CN113603805B - Preparation method of nitraria tangutorum polysaccharide - Google Patents
Preparation method of nitraria tangutorum polysaccharide Download PDFInfo
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- CN113603805B CN113603805B CN202111034534.2A CN202111034534A CN113603805B CN 113603805 B CN113603805 B CN 113603805B CN 202111034534 A CN202111034534 A CN 202111034534A CN 113603805 B CN113603805 B CN 113603805B
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- 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
Abstract
The invention discloses a method for purifying nitraria polysaccharide, which comprises the following steps: taking nitraria tangutorum bobr extracting solution, carrying out microfiltration, decoloring with macroporous resin, carrying out ultrafiltration twice and carrying out nanofiltration once to obtain nitraria tangutorum bobr polysaccharide. The invention also provides a preparation method of the nitraria tangutorum bobr polysaccharide. Experiments prove that the extraction method is simple and convenient to operate, the purity of the nitraria tangutorum polysaccharide obtained through separation and purification is more than 60%, and the total yield is more than 45%. Is suitable for industrialized mass production.
Description
Technical Field
The invention particularly relates to a preparation method of nitraria tangutorum bobr polysaccharide.
Background
The nitraria tangutorum bobr (Nitraria tangutorun Bobr) is of the genus nitraria of the family tribulidae, has homology of medicine and food of fruits, is rich in polysaccharide, and has great development and utilization value due to research shows that the nitraria tangutorum bobr polysaccharide has biological activities such as immunoregulation and antioxidation.
The traditional method for extracting the nitraria tangutorum polysaccharide is to prepare crude polysaccharide by adopting an ethanol precipitation method and purify the crude polysaccharide by using column chromatography, and is not suitable for industrial mass production due to complex process, high energy consumption and high organic reagent consumption. The method is characterized by that although it uses hot water to make extraction, macroporous resin decoloration and deproteinization, and its process is simple, and does not use a large quantity of organic solvent, and because of the enzyme added in the extraction process, the production cost is raised, and its obtained nitraria polysaccharide has low yield and low purity, and can not be converted into industrial production.
Disclosure of Invention
In order to solve the problems, the invention provides a method for purifying nitraria polysaccharide, which is characterized by comprising the following steps: it comprises the following steps:
taking nitraria tangutorum bobr extracting solution, carrying out microfiltration, decoloring with macroporous resin, carrying out ultrafiltration twice and carrying out nanofiltration once to obtain nitraria tangutorum bobr polysaccharide.
Further, the microfiltration is carried out by using a microfiltration membrane with the pore diameter of 0.05-0.2 mu m, and the microfiltration membrane is preferably 0.1 mu m; the pressure of the filtration by the microfiltration membrane is 0.2-0.4Mpa.
Further, the macroporous resin decolorization is that the filter membrane permeate liquid after microfiltration is applied to a macroporous resin column at the flow rate of 1-2BV/h and then is washed with water at the flow rate of 3-5BV at the flow rate of 1-2 BV/h.
Still further, the macroporous resin is DM21D or X-5 macroporous resin; the water is deionized water.
Further, the ultrafiltration is carried out twice by passing the chromatographic liquid after the macroporous resin decolorization through an ultrafiltration membrane with the interception molecular weight of 100000Da under the pressure of 0.25-0.75Mpa, then washing the interception liquid with water until the permeation liquid is free of polysaccharide, and then passing the permeation liquid through the ultrafiltration membrane with the interception molecular weight of 3000Da under the pressure of 0.1-0.2 Mpa.
Further, the nanofiltration is that the permeate liquid after ultrafiltration twice passes through an ultrafiltration membrane with the molecular weight cut-off of 300Da under the pressure of 1.5-2.5 Mpa.
Further, the temperature of the circulating liquid of ultrafiltration or nanofiltration is controlled to be 20-40 ℃.
The invention also provides a preparation method of the nitraria polysaccharide, which comprises the following steps:
1) Soaking fructus Hippophae in water for 12-24 hr, removing pericarp, extracting, filtering the extractive solution, and centrifuging;
2) And (3) taking the supernatant obtained in the step (1), purifying according to the method, and freeze-drying to obtain the product.
Further, the mass-volume ratio of the nitraria tangutorum bobr to the water in the step 1) is 1g: 6-7 ml; the water is distilled water.
Further, the extraction in the step 1) is leaching, wherein the leaching temperature is 70-90 ℃, preferably 80 ℃, and the times are 2 times, and each time is 1-2 hours; the centrifugal speed is 7000-9000 r/min, the time is 0.2-1 h, and the centrifugal speed is 8000r/min and the time is 0.5 h; the freeze drying time is 48-60 h, and the temperature is below-40 ℃.
According to the preparation method of the nitraria tangutorum bobr polysaccharide, through specific process steps, the polysaccharide components can be effectively reserved through precise parameter control, and the nitraria tangutorum bobr polysaccharide with high safety and purity is obtained. Experiments prove that the extraction method is simple and convenient to operate, the purity of the nitraria tangutorum polysaccharide obtained through separation and purification is more than 60%, and the total yield is more than 45%. Is suitable for industrialized mass production.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Detailed Description
EXAMPLE 1 preparation of the Nitraria polysaccharide of the invention
1. Extracting: 580g of nitraria tangutorum bobr (dry product) is weighed, 3600ml of distilled water is added, and the mixture is soaked for 12 hours, and the pericarp is removed. Extracting at 80deg.C for 2 hr for 2 times. Mixing the obtained extracts for 2 times, and filtering and centrifuging (8000 r/min,0.5 hr) to obtain extractive solution.
2. Microfiltration membrane: filtering the nitraria tangutorum bobr extracting solution obtained in the step 1) through a micro-filtration membrane system to remove solids, thereby obtaining a filter membrane permeate. The microfiltration operating parameters were: the pressure is 0.2Mpa and the temperature is normal temperature.
3. Decoloring with macroporous resin:
and (3) decoloring by selecting DM21D macroporous resin, performing pretreatment operations (soaking, acid washing, alkali washing and flushing) on the macroporous resin in advance, and loading the resin into a chromatographic column. The sample is micro-filtration membrane permeate, and the sample feeding flow rate is controlled to be 1 BV/h. When the feeding is completed, deionized water is used for cleaning, the cleaning flow rate is 1BV/h, the dosage is 3BV, and the chromatographic column polysaccharide is cleaned to obtain chromatographic liquid.
4. Ultrafiltration membrane (molecular weight 100000 Da)
Separating and purifying the chromatographic liquid in the step 3) by an ultrafiltration membrane (molecular weight is 100000 Da) to remove macromolecular impurities such as protein, pectin, cellulose and the like. The operating parameters are: the pressure is 0.25Mpa, and the temperature of the circulating liquid is controlled at 20 ℃. After the feed is completed, deionized water is added to wash the retentate, and the residual polysaccharide is recovered until the permeate is stopped when no polysaccharide is detected (polysaccharide content is detected using the phenol sulfuric acid method known in the art).
5. Ultrafiltration membrane (molecular weight 3000 Da)
Separating and purifying the permeate from the step 4) by an ultrafiltration membrane (with a molecular weight of 3000 Da) to remove small molecular impurities such as monosaccharides, oligosaccharides, amino acids and the like. The operating parameters are: the pressure is 0.1Mpa, and the temperature of the circulating liquid is controlled at 20 ℃.
6. Nanofiltration membrane (molecular weight 300 Da)
Concentrating the trapped liquid in the step 5) through a nanofiltration membrane (with the molecular weight of 300 Da), and removing impurities such as inorganic salts and the like, wherein the operation parameters are as follows: the pressure is 1.5Mpa, and the temperature of the circulating liquid is controlled at 20 ℃. The concentration multiple is 4-10 times. 7. Freeze drying
And (3) drying the concentrated solution in a freeze dryer for 48 hours at the temperature below minus 40 ℃. And after drying, rapidly filling into a sealing bag, and drying and storing at a low temperature in a dark place. The final polysaccharide yield was 46% and polysaccharide content was 60%.
EXAMPLE 2 preparation of the Nitraria polysaccharide of the invention
1. Extracting: 580g of nitraria tangutorum bobr (dry product) is weighed, 3600ml of distilled water is added, and the mixture is soaked for 12 hours, and the pericarp is removed. Extracting at 80deg.C for 2 hr for 2 times. Mixing the obtained extracts for 2 times, filtering, and centrifuging (8000 r/min,0.5 hr) to obtain extractive solution.
2. Microfiltration membrane: filtering the nitraria tangutorum bobr extracting solution obtained in the step 1) through a micro-filtration membrane system to remove solids, thereby obtaining a filter membrane permeate. The microfiltration operating parameters were: the pressure is 0.3Mpa and the temperature is normal temperature.
3. Decoloring with macroporous resin:
and (3) decoloring the macroporous resin X-5, performing pretreatment operations (soaking, acid washing, alkali washing and flushing) on the macroporous resin in advance, and then loading the resin into a chromatographic column. And the sample is taken as a microfiltration membrane permeate, the sample loading flow rate is controlled to be 1.5BV/h, and when the feeding is finished, deionized water is used for cleaning, the cleaning flow rate is 1.5BV/h, and the dosage is 4BV, so that the chromatographic liquid is obtained.
4. Ultrafiltration membrane (molecular weight 100000 Da)
Separating and purifying the chromatographic liquid in the step 3) by an ultrafiltration membrane (molecular weight is 100000 Da) to remove macromolecular impurities such as protein, pectin, cellulose and the like. The operating parameters are: the pressure is 0.5Mpa, and the temperature of the circulating liquid is controlled at 30 ℃. After the feeding is completed, deionized water is added to clean the trapped fluid, and residual polysaccharide is recovered until the permeate is substantially free of polysaccharide.
5. Ultrafiltration membrane (molecular weight 3000 Da)
Separating and purifying the permeate from the step 4) by an ultrafiltration membrane (with a molecular weight of 3000 Da) to remove small molecular impurities such as monosaccharides, oligosaccharides, amino acids and the like. The operating parameters are: the pressure is 0.15Mpa, and the temperature of the circulating liquid is controlled at 30 ℃.
6. Nanofiltration membrane (molecular weight 300 Da)
Concentrating the trapped liquid in the step 5) through a nanofiltration membrane (with the molecular weight of 300 Da), and removing impurities such as inorganic salts and the like, wherein the operation parameters are as follows: the pressure is 2.0Mpa, and the temperature of the circulating liquid is controlled at 30 ℃. The concentration multiple is 4-10 times.
7. Freeze drying
And (3) drying the concentrated solution in a freeze dryer for 50 hours at the temperature below minus 40 ℃. And after drying, rapidly filling into a sealing bag, and drying and storing at a low temperature in a dark place. The final polysaccharide yield was 48% and polysaccharide content was 63.9%.
Example 3 preparation of the present invention understand the thorn polysaccharide
1. Extracting: 580g of nitraria tangutorum bobr (dry product) is weighed, 3600ml of distilled water is added, and the mixture is soaked for 12 hours, and the pericarp is removed. Extracting at 80deg.C for 2 hr for 2 times. Mixing the obtained extracts for 2 times, filtering, and centrifuging (8000 r/min,0.5 hr) to obtain extractive solution.
2. Microfiltration membrane: filtering the treated nitraria tangutorum bobr extracting solution by a micro-filtration membrane system to remove solid matters, thereby obtaining a filter membrane permeate. The microfiltration operating parameters were: the pressure is 0.4Mpa and the temperature is normal temperature.
3. Decoloring with macroporous resin:
and (3) decoloring DM21D or X-5 macroporous resin, pre-treating the macroporous resin (soaking, pickling, alkali washing and flushing) in advance, and loading the resin into a chromatographic column. The sample is micro-filtration membrane permeate, and the sample feeding flow rate is controlled to be 2 BV/h. When the feeding is completed, deionized water is used for cleaning, the cleaning flow rate is 1-2BV/h, and the dosage is 5BV, so that the chromatographic liquid is obtained.
4. Ultrafiltration membrane (molecular weight 100000 Da)
Separating and purifying the chromatographic solution of the previous step by ultrafiltration membrane (molecular weight of 100000 Da) to remove macromolecular impurities such as protein, pectin, cellulose, etc. The operating parameters are: the pressure is 0.75Mpa, and the temperature of the circulating liquid is controlled at 40 ℃. After the feeding is completed, deionized water is added to clean the trapped fluid, and residual polysaccharide is recovered until the permeate is substantially free of polysaccharide.
5. Ultrafiltration membrane (molecular weight 3000 Da)
Separating and purifying the permeate from the previous step by ultrafiltration membrane (molecular weight of 3000 Da) to remove small molecular impurities such as monosaccharide, oligosaccharide, amino acid, etc. The operating parameters are: the pressure is 0.2Mpa, and the temperature of the circulating liquid is controlled at 40 ℃.
6. Nanofiltration membrane (molecular weight 300 Da)
Concentrating the trapped liquid in the previous step through a nanofiltration membrane (molecular weight is 300 Da), and removing impurities such as inorganic salts, wherein the operation parameters are as follows: the pressure is 2.5Mpa, and the temperature of the circulating liquid is controlled at 40 ℃. The concentration multiple is 4-10 times.
7. Freeze drying
And (3) drying the concentrated solution in a freeze dryer for 60 hours at the temperature below minus 40 ℃. And after drying, rapidly filling into a sealing bag, and drying and storing at a low temperature in a dark place. The final polysaccharide yield was 46% and polysaccharide content was 66%. The advantageous effects of the present invention are described below by way of test examples.
Experimental example preparation of Nitraria polysaccharide
1. The invention discloses a nitraria polysaccharide extraction method
1.1 specific process flow:
earlier studies show that the quality of the final product, i.e., the nitraria polysaccharide, can be improved by combining macroporous resin with filter membrane filtration to purify the nitraria polysaccharide extract, but the sequence of purification steps can significantly influence the yield and purity of the polysaccharide, and after continuous attempts, the following process flow of nitraria polysaccharide preparation is finally determined:
nitraria tangutorum bobr extracting, micro-filtering, decoloring with macroporous resin, ultrafiltering (molecular weight of 100000 Da), ultrafiltering (molecular weight of 3000 Da), nanofiltration (molecular weight of 300 Da), freeze drying, and making into polysaccharide product
1.2 specific preparation method
In the same manner as in example 1,
2. contrast method
2.1 process flow:
nitraria tangutorum bobr, extracting, alcohol precipitating, deproteinizing by Sevage, decoloring by macroporous resin, freeze drying and obtaining a polysaccharide finished product;
2.2 specific preparation method:
1) Extracting: 580g of nitraria tangutorum bobr (dry product) is weighed, 3600ml of distilled water is added, and the mixture is soaked for 12 hours, and the pericarp is removed. Extracting at 80deg.C for 2 hr for 2 times. Mixing the obtained extracts for 2 times, and filtering and centrifuging (8000 r/min,0.5 hr) to obtain extractive solution. ,
2) Alcohol precipitation: precipitating the extractive solution with 80% ethanol overnight to obtain crude polysaccharide, and dissolving with water until no precipitate exists.
3) Deproteinization: 300mL of the crude polysaccharide solution was mixed with 100mL of the LSveag reagent (3:l ratio), shaken well in a separatory funnel for 30 minutes, and allowed to stand for 3-6 hours to separate. The lower white precipitate was a protein layer, which was removed by pipetting.
4) Decoloring: and (3) decoloring DM21D or X-5 macroporous resin, pre-treating the macroporous resin (soaking, pickling, alkali washing and flushing) in advance, and loading the resin into a chromatographic column. The sample is deproteinized solution, and the sample feeding flow rate is controlled to be 2 BV/h. When the feeding was completed, the mixture was washed with deionized water at a washing flow rate of 2BV/h to obtain a chromatographic liquid.
5) And (3) freeze drying: and (3) drying the concentrated solution in a freeze dryer for 60 hours at the temperature below minus 40 ℃. And after drying, rapidly filling into a sealing bag, and drying and storing at a low temperature in a dark place.
3. Detection of
3.1 detection method
Detecting the polysaccharide content in the intermediate nitraria extract and the finished nitraria polysaccharide freeze-dried powder extracted by the three methods by using a phenol-sulfuric acid method, wherein the detection steps of the phenol-sulfuric acid method are as follows:
1) Preparing a standard curve: accurately weighing 10mg of standard glucan in a 100ml volumetric flask, adding water to the scale, respectively sucking 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2, supplementing distilled water to 2.0ml, adding 1.0ml of 5% phenol and 5.0ml of concentrated sulfuric acid, shaking uniformly, standing for 30 minutes, measuring absorbance at 490nm, taking 2.0ml of water as blank according to the same color development operation, taking the abscissa as polysaccharide micrograms, and taking the ordinate as optical density value, thus obtaining a standard curve.
2) Dissolving or diluting polysaccharide sample with distilled water to obtain solution, adding 1.0ml of 5% phenol into 2ml of solution, rapidly adding 5.0ml of concentrated sulfuric acid, shaking on vortex instrument, mixing well, standing for 30 min, measuring absorbance at 490nm, and introducing the absorbance into standard curve to obtain polysaccharide content.
3) Calculating the yield and purity of the nitraria polysaccharide under each method
Yield = polysaccharide mass in finished product/polysaccharide mass in extract 100%
Purity = mass of polysaccharide in finished product/mass of polysaccharide in finished product 100%
4. Detection result
4.1 product comparison
The detection results of the nitraria polysaccharide prepared by each method are shown in table 1
TABLE 1 comparison of detection results of Nitraria tangutica polysaccharide
As can be seen from table 1: the polysaccharide yield and the purity of the finished polysaccharide are higher, and the organic reagent is less in use and the energy consumption is low in the process.
4.2 process comparison:
the specific process flow is shown in Table 2
Claims (8)
1. A method for purifying nitraria polysaccharide is characterized in that: it comprises the following steps:
taking nitraria tangutorum bobr extracting solution, carrying out microfiltration, decoloring with macroporous resin, carrying out ultrafiltration twice and nanofiltration once to obtain nitraria tangutorum bobr polysaccharide;
the microfiltration is carried out by using a microfiltration membrane with the pore diameter of 0.1 mu m;
the twice ultrafiltration is that the chromatographic liquid after the macroporous resin decolorization passes through an ultrafiltration membrane with the interception molecular weight of 100000Da at the pressure of 0.25-0.75Mpa, then the interception liquid is washed by water until the permeation liquid has no polysaccharide, and the permeation liquid passes through an ultrafiltration membrane with the interception molecular weight of 3000Da at the pressure of 0.1-0.2 Mpa;
the nanofiltration is carried out by ultrafiltering the permeate liquid twice, and then passing the permeate liquid through an ultrafiltration membrane with the interception molecular weight of 300Da under the pressure of 1.5-2.5 Mpa.
2. The purification method according to claim 1, wherein: the pressure of the filtration by the microfiltration membrane is 0.2-0.4Mpa.
3. The purification method according to claim 1, wherein: the macroporous resin decolorization is that the micro-filtered permeate is applied to a macroporous resin column at the flow rate of 1-2BV/h, and then is washed with 3-5BV water at the flow rate of 1-2 BV/h.
4. A purification process according to claim 3, characterized in that: the macroporous resin is DM21D or X-5 macroporous resin; the water is deionized water.
5. The purification method according to claim 1, wherein: the temperature of the circulating liquid of ultrafiltration or nanofiltration is controlled to be 20-40 ℃.
6. A preparation method of nitraria polysaccharide is characterized by comprising the following steps: it comprises the following steps:
1) Soaking fructus Hippophae in water for 12-24 hr, removing pericarp, extracting, filtering the extractive solution, and centrifuging;
2) Taking the supernatant obtained in the step 1), purifying by the method of any one of claims 1-4, and freeze-drying.
7. The method of manufacture of claim 1, wherein: the mass volume ratio of the nitraria tangutorum bobr to the water in the step 1) is 1g: 6-7 ml; the water is distilled water.
8. The method of manufacture of claim 1, wherein: the extraction in the step 1) is carried out at the temperature of 70-90 ℃ for 2 times, each time for 1-2 hours; the centrifugal speed is 7000-9000 r/min, and the time is 0.2-1 hour; the freeze drying time is 48-60 h, and the temperature is below-40 ℃.
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