Method for simultaneously preparing arabinogalactan and dihydroquercetin based on larch

Abstract

The invention relates to a method for simultaneously preparing arabinogalactan and dihydroquercetin on the basis of larch, which comprises the following steps: selecting root wood after rotation of larch, clearly crushing the root wood to obtain the used raw material small sawdust, performing enzymolysis and pressurized water extraction, and simultaneously combining membrane separation and membrane concentration technologies to obtain the arabinogalactan and dihydroquercetin. Meanwhile, an arabinogalactan product and 99% of dihydroquercetin product are prepared. Extraction adopts enzymolysis, pressurization and low-temperature water extraction, the tight structure of the raw material is destroyed through cellulase and hemicellulase, the seepage force and penetrating force of water are increased through pressurization, the extraction efficiency is improved, then existing dihydroquercetin glycosides are subjected to enzymolysis through beta-glucosidase to form free dihydroquercetin, the yield is improved, and the method is suitable for industrial production. The method is simple in operation, combines membrane separation and membrane concentration technologies, avoids the defects of traditional normal-pressure water extraction, and compared with traditional organic reagent extraction and ether ester extraction, the method does not use organic reagents in the whole process, avoids environmental pollution, and is safe, feasible and low in cost.

Description

Technical Field

[0001] The invention belongs to the technical field of preparation of natural products, and particularly relates to a method for simultaneously preparing arabinogalactan and dihydroquercetin based on larch. Background Art

[0002] Larix is ​​a tree of the genus Larix, Pinaceae, up to 35 meters tall and 90 centimeters in diameter at breast height. It is the main tree species in the coniferous forest of Greater Khingan Range in China, with abundant timber reserves. A large number of tree roots remain after annual rotation felling, resulting in a huge waste of resources. Countries with abundant larch resources, such as the United States, Canada, and Russia, extract dihydroquercetin from them to achieve the purpose of comprehensive utilization. Dihydroquercetin is a scarce resource in the world and is an extremely precious ingredient for medicines and functional foods. Because yew and yellow fir have been listed as endangered species worldwide and are prohibited from being cut down, and larch is only distributed in eastern Siberia, Russia, northeastern Mongolia, northeastern China, and North Korea, and has a long growth cycle, the resources available for production are extremely scarce and scarce.

[0003] In addition to dihydroquercetin, larch can also be used to extract arabinogalactan, a neutral polysaccharide composed of arabinose and galactose. Arabinogalactan is found in large quantities in the xylem of coniferous trees, and the content in larch is as high as 25%.

[0004] Dihydroquercetin, also known as taxol and taxafoetida, is a dihydroflavonol compound belonging to the vitamin P family. It was first discovered by Hungarian biochemist Albert Szent-Gyorgyi in 1938. In 2017, dihydroquercetin was approved by the European Union as a new food ingredient for the EU market. In the United States and Russia, dihydroquercetin was initially listed as a pharmaceutical raw material, and later expanded to the food field with development; Japan, South Korea, Thailand, Canada and other countries also use dihydroquercetin as a raw material for medicinal, functional foods, special diets, cosmetics and other fields. According to data, by 2027, the global demand for dihydroquercetin will be 70 tons. In April 2021, dihydroquercetin was officially approved by the National Health Commission as a new food raw material. Dihydroquercetin was approved as a new food raw material in China, filling the gap in the domestic dihydroquercetin market. It will be widely used in food, health food, cosmetics, pharmaceutical industry, industry, agriculture and other fields, and its development prospects are very broad.

[0005] The existing US patent (US2744919A) uses alcohol extraction, ether extraction, crystallization and other means to prepare dihydroquercetin; the Russian patent (RU2184561C1) uses organic solvent high temperature reflux extraction, acid hydrolysis, crystallization and other means to prepare dihydroquercetin; the Chinese patent CN100417643C uses 70-90℃ hot water extraction, cooling, methyl tert-butyl ether extraction and other means to prepare dihydroquercetin. In patents such as CN101851221B, CN103360359B, CN116354918B, and CN116987056A, hot water, ethanol, petroleum ether, acetonitrile and other organic solvents are basically used for extraction, and dihydroquercetin is prepared by ether and ester solvent extraction and other means. At present, the production of dihydroquercetin is mainly prepared by extraction with hot water and aqueous ethanol, extraction with ether and ester solvents, crystallization and recrystallization; or extraction with hot water and aqueous ethanol, purification with macroporous adsorption resin, extraction, crystallization and other means. However, these methods have more or less defects, such as cumbersome production process and high loss; excessive use of organic solvents and high toxicity; high temperature and high energy consumption during the production process; therefore, the development of safe, efficient, simple to operate and environmentally friendly preparation technology is of great significance for the wide application of dihydroquercetin in the pharmaceutical and food industries. Summary of the invention

[0006] The purpose of the present invention is to solve the above-mentioned problems. The present application proposes a method for simultaneously preparing arabinogalactan and dihydroquercetin based on larch. Arabinogalactan and 99% dihydroquercetin are prepared simultaneously by enzymatic hydrolysis, pressurized water purification and membrane separation and membrane concentration technology.

[0007] To achieve the above object, the present invention provides the following technical solution: a method for simultaneously preparing arabinogalactan and dihydroquercetin based on larch, using root wood after rotation felling of larch, comprising the following steps: S1. Raw materials: Wash and crush the root wood of larch after rotation felling to obtain small wood chips; S2. Fermentation: Use water with a content of about 10% sawdust, add 0.5-1% sawdust-based cellulase, hemicellulase, and β-glucosidase, mix well, and evenly spray on the sawdust. Leave it to stand at room temperature for 3-10 days for natural fermentation. S3. Extraction: After the fermentation of small sawdust, add 10 times the amount of drinking water, pressurize to 0.5-0.8MPa by compressed air at room temperature, and maintain the pressure for 2-5 hours; S4. Filtration: After the extraction is completed, plate and frame filtration, 0.1um precision filtration, and ultrafiltration are used in sequence to remove insoluble large particle impurities and large molecular proteins and sugars to obtain a clear solution; S5. Separation of arabinogalactan: After the filtration in step S4, the clear liquid is further filtered through a nanofiltration membrane, and dihydroquercetin and small molecular impurities of small molecular structure pass through the nanofiltration membrane, and arabinogalactan of large molecular structure is retained on the nanofiltration membrane, and arabinogalactan and dihydroquercetin are separated, and the retained arabinogalactan is collected in a container, stirred, and 1 to 2 times of edible alcohol is slowly added during the stirring process, centrifuged, and freeze-dried to obtain arabinogalactan; S6. Purification of dihydroquercetin: The clear solution containing dihydroquercetin in step S5 is passed through a nucleopore membrane of a certain pore size to remove impurities with a molecular weight greater than that of dihydroquercetin, and then passed through the nucleopore membrane to remove impurities with a molecular weight less than that of dihydroquercetin, and the portion containing dihydroquercetin is collected, refrigerated for crystallization, and the solid matter is freeze-dried after filtration to obtain a dihydroquercetin product with a content greater than 99%.

[0008] Furthermore: the cellulase and hemicellulase in step S2 can destroy the compact structure of the raw material, and the β-glucosidase enzymatically hydrolyzes the free dihydroquercetin glycoside into free dihydroquercetin.

[0009] Further: in step S6, the pore diameter of the nucleopore membrane for filtering out impurities with a molecular weight greater than that of dihydroquercetin is selected to be 0.2-10 microns, and the diameter of the nucleopore membrane for removing impurities with a molecular weight less than that of dihydroquercetin is selected to be less than 0.2 microns.

[0010] Compared with the prior art, the present invention has the following beneficial effects: The preparation method of the present invention is extracted by enzymolysis and pressurized water, and is combined with membrane separation and membrane concentration technology to prepare arabinogalactan products and 99% dihydroquercetin products at the same time. The extraction adopts enzymolysis, pressurization, and low-temperature water extraction, and the tight structure of the raw material is destroyed by cellulase and hemicellulase, and the permeability and penetration of water are increased by pressurization, thereby improving the extraction efficiency, and then the existing dihydroquercetin glycosides are hydrolyzed into free dihydroquercetin by beta-glucosidase, thereby improving the yield, and then combined with membrane separation and membrane concentration technology to prepare arabinogalactan and 99% dihydroquercetin products at the same time. The shortcomings of traditional normal pressure water extraction are avoided, and compared with traditional organic reagent extraction and ether ester extraction, the present method does not use organic reagents throughout the whole process, thereby avoiding environmental pollution, being safe and feasible, and having low cost.

[0011] Moreover, the preparation method of the present invention does not require steam throughout the entire process, is produced at room temperature, and is freeze-dried at low temperature. The product has good quality and high yield. The production process equipment is simple, easy to operate, safe and reliable, pollution-free, highly efficient, and low cost. DETAILED DESCRIPTION

[0012] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments. However, the embodiments are only intended to illustrate the present invention and not to limit the present invention.

[0013] Dihydroquercetin, also known as taxol and taxifolin, is a dihydroflavonol compound belonging to the vitamin P family.

[0014] Arabinogalactan is a neutral polysaccharide composed of arabinose and galactose.

[0015] The invention discloses a method for simultaneously preparing arabinogalactan and dihydroquercetin based on larch, and the root wood after rotation felling of larch is selected. Embodiment 1

[0016] S1: Clean and crush the root wood of larch after rotation felling to obtain small wood chips; S2: Fermentation: Take 1000g of small sawdust, use 100ml of water, add 5g of cellulase, 5g of hemicellulase, and 5g of β-glucosidase, mix well, spray evenly on the sawdust, and then leave it to stand at room temperature for 5 days for natural fermentation; S3: Extraction: Add 10 times the amount of drinking water to the fermented sawdust, pressurize to 0.5MPa with compressed air at room temperature, and maintain the pressure for 3 hours; S4. Filtration: After the extraction is completed, plate and frame filtration, 0.1um precision filtration, and ultrafiltration are used in sequence to remove insoluble large particle impurities and large molecular proteins and sugars to obtain a clear solution; S5. Separation of arabinogalactan: After the filtration in step S4, the clear liquid is further filtered through a nanofiltration membrane, and dihydroquercetin and small molecular impurities of small molecular structure pass through the nanofiltration membrane, and arabinogalactan of large molecular structure is retained on the nanofiltration membrane, and arabinogalactan and dihydroquercetin are separated. The retained arabinogalactan is collected in a container, stirred, and 1 times of edible alcohol is slowly added during the stirring process, centrifuged, and freeze-dried to obtain 51g of arabinogalactan; S6. Purification of dihydroquercetin: The clear solution containing dihydroquercetin in step S5 is passed through a nucleopore membrane of a certain pore size to remove impurities with a molecular weight greater than dihydroquercetin, and then passed through the nucleopore membrane to remove impurities with a molecular weight less than dihydroquercetin, and the portion containing dihydroquercetin is collected, refrigerated and crystallized, and the solid matter is freeze-dried after filtration to obtain 26.4 g of a dihydroquercetin product with a content greater than 99.4%. Embodiment 2

[0017] S1: Clean and crush the root wood of larch after rotation felling to obtain small wood chips; S2: Fermentation: Take 500kg of small sawdust, use 50kg of water, add 3kg of cellulase, 3kg of hemicellulase, and 3kg of β-glucosidase, mix well, spray evenly on the sawdust, and then leave it to stand at room temperature for 8 days for natural fermentation; S3: Extraction: Add 10 times the amount of drinking water to the fermented sawdust, pressurize to 0.7MPa with compressed air at room temperature, and maintain the pressure for 4 hours; S4. Filtration: After the extraction is completed, plate and frame filtration, 0.1um precision filtration, and ultrafiltration are used in sequence to remove insoluble large particle impurities and large molecular proteins and sugars to obtain a clear solution; S5. Separation of arabinogalactan: After the filtration in step S4, the clear liquid is further filtered through a nanofiltration membrane, and dihydroquercetin and small molecular impurities of small molecular structure pass through the nanofiltration membrane, and arabinogalactan of large molecular structure is retained on the nanofiltration membrane, and arabinogalactan and dihydroquercetin are separated. The retained arabinogalactan is collected in a container, stirred, and 1 times of edible alcohol is slowly added during the stirring process. Centrifugation and freeze-drying are performed to obtain 26.3 g of arabinogalactan; S6. Purification of dihydroquercetin: The clear solution containing dihydroquercetin in step S5 is passed through a nucleopore membrane of a certain pore size to remove impurities with a molecular weight greater than dihydroquercetin, and then passed through the nucleopore membrane to remove impurities with a molecular weight less than dihydroquercetin, and the portion containing dihydroquercetin is collected, refrigerated and crystallized, and the solid matter is freeze-dried after filtration to obtain 14.9 kg of a dihydroquercetin product with a content greater than 99.6%. Embodiment 3

[0018] S1: Clean and crush the root wood of larch after rotation felling to obtain small wood chips; S2: Fermentation: Take 1000kg of small sawdust, use 100kg of water, add 8g of cellulase, 8kg of hemicellulase, and 8kg of β-glucosidase, mix well, spray evenly on the sawdust, and then leave it to stand at room temperature for 10 days for natural fermentation; S3: Extraction: Add 10 times the amount of drinking water to the fermented sawdust, pressurize to 0.8MPa with compressed air at room temperature, and maintain the pressure for 3.5 hours; S4. Filtration: After the extraction is completed, plate and frame filtration, 0.1um precision filtration, and ultrafiltration are used in sequence to remove insoluble large particle impurities and large molecular proteins and sugars to obtain a clear solution; S5. Separation of arabinogalactan: After the filtration in step S4, the clear liquid is further filtered through a nanofiltration membrane, and dihydroquercetin and small molecular impurities of small molecular structure pass through the nanofiltration membrane, and arabinogalactan of large molecular structure is retained on the nanofiltration membrane, and arabinogalactan and dihydroquercetin are separated. The retained arabinogalactan is collected in a container, stirred, and 1 times of edible alcohol is slowly added during the stirring process. Centrifugation and freeze-drying are performed to obtain 64.8 g of arabinogalactan; S6. Purification of dihydroquercetin: The clear solution containing dihydroquercetin in step S5 is passed through a nucleopore membrane of a certain pore size to remove impurities with a molecular weight greater than dihydroquercetin, and then passed through the nucleopore membrane to remove impurities with a molecular weight less than dihydroquercetin, and the portion containing dihydroquercetin is collected, refrigerated and crystallized, and the solid matter is freeze-dried after filtration to obtain 31.6 kg of a dihydroquercetin product with a content greater than 99.5%.

[0019] In step S6, the pore size of the nucleopore membrane for filtering out impurities with a molecular weight greater than that of dihydroquercetin is 0.2-10 microns, and the pore size of the nucleopore membrane for removing impurities with a molecular weight less than that of dihydroquercetin is less than 0.2 microns.

[0020] The preparation method of the present invention is extracted by enzymolysis and pressurized water, and is combined with membrane separation and membrane concentration technology to prepare arabinogalactan products and 99% dihydroquercetin products at the same time. The extraction adopts enzymolysis, pressurization, and low-temperature water extraction, and the tight structure of the raw material is destroyed by cellulase and hemicellulase, and the permeability and penetration of water are increased by pressurization, thereby improving the extraction efficiency, and then the existing dihydroquercetin glycosides are hydrolyzed into free dihydroquercetin by beta-glucosidase, thereby improving the yield, and then combined with membrane separation and membrane concentration technology to prepare arabinogalactan and 99% dihydroquercetin products at the same time. The shortcomings of traditional normal pressure water extraction are avoided, and compared with traditional organic reagent extraction and ether ester extraction, the present method does not use organic reagents throughout the whole process, thereby avoiding environmental pollution, being safe and feasible, and having low cost.

[0021] Moreover, the preparation method of the present invention does not require steam throughout the entire process, is produced at room temperature, and is freeze-dried at low temperature. The product has good quality and high yield. The production process equipment is simple, easy to operate, safe and reliable, pollution-free, highly efficient, and low cost.

[0022] The contents not described in detail in the present invention are all prior art.

[0023] The above description is only a preferred embodiment of the present invention, and is not limited to the description in the specification and implementation. Therefore, any equivalent changes or modifications made according to the structure, features and principles described in the scope of the present invention should be included in the scope of the present invention.

Claims

1. A method for simultaneously preparing arabinogalactan and dihydroquercetin based on larch, using root wood after rotation felling of larch, characterized in that: The following steps are involved: S1. Raw materials: Wash and crush the root wood of larch after rotation felling to obtain small wood chips; S2. Fermentation: Take a certain amount of small sawdust, use about 10% of the water in the sawdust, add 0.5~1% of the cellulase, hemicellulase, and β-glucosidase in the sawdust, mix well, spray evenly on the sawdust, and leave it to stand at room temperature for 3~10 days for natural fermentation; S3. Extraction: After the fermentation of small sawdust, add 10 times the amount of drinking water, pressurize to 0.5-0.8MPa by compressed air at room temperature, and maintain the pressure for 2-5 hours; S4. Filtration: After the extraction is completed, plate and frame filtration, 0.1um precision filtration, and ultrafiltration are used in sequence to remove insoluble large particle impurities and large molecular proteins and sugars to obtain a clear solution; S5. Separation of arabinogalactan: After the filtration in step S4, the clear liquid is further filtered through a nanofiltration membrane, and dihydroquercetin and small molecular impurities of small molecular structure pass through the nanofiltration membrane, and arabinogalactan of large molecular structure is retained on the nanofiltration membrane, and arabinogalactan and dihydroquercetin are separated, and the retained arabinogalactan is collected in a container, stirred, and 1 to 2 times of edible alcohol is slowly added during the stirring process, centrifuged, and freeze-dried to obtain arabinogalactan; S6. Purification of dihydroquercetin: The clear solution containing dihydroquercetin in step S5 is passed through a nucleopore membrane of a certain pore size to remove impurities with a molecular weight greater than that of dihydroquercetin, and then passed through the nucleopore membrane to remove impurities with a molecular weight less than that of dihydroquercetin, and the portion containing dihydroquercetin is collected, refrigerated for crystallization, and the solid matter is freeze-dried after filtration to obtain a dihydroquercetin product with a content greater than 99%.

2. The method for simultaneously preparing arabinogalactan and dihydroquercetin based on larch according to claim 1, characterized in that: The cellulase and hemicellulase in step S2 can destroy the compact structure of the raw material, and the β-glucosidase enzymatically hydrolyzes the free dihydroquercetin glycoside into free dihydroquercetin.

3. The method for simultaneously preparing arabinogalactan and dihydroquercetin based on larch according to claim 1, characterized in that: In step S6, the pore size of the nucleopore membrane for filtering out impurities with a molecular weight greater than that of dihydroquercetin is 0.2-10 microns, and the pore size of the nucleopore membrane for removing impurities with a molecular weight less than that of dihydroquercetin is less than 0.2 microns.