in guluronic acid (rich in G).
[049] As one of the preferred embodiments, the present invention also provides a process for preparing active polysaccharides using Pelvetia carrageenan. The process is as follows: place dry carrageenan powder in a microwave extraction chamber, add a solution of oxalic acid-hydrochloric acid mixed 0.5 to 2 times the same, in which the oxalic acid content in the mixed solution is 20% and the hydrochloric acid content is 0.1%, at a microwave power density of 1 to 2 kW / kg under pressure from 500 mmHg to 760 mmHg, maintaining the reflux of the acid solution mixed for 15 to 25 minutes and then evaporate the solution to dryness under reduced pressure. An ethanol solution of 3 to 5 times the weight of carrageenan powder is added to the reaction chamber. The mixer is stirred for 30 to 60 minutes and filtered. The filter residue, after drying, is extracted with water of 4 to 6 times the weight of the residue, is extracted at 70 ° C for 40 minutes and is filtered; the above extraction process is repeated again. The two filtrate solutions, after filtration, are combined, neutralized with a sodium hydroxide solution to neutrality and concentrated to 1/5 of the extract volume; then, the ethanol is added to the solution until the ethanol content of 35% and the solution remains for 4 to 8 hours and is filtered; and the filtered cake is washed with anhydrous ethanol and ether, and is dried to obtain polysaccharide A; ethanol continues to be added to the filtrate until it reaches 65% ethanol content in the solution, which remains in place for 4 to 8 hours and is filtered; and the filtered cake is washed with anhydrous ethanol and ether, and dried to obtain polysaccharide B. Ethanol continues to be added to the filtrate to an 85% ethanol content of the solution and the solution is left to stand for 4 to 8 hours and is filtered; and the filtered cake is washed with anhydrous ethanol and ether, and is dried to obtain the polysaccharide C. The carrageenan residue, after the aqueous extraction above, is added with a solution of sodium carbonate at a temperature of 35 to 60 ° C for alkaline digestion reaction for 40 to 60 minutes, filtered, the filtrate has the pH adjusted to neutrality with concentrated hydrochloric acid and is subjected to precipitation with alcohol to obtain polysaccharide D.
[050] Where polysaccharide A is sodium alginate rich in manuronic acid (rich in M), polysaccharide B is fucoidan, polysaccharide C is laminarane and polysaccharide D is sodium alginate rich in guluronic acid (rich in G) .
[051] As one of the preferred embodiments, the present invention also provides a process for preparing active polysaccharides using Undaria wakame. The process is as follows: place dry wakame powder in a microwave extraction chamber, add a solution of formic acid and hydrochloric acid of 0.5 to 2.5 times its weight, in which the content formic acid content is 80% and the hydrochloric acid content is 0.5% at a microwave power density of 2 to 4 kW / kg under pressure from 500 mmHg to 760 mmHg, maintaining the reflux of the mixed acid solution for 10 to 30 min and then evaporate under reduced pressure the mixed acid solution to dryness. An ethanol solution of 3 to 5 times the weight of the wakame powder is added to the reaction chamber. The mixer is stirred and washed for 30 to 60 minutes and filtered. The filter residue, after drying, is extracted with water of 4 to 6 times the weight of the residue, is extracted at 60 ° C for 40 minutes and is filtered; the above extraction process is repeated again. The two filtrate solutions, after filtration, are combined, neutralized with a sodium hydroxide solution to neutrality and concentrated to 1/5 of the extract volume; then, the ethanol is added to the solution until the ethanol content of 35% and the solution remains for 4 to 8 hours and is filtered; and the filtered cake is washed with anhydrous ethanol and ether, and is dried to obtain polysaccharide A; ethanol continues to be added to the filtrate until it reaches 65% ethanol content in the solution, and the solution remains for 4 to 8 hours and is filtered; and the filtered cake is washed with anhydrous ethanol and ether, and is dried to obtain polysaccharide B. Ethanol continues to be added to the filtrate to an 85% ethanol content of the solution and it is left to stand for 4 to 8 hours and is filtered; and the filtered cake is washed with anhydrous ethanol and ether, and is dried to obtain polysaccharide C. The wakame residue, after the aqueous extraction above, is added with a solution of sodium carbonate at a temperature of 35 to 60 ° C for alkaline digestion reaction for 40 to 60 minutes, filtered, the filtrate has the pH adjusted to neutrality with concentrated hydrochloric acid and is subjected to precipitation with alcohol to obtain polysaccharide D.
[052] Where polysaccharide A is sodium alginate rich in manuronic acid (rich in M), polysaccharide B is fucoidan, polysaccharide C is laminarane and polysaccharide D is sodium alginate rich in guluronic acid (rich in G) .
[053] As one of the preferred embodiments, the present invention also provides a process for preparing active polysaccharides using Maerocystis kelp. The process is as follows: put dry kelp powder in a microwave extraction chamber, add 80% to 95% acetic acid solution from 0.3 to 1.2 times its weight, in a density microwave power from 1 to 4 kW / kg under pressure from 500 mmHg to 760 mmHg, maintaining the reflux of the acetic acid solution for 30 to 40 minutes and then evaporating under reduced pressure the acetic acid solution until dryness. An ethanol solution of 3 to 5 times the weight of the kelp powder is added to the reaction chamber. The mixer is stirred and washed for 30 to 60 minutes and filtered. The filter residue, after drying, is extracted with water of 4 to 6 times the weight of the residue, is extracted at 60 ° C for 40 minutes and is filtered; the above extraction process is repeated again. The two filtrate solutions, after filtration, are combined, neutralized with a sodium hydroxide solution to neutrality and concentrated to 1/5 of the extract volume; then, the ethanol is added to the solution until the ethanol content of 35% and the solution remains for 4 to 8 hours and is filtered; and the filtered cake is washed with anhydrous ethanol and ether, and is dried to obtain polysaccharide A; ethanol continues to be added to the filtrate until it reaches 65% ethanol content in the solution, which remains in place for 4 to 8 hours and is filtered; and the filtered cake is washed with anhydrous ethanol and ether, and is dried to obtain polysaccharide B. Ethanol continues to be added to the filtrate to an 85% ethanol content of the solution and it is left to stand for 4 to 8 hours and is filtered; and the filtered cake is washed with anhydrous ethanol and ether, and is dried to obtain polysaccharide C. The kelp residue, after the aqueous extraction above, is added with a solution of sodium carbonate at a temperature of 35 to 60 ° C for alkaline digestion reaction for 40 to 60 minutes, filtered, the filtrate has the pH adjusted to neutrality with concentrated hydrochloric acid and is subjected to precipitation with alcohol to obtain polysaccharide D.
[054] Where polysaccharide A is sodium alginate rich in manuronic acid (rich in M), polysaccharide B is fucoidan, polysaccharide C is laminarane and polysaccharide D is sodium alginate rich in guluronic acid (rich in G) .
[055] The present invention also provides brown algae polysaccharides prepared using the process of the invention, wherein said brown algae polysaccharides are obtained using the process of the invention.
[056] As one of the preferred embodiments, the present invention also provides an algin with a fragment rich in guluronic acid (rich in G) or an algin with a fragment rich in manuronic acid (rich in M) prepared using the process of the invention.
[057] As one of the preferred modalities, algin with a fragment rich in guluronic acid (rich in G) is preferably algin from japonica with a fragment rich in guluronic acid (rich in G), fusiform algin, algin of focus, algin of carrageenan, wakame algin or kelp algin.
[058] As one of the preferred modalities, algin with a fragment rich in manuronic acid (rich in M) is preferably alginate of japonica with a fragment rich in manuronic acid (rich in M), fusiform algin, focus algin, algin of carrageenan, wakame algin or kelp algin.
[059] The present invention also provides a process for preparing sodium alginate with a fragment rich in manuronic acid (rich in M), in which the said process is: add sodium carbonate to algin with a fragment rich in manuronic acid (rich in M M) prepared using the process of the invention to be converted into sodium alginate with a fragment rich in manuronic acid (rich in M).
[060] The present invention also provides a sodium alginate with a fragment rich in manuronic acid (rich in M) prepared using the process of the invention.
[061] The present invention has the following characteristics:
[062] Firstly, the use of a microwave with an organic acid directly on raw materials of brown algae, the use of organic acid cleaves various connections between the polysaccharides and the organic macromolecules (including cellulose and algin) of the cell walls of the raw materials from brown algae, in order to promote the release of polysaccharides from said medicinal materials and improve the rate of extraction of polysaccharides; in addition to organic acid, and in addition to the degradation effects of H + ions on polysaccharides, organic acid radical ions can protect polysaccharide molecules by forming hydrogen bonds with the polysaccharide hydroxyl groups.
[063] Second, microwave-intensified organic acid can further moderate the released polysaccharides, thereby significantly increasing the water solubility of the polysaccharides. Polysaccharides with relatively centralized molecular weight distribution and good solubility in water are obtained and the entire process achieves efficient extraction and restructuring of brown algae polysaccharides.
[064] Thirdly, brown algae polysaccharides with different molecular structures have varying sensitivity to microwave radiation and organic acids. The molecular weight of fragments rich in M, which are relatively sensitive to the degradation effect of organic acids under microwave radiation in brown algae decreases significantly, their water solubility increases significantly and they can be successfully extracted without hydrolysis.
[065] Fragments rich in G that are not sensitive to the effects of organic acid degradation under microwave radiation remain in the brown algae residue, and can be extracted through the process of alkaline digestion and thus the process of separation of algin rich in M and algin rich in G is simplified.
[066] Fourthly, microwave heating can ensure that the internal and external parts of the materials are heated simultaneously and sufficiently overcome a series of insurmountable problems such as non-uniform heating of materials and high energy consumption in conventional heating methods.
[067] In comparison with the prior art, the present invention also has the following advantages: 1. The present invention saves time, uses less acids and has an easy and efficient recycling, in addition to energy saving effects. The use of microwave heating technology effectively overcomes the heating problem, which is difficult to avoid in conventional heating methods, significantly reducing the amount of organic acids used and the processing time, especially in the acid removal distillation process , and can solve the problem of non-uniform heating, which is not overcome in conventional heating methods. This feature in large-scale production has been particularly notable. 2. Microwave-enhanced organic acids can also moderately adjust the molecular structure of released brown algae polysaccharides, including moderate algin degradation, improving their water solubility and separating M-rich fragments from fragments rich in G. The sulfur content of the polysaccharides in brown algae containing sulfur can be moderately reduced. The present process uses acids that act directly on medicinal raw materials, overcoming many deficiencies in the extraction of polysaccharides in existing processes. Currently, research on various modifications to brown algae polysaccharides basically uses brown algae polysaccharides as raw materials and cannot overcome many problems related to polysaccharide extraction. 3. The polysaccharide products obtained by the present invention have a narrow molecular weight distribution, a high degree of purity, good water solubility and good activity. The process of the present invention has distinct advantages in large-scale industrial production. Brief Description of Drawings
[068] Figure 1 is a process flow chart of the present invention. Modalities
[069] The present invention will be further described by the following Examples and Experimental Examples, but the present invention is not limited to them.
[070] The process of the present invention is as follows: 1) placing raw materials of brown algae after washing, drying and purification in a microwave reactor chamber, adding an organic acid or an acid mixing solution to it organic / inorganic and shake sufficiently to wet the powder well; 2) treat with microwaves, apply microwave power using the cooperative effects between microwaves, organic acid molecules and brown algae polysaccharides, selectively cut the glycosidic bonds of brown algae polysaccharides, in order to achieve controllable degradation the same; 3) use distillation under reduced pressure by microwave heating to remove most organic acids or the mixed organic / inorganic acid solution and wash sufficiently with an organic solvent to remove a small amount of residual acid to complete the pre- microwave treatment of brown algae; 4) add about 5 to 8 times of water to the brown algae extract pretreated with microwave, in which the extraction solution, after concentration, is subjected to gradual alcohol precipitation to obtain the excellent algine with fragments rich in water-soluble manuronic acid, fucoidan and laminarane; 5) submit the raw material residue of brown algae pretreated with a microwave after the aqueous extraction to the alkaline digestion process to obtain algin with fragments rich in guluronic acid (rich in G).
[071] The entire process is shown in Figure 1.
[072] To evaluate the new process, there are control processes designed according to the references in each of the modalities, and by comparing data on the corresponding extraction yields of sodium alginate, fucoidan and laminarane, the total water consumption in the process, the viscosity of the sodium alginate products, the reaction time, the amount of organic acids (or mixed acid) and the fucoidan sulfate content, from the control process, the new process and the heating method conventional in each of the examples are listed. Sodium alginate products are purified by a suitable method to obtain monomer polysaccharides with a uniform composition, in which their polymanuronic acid (M) and guluronic acid (G) fragments are measured by a 1H NMR method to obtain the ratio M / G (see “sugar complex biochemical research”), the molecular weight of sodium alginate is determined using high-performance liquid chromatography (Wenjing Tai, Guangli Yu, Jiandong Wu, Xia Zhao, Extract and physicochemical properties of four types of algae polysaccharides, Journal of Ocean University of China, 2010, 40 (5): 23-26) and the fucoidan sulfate content is measured by nephelometry (Jianbo Cong, Changzhen Wang, Yan Li, etc., Measurement of sulfate group content of brown algae sulfated polysaccharides - study of barium sulfate nephelometry [J] Pharmaceutical Journal of Chinese People's Liberation Army, 2003,19 (3): 181). Comparative Example 1
[073] Control process: 100 g of Japanese powder raw material after being washed, dried and sprayed is weighed and placed in a 2L beaker, and 1L of water and 20 g of sodium carbonate are added to the beaker . The mixer is stirred in a water bath at 70 ° C for 1 hour and then diluted with 80 to 100 L of water, followed by sufficient stirring and filtration. The filtrate is adjusted to pH 2.0 by the addition of hydrochloric acid and is centrifuged. The precipitate is converted to sodium alginate by the addition of sodium carbonate. The resulting supernatant is subjected to gradual ethanol precipitation to obtain fucoidan (by adding ethanol until the alcohol content reaches 65% by weight) and laminarane (by adding ethanol until the alcohol content reaches 85% by weight). The products obtained through each of the stages are dried to calculate their extraction yields, where the extraction yield is the percentage by weight of the product of each stage and raw material of Japanese powder. All analytical results are shown in Table 1. Example 1: 1) 1.5 kg of dry Japanese powder is placed in a reaction wave microwave oven; 2) 0.5 L of anhydrous acid and 0.25 L of water are mixed to prepare 0.75 L of 70% formic acid solution; 3) 0.75 L of formic acid in step 2) is added to the microwave reaction chamber in step 1), and the mixer is sufficiently agitated to uniformly humidify the Japanese powder; 4) the material moistened in step 3) is subjected to irradiation in the continuous microwave power of 5 kW until the liquid begins to reflux, that is, the organic acid solution is vaporized and then the microwave is switched to a pulsed microwave power operation mode, where the service ratio is 5 seconds / 5 seconds (that is, the relationship between the on and off time), the peak power is 10 kW; after 12 min, aspiration is performed (the working pressure in the reaction chamber is 20 mmHg) to perform the distillation under reduced pressure until there is no liquid in the microwave reaction chamber; the microwave pre-treatment of Japanese powder is completed; 5) 5 L of absolute ethanol is added to 1.5 kg of pre-treated Japanese powder, sufficiently stirred and filtered, in which the filtrate is distilled to recover ethanol, and the residue after being dried is pre-treated Japanese powder with microwave; 6) 100 g of Japanese powder pre-treated with microwave in step 5) are placed in a 1 L beaker, 500 mL of distilled water is added to this and the mixer is placed in a hot water bath at 70 ° C for 40 min for extraction and then filtered. The above procedure is repeated once. The two filtrates are combined and their pH is adjusted to neutrality, and the residue is kept for further processing; 7) The filtrate in step 6) is concentrated and subjected to gradual ethanol precipitation to obtain sodium alginate with fragments rich in M (by adding ethanol until the alcohol content reaches 30% by weight), fucoidan (by adding ethanol until the alcohol content reaches 65% by weight) and laminarane (by adding ethanol until the alcohol content reaches 85% by weight); 8) 500 ml of water and 6 g of sodium carbonate are added to the residue in step 6), the mixer is stirred in a hot water bath at 70 ° C for 40 min and filtered. The filtrate is neutralized and concentrated, and is subjected to precipitation with alcohol to produce sodium alginate with fragments rich in G.
[074] Data including product extraction yield at each stage, total water consumption in the process, viscosity of sodium alginate products, fucoidan sulfate content, etc. are listed in Table 1.
[075] Table 1 Results of the comparison of the polysaccharide extraction yield of each stage, total amount of water consumption, sodium alginate viscosity and fucoidan sulfate content between the use of dry powder pre-treated with micro- waves and use of untreated dry Japanese powder raw materials:
Comparative Example 2
[076] Control process: 100 g of powdered fusiform raw material after being washed, dried and sprayed is weighed and placed in a 2L beaker, and 1L of water and 25 g of sodium carbonate are added to the beaker . The mixer is stirred in a water bath at 70 ° C for 1 hour and then diluted with 80 to 100 L of water, followed by sufficient stirring and filtration. The filtrate is adjusted to pH 2.0 by the addition of hydrochloric acid and is centrifuged. The precipitate is converted to sodium alginate by the addition of sodium carbonate. The resulting supernatant is subjected to precipitation with ethanol of classification to obtain fucoidan (by adding ethanol until the alcohol content reaches 65% by weight) and laminarane (by adding ethanol until the alcohol content reaches 85% by weight). The parameters in relation to the products are measured according to Comparative Example 1. All analysis results are shown in Table 2. Example 2: 1) 1.5 Kg of dry powder spindle is placed in a micro reaction chamber propagation wave waves; 2) 180 g of oxalic acid and 1.5 L of water are mixed to prepare a 10% solution of oxalic acid; 3) 1.5 L of oxalic acid solution in step 2) is added to the microwave reaction chamber in step 1) and the mixer is sufficiently agitated to uniformly humidify the fusiform powder; 4) the material moistened in step 3) is subjected to irradiation in the continuous microwave power of 3 kW until the liquid begins to reflux, that is, the organic acid solution is vaporized and then the microwave is switched to a pulsed microwave power operation mode, where the service ratio is 5 seconds / 5 seconds, the peak power being 5 kW; after 15 min, aspiration is performed (working pressure of the reaction chamber equal to 100 mmHg) for distillation under reduced pressure to remove the organic acid solution until there is no more liquid in the microwave reaction chamber; the microwave pretreatment of the spindle powder is completed; 5) 5 L of absolute ethanol are added to 1.5 kg of fusiform powder pre-treated in step 4), sufficiently stirred and filtered, in which the filtrate is distilled to recover ethanol and oxalic acid, and the residue after drying is fusiform powder pretreated with microwave; 6) 100 g of fusiform powder pretreated with microwave in step 5) are placed in a 1 L beaker, 500 ml of distilled water is added to this and the mixer is placed in a hot water bath at 70 ° C for 40 min for extraction and then filtered. The above procedure is repeated once. The two filtrates are combined and their pH is adjusted to neutrality, and the residue is kept for further processing;
[077] Steps 7) and 8) are the same as in Example 1.
[078] Data including product extraction yield at each stage, total water consumption in the process, viscosity of sodium alginate products, fucoidan sulfate content, etc. are listed in Table 2.
[079] Table 2 Results of the comparison of the polysaccharide extraction yield of each stage, total amount of water consumption, sodium alginate viscosity and fucoidan sulfate content between the use of pre-treated dry fusiform powder waves and use of untreated dry fusiform powder raw materials:
Comparative Example 3
[080] Control process: 100 g of raw kelp powder after being washed, dried and sprayed is weighed and placed in a 2L beaker, and 1L of water and 25 g of sodium carbonate are added to the beaker . The mixer is stirred in a water bath at 70 ° C for 1 hour and then diluted with 80 to 100 L of water, followed by sufficient stirring and filtration. The filtrate is adjusted to pH 2.0 by the addition of hydrochloric acid and is centrifuged. The precipitate is converted to sodium alginate by the addition of sodium carbonate. The resulting supernatant is subjected to gradual ethanol precipitation to obtain fucoidan (by adding ethanol until the alcohol content reaches 65% by weight) and laminarane (by adding ethanol until the alcohol content reaches 85% by weight). The parameters in relation to the products are measured according to Comparative Example 1. All analysis results are shown in Table 3. Example 3: 1) 1.5 Kg of dry kelp powder is placed in a micro reaction chamber resonant wave waves; 2) 0.4 L of acetic anhydride and 0.1 L of water are mixed to prepare 0.5 L of an 80% acetic acid solution; 3) 0.5 L of acetic acid solution in step 2) is added to the microwave reaction chamber in step 1) and the mixer is sufficiently agitated to uniformly humidify the powdered kelp; 4) the humidified material in step 3) is subjected to irradiation in the continuous microwave power of 2 kW until the liquid begins to reflux, that is, the organic acid solution is vaporized and then the microwave it is switched to a pulsed microwave power operation mode, where the service ratio is 5 seconds / 5 seconds, the peak power being 4 kW; after 35 min, aspiration is carried out (working pressure of the reaction chamber equal to 200 mmHg) for distillation under reduced pressure to remove the organic acid solution until there is no more liquid in the microwave reaction chamber; 5) 5 L of absolute ethanol are added in the microwave reaction chamber in step 4), sufficiently stirred and filtered, in which the filtrate is distilled to recover the ethanol, and the residue after drying is the pre-dried kelp powder. microwave-treated; 6) 100 g of kelp powder pre-treated with microwave powder in step 5) are placed in a 1 L beaker, 500 ml of distilled water is added to this and the mixer is placed in a hot water bath at 70 ° C for 40 min for extraction and then filtered. The above procedure is repeated once. The two filtrates are combined and their pH is adjusted to neutrality, and the residue is kept for further processing;
[081] Steps 7) and 8) are the same as in Example 1.
[082] Data including product extraction yield at each stage, total water consumption in the process, viscosity of sodium alginate products, fucoidan sulfate content, etc. are listed in Table 3.
[083] Table 3 Results of the comparison of the polysaccharide extraction yield of each stage, total amount of water consumption, sodium alginate viscosity and fucoidan sulfate content between the use of kelp powder pretreated with microwave and use of untreated dry powder kelp raw materials:
Comparative Example 4
[084] Control process: 100 g of raw wakame powder after being washed, dried and sprayed is weighed and placed in a 2L beaker, and 1L of water and 25 g of sodium carbonate are added to the beaker . The mixer is stirred in a water bath at 70 ° C for 1 hour and then diluted with 80 to 100 L of water, followed by sufficient stirring and filtration. The filtrate is adjusted to pH 2.0 by the addition of hydrochloric acid and is centrifuged. The precipitate is converted to sodium alginate by the addition of sodium carbonate. The resulting supernatant is subjected to gradual ethanol precipitation to obtain fucoidan (by adding ethanol until the alcohol content reaches 65% by weight) and laminarane (by adding ethanol until the alcohol content reaches 85% by weight). The parameters in relation to the products are measured according to Comparative Example 1. All analysis results are shown in Table 4. Example 4:
[085] The differences from Example 1 are: 1) 1.5 kg of dry powdered wakame after being dried, decontaminated and sprayed is placed in a resonant microwave reaction chamber; 2) 0.8 L of anhydrous formic acid, 0.15 L of water and 0.05 L of 20% hydrochloric acid solution are mixed to prepare 1 L of formic acid - hydrochloric acid mixture solution, in which the content formic acid is 83% and the hydrochloric acid content is 0.1%; 3) 1 L of mixed acid solution in step 2) is added to the microwave reaction chamber in step 1) and the mixer is sufficiently stirred to uniformly moisten the powdered wakame; 4) the material moistened in step 3) is subjected to irradiation in the continuous microwave power of 3.5 kW until the liquid begins to reflux, that is, the organic acid solution is vaporized and then the micro -waves is switched to a pulsed microwave power operation mode, where the service ratio is 5 seconds / 5 seconds, the peak power being 7 kW; after 13 min, aspiration is carried out (working pressure of the reaction chamber equal to 150 mmHg) for distillation under reduced pressure to remove the organic acid solution until there is no more liquid in the microwave reaction chamber; 5) 5 L of absolute ethanol are added to the microwave reaction chamber in step 4), sufficiently stirred and filtered, in which the filtrate is distilled to recover the ethanol, and the residue after drying is the pre-wakame powder. microwave-treated; 6) 100 g of microwaved powdered wakame in step 5) are placed in a 1 L beaker, 500 ml of distilled water is added to this and the mixer is placed in a hot water bath at 70 ° C for 40 min for extraction and then filtered. The above procedure is repeated once. The two filtrates are combined and their pH is adjusted to neutrality, and the residue is kept for further processing;
[086] Steps 7) and 8) are the same as in Example 1.
[087] Data including product extraction yield at each stage, total water consumption in the process, viscosity of sodium alginate products, fucoidan sulfate content, etc. are listed in Table 4.
[088] Table 4 Results of the comparison of the polysaccharide extraction yield of each stage, total amount of water consumption, sodium alginate viscosity and fucoidan sulfate content between the use of wakame in dry powder pretreated with micro- waves and use of untreated dry powder wakame raw materials:
Comparative Example 5
[089] Control process: 100 g of raw fucus powder after being washed, dried and sprayed is weighed and placed in a 2L beaker, and 1L of water and 25 g of sodium carbonate are added to the beaker . The mixer is stirred in a water bath at 70 ° C for 1 hour and then diluted with 80 to 100 L of water, followed by sufficient stirring and filtration. The filtrate is adjusted to pH 2.0 by the addition of hydrochloric acid and is centrifuged. The precipitate is converted to sodium alginate by the addition of sodium carbonate. The resulting supernatant is subjected to gradual ethanol precipitation to obtain fucoidan (by adding ethanol until the alcohol content reaches 65% by weight) and laminarane (by adding ethanol until the alcohol content reaches 85% by weight). The parameters in relation to the products are measured according to Comparative Example 1. All analysis results are shown in Table 5. Example 5:
[090] The differences with respect to Example 1 are: 1) 1.5 kg of dry powdered fucus after being dried, decontaminated and sprayed is placed in a resonant microwave reaction chamber; 2) 1.8 L of anhydrous propanoic acid and 0.2 L of water are mixed to prepare 2 L of 90% propanoic acid solution; 3) 2 L of propanoic acid solution in step 2) are added to the microwave reaction chamber in step 1) and the mixer is sufficiently stirred to uniformly moisten the fucus powder; 4) the material moistened in step 3) is subjected to irradiation in the continuous microwave power of 4 kW until the liquid begins to reflux, that is, the organic acid solution is vaporized and then the power of the micro -waves is switched to 2 kW; after 30 min, aspiration is performed (working pressure of the reaction chamber equal to 150 mmHg) for distillation under reduced pressure to remove organic acid until there is no more liquid in the microwave reaction chamber; 5) 5 L of absolute ethanol are added in the microwave reaction chamber in step 4), sufficiently stirred and filtered, in which the filtrate is distilled to recover the ethanol, and the residue after drying is the pre-dried fucus powder. microwave-treated; 6) 100 g of powdered fucus pre-treated with microwave in step 5) are placed in a 1 L beaker, 500 ml of distilled water is added to this and the mixer is placed in a hot water bath at 70 ° C for 40 min for extraction and then filtered. The above procedure is repeated once. The two filtrates are combined and their pH is adjusted to neutrality, and the residue is kept for further processing;
[091] Steps 7) and 8) are the same as in Example 1.
[092] Data including product extraction yield at each stage, total water consumption in the process, viscosity of sodium alginate products, fucoidan sulfate content, etc. are listed in Table 5.
[093] Table 5 Results of the comparison of the polysaccharide extraction yield of each stage, total amount of water consumption, sodium alginate viscosity and fucoidan sulfate content between the use of pre-treated dry powdered fucus waves and use of raw raw powdered fucus raw materials:
Comparative Example 6
[094] Control process: 100 g of powdered carrageenan raw material after being washed, dried and pulverized is weighed and placed in a 2L beaker, and 1L of water and 25 g of sodium carbonate are added to the beaker . The mixer is stirred in a water bath at 70 ° C for 1 hour and then diluted with 80 to 100 L of water, followed by sufficient stirring and filtration. The filtrate is adjusted to pH 2.0 by the addition of hydrochloric acid and is centrifuged. The precipitate is converted to sodium alginate by the addition of sodium carbonate. The resulting supernatant is subjected to gradual ethanol precipitation to obtain fucoidan (by adding ethanol until the alcohol content reaches 65% by weight) and laminarane (by adding ethanol until the alcohol content reaches 85% by weight). The parameters in relation to the products are measured according to Comparative Example 1. All analysis results are shown in Table 6. Example 6:
[095] The differences with respect to Example 1 are: 1) 1.5 Kg of dry powdered carrageenan, after being dried, decontaminated and pulverized, is placed in a resonant microwave reaction chamber; 2) 300 g of anhydrous oxalic acid and 1.5 L of 0.12% hydrochloric acid solution are mixed to prepare about 1.5 L of mixed oxalic acid - hydrochloric acid solution, where the oxalic acid content is 20% and the hydrochloric acid content is 0.1%; 3) 1.5 L of mixed oxalic acid-hydrochloric acid solution in step 2) is added to the microwave reaction chamber in step 1) and the mixer is sufficiently agitated to uniformly humidify the powdered carrageenan; 4) the humidified material in step 3) is subjected to irradiation in the continuous microwave power of 3 kW until the liquid begins to reflux, that is, the organic acid solution is vaporized and then the power of the micro -waves is switched to 1.5 kW; after 30 min, aspiration is performed (working pressure of the reaction chamber equal to 100 mmHg) for distillation under reduced pressure until there is no more liquid in the microwave reaction chamber; 5) 5 L of absolute ethanol are added to the microwave reaction chamber in step 4), sufficiently stirred and filtered, in which the filtrate is distilled to recover the ethanol and oxalic acid, and the residue after drying is the carrageenan in microwave pretreated powder; 6) 100 g of carrageenan powder pretreated with microwave in step 5) are placed in a 1 L beaker, 500 ml of distilled water is added to this and the mixer is placed in a hot water bath at 70 ° C for 40 min for extraction and then filtered. The above procedure is repeated once. The two filtrates are combined and their pH is adjusted to neutrality, and the residue is kept for further processing;
[096] Steps 7) and 8) are the same as in Example 1.
[097] Data including product extraction yield at each stage, total water consumption in the process, viscosity of sodium alginate products, fucoidan sulfate content, etc. are listed in Table 6.
[098] Table 6 Results of the comparison of the polysaccharide extraction yield for each stage, total amount of water consumption, sodium alginate viscosity and fucoidan sulfate content between the use of microwaved powdered carrageenan and use of raw raw powdered carrageenan raw materials:

[099] The results of the examples show that the present invention uses the microwave chemical method to pre-treat raw materials from brown algae and then uses, respectively, aqueous extraction and alkaline digestion to obtain uronic acid and oligosaccharide oligosaccharides of fucoidan with different structures and to make the sulfate content in fucoidan stay in a moderate range, while overcoming the disadvantages of high water consumption, high level of heavy pollution and many others in the existing processes.
[100] Experiment 1 Effects of Japanese brown algae polysaccharides on blood glucose levels in diabetic mice
[101] Experimental animals: male Kunming mice, weighing (22 ± 2) g, supplied by Experimental Animal Center of Military Medical Sciences, Beijing
[102] Experimental materials: streptozotocin (STZ), available from Sigma, and Japanese brown algae polysaccharides prepared in Example 1.
[103] Main instruments and equipment: UV-Vis spectrophotometer TU-1810, by Beijing Purkinje General Instrument Co., Ltd .; electronic scale BS-124S, Sartorius AG, Germany; centrifuge TGL-16G-A, Shanghai Anting Scientific Instrument Factory; HPX-9052MBE electric oven, Shanghai Haibo Motion Industries Limited; superfine homogenizer F6 / 10, Shanghai Fluko Fluid Machinery Manufacturing Co., Ltd.
[104] Experimental method: mice are injected intraperitoneally with streptozotocin (STZ) 50 mg / kg, and those with blood glucose levels> 11.1 mmol / L are used as a model for diabetes. 20 model mice, according to the blood glucose level, are randomly assigned to the model group and the brown algae polysaccharide group. Another 10 normal mice are used as a control group. The mice in each group are treated by gavage daily at 9:00 am, in which the brown algae polysaccharide group receives a dose of 500 mg / kg, the model group and the control group receive a corresponding volume of water distilled. The treatment continues for 32 days and the mice are fasted, but fed with water for 12 hours on the 21st day and on the 32nd day. Blood samples are collected from the orbital cavity, and serum is separated to detect the fasting blood glucose values of the mice.
[105] Statistical method: the statistical method uses the SPSS statistical software, the data are all represented in ± if a comparative analysis between the groups is analyzed by variance. The results are shown in the table below:

[106] The brown algae polysaccharides of the present invention can effectively and significantly reduce the blood glucose level in diabetic mice.

权利要求:
Claims (9)
[0001]
1. PROCESS OF EXTRACTION OF POLYSACARIDE FROM BROWN ALGAE by means of a chemical method with microwave, characterized by the fact that the process comprises the following steps: 1) placing raw materials of brown algae after washing, drying and spraying in a microwave reactor chamber, add an organic acid or an organic / inorganic acid mixture solution and stir sufficiently to moisten the powder well; 2) treat with microwaves, apply microwave power using the cooperative effects between microwaves, organic acid molecules and brown algae polysaccharides, selectively cut the glycosidic bonds of brown algae polysaccharides, in order to achieve controllable degradation the same; 3) use distillation under reduced pressure by microwave heating to remove most organic acids or the mixed organic / inorganic acid solution and wash sufficiently with an organic solvent to remove a small amount of residual acid to complete the pre- microwave treatment of brown algae; 4) add about 5 to 8 times of water to the brown algae extract pretreated with microwave, in which the extraction solution, after concentration, is subjected to gradual alcohol precipitation to obtain the excellent algine with fragments rich in water-soluble manuronic acid (rich in M), fucoidan and laminaran; and 5) submit the raw material residue of brown algae pretreated with a microwave after the aqueous extraction to the alkaline digestion process to obtain algin with fragments rich in guluronic acid (rich in G).
[0002]
2. PROCESS, according to claim 1, characterized by the fact that the organic acid is selected from oxalic acid from a concentration in weight of 5% to 50%; acetic acid of a concentration by weight of 10% to 99%, or propionic acid of a concentration by weight of 10% to 99%.
[0003]
3. PROCESS, according to claim 1, characterized by the fact that the microwave power application mode is a continuous microwave mode or a combination of continuous microwave mode and microwave mode pulsed; where, in the case of using the combination of continuous microwave and pulsed microwave, continuous microwave irradiation is first used until the acid solution is refluxed and then it is switched to microwave irradiation waves pulsed for 5 min to 120 min.
[0004]
4. PROCESS, according to claim 1, characterized by the fact that, in said step 2), in the case of continuous microwave, the mass power density is 1 kW per kilogram of material at 5 kilowatts per kilogram of material; in the case of pulsed microwaves, the mass power density is 2 kilowatts per kilogram of material to 10 kilowatts per kilogram of material, the service ratio is A / B, where A = 1 s to 100 s and B = 1 s 100 s.
[0005]
5. PROCESS, according to claim 1, characterized by the fact that brown algae are selected from Japanese Laminaria (Laminaria), Sargassum (Sargassum) sargassum, sea millet, fusiform, seaweed, creeping sargasso rods, Fucus (Fucus ) fucus, fucus, Pelvetia carragena (Pelvetia), Undaria (Undaria) wakame and / or Maerocystis (Maerocystis) kelp.
[0006]
6. PROCESS OF EXTRACTION OF POLYSACARIDES FROM BROWN ALGAE, by means of a chemical method with microwave, according to one of the claims 1-5, in which the process comprises the following steps: 1) 1.5 Kg of japonica in dry powder is placed in a reaction microwave wave propagation chamber; 2) 0.5 L of anhydrous acid and 0.25 L of water are mixed to prepare 0.75 L of 70% formic acid solution; 3) 0.75 L of formic acid in step 2) is added to the microwave reaction chamber in step 1), and the mixer is sufficiently agitated to uniformly humidify the Japanese powder; 4) the material moistened in step 3) is subjected to irradiation in the continuous microwave power of 5 kW until the liquid begins to reflux, that is, the organic acid solution is vaporized and then the microwave is switched to a pulsed microwave power operation mode, where the service ratio is 5 seconds / 5 seconds (that is, the relationship between the on and off time), the peak power is 10 kW; after 12 min, aspiration is performed (the working pressure in the reaction chamber is 20 mmHg) to perform the distillation under reduced pressure until there is no liquid in the microwave reaction chamber; the microwave pre-treatment of Japanese powder is completed; 5) 5 L of absolute ethanol is added to 1.5 kg of pre-treated Japanese powder, sufficiently stirred and filtered, in which the filtrate is distilled to recover ethanol, and the residue after being dried is pre-treated Japanese powder with microwave; 6) 100 g of Japanese powder pre-treated with microwave in step 5) are placed in a 1 L beaker, 500 mL of distilled water is added to this and the mixer is placed in a hot water bath at 70 ° C for 40 min for extraction and then filtered; the above procedure is repeated once; the two filtrates are combined and their pH is adjusted to neutrality, and the residue is kept for further processing; 7) The filtrate in step 6) is concentrated and subjected to gradual ethanol precipitation to obtain sodium alginate with fragments rich in M (by adding ethanol until the alcohol content reaches 30% by weight), fucoidan (by adding ethanol until the alcohol content reaches 65% by weight) and laminarane (by adding ethanol until the alcohol content reaches 85% by weight); and 8) 500 ml of water and 6 g of sodium carbonate are added to the residue in step 6), the mixer is stirred in a hot water bath at 70 ° C for 40 min and filtered. The filtrate is neutralized and concentrated, and is subjected to precipitation with alcohol to produce sodium alginate with fragments rich in G.
[0007]
7. PROCESS OF EXTRACTION OF POLYSACARIDES FROM BROWN ALGAE, by means of a chemical method with microwave, according to one of the claims 1-5, in which the process comprises the following steps: 1) 1.5 Kg of fusiform in dry powder is placed in a reaction microwave wave propagation chamber; 2) 180 g of oxalic acid and 1.5 L of water are mixed to prepare a 10% solution of oxalic acid; 3) 1.5 L of oxalic acid solution in step 2) is added to the microwave reaction chamber in step 1) and the mixer is sufficiently agitated to uniformly humidify the fusiform powder; 4) the material moistened in step 3) is subjected to irradiation in the continuous microwave power of 3 kW until the liquid begins to reflux, that is, the organic acid solution is vaporized and then the microwave is switched to a pulsed microwave power operation mode, where the service ratio is 5 seconds / 5 seconds, the peak power being 5 kW; after 15 min, aspiration is performed (working pressure of the reaction chamber equal to 100 mmHg) for distillation under reduced pressure to remove the organic acid solution until there is no more liquid in the microwave reaction chamber; the microwave pretreatment of the spindle powder is completed; 5) 5 L of absolute ethanol are added to 1.5 kg of fusiform powder pre-treated in step 4), sufficiently stirred and filtered, in which the filtrate is distilled to recover ethanol and oxalic acid, and the residue after drying is fusiform powder pretreated with microwave; 6) 100 g of fusiform powder pretreated with microwave in step 5) are placed in a 1 L beaker, 500 ml of distilled water is added to this and the mixer is placed in a hot water bath at 70 ° C for 40 min for extraction and then it is filtered; the above procedure is repeated once; the two filtrates are combined and their pH is adjusted to neutrality, and the residue is kept for further processing; 7) and 8) subsequent steps 7) and 8) are as defined in claim 6.
[0008]
8. BROWN ALGAE POLYSACARIDE EXTRACTION PROCESS, using a microwave microwave method, according to one of claims 1-5, in which the process comprises the following steps: 1) 1.5 kg of kelp in dry powder is placed in a microwave resonant wave reaction chamber; 2) 0.4 L of acetic anhydride and 0.1 L of water are mixed to prepare 0.5 L of an 80% acetic acid solution; 3) 0.5 L of acetic acid solution in step 2) is added to the microwave reaction chamber in step 1) and the mixer is sufficiently agitated to uniformly humidify the powdered kelp; 4) the humidified material in step 3) is subjected to irradiation in the continuous microwave power of 2 kW until the liquid begins to reflux, that is, the organic acid solution is vaporized and then the microwave it is switched to a pulsed microwave power operation mode, where the service ratio is 5 seconds / 5 seconds, the peak power being 4 kW; after 35 min, aspiration is carried out (working pressure of the reaction chamber equal to 200 mmHg) for distillation under reduced pressure to remove the organic acid solution until there is no more liquid in the microwave reaction chamber; 5) 5 L of absolute ethanol are added in the microwave reaction chamber in step 4), sufficiently stirred and filtered, in which the filtrate is distilled to recover the ethanol, and the residue after drying is the pre-dried kelp powder. microwave-treated; 6) 100 g of kelp powder pre-treated with microwave powder in step 5) are placed in a 1 L beaker, 500 ml of distilled water is added to this and the mixer is placed in a hot water bath at 70 ° C for 40 min for extraction and then filtered; the above procedure is repeated once; the two filtrates are combined and their pH is adjusted to neutrality, and the residue is kept for further processing; 7) and 8) subsequent steps 7) and 8) are as defined in claim 6.
[0009]
9. BROWN ALGAE POLYSACARIDE EXTRACTION PROCESS, by means of a chemical microwave method, according to one of claims 1-5, in which the process comprises the following steps: 1) 1.5 Kg of wakame in dry powder after being dried, decontaminated and sprayed is placed in a microwave resonant wave reaction chamber; 2) 0.8 L of anhydrous formic acid, 0.15 L of water and 0.05 L of 20% hydrochloric acid solution are mixed to prepare 1 L of formic acid - hydrochloric acid mixture solution, in which the content formic acid is 83% and the hydrochloric acid content is 0.1%; 3) 1 L of mixed acid solution in step 2) is added to the microwave reaction chamber in step 1) and the mixer is sufficiently stirred to uniformly moisten the powdered wakame; 4) the material moistened in step 3) is subjected to irradiation in the continuous microwave power of 3.5 kW until the liquid begins to reflux, that is, the organic acid solution is vaporized and then the micro -waves is switched to a pulsed microwave power operation mode, where the service ratio is 5 seconds / 5 seconds, the peak power being 7 kW; after 13 min, aspiration is carried out (working pressure of the reaction chamber equal to 150 mmHg) for distillation under reduced pressure to remove the organic acid solution until there is no more liquid in the microwave reaction chamber; 5) 5 L of absolute ethanol are added to the microwave reaction chamber in step 4), sufficiently stirred and filtered, in which the filtrate is distilled to recover the ethanol, and the residue after drying is the pre-wakame powder. microwave-treated; 6) 100 g of microwaved powdered wakame in step 5) are placed in a 1 L beaker, 500 ml of distilled water is added to this and the mixer is placed in a hot water bath at 70 ° C for 40 min for extraction and then filtered; the above procedure is repeated once; the two filtrates are combined and their pH is adjusted to neutrality, and the residue is kept for further processing; and 7) and 8) the subsequent steps 7) and 8) are as defined in claim 6.

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KR20140087052A|2014-07-08|

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JP5878241B2|2016-03-08|

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EP2778178A1|2014-09-17|

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法律状态:
2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|

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2019-10-01| B06U| Preliminary requirement: requests with searches performed by other patent offices: suspension of the patent application procedure|

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2020-09-01| B09A| Decision: intention to grant|

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2020-12-15| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 01/11/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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CN201110348242.6|2011-11-07|

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CN2011103482426A|CN102417549B|2011-11-07|2011-11-07|Efficient extracting method for active polysaccharide of brown alga based on microwave chemistry|

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PCT/CN2012/083932|WO2013067896A1|2011-11-07|2012-11-01|Method for extracting brown algae polysaccharide via microwave chemical process|

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