CN108239177B - Method for purifying and preparing high-quality biological polysaccharide - Google Patents
Method for purifying and preparing high-quality biological polysaccharide Download PDFInfo
- Publication number
- CN108239177B CN108239177B CN201611215234.3A CN201611215234A CN108239177B CN 108239177 B CN108239177 B CN 108239177B CN 201611215234 A CN201611215234 A CN 201611215234A CN 108239177 B CN108239177 B CN 108239177B
- Authority
- CN
- China
- Prior art keywords
- glucan
- beta
- fermentation
- schizophyllum commune
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Sustainable Development (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for purifying and preparing high-quality biological polysaccharide. The preparation method of the beta-glucan provided by the invention comprises the following steps: (1) mixing Schizophyllum commune fermentation liquor with water, and performing digestion to obtain a digestion solution; (2) carrying out solid-liquid separation on the digestion solution obtained in the step (1) and taking clear liquid; (3) adding activated carbon into the clear liquid obtained in the step (2), and performing adsorption treatment to obtain a carbon-containing mixture; carrying out solid-liquid separation on the carbon-containing mixture, and taking clear liquid; (4) mixing the clear liquid obtained in the step (3) with ethanol for alcohol precipitation, and collecting precipitated solids; (5) and (4) drying the solid in the step (4) to obtain the beta-glucan. The method does not use an enzymolysis process and has lower cost. The functional structure of the beta-glucan can not be damaged without using an acidolysis or alkaline hydrolysis process. The prepared beta-glucan solution has high viscosity and high light transmittance and is suitable for various applications.
Description
Technical Field
The invention relates to a preparation method of beta-glucan, in particular to a method for purifying and preparing high-quality biological polysaccharide.
Background
Polysaccharides are natural high molecular compounds obtained by polymerizing more than 10 monosaccharide molecules, and are widely distributed in animals, plants, microorganisms (bacteria and fungi) and algae lichens. The polysaccharide as an important bioactive substance has the effects of regulating immunity, resisting tumors, reducing glycolipid, delaying senescence and the like, and has wide application prospects in the fields of medical care, food, animal breeding and the like.
Beta-glucan is a natural polysaccharide. A considerable variety of β -glucans are found in the natural environment, usually in the cell walls of specific species of bacteria, yeasts, fungi (ganoderma lucidum) and also in the coating of seeds of higher plants. The mushroom glucan which takes the beta-1, 3-glycosidic bond as the framework has the functions of enhancing immunity, resisting tumor, bacteria and virus, and the like, has obvious effects on the aspects of moisture preservation, wrinkle resistance, oxidation resistance, ultraviolet resistance, wound healing promotion and the like, and in addition, compared with the traditional yeast or cereal glucan, the mushroom glucan has more uniform and stable structure, higher molecular weight and more excellent water solubility. Therefore, the mushroom beta-glucan solution with high viscosity and high light transmittance is widely applied to the fields of food, cosmetics and pharmacy, and has important significance in preparing the mushroom beta-glucan solution with high viscosity and high light transmittance.
At present, two main methods for extracting beta-glucan are provided, one is to directly extract beta-glucan from cereals such as oat or fruiting body fungi such as mushroom; and the other is to obtain the beta-glucan by extracting and processing fermentation liquor through liquid fermentation of fungi or bacteria. The beta-glucan is prepared by a fermentation method, impurities such as nucleic acid, protein and the like generated in the fermentation process need to be removed during purification, acid-base treatment or enzyme treatment is used for removing the nucleic acid and the protein in the traditional method, the cost is high, and the glucan is damaged by the acid-base treatment.
Disclosure of Invention
The invention aims to provide a method for purifying and preparing high-quality beta-glucan, the method is simple and convenient to operate, acid-base and enzymolysis treatment is not needed, and the prepared beta-glucan solution has high viscosity and high light transmittance.
The invention provides a preparation method of beta-glucan, which comprises the following steps:
(1) mixing Schizophyllum commune fermentation liquor with water, and performing digestion to obtain a digestion solution;
(2) carrying out solid-liquid separation on the digestion solution obtained in the step (1) and taking clear liquid;
(3) adding activated carbon into the clear liquid obtained in the step (2), and performing adsorption treatment to obtain a carbon-containing mixture; carrying out solid-liquid separation on the carbon-containing mixture, and taking clear liquid;
(4) mixing the clear liquid obtained in the step (3) with ethanol for alcohol precipitation, and collecting precipitated solids;
(5) and (4) drying the solid in the step (4) to obtain the beta-glucan.
In the above preparation method, in the step (1), the Schizophyllum commune fermentation broth can be prepared by a conventional liquid fermentation method, and can be a Schizophyllum commune (Schizophyllum commune Fr.) fermentation broth prepared under a common fermentation condition or after the fermentation condition is optimized. The preparation method specifically comprises the following steps:
(a) and (3) activation of the Schizophyllum commune strain: preparing a plate culture medium from 200g/L of potatoes, 20-30 g/L of glucose, 10-20 g/L of sodium chloride and 15-20 g/L of agar, inoculating a Schizophyllum commune strain to the plate culture medium, and culturing in a constant-temperature incubator at 20-30 ℃ for 4-10 days to obtain plate mycelia;
(b) seed activation: putting a liquid culture medium prepared from 50-200 g/L of potato starch, 20-60 g/L of glucose, 1-10 g/L of yeast extract powder and water into a shake flask, wherein the liquid filling amount is 1/5-1/3, inoculating the flat plate mycelium obtained in the step (a) into the shake flask, and performing shake culture in a constant-temperature shaking table at 20-30 ℃ at 100-220 rpm for 4-10 days to obtain a seed solution;
(c) fermentation culture: adding a fermentation culture medium prepared from 20-80 g/L glucose, 50-100 g/L sucrose, 5-30 g/L soybean meal, 1-10 g/L yeast extract powder, 0.1-2 g/L potassium dihydrogen phosphate, 0.1-2 g/L magnesium sulfate heptahydrate, 0.1-2 g/L ammonium sulfate, 1-10 g/L potassium nitrate and water into a fermentation tank, sterilizing at 115-128 ℃ for 15-30 minutes, inoculating the seed solution obtained in the step (b) into the fermentation tank, stirring at 100-600 rpm at a constant temperature of 20-30 ℃, and performing aeration fermentation culture for 5-10 days.
The volume ratio of the Schizophyllum commune fermentation liquor to water can be 1: (0.1-100), specifically 1: (1-10) and 1: (1-5) and 1: (1-4) and 1: (3-5) and 1: (3-4) 1: 1. 1: 3. 1: 5 or 1: 10; the temperature of the immersion boiling can be 30-121 ℃, specifically 50-90 ℃, 50-70 ℃, 60-90 ℃, 50 ℃, 60 ℃, 70 ℃ or 90 ℃, and the time can be 0.2-120 h, specifically 0.5-8 h, 0.5-4 h, 2-8 h, 0.5h, 2h, 4h or 8 h.
In the above-mentioned production method, in the step (2), the solid-liquid separation may be performed by at least one of centrifugation, positive pressure filtration and negative pressure filtration, and specifically may be any of the following 1) to 3): 1) centrifuging at 500-13000 rpm (such as 4000rpm) for 1-120 min (such as 5 min); 2) filtering with 100-1500 mesh (such as 300 mesh) filter cloth under negative pressure; 3) centrifuging at 500-13000 rpm (such as 4000rpm) for 3-60 min (such as 5min), and vacuum filtering with 100-1500 mesh (such as 300 mesh) filter cloth. And (4) performing suction filtration, wherein a filter aid can be added or not added. If filter aid is added, it can be 0.1-10% of active carbon, diatomite, pearlite and cellulose, etc.
In the preparation method, in the step (3), the activated carbon may be wood activated carbon and/or shell activated carbon, and the shell activated carbon may be coconut shell activated carbon; the adding volume of the wood activated carbon and the shell activated carbon is 0.1-10% of that of the clear liquid, and specifically can be 0.75-2.5%, 0.75-2%, 1-2.5%, 0.75%, 1%, 2% or 2.5%; the granularity of the activated carbon can be 8-400 meshes, and specifically, the granularity of the wood activated carbon can be 8-300 meshes, 8-30 meshes, 20-60 meshes, 200 meshes or 300 meshes; the granularity of the shell activated carbon can be 8-200 meshes, specifically 8-16 meshes and 40-60 meshes.
The temperature of the adsorption treatment can be 4-99 ℃, specifically 4-80 ℃, 4 ℃, 40 ℃, 50 ℃ or 80 ℃, and the treatment time can be 0.1-24 hours, specifically 4-16 hours, 4 hours or 16 hours. The adsorption treatment can be carried out by stirring and comprises three adsorption modes, namely 1) standing adsorption, and after the stirring and the uniform mixing, continuously standing adsorption; 2) continuously stirring and adsorbing; 3) intermittently stirring and adsorbing, and only stirring and uniformly mixing, and then intermittently stirring once every 10-60 min (such as 30 min). The stirring speed can be 1-1000 rpm, specifically 300-350 rpm, 300rpm or 350 rpm.
The solid-liquid separation method can be at least one of centrifugation, positive pressure filtration and negative pressure filtration, specifically can be negative pressure filtration by using a 100-30000 mesh (such as 300 mesh and 5000 mesh) filtration device, such as 300 mesh filter cloth and 5000 mesh filter plate in sequence.
In the preparation method, in the step (4), the volume ratio of the clear liquid to the ethanol may be 1: (0.1-100), specifically 1: (1-10) and 1: 3. 1: 1. 1: 5 or 1: 10. the ethanol is added in the form of ethanol aqueous solution with volume concentration of 70-100% (such as edible ethanol with volume concentration of 95%). The alcohol precipitation may be performed more than once, and for example, the alcohol precipitation may further include the steps of re-dissolving the solid in water to obtain a β -glucan solution, mixing the β -glucan solution with ethanol to perform alcohol precipitation again, and collecting the re-precipitated solid. In the secondary alcohol precipitation, the volume ratio of the beta-glucan solution to the ethanol may be 1: (0.1-100), specifically 1: 2. 1: 1; the ethanol is added in the form of an aqueous solution of ethanol with a volume concentration of 70-100% (e.g. edible ethanol with a volume concentration of 90-95%).
In the above preparation method, in the step (5), the drying may be performed by any one of thermal drying, reduced pressure drying, spray drying and low temperature freeze drying, and specifically, the drying may be performed at 30 to 130 ℃ (e.g., 40 to 60 ℃, 40 ℃, 50 ℃ or 60 ℃) to constant weight.
The beta-glucan prepared by the preparation method is also within the protection scope of the invention. The water solution prepared from the beta-glucan has high viscosity and high light transmittance.
The invention also provides a preparation method of the beta-glucan solution, which comprises the following steps: and (2) crushing the beta-glucan, dissolving the beta-glucan in water, stirring, filtering, and collecting clear liquid to obtain the beta-glucan solution.
In the above preparation method, the dissolving may be performed at normal temperature (15-30 ℃) or in a heating state, and the heating temperature may be 30-121 ℃, specifically 40-80 ℃, 40 ℃, 70 ℃ or 80 ℃. The agitation may be 1) not continuous; 2) the stirring is continued at a rotation speed of 1-1000 rpm (e.g., 600-800 rpm, 600rpm, or 800rpm) for 0.2-8 h (e.g., 2-6 h, 2h, 4h, or 6 h).
The filtration can be performed by at least one of positive pressure filtration, negative pressure filtration and membrane filtration, for example, negative pressure filtration with a filter membrane of 1-150 μm (e.g., 5-10 μm, 5 μm or 10 μm) or negative pressure filtration with a filter cloth or filter plate of 100-30000 mesh (e.g., 300 mesh).
The concentration of the beta-glucan solution can be 0.01-10.0%, specifically 0.5-5.0%, 0.5%, 0.8%, 1.0%, 2.0% or 5.0%.
The beta-glucan solution prepared by the preparation method is also within the protection scope of the invention. Wherein the viscosity of the beta-glucan solution with the concentration of 0.5 percent can reach 600mPa & s, and the light transmittance can reach more than 90 percent; the viscosity of the beta-glucan solution with the concentration of 0.8% can reach 1600 mPas, and the light transmittance can reach more than 85%.
The invention has the following beneficial effects:
(1) the enzymolysis process is not used, and the cost is lower.
(2) The functional structure of the beta-glucan can not be damaged without using an acidolysis or alkaline hydrolysis process.
(3) The equipment and materials are simple and common, the process is simple and easy to implement, and the method is suitable for industrialization.
(4) The beta-glucan solution prepared by the method has high viscosity and high light transmittance, and is suitable for various applications.
(5) The beta-glucan dry product prepared by the method has strong water solubility, and the beta-glucan solution obtained after dissolution has large viscosity and high light transmittance.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The fermentation broth in the following examples was prepared by the following steps:
(1) and (3) activation of the Schizophyllum commune strain: preparing a plate culture medium from 200g/L of potato, 30g/L of glucose, 10g/L of sodium chloride and 20g/L of agar, inoculating Schizophyllum commune strains on the plate culture medium, and culturing in a constant-temperature incubator at 25 ℃ for 7 days to obtain plate mycelia;
(2) seed activation: putting a liquid culture medium prepared from 100g/L of potato starch, 40g/L of glucose, 2g/L of yeast extract powder and water into a shake flask, wherein the liquid filling amount is 1/3, inoculating the flat plate mycelium obtained in the step (1) into the shake flask, and performing shake culture in a constant-temperature shaking table at 25 ℃ and 160rpm for 7 days to obtain a seed solution;
(3) fermentation culture: adding fermentation culture medium prepared from 50g/L glucose, 50g/L sucrose, 5g/L soybean powder, 2g/L yeast extract powder, 0.5g/L potassium dihydrogen phosphate, 0.5g/L magnesium sulfate heptahydrate, 0.5g/L ammonium sulfate, 6g/L potassium nitrate and water into a fermentation tank, sterilizing at 121 deg.C for 15 min, inoculating the seed solution obtained in step (2) into the fermentation tank, stirring at 25 deg.C and 300rpm, and performing aerobic fermentation with 4Lpm for 8 days.
The vacuum pump for the negative pressure filter in the following examples was purchased from Auto Science, model AP-9925. SCP-321# Filter Panel was purchased from Shenyang great wall Filter Board, Inc.
In the following examples, the concentration of a β -Glucan solution was measured by a conventional phenol sulfate method and a β -Glucan Assay Kit (Yeast & Mushroom)) available from Megazyme.
The method for measuring the light transmittance of a β -glucan solution in the following examples is as follows:
(1) placing a beta-glucan solution sample in a glass cuvette;
(2) adjusting the detection wavelength to 600nm by using a spectrophotometer;
(3) detecting the light transmittance of the beta-glucan solution sample at the wavelength;
the dynamic viscosity was measured as follows:
(1) placing 200ml of beta-glucan solution sample in a 250ml beaker;
(2) placing the beaker containing the beta-glucan solution sample in a water bath kettle, and keeping the temperature at 25 ℃;
(3) the kinetic viscosity at 25 ℃ was measured using a rotational viscometer by selecting the corresponding spindle depending on the concentration of the sample of the beta-glucan solution.
Example 1 purification preparation of high viscosity, high light transmittance beta-glucan solution
Purifying to prepare a high-viscosity high-light-transmittance beta-glucan solution according to the following steps:
(1) mixing Schizophyllum commune fermentation broth with 4 times of distilled water, and soaking at 60 deg.C for 8 hr.
(2) Centrifuging the decoction at 4000rpm for 5min, and collecting supernatant; filtering the supernatant with 300 mesh filter cloth under negative pressure, and collecting the filtrate for use.
(3) And (3) heating the digestion solution filtered in the step (2) to 50 ℃, and simultaneously adding 200-mesh wood activated carbon and 8-16-mesh coconut shell activated carbon into the digestion solution, wherein the adding volume of each activated carbon is 1% of the volume of the digestion solution. Continuously stirring for 4h at 50 ℃ and 350rpm, and cooling for later use; and (3) carrying out negative pressure suction filtration on the cooled and standby immersion clear liquid mixed with the activated carbon by using 300-mesh filter cloth and an SCP-321# filter plate (the pore size is about 1.5 mu m) in sequence, and taking the filtered clear liquid for standby.
(4) And (4) mixing the clear liquid obtained after filtration in the step (3) with edible ethanol with the concentration of 95% (volume ratio is 1: 3), and stirring until the beta-glucan is separated out.
(5) And (5) placing the precipitate obtained in the step (4) in a tray with a hole, and drying the precipitate by using an electric heating oven at 40 ℃ until the weight is constant to obtain the dried beta-glucan.
(6) Crushing the dried beta-glucan obtained in the step (5), weighing 5g of crushed beta-glucan, dissolving the crushed beta-glucan in 1000ml of ultrapure water, and stirring the crushed beta-glucan at 600rpm for 2 hours until the beta-glucan is fully dissolved to obtain a beta-glucan solution; and (3) carrying out negative pressure suction filtration on the beta-glucan solution by using a 5-micron filter membrane to obtain the beta-glucan solution with high viscosity and high light transmittance, wherein the concentration of the beta-glucan solution is 0.5%.
The light transmittance of the beta-glucan solution with the mass concentration of 0.5% at the wavelength of 600nm can reach 92.2%, and the dynamic viscosity can reach 710mPa & s (30 rpm of a No. 2 rotor).
Example 2 purification preparation of high viscosity, high light transmittance beta-glucan solution
Purifying to prepare a high-viscosity high-light-transmittance beta-glucan solution according to the following steps:
(1) mixing Schizophyllum commune fermentation broth with 1 volume of distilled water, and soaking and decocting at 90 deg.C for 0.5 h.
(2) Centrifuging the Schizophyllum commune fermentation digestion liquid obtained in the step (1) at 3000rpm for 60min, and taking supernatant; adding 2% diatomite into the supernatant, filtering with 300 mesh filter cloth under negative pressure, and collecting the filtrate for use.
(3) Adding 200-mesh wood activated carbon accounting for 2% of the volume of the digestion solution into the digestion solution filtered in the step (2), stirring at 300rpm at room temperature for 5min, standing at 4 ℃ for 16h after uniformly stirring; and (3) performing negative pressure suction filtration on the mixed solution containing the activated carbon by using 300-mesh filter cloth and an SCP-321# (the pore size is about 1.5 mu m) filter plate in sequence, and taking the filtered clear solution for later use.
(4) And (3) mixing the filtered clear liquid obtained in the step (3) with edible ethanol with the concentration of 95% (volume ratio is 1: 1), and stirring until the beta-glucan is separated out. An appropriate amount of pure water was added to the precipitate to make up the total volume to the volume of the supernatant before the alcohol precipitation, and stirring was continued until the alcohol precipitate was sufficiently dissolved. Mixing the dissolved beta-glucan solution with 95% edible ethanol (volume ratio of 1: 2) again, and stirring until the beta-glucan is separated out. Transferring the beta-glucan precipitate into new edible ethanol with the concentration of 95%, and soaking overnight to obtain the beta-glucan precipitate washed by ethanol.
(5) And (4) placing the precipitate obtained in the step (4) in a tray, and placing the tray in an electric heating oven to dry at 40 ℃ until the weight is constant, so as to obtain a dried beta-glucan.
(6) Crushing the dried beta-glucan obtained in the step (5), weighing 8g of the crushed beta-glucan, dissolving the crushed beta-glucan in 1000ml of ultrapure water preheated to 40 ℃, and stirring the crushed beta-glucan at 600rpm for 4 hours until the beta-glucan is fully dissolved to obtain a beta-glucan solution; and (3) performing negative pressure suction filtration by using a filter membrane of 10 mu m to obtain the beta-glucan solution with high viscosity and high light transmittance and concentration of 0.8%.
The light transmittance of the beta-glucan solution with the mass concentration of 0.8% at the wavelength of 600nm can reach 90.0%, and the dynamic viscosity can reach 2480mPa & s (30 rpm of a 4# rotor).
Example 3 purification preparation of high viscosity, high light transmittance beta-glucan solution
Purifying to prepare a high-viscosity high-light-transmittance beta-glucan solution according to the following steps:
(1) mixing Schizophyllum commune fermentation liquid with 5 times volume of tap water, and soaking and decocting at 70 deg.C for 4 hr.
(2) And (2) carrying out negative pressure filtration on the schizophyllum commune fermentation digestion solution obtained in the step (1) by using 8 layers of gauze, and taking and filtering out clear liquid.
(3) And (3) adding 200-mesh wood activated carbon and 8-16-mesh coconut shell activated carbon into the filtered digestion solution obtained in the step (2), wherein the adding volume of each activated carbon is 0.75% of the volume of the digestion solution. Stirring at 300rpm at room temperature for 5min until the activated carbon is uniformly distributed, heating the mixed solution to 50 ℃, and standing for 4h at 50 ℃; then, vacuum filtration is carried out by using 300-mesh filter cloth and an SCP-321# filter plate (the aperture is about 1.5 mu m) in sequence, and the filtered clear liquid is taken for standby.
(4) Mixing the clear liquid filtered in the step (3) with edible ethanol with the concentration of 95% (volume ratio is 1: 5), stirring until beta-glucan is separated out, and fishing out the precipitate for later use.
(5) And (5) placing the precipitate obtained in the step (4) in a tray with a hole, and drying the precipitate by using an electric heating oven at 50 ℃ until the weight is constant to obtain the dried beta-glucan.
(6) Crushing the dried beta-glucan obtained in the step (5), weighing 10g, dissolving in 1000mL of ultrapure water to prepare a beta-glucan solution, then sterilizing for 30min by high-pressure steam at 121 ℃, standing and cooling for later use; heating the sterilized beta-glucan solution to 80 ℃ in a water-proof manner, and stirring at 600rpm for 6 hours until the beta-glucan is fully dissolved; and then carrying out negative pressure suction filtration by using 300-mesh filter cloth to obtain the beta-glucan solution with high viscosity and high light transmittance and the concentration of 1.0%.
The light transmittance of the beta-glucan solution with the mass concentration of 1.0% at the wavelength of 600nm can reach 81.1%, and the dynamic viscosity can reach 3100 mPas (30 rpm of a 4# rotor).
Example 4 purification preparation of high viscosity, high light transmittance beta-glucan solution
Purifying to prepare a high-viscosity high-light-transmittance beta-glucan solution according to the following steps:
(1) mixing Schizophyllum commune fermentation liquid with 10 times of tap water, and soaking and decocting at 50 deg.C for 2 hr.
(2) And (2) centrifuging the schizophyllum commune fermentation liquor obtained in the step (1) at 5000rpm for 10min, and taking supernatant for later use.
(3) Adding 300-mesh active carbon into the digestion solution after centrifugal separation in the step (2), wherein the adding volume of the active carbon is 2.5% of the volume of the digestion solution. Stirring at 300rpm at room temperature for 5min, heating to 80 deg.C, maintaining the temperature at 80 deg.C, standing for 4h, and intermittently stirring once every 30 min; adding 0.5% diatomite into the above decoction mixed with activated carbon, mixing, vacuum filtering with 300 mesh filter cloth and SCP-321# filter plate (pore size about 1.5 μm), and collecting the filtered supernatant.
(4) And (4) mixing the clear liquid obtained after filtration in the step (3) with edible ethanol with the concentration of 95% (volume ratio of 1: 10), and stirring until the beta-glucan is separated out. Dissolving the precipitate in pure water with the same volume as the precipitate, and continuously stirring at 80 ℃ until the alcohol precipitate is fully dissolved. Mixing the dissolved beta-glucan solution with 95% edible ethanol (volume ratio of 1: 1) again, and stirring until the beta-glucan is separated out.
(5) And (3) placing the precipitate obtained in the step (4) in a tray with a hole, and drying the precipitate by using an electric heating oven at 60 ℃ until the weight is constant to obtain the dried beta-glucan.
(6) Crushing the dried beta-glucan obtained in the step (5), weighing 5g and 8g of crushed beta-glucan, respectively dissolving the crushed beta-glucan and the crushed beta-glucan in 1000mL of ultrapure water, and then sterilizing the mixture for 20min by high-pressure steam at 115 ℃; heating the sterilized beta-glucan solution to 70 ℃ in a water-proof way, maintaining 800rpm and stirring for 4 hours until the beta-glucan is fully dissolved; then a filter membrane with the thickness of 10 mu m is used for vacuum filtration, and beta-glucan solutions with the concentration of 0.5 percent and 0.8 percent, high viscosity and high light transmittance can be respectively obtained.
The light transmittance of the beta-glucan solution with the mass concentration of 0.5% under the wavelength of 600nm can reach 93.1%, and the dynamic viscosity can reach 740mPa & s (30 rpm of a 2# rotor); the light transmittance of the beta-glucan solution with the mass concentration of 0.8% at the wavelength of 600nm can reach 87.5%, and the dynamic viscosity can reach 2150mPa & s (30 rpm of a No. 2 rotor).
Claims (5)
1. A method for preparing beta-glucan, which is characterized by comprising the following steps:
(1) mixing Schizophyllum commune fermentation liquor with distilled water of 4 times volume, and soaking and boiling at 60 deg.C for 8 hr;
(2) centrifuging the decoction at 4000rpm for 5min, and collecting supernatant; filtering the supernatant with 300 mesh filter cloth under negative pressure, and collecting the filtrate;
(3) heating the digestion solution filtered in the step (2) to 50 ℃, and simultaneously adding 200-mesh wood activated carbon and 8-16-mesh coconut shell activated carbon into the digestion solution, wherein the adding volume of each activated carbon is 1% of the volume of the digestion solution; continuously stirring for 4h at 50 ℃ and 350rpm, and cooling for later use; carrying out negative pressure suction filtration on the cooled and standby immersion clear liquid mixed with the activated carbon by using 300-mesh filter cloth and an SCP-321# filter plate in sequence, and taking the filtered clear liquid for standby;
(4) mixing the clear liquid filtered in the step (3) with edible ethanol with the concentration of 95%, wherein the volume ratio is 1: 3, stirring until the beta-glucan is separated out; and
(5) placing the precipitate obtained in the step (4) in a tray with holes, and drying the precipitate by using an electric heating oven at 40 ℃ until the weight is constant to obtain dried beta-glucan;
the Schizophyllum commune fermentation liquor is prepared by the following steps:
(a) and (3) activation of the Schizophyllum commune strain: preparing a plate culture medium from 200g/L of potato, 30g/L of glucose, 10g/L of sodium chloride and 20g/L of agar, inoculating Schizophyllum commune strains on the plate culture medium, and culturing in a constant-temperature incubator at 25 ℃ for 7 days to obtain plate mycelia;
(b) seed activation: putting a liquid culture medium prepared from 100g/L of potato starch, 40g/L of glucose, 2g/L of yeast extract powder and water into a shake flask, wherein the liquid filling amount is 1/3, inoculating the flat plate mycelium obtained in the step (a) into the shake flask, and performing shake culture in a constant-temperature shaking table at 25 ℃ and 160rpm for 7 days to obtain a seed solution;
(c) fermentation culture: adding a fermentation culture medium prepared from 50g/L glucose, 50g/L sucrose, 5g/L soybean meal, 2g/L yeast extract powder, 0.5g/L potassium dihydrogen phosphate, 0.5g/L magnesium sulfate heptahydrate, 0.5g/L ammonium sulfate, 6g/L potassium nitrate and water into a fermentation tank, sterilizing at 121 ℃ for 15 minutes, inoculating the seed solution obtained in the step (b) into the fermentation tank, stirring at a constant temperature of 300rpm at 25 ℃ for 4Lpm, and performing aeration fermentation culture for 8 days to obtain the schizophyllum commune fermentation broth.
2. A method for preparing beta-glucan, which is characterized by comprising the following steps:
(1) mixing Schizophyllum commune fermentation liquor with distilled water of 1 time volume, and soaking and boiling at 90 deg.C for 0.5 h;
(2) centrifuging the Schizophyllum commune fermentation digestion liquid obtained in the step (1) at 3000rpm for 60min, and taking supernatant; adding 2% of diatomite into the supernatant, filtering with 300-mesh filter cloth under negative pressure, and taking the filtered supernatant for later use;
(3) adding 200-mesh wood activated carbon accounting for 2% of the volume of the digestion solution into the digestion solution filtered in the step (2), stirring at 300rpm at room temperature for 5min, standing at 4 ℃ for 16h after uniformly stirring; sequentially performing negative pressure suction filtration on the mixed solution containing the activated carbon by using 300-mesh filter cloth and an SCP-321# filter plate, and taking the filtered clear solution for later use;
(4) mixing the clear liquid filtered in the step (3) with edible ethanol with the concentration of 95%, wherein the volume ratio is 1: 1, stirring until the beta-glucan is separated out; adding a proper amount of pure water into the precipitate, supplementing the pure water until the total volume reaches the volume of a clear solution before alcohol precipitation, and continuously stirring until the alcohol precipitation is fully dissolved; mixing the dissolved beta-glucan solution with 95% edible ethanol again, wherein the volume ratio is 1: 2, stirring until the beta-glucan is separated out; transferring the beta-glucan precipitate into new edible ethanol with the concentration of 95%, and soaking overnight to obtain the beta-glucan precipitate washed by ethanol; and
(5) placing the precipitate obtained in the step (4) in a tray, and placing the tray in an electric heating oven to dry at 40 ℃ until the weight is constant to obtain a dried beta-glucan;
the Schizophyllum commune fermentation liquor is prepared by the following steps:
(a) and (3) activation of the Schizophyllum commune strain: preparing a plate culture medium from 200g/L of potato, 30g/L of glucose, 10g/L of sodium chloride and 20g/L of agar, inoculating Schizophyllum commune strains on the plate culture medium, and culturing in a constant-temperature incubator at 25 ℃ for 7 days to obtain plate mycelia;
(b) seed activation: putting a liquid culture medium prepared from 100g/L of potato starch, 40g/L of glucose, 2g/L of yeast extract powder and water into a shake flask, wherein the liquid filling amount is 1/3, inoculating the flat plate mycelium obtained in the step (a) into the shake flask, and performing shake culture in a constant-temperature shaking table at 25 ℃ and 160rpm for 7 days to obtain a seed solution;
(c) fermentation culture: adding a fermentation culture medium prepared from 50g/L glucose, 50g/L sucrose, 5g/L soybean meal, 2g/L yeast extract powder, 0.5g/L potassium dihydrogen phosphate, 0.5g/L magnesium sulfate heptahydrate, 0.5g/L ammonium sulfate, 6g/L potassium nitrate and water into a fermentation tank, sterilizing at 121 ℃ for 15 minutes, inoculating the seed solution obtained in the step (b) into the fermentation tank, stirring at a constant temperature of 300rpm at 25 ℃ for 4Lpm, and performing aeration fermentation culture for 8 days to obtain the schizophyllum commune fermentation broth.
3. A method for preparing beta-glucan, which is characterized by comprising the following steps:
(1) mixing Schizophyllum commune fermentation liquor with tap water of 5 times volume, and soaking and boiling at 70 deg.C for 4 hr;
(2) carrying out negative pressure filtration on the schizophyllum commune fermentation digestion solution obtained in the step (1) by using 8 layers of gauze, and taking and filtering out clear liquid;
(3) adding 200 meshes of wood activated carbon and 8-16 meshes of coconut shell activated carbon into the filtered digestion solution obtained in the step (2), wherein the addition volume of each activated carbon is 0.75% of the volume of the digestion solution; stirring at 300rpm at room temperature for 5min until the activated carbon is uniformly distributed, heating the mixed solution to 50 ℃, and standing for 4h at 50 ℃; then, carrying out negative pressure suction filtration by using 300-mesh filter cloth and an SCP-321# filter plate in sequence, and taking the filtered clear liquid for later use;
(4) mixing the clear liquid filtered in the step (3) with edible ethanol with the concentration of 95%, wherein the volume ratio is 1: 5, stirring until the beta-glucan is separated out, and fishing out the precipitate for later use; and
(5) placing the precipitate obtained in the step (4) in a tray with holes, and drying the precipitate by using an electric heating oven at 50 ℃ until the weight is constant to obtain dried beta-glucan;
the Schizophyllum commune fermentation liquor is prepared by the following steps:
(a) and (3) activation of the Schizophyllum commune strain: preparing a plate culture medium from 200g/L of potato, 30g/L of glucose, 10g/L of sodium chloride and 20g/L of agar, inoculating Schizophyllum commune strains on the plate culture medium, and culturing in a constant-temperature incubator at 25 ℃ for 7 days to obtain plate mycelia;
(b) seed activation: putting a liquid culture medium prepared from 100g/L of potato starch, 40g/L of glucose, 2g/L of yeast extract powder and water into a shake flask, wherein the liquid filling amount is 1/3, inoculating the flat plate mycelium obtained in the step (a) into the shake flask, and performing shake culture in a constant-temperature shaking table at 25 ℃ and 160rpm for 7 days to obtain a seed solution;
(c) fermentation culture: adding a fermentation culture medium prepared from 50g/L glucose, 50g/L sucrose, 5g/L soybean meal, 2g/L yeast extract powder, 0.5g/L potassium dihydrogen phosphate, 0.5g/L magnesium sulfate heptahydrate, 0.5g/L ammonium sulfate, 6g/L potassium nitrate and water into a fermentation tank, sterilizing at 121 ℃ for 15 minutes, inoculating the seed solution obtained in the step (b) into the fermentation tank, stirring at a constant temperature of 300rpm at 25 ℃ for 4Lpm, and performing aeration fermentation culture for 8 days to obtain the schizophyllum commune fermentation broth.
4. A method for preparing beta-glucan, which is characterized by comprising the following steps:
(1) mixing Schizophyllum commune fermentation liquor with 10 times of tap water, and soaking and boiling at 50 deg.C for 2 hr;
(2) centrifuging the Schizophyllum commune fermentation liquor obtained in the step (1) at 5000rpm for 10min, and taking supernatant for later use;
(3) adding 300-mesh active carbon into the digestion solution after centrifugal separation in the step (2), wherein the addition volume is 2.5% of the volume of the digestion solution; stirring at 300rpm at room temperature for 5min, heating to 80 deg.C, maintaining the temperature at 80 deg.C, standing for 4h, and intermittently stirring once every 30 min; adding 0.5% of diatomite into the boiled clear liquid mixed with the active carbon, uniformly mixing, sequentially performing negative pressure suction filtration by using 300-mesh filter cloth and an SCP-321# filter plate, and taking the filtered clear liquid for later use;
(4) mixing the clear liquid filtered in the step (3) with edible ethanol with the concentration of 95%, wherein the volume ratio is 1: 10, stirring until the beta-glucan is separated out; dissolving the precipitate in pure water with the same volume as the precipitate, and continuously stirring at 80 ℃ until the alcohol precipitate is fully dissolved; mixing the dissolved beta-glucan solution with 95% edible ethanol again, wherein the volume ratio is 1: 1, stirring until the beta-glucan is separated out; and
(5) placing the precipitate obtained in the step (4) in a tray with holes, and drying the precipitate by using an electric heating oven at 60 ℃ until the weight is constant to obtain dried beta-glucan;
the Schizophyllum commune fermentation liquor is prepared by the following steps:
(a) and (3) activation of the Schizophyllum commune strain: preparing a plate culture medium from 200g/L of potato, 30g/L of glucose, 10g/L of sodium chloride and 20g/L of agar, inoculating Schizophyllum commune strains on the plate culture medium, and culturing in a constant-temperature incubator at 25 ℃ for 7 days to obtain plate mycelia;
(b) seed activation: putting a liquid culture medium prepared from 100g/L of potato starch, 40g/L of glucose, 2g/L of yeast extract powder and water into a shake flask, wherein the liquid filling amount is 1/3, inoculating the flat plate mycelium obtained in the step (a) into the shake flask, and performing shake culture in a constant-temperature shaking table at 25 ℃ and 160rpm for 7 days to obtain a seed solution;
(c) fermentation culture: adding a fermentation culture medium prepared from 50g/L glucose, 50g/L sucrose, 5g/L soybean meal, 2g/L yeast extract powder, 0.5g/L potassium dihydrogen phosphate, 0.5g/L magnesium sulfate heptahydrate, 0.5g/L ammonium sulfate, 6g/L potassium nitrate and water into a fermentation tank, sterilizing at 121 ℃ for 15 minutes, inoculating the seed solution obtained in the step (b) into the fermentation tank, stirring at a constant temperature of 300rpm at 25 ℃ for 4Lpm, and performing aeration fermentation culture for 8 days to obtain the schizophyllum commune fermentation broth.
5. The beta-glucan produced by the production method according to any one of claims 1 to 4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611215234.3A CN108239177B (en) | 2016-12-26 | 2016-12-26 | Method for purifying and preparing high-quality biological polysaccharide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611215234.3A CN108239177B (en) | 2016-12-26 | 2016-12-26 | Method for purifying and preparing high-quality biological polysaccharide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108239177A CN108239177A (en) | 2018-07-03 |
| CN108239177B true CN108239177B (en) | 2021-06-29 |
Family
ID=62704838
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611215234.3A Active CN108239177B (en) | 2016-12-26 | 2016-12-26 | Method for purifying and preparing high-quality biological polysaccharide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108239177B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110090223B (en) * | 2018-11-30 | 2022-12-20 | 浙江立恩生物科技有限公司 | Solid dispersion of beta-glucan and preparation method thereof |
| CN111440832B (en) * | 2019-01-17 | 2024-03-19 | 浙江立恩生物科技有限公司 | Fungus culture method for high-yield polysaccharide |
| CN111778301B (en) * | 2020-08-05 | 2021-06-04 | 上海珈凯生物科技有限公司 | Preparation process of beta-glucan |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103214593B (en) * | 2008-12-31 | 2015-09-09 | 陈秀男 | The preparation method of beta-glucan |
| CN101643759B (en) * | 2009-07-31 | 2012-02-01 | 中国农业科学院农产品加工研究所 | Method for preparing Schizophyllum commune Fr polysaccharides and dedicated culture medium thereof |
| CN105255964B (en) * | 2015-10-30 | 2019-01-18 | 宁波希诺亚海洋生物科技有限公司 | A kind of production method of beta glucan |
-
2016
- 2016-12-26 CN CN201611215234.3A patent/CN108239177B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN108239177A (en) | 2018-07-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102824377B (en) | Method for extracting functional ingredients from lucid ganoderma sporocarp | |
| CN108239177B (en) | Method for purifying and preparing high-quality biological polysaccharide | |
| CN107226871B (en) | preparation method of highland barley β -glucan | |
| CN107868805B (en) | Longan polysaccharide degraded by lactobacillus fermentation and preparation method thereof | |
| CN110272506A (en) | A kind of extracting method of Fuscoporia obliqua polysaccharide | |
| CN109157670A (en) | A kind of composite antibacterial gel rubber material | |
| CN106832041A (en) | A kind of method that biologic enzymolysis method extracts fucoidin | |
| CN108948227A (en) | A kind of method that high-voltage pulse extracts okra pectin | |
| CN107936129A (en) | A kind of preparation process of complete series molecular weight tremella polysaccharides | |
| CN109576324A (en) | A kind of astragalus polyose and its biological extraction method | |
| PL103045B1 (en) | THE METHOD OF MAKING MULTIPLE SUGAR | |
| CN102532334B (en) | Method for producing flaxseed mucilage | |
| CN102838689A (en) | Production process for extracting lentinan by fermentation method and product thereof | |
| CN105861597A (en) | Novel marine polysaccharide-chitooligosaccharide production technique | |
| CN103554287B (en) | A kind of extracting method of King Boletus mycelia polysaccharide | |
| CN110922499B (en) | Selenium-enriched sparassis crispa polysaccharide and preparation method and application thereof | |
| CN107736538A (en) | A kind of black fungus small-molecular peptides solid beverage and preparation method thereof | |
| CN107987178A (en) | A kind of Radix Isatidis preprocess method for being used for fermentation extraction Banlangen Polysaccharide | |
| CN110872385B (en) | Preparation method of starch/bacterial cellulose composite material | |
| CN103504286A (en) | Method for extracting grifola frondosa mycelium based on membrane technology | |
| CN105695528A (en) | Method for selectively removing monosaccharides and producing oligosaccharides by using yeast culture | |
| CN110423285A (en) | A kind of Herba portulacae extracts the environment protection method of polysaccharide | |
| CN107227327B (en) | Preparation method of mussel oligosaccharide, and product and application thereof | |
| CN111647092B (en) | Method for improving pachyman yield by utilizing semi-solid fermentation of trichoderma reesei and application of method | |
| CN111718429B (en) | Mushroom edible fungus polysaccharide and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Method for purifying and preparing high quality biological polysaccharide Effective date of registration: 20211102 Granted publication date: 20210629 Pledgee: Pinghu Rural Commercial Bank of Zhejiang, Limited by Share Ltd. Pledgor: ZHEJIANG GLLION BIOSCIENCE Co.,Ltd. Registration number: Y2021330002136 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20230907 Granted publication date: 20210629 Pledgee: Pinghu Rural Commercial Bank of Zhejiang, Limited by Share Ltd. Pledgor: ZHEJIANG GLLION BIOSCIENCE Co.,Ltd. Registration number: Y2021330002136 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right |