CN113563488A - Preparation method of pharmaceutical-grade micromolecular marine organism polysaccharide - Google Patents
Preparation method of pharmaceutical-grade micromolecular marine organism polysaccharide Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
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Abstract
The invention discloses a preparation method of pharmaceutical-grade micromolecule marine organism polysaccharide, which comprises the following steps: (1) adding endoglucanase and/or endoglucanase into the Schizophyllum commune fermentation inoculation stage, and obtaining marine fungus fermentation liquor after fermentation is completed; (2) homogenizing the marine fungus fermentation liquor under high pressure to break the wall of the mycelium to obtain marine organism polysaccharide homogeneous slurry; (3) adjusting the pH value of the marine organism polysaccharide homogenate, and adding complex enzyme for enzymolysis; (4) heating the marine organism polysaccharide homogenate liquid subjected to the enzymolysis by the compound enzyme in the step (3) to inactivate the enzyme, filtering a product subjected to inactivation, adding absolute ethyl alcohol into the filtrate, uniformly stirring and filtering, adding water into an alcohol precipitate obtained by filtering, heating, stirring and redissolving, passing the alcohol precipitate redissolved solution through a resin column, further removing pigments and proteins in the marine organism polysaccharide by using an ion exchange layer, performing chromatographic fractionation by using a preparative gel column to obtain marine organism polysaccharide filtrate with different molecular weights, and drying.
Description
Technical Field
The invention relates to the technical field of marine bioengineering, in particular to a preparation method of pharmaceutical-grade micromolecule marine organism polysaccharide.
Background
Biological polysaccharides (polysaccharides) have multiple functions of immunoregulation, tumor resistance, inflammation resistance, oxidation resistance, radiation resistance, cholesterol reduction, blood sugar reduction and the like, wherein the antitumor and immunoregulation functions of the polysaccharides are concerned and become one of the hot spots of tumor treatment. At present, more and more bioactive polysaccharides with novel structures and unique effects are obtained from marine organisms, and are expected to become new drug resources in tumor treatment. Biological polysaccharides in the ocean are mainly divided into three major groups: marine animal polysaccharides, algal polysaccharides, and polysaccharides produced by the metabolism of marine microorganisms. The marine animal polysaccharide includes polysaccharide and acidic mucopolysaccharide contained in shellfish, Stichopus japonicus, sea urchin, etc. Algal polysaccharides refer to various high molecular carbohydrates contained in seaweeds, including alginic acid, algin, sulfated polysaccharides in green algae, agar in red algae, carrageenan, and the like. Marine microbial polysaccharides refer mainly to microbial extracellular polysaccharides, which are mostly isolated from bacteria in sea mud, seawater and seaweeds.
The marine microbial polysaccharide is polysaccharide generated by bacteria, fungi (without large-scale fungi) and other microorganisms, has short production period, is not limited by seasons, regions and conditions of plant diseases and insect pests, has rich and cheap raw material sources and relatively low cost, can be subjected to large-scale industrial production under the condition of manual control, has high yield and simple purification, does not pollute the environment, and has stronger market competitiveness and wide development prospect.
The marine fungus polysaccharide is not easy to dissolve in water, has large viscosity and wide molecular weight distribution range, the fermentation liquor contains a large amount of mycelia, the components and the structure are complex, and the problem to be solved urgently is to find an effective and reasonable preparation method of the pharmaceutical-grade marine fungus polysaccharide. At present, the extraction technology of edible and medicinal fungi polysaccharide mainly comprises the following steps: (1) water extraction and alcohol precipitation; (2) alkali extraction; (3) acid extraction method: (4) microwave extraction; (5) an enzyme extraction method; (6) ultrasonic extraction, etc. The extraction rate of the water extraction and alcohol precipitation method in the methods is low; the alkali (or acid) extraction method strictly controls the pH value in the extraction process, otherwise, the breakage of glycosidic bonds in the polysaccharide can be caused; ultrasonic and microwave extraction methods have a great influence on the composition and activity of polysaccharides. The enzyme engineering technology is a biological engineering technology used for extracting active ingredients of natural plants in recent years, and in the process of extracting marine polysaccharide, the extraction conditions can be reduced by using enzyme, tissues are decomposed under mild conditions, and the extraction rate of polysaccharide is improved.
Studies show that the distribution of marine organism polysaccharide is not completely linear, small molecules below 10kD are more, polysaccharide content of 10kD-100kD is less, macromolecular polysaccharide above 100kD is more, and the macromolecular polysaccharide is often combined with protein. In order to obtain more small-molecular polysaccharides, on one hand, separation needs to be completed, and on the other hand, hydrolysis and protein removal of the large-molecular polysaccharides are considered.
Disclosure of Invention
The invention aims to solve the defects in the prior art, and provides a preparation method of the pharmaceutical-grade micromolecular marine organism polysaccharide, so that the aims of reducing the production cost, improving the activity and purity of the biological polysaccharide and realizing the production of the pharmaceutical-grade micromolecular marine organism polysaccharide in a factory are fulfilled.
In order to achieve the purpose, the invention adopts the following technical scheme:
a preparation method of pharmaceutical-grade micromolecular marine organism polysaccharide comprises the following steps:
(1) adding endoglucanase and/or endoglucanase into the Schizophyllum commune fermentation inoculation stage, and obtaining marine fungus fermentation liquor after fermentation is completed;
(2) homogenizing the marine fungus fermentation liquor under high pressure to break the wall of the mycelium to obtain marine organism polysaccharide homogeneous slurry;
(3) adjusting the pH value of the marine organism polysaccharide homogenate, and adding complex enzyme for enzymolysis; adding complex enzyme for enzymolysis to realize the functions of decoloring, deproteinizing, desugaring, defibrinating, defatting, improving the content of micromolecular marine organism polysaccharide and improving the purity of the marine organism polysaccharide;
(4) heating the marine organism polysaccharide homogenate liquid subjected to the enzymolysis by the complex enzyme in the step (3) to inactivate the enzyme, filtering a product subjected to inactivation, adding absolute ethyl alcohol into the filtrate, uniformly stirring and filtering, adding water into an alcohol precipitate obtained by filtering, heating, stirring and redissolving, passing the alcohol precipitation redissolved solution through a resin column, further removing pigments and proteins in the marine organism polysaccharide by using an ion exchange layer, performing chromatographic fractionation by using a preparative gel column to obtain marine organism polysaccharide filtrate with different molecular weights, and drying the marine organism polysaccharide filtrate to obtain the pharmaceutical-grade micromolecule marine organism polysaccharide.
As a preferred embodiment, in the step (1), in the schizophyllum commune fermentation inoculation stage, 1-8 per mill of endoglucanase and/or 0.5-5 per mill of endoglucanase is added according to the total feeding amount of fermentation materials, the fermentation temperature is 28-30 ℃, and the fermentation period is 5-7 days. The secondary seed tank can be expanded to 10 tons of fermentation tank.
As a preferred embodiment, in the step (2), the homogenization pressure of the high-pressure homogenized marine fungus fermentation broth is 85-120MP, the homogenization temperature is 10-30 ℃, and the homogenization times are 2-5 times. Homogenizing under high pressure to obtain uniform slurry of marine organism polysaccharide with good fluidity and low viscosity.
As a preferred embodiment, in the step (3), sodium hydroxide with the concentration of 40% -60% is adopted to adjust the pH value of the marine organism polysaccharide homogenate to 6-8, and then complex enzyme is added for enzymolysis.
As a preferable embodiment, in the step (3), the complex enzyme is formed by compounding neutral protease and/or glucanase and/or glucose oxidase and/or sucrase and/or cellulase and/or lipase.
As a preferred embodiment, in the step (3), the addition amount of each enzyme in the complex enzyme is 1-5 per mill of neutral protease and/or 1-10 per mill of glucanase, and/or 1-10 per mill of glucose oxidase, and/or 1-5 per mill of sucrase, and/or 1-5 per mill of cellulase, and/or 1-3 per mill of lipase, the enzymolysis time is 2-6h, and the enzymolysis temperature is 40-60 ℃.
As a preferred embodiment, in the step (4), the heating and enzyme inactivation temperature is 80-120 ℃, and the enzyme inactivation time is 15-30 min.
In the step (4), the product after enzyme deactivation is filtered by adopting plate and frame filtration, absolute ethyl alcohol is added into the filtrate, the filtrate is uniformly stirred and filtered, and the volume of the added absolute ethyl alcohol is 1-3 times of the volume of the filtrate; adding water into the alcohol precipitate in an amount of 1-3 times the weight of the alcohol precipitate, heating to dissolve at 75-90 deg.C, and stirring for 1-3 hr.
As a preferable embodiment, in the step (4), the alcohol precipitation compound solution passes through a resin column, and pigment and protein in the marine organism polysaccharide are removed by adopting a weak acid and weak base type anion-cation exchange resin series connection method.
In the above method for preparing a pharmaceutical-grade small-molecule marine organism polysaccharide, as a preferred embodiment, in step (4), the molecular weight of the obtained marine organism polysaccharide is: less than 1KD, 1-10KD, 10-50KD and 50-100 KD; the drying is low-temperature spray drying or freeze vacuum drying. The obtained medicinal small molecular marine organism polysaccharide has strong immunological activity, anti-tumor activity, high water solubility, low viscosity, easy absorption by human body, high purity (95.0-99.9%), and four kinds of marine organism polysaccharides with gradient molecular weight. As a preferred embodiment, the temperature of the freeze-drying is from-20 ℃ to-40 ℃.
Compared with the prior art, the invention has the beneficial effects that:
the invention adds endoglucanase and/or endoglucanase in the initial fermentation stage of the Schizophyllum commune strain to improve the yield and purity of polysaccharide and lower molecular weight by utilizing enzymatic reaction, and the marine fungus fermentation liquor is repeatedly homogenized under high pressure to reduce the viscosity of the polysaccharide, enhance the fluidity of the homogenate liquid and facilitate the post-treatment of the material, the homogenate liquid is fed by a one-pot method, and most of the impurities such as protein, pigment, monosaccharide, oligosaccharide, cellulose, fat and the like are removed by compounding different enzymes and carrying out enzymolysis once, the polysaccharide in the cell wall is released to further improve the yield of the small molecular marine organism polysaccharide, the small molecular marine organism polysaccharide is secondarily purified by a series connection method of weak acid and weak base type anion and cation exchange resins along with filtration and alcohol redissolution, and then is classified by preparative gel column chromatography to obtain four kinds of small molecular marine organism polysaccharides with high purity molecular weights less than 1kD, 1-10kD, 10-50kD and 50-100 kD.
The marine organism polysaccharide prepared by the invention has small molecular weight, better immunity enhancing function and anti-tumor activity, low viscosity, easy water solubility, easy absorption by human bodies and high purity.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely a few embodiments of the invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
The endoglucanase and the endoglucanase described in the embodiments of the present invention are commercially available products.
The storage unit of the schizophyllum commune is as follows: china center for type culture Collection, Address: eight Wuhan university No. 299 in Wuhan district, Wuhan city, Hubei province, the preservation date is: 18/2016, accession number: CCTCC NO: m2016055, classification name: schizophyllum commune XNY20151228 (Schizaphyls commne Fr. XNY20151228).
Example 1
In the Schizophyllum commune fermentation inoculation stage, no endoglucanase and endoglucanase are added, the secondary seed tank is expanded to a 10 ton fermentation tank, the fermentation temperature is 28 ℃, the fermentation period is 7 days, the polysaccharide content of the fermentation liquor filtrate is 4.5 per mill, the purity is 62 percent, and the biological polysaccharide with the molecular weight of 1-10kD and more than 100kD is mainly used.
Example 2
In the Schizophyllum commune fermentation inoculation stage, adding endoglucanase 4 per mill of total feed of fermentation material, performing enlarged culture in the secondary seed tank to 10 ton fermentation tank at 28 deg.C for 6 days, filtering the fermentation liquor to obtain supernatant with polysaccharide content of 6.1 per mill and purity of 68%, and mainly using biological polysaccharide with molecular weight less than 1kD and 1-10 kD.
Example 3
In the Schizophyllum commune fermentation inoculation stage, adding endo-cellulase 5 per mill based on total feeding of fermentation materials, performing enlarged culture in a secondary seed tank to 10 ton fermentation tank at 28 deg.C for 5 days, wherein the content of polysaccharide in the filtrate of the fermentation liquid is 9.7 per mill, the purity is 73%, and the biological polysaccharide with molecular weight of 1-10kD and 10-50kD is used as main material.
Example 4
In the Schizophyllum commune fermentation inoculation stage, 4 per mill and 3 per mill of endoglucanase and endoglucanase are added based on the total feeding of fermentation materials, the secondary seed tank is expanded to a 10 ton fermentation tank, the fermentation temperature is 28 ℃, the fermentation period is 6 days, the polysaccharide content of the fermentation liquor filtered supernatant is 17.3 per mill, the purity is 81 percent, and the biological polysaccharide with the molecular weight less than 1kD, 1-10kD and 10-50kD is mainly used.
Physical and chemical indexes of fermentation liquids of examples 1 to 4 are shown in Table 1
TABLE 1 physicochemical indices of fermentation broths of examples 1 to 4
It can be seen from the results of fermentation in Table 1 that the marine fungus fermentation broth with smaller molecular weight, higher purity and higher yield can be obtained by enzymatic fermentation.
Example 5
The marine fungus fermentation broth fermented by the enzymatic reaction in example 4 was homogenized at high pressure of 85MPa and 15 ℃ for 3 times. Adjusting the pH value of the prepared marine organism polysaccharide homogenate to 6.8 by using NaOH (the concentration is 50 percent), adding complex enzyme, wherein the addition amount of various enzymes in the complex enzyme is calculated by the added marine fungus fermentation liquid, 2 per mill of neutral protease, 2 per mill of dextranase and 4 per mill of glucose oxidase are added, the total enzyme addition amount is 8 per mill, the enzymolysis time is 6 hours, and the enzymolysis temperature is 40 ℃. Heating and inactivating the enzymolysis liquid after enzymolysis, wherein the inactivation temperature is 80 ℃, and the inactivation time is 30 min.
Filtering the enzyme inactivated product by adopting plate and frame filtration, adding absolute ethyl alcohol into the filtrate, uniformly stirring and filtering, wherein the volume of the added absolute ethyl alcohol is 1.2 times of the volume of the filtrate;
adding water into the alcohol precipitate for heating and redissolving, wherein the amount of the water added into the alcohol precipitate is 1.2 times of the weight of the alcohol precipitate, the heating and redissolving temperature is 75 ℃, and the stirring time is 3 hours.
Passing the alcohol precipitation complex solution through a resin column, further removing pigment and protein in the marine organism polysaccharide by using a weak acid weak base type anion and cation exchange resin series connection method, carrying out chromatographic fractionation on a preparative gel column to obtain marine organism polysaccharide clear liquid with 4 molecular weight gradients, and carrying out freeze drying on the obtained marine organism polysaccharide clear liquid to prepare the micromolecule marine organism polysaccharide (the purity is 95.6, and the molecular weight is mainly distributed less than 1kD, 1-10kD and 10-50 kD).
Example 6
The marine fungus fermentation broth fermented by the enzymatic reaction of example 4 was homogenized at a high pressure of 90mPa and a homogenization temperature of 30 ℃ for 2 times. Adjusting the pH value of the prepared marine organism polysaccharide homogenate to 6.5 by using NaOH (the concentration is 55 percent), adding complex enzyme, wherein the addition amount of various enzymes in the complex enzyme is calculated by the added marine fungus fermentation liquid, 2 per mill of neutral protease, 2 per mill of dextranase, 2 per mill of glucose oxidase and 2 per mill of sucrase are added, the total enzyme addition amount is 8 per mill, the enzymolysis time is 4 hours, and the enzymolysis temperature is 48 ℃.
Heating and inactivating the enzymolysis liquid after enzymolysis, wherein the inactivation temperature is 90 ℃, and the inactivation time is 20 min. Filtering the enzyme inactivated product by adopting plate and frame filtration, adding absolute ethyl alcohol into the filtrate, uniformly stirring and filtering, wherein the volume of the added absolute ethyl alcohol is 1.5 times of the volume of the filtrate; adding water into the alcohol precipitate for heating and redissolving, wherein the amount of the water added into the alcohol precipitate is 2 times of the weight of the alcohol precipitate, the heating temperature is 80 ℃, and the stirring time is 3 hours.
And (3) enabling the alcohol precipitation complex solution to pass through a resin column, removing pigments and proteins in the marine organism polysaccharide by adopting a weak acid and weak base type anion-cation exchange resin series connection method, and carrying out chromatographic fractionation on a preparative gel column to obtain the marine organism polysaccharide clear solution with 4 molecular weight gradients. Spray drying the obtained marine organism polysaccharide clear liquid to prepare small molecular marine organism polysaccharide (with purity of 96.7, molecular weight distribution of less than 1kD, 1-10kD and 10-50 kD).
Example 7
The marine fungus fermentation broth fermented by the enzymatic reaction of example 4 was homogenized at a high pressure of 100mPa and a homogenization temperature of 25 ℃ for 3 times. Adjusting the pH value of the prepared marine organism polysaccharide homogenate to 7.0 by using NaOH (the concentration is 60%), adding a complex enzyme, wherein the addition amount of various enzymes in the complex enzyme is calculated by the added marine fungus fermentation liquid, and 3 permillage of neutral protease, 5 permillage of dextranase, 5 permillage of glucose oxidase, 2 permillage of sucrase and 3 permillage of cellulase are added, the total enzyme addition amount is 18 permillage, the enzymolysis time is 5 hours, and the enzymolysis temperature is 50 ℃.
Heating and inactivating the enzymolysis liquid after enzymolysis, wherein the inactivation temperature is 85 ℃, and the inactivation time is 30 min. Filtering the enzyme inactivated product by adopting plate and frame filtration, adding absolute ethyl alcohol into the filtrate, uniformly stirring and filtering, wherein the volume of the added absolute ethyl alcohol is 1.3 times of the volume of the filtrate; adding water into the alcohol precipitate for heating and redissolving, wherein the amount of the water added into the alcohol precipitate is 2.5 times of the weight of the alcohol precipitate, the heating and redissolving temperature is 85 ℃, and the stirring time is 2 hours.
And (3) enabling the alcohol precipitation complex solution to pass through a resin column, removing pigments and proteins in the marine organism polysaccharide by adopting a weak acid and weak base type anion-cation exchange resin series connection method, carrying out chromatographic fractionation on the preparative gel column to obtain marine organism polysaccharide clear liquid with 4 molecular weight gradients, and carrying out spray drying on the obtained marine organism polysaccharide clear liquid to prepare the micromolecule marine organism polysaccharide (the purity is 98.3, and the molecular weight is mainly distributed to be less than 1kD, 1-10kD and 10-50 kD).
Example 8
The marine fungus fermentation broth fermented by the enzymatic reaction of the example 4 is homogenized for 4 times under high pressure, wherein the homogenization pressure is 85MPa, and the homogenization temperature is 20 ℃. Regulating the pH value of the prepared marine organism polysaccharide homogenate to 7.3 by using NaOH (the concentration is 56%), adding a complex enzyme, wherein the addition amount of various enzymes in the complex enzyme is calculated by the added marine fungus fermentation liquid, and 4 per mill of neutral protease, 5 per mill of dextranase, 6 per mill of glucose oxidase, 1 per mill of sucrase, 2 per mill of cellulase and 2 per mill of lipase are added, the total enzyme addition amount is 20 per mill, the enzymolysis time is 4 hours, and the enzymolysis temperature is 52 ℃.
Heating and inactivating the enzymolysis liquid after enzymolysis, wherein the inactivation temperature is 115 ℃, and the inactivation time is 20 min. Filtering the enzyme inactivated product by adopting plate and frame filtration, adding absolute ethyl alcohol into the filtrate, uniformly stirring and filtering, wherein the volume of the added absolute ethyl alcohol is 1.3 times of the volume of the filtrate; adding water into the alcohol precipitate for heating and redissolving, wherein the amount of the water added into the alcohol precipitate is 3 times of the weight of the alcohol precipitate, the heating and redissolving temperature is 90 ℃, and the stirring time is 1.5 h.
And (3) enabling the alcohol precipitation complex solution to pass through a resin column, removing pigments and proteins in the marine organism polysaccharide by adopting a weak acid and weak base type anion-cation exchange resin series connection method, carrying out chromatographic fractionation on a preparative gel column to obtain marine organism polysaccharide clear liquid with 4 molecular weight gradients, and carrying out spray drying on the obtained marine organism polysaccharide clear liquid to prepare the micromolecule marine organism polysaccharide (the purity is 99.2, and the molecular weight is mainly distributed to be less than 1kD and 1-10 kD).
Example 9
The marine fungus fermentation broth fermented by the enzymatic reaction of example 4 was homogenized at a high pressure of 110MPa and a homogenization temperature of 15 ℃ for 5 times. Adjusting the pH value of the prepared marine organism polysaccharide homogenate to 7.5 by using NaOH (the concentration is 54%), adding a complex enzyme, wherein the addition amount of various enzymes in the complex enzyme is calculated by the added marine fungus fermentation liquid, 5 per mill of neutral protease, 6 per mill of dextranase, 6 per mill of glucose oxidase, 2 per mill of sucrase, 3 per mill of cellulase and 2 per mill of lipase are added, the total enzyme addition amount is 24 per mill, the enzymolysis time is 5, and the enzymolysis temperature is 48 ℃. Heating and inactivating the enzymolysis liquid after enzymolysis, wherein the inactivation temperature is 110 ℃, and the inactivation time is 20 min.
Filtering the enzyme inactivated product by adopting plate and frame filtration, adding absolute ethyl alcohol into the filtrate, uniformly stirring and filtering, wherein the volume of the added absolute ethyl alcohol is 1.2 times of the volume of the filtrate; adding water into the alcohol precipitate for heating and redissolving, wherein the amount of the water added into the alcohol precipitate is 2 times of the weight of the alcohol precipitate, the heating and redissolving temperature is 85 ℃, and the stirring time is 3 hours.
And (3) enabling the alcohol precipitation complex solution to pass through a resin column, removing pigments and proteins in the marine organism polysaccharide by adopting a weak acid and weak base type anion-cation exchange resin series connection method, carrying out chromatographic fractionation on a preparative gel column to obtain marine organism polysaccharide clear liquid with 4 molecular weight gradients, and carrying out spray drying on the obtained marine organism polysaccharide clear liquid to prepare the micromolecule marine organism polysaccharide (the purity is 99.5, and the molecular weight is mainly distributed to be less than 1kD and 1-10 kD).
Example 10
The marine fungus fermentation broth fermented by the enzymatic reaction in the example 4 is homogenized for 2 times under high pressure, wherein the homogenization pressure is 120MPa, and the homogenization temperature is 10 ℃. Adjusting the pH value of the prepared marine organism polysaccharide homogenate to 7.4 by using NaOH (the concentration is 52%), adding a complex enzyme, wherein the addition amount of various enzymes in the complex enzyme is calculated by the added marine fungus fermentation liquid, 5 per mill of neutral protease, 7 per mill of dextranase, 7 per mill of glucose oxidase, 3 per mill of sucrase, 3 per mill of cellulase and 2 per mill of lipase are added, the total enzyme addition amount is 27 per mill, the enzymolysis time is 6 hours, and the enzymolysis temperature is 46 ℃. Heating and inactivating the enzymolysis liquid after enzymolysis, wherein the inactivation temperature is 110 ℃, and the inactivation time is 20 min.
Filtering the enzyme inactivated product by adopting plate and frame filtration, adding absolute ethyl alcohol into the filtrate, uniformly stirring and filtering, wherein the volume of the added absolute ethyl alcohol is 1.1 times of the volume of the filtrate; adding water into the alcohol precipitate for heating and redissolving, wherein the amount of the water added into the alcohol precipitate is 2.5 times of the weight of the alcohol precipitate, the heating and redissolving temperature is 85 ℃, and the stirring time is 3 hours. .
And (3) enabling the alcohol precipitation complex solution to pass through a resin column, removing pigments and proteins in the marine organism polysaccharide by adopting a weak acid and weak base type anion-cation exchange resin series connection method, carrying out chromatographic fractionation on a preparative gel column to obtain marine organism polysaccharide clear liquid with 4 molecular weight gradients, and carrying out freeze drying on the obtained marine organism polysaccharide clear liquid to prepare the micromolecule marine organism polysaccharide (the purity is 99.9, and the molecular weight is mainly distributed to be less than 1kD and 1-10 kD).
TABLE 2 physicochemical indices of examples 6 to 10
From table 2, it can be found that the purity of the marine organism polysaccharide obtained after purification is further improved (up to 99.9% purity) with the increase of the enzyme types and the total enzyme addition amount; meanwhile, the molecular weight of the prepared marine organism polysaccharide is obviously reduced, and the marine organism polysaccharide is easier to be absorbed by a human body.
The data in tables 1 and 2 are combined to show that fermentation is carried out by an enzymatic reaction; the fermentation liquor is treated by high-pressure homogenization to reduce the viscosity and improve the yield; removing impurities, decolorizing, improving purity, and reducing molecular weight by multienzyme complex enzymolysis; filtering the absolute ethyl alcohol to extract and redissolve; secondary purification by anion and cation exchange resin; the marine organism polysaccharide prepared by the preparation type gel column chromatographic fractionation and other processes has the advantages of stronger immunological activity, anti-tumor activity, easy water solubility, low viscosity, easy absorption by human bodies, high purity, small molecular weight and the like.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (10)
1. A preparation method of a pharmaceutical-grade micromolecular marine organism polysaccharide is characterized by comprising the following steps:
(1) adding endoglucanase and/or endoglucanase into the Schizophyllum commune fermentation inoculation stage, and obtaining marine fungus fermentation liquor after fermentation is completed;
(2) homogenizing the marine fungus fermentation liquor under high pressure to break the wall of the mycelium to obtain marine organism polysaccharide homogeneous slurry;
(3) adjusting the pH value of the marine organism polysaccharide homogenate, and adding complex enzyme for enzymolysis;
(4) heating the marine organism polysaccharide homogenate liquid subjected to the enzymolysis by the complex enzyme in the step (3) to inactivate the enzyme, filtering a product subjected to inactivation, adding absolute ethyl alcohol into the filtrate, uniformly stirring and filtering, adding water into an alcohol precipitate obtained by filtering, heating, stirring and redissolving, passing the alcohol precipitation redissolved solution through a resin column, further removing pigments and proteins in the marine organism polysaccharide by using an ion exchange layer, performing chromatographic fractionation by using a preparative gel column to obtain marine organism polysaccharide filtrate with different molecular weights, and drying the marine organism polysaccharide filtrate to obtain the pharmaceutical-grade micromolecule marine organism polysaccharide.
2. The method for preparing a pharmaceutical-grade small molecular marine organism polysaccharide according to claim 1, wherein in the step (1), 1-8 per mill of endoglucanase and/or 0.5-5 per mill of endoglucanase is added based on the total feed of the fermented materials in the Schizophyllum commune fermentation inoculation stage, the fermentation temperature is 28-30 ℃, and the fermentation period is 5-7 days.
3. The method for preparing a pharmaceutical-grade small molecular marine organism polysaccharide according to claim 1, wherein in the step (2), the high-pressure homogenization pressure of the marine fungus fermentation broth is 85-120MPa, the homogenization temperature is 10-30 ℃, and the homogenization times are 2-5 times.
4. The method for preparing the pharmaceutical-grade micromolecular marine organism polysaccharide as claimed in claim 1, wherein in the step (3), sodium hydroxide with the concentration of 40% -60% is adopted to adjust the pH of the marine organism polysaccharide homogenate to 6-8, and then complex enzyme is added for enzymolysis.
5. The method for preparing the pharmaceutical-grade small-molecule marine organism polysaccharide according to claim 1, wherein in the step (3), the complex enzyme is compounded by neutral protease and/or glucanase and/or glucose oxidase and/or sucrase and/or cellulase and/or lipase.
6. The method for preparing a pharmaceutical-grade small-molecule marine organism polysaccharide as claimed in claim 5, wherein in step (3), the amount of each enzyme added in the complex enzyme is 1-5% o neutral protease and/or 1-10% o glucanase, and/or 1-10% o glucose oxidase, and/or 1-5% o sucrase, and/or 1-5% o cellulase, and/or 1-3% o lipase, the enzymolysis time is 2-6h, and the enzymolysis temperature is 40-60 ℃.
7. The method for preparing a pharmaceutical-grade small molecular marine organism polysaccharide according to claim 1, wherein in the step (4), the temperature for heating and enzyme inactivation is 80-120 ℃, and the time for enzyme inactivation is 15-30 min.
8. The method for preparing a pharmaceutical-grade small molecular marine organism polysaccharide according to claim 1, wherein in the step (4), the product after enzyme deactivation is filtered by adopting plate and frame filtration, absolute ethyl alcohol is added into the filtrate, the mixture is stirred and filtered evenly, and the volume of the added absolute ethyl alcohol is 1-3 times of the volume of the filtrate; adding water into the alcohol precipitate in an amount of 1-3 times the weight of the alcohol precipitate, heating to dissolve at 75-90 deg.C, and stirring for 1-3 hr.
9. The method for preparing a pharmaceutical-grade small molecular marine organism polysaccharide according to claim 1, wherein in the step (4), the alcohol precipitation solution is passed through a resin column, and pigments and proteins in the marine organism polysaccharide are removed by a weak acid and weak base type anion and cation exchange resin series connection method.
10. The method for preparing the pharmaceutical-grade small-molecule marine organism polysaccharide according to claim 1, wherein in the step (4), the molecular weight of the obtained marine organism polysaccharide is as follows: less than 1KD, 1-10KD, 10-50KD and 50-100 KD; the drying is low-temperature spray drying or freeze vacuum drying.
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