CN110964767A - Method for extracting aster polysaccharide and aster polypeptide - Google Patents

Method for extracting aster polysaccharide and aster polypeptide Download PDF

Info

Publication number
CN110964767A
CN110964767A CN201911392161.9A CN201911392161A CN110964767A CN 110964767 A CN110964767 A CN 110964767A CN 201911392161 A CN201911392161 A CN 201911392161A CN 110964767 A CN110964767 A CN 110964767A
Authority
CN
China
Prior art keywords
aster
enzymolysis
liquid
polysaccharide
mixing
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.)
Pending
Application number
CN201911392161.9A
Other languages
Chinese (zh)
Inventor
郭强
贺佩
贾志军
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hebei Zhongke Tongchuang Technology Development Co ltd
Original Assignee
Hebei Zhongke Tongchuang Technology Development Co ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Hebei Zhongke Tongchuang Technology Development Co ltd filed Critical Hebei Zhongke Tongchuang Technology Development Co ltd
Priority to CN201911392161.9A priority Critical patent/CN110964767A/en
Publication of CN110964767A publication Critical patent/CN110964767A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • 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
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • C07K1/303—Extraction; Separation; Purification by precipitation by salting out
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • 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

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biotechnology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Sustainable Development (AREA)
  • Materials Engineering (AREA)
  • Polymers & Plastics (AREA)
  • Water Supply & Treatment (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention provides a method for extracting aster polysaccharide and aster polypeptide, belonging to the technical field of plant extraction, and the method comprises the following steps: 1) mixing radix Asteris with water, pulping, and ultrasonically treating to obtain radix Asteris slurry; 2) mixing the aster slurry and cellulase for first enzymolysis; 3) treating the first enzymolysis liquid at 30-50 ℃ for 30-50 min, and then carrying out solid-liquid separation; 4) adjusting the pH value of the first clear liquid to 4-7, mixing the first clear liquid with a sulfate mixture, and salting out and layering to obtain an upper-layer second clear liquid and a lower-layer precipitation liquid; 5) mixing the lower-layer precipitation solution with alkaline protease to perform second enzymolysis to obtain a second enzymolysis solution, and performing ultrafiltration, nanofiltration and drying on the second enzymolysis solution to obtain the aster polypeptide; 6) drying the second supernatant in the step 4) to obtain the aster polysaccharide. The method can realize simultaneous extraction of the aster polysaccharide and the aster polypeptide, and has high yield.

Description

Method for extracting aster polysaccharide and aster polypeptide
Technical Field
The invention belongs to the technical field of plant extraction, and particularly relates to a method for extracting aster polysaccharide and aster polypeptide.
Background
Aster is the dried root and rhizome of Aster (Aster tataricus L.) of Aster of Compositae, is a common Chinese medicinal material, also called Aster, Aster tataricus, Senecio cannabifolius Rolfe root, etc., originally recorded in Shennong Ben Cao Jing, listed as a Chinese product, and mainly produced in Hebei Anguo and Anhui Bozhou. Radix Asteris has effects of moistening lung, invigorating qi, eliminating phlegm and relieving cough, and can be used for treating excessive phlegm, cough due to overstrain, cough, etc. The active ingredients in the aster are very rich and mainly comprise terpenes and glycosides thereof, peptides, sterols, flavonoids, anthraquinones, coumarins, micromolecular polysaccharides and the like, wherein the triterpenoid saponin is the main characteristic ingredient of the aster, and the cyclic peptide is a specific chemical ingredient of the aster and is the main ingredient of the aster with the anti-tumor activity.
Although there are many kinds of active ingredients in aster, they have not been fully utilized at present, and there is a lack of a method for comprehensive utilization. There is no method for extracting active ingredients of polysaccharide and polypeptide from radix Asteris with good extraction effect and high yield.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for extracting aster polysaccharide and aster polypeptide, which can simultaneously extract aster polysaccharide and aster polypeptide with high yield; the aster polysaccharide extracted by the method is micromolecular polysaccharide, and the aster polypeptide is micromolecular peptide; the aster small molecular peptide and the aster small molecular polysaccharide can be used as raw materials of health-care food and functional food; the method realizes the extraction of the micromolecular polypeptide and the micromolecular polysaccharide from the aster at one time, simplifies the production link, saves the time and improves the full fruit utilization of the aster.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a method for extracting aster polysaccharide and aster polypeptide, which comprises the following steps:
1) mixing radix Asteris with water, pulping, and ultrasonically treating to obtain radix Asteris slurry;
2) mixing the aster slurry and cellulase, and carrying out first enzymolysis to obtain a first enzymolysis liquid;
3) treating the first enzymolysis liquid at 30-50 ℃ for 30-50 min, then carrying out solid-liquid separation, and collecting liquid-phase components to obtain a first clear liquid;
4) adjusting the pH value of the first clear liquid to 4-7, mixing the first clear liquid with a sulfate mixture, and salting out and layering to obtain an upper-layer second clear liquid and a lower-layer precipitation liquid;
5) mixing the lower-layer precipitation solution with alkaline protease to perform second enzymolysis to obtain a second enzymolysis solution, and performing ultrafiltration, nanofiltration and drying on the second enzymolysis solution to obtain the aster polypeptide;
6) drying the second supernatant in the step 4) to obtain the aster polysaccharide.
Preferably, the power of the ultrasonic treatment in the step 1) is 20-40 KHz, and the time of the ultrasonic treatment is 15-35 min.
Preferably, the mass ratio of the cellulase to the aster in the step 2) is 1 (3000-5000).
Preferably, the temperature of the first enzymolysis is 30-50 ℃, the time of the first enzymolysis is 2-4 h, and the pH value of the first enzymolysis is 4.5-6.5.
Preferably, the solid-liquid separation in the step 3) is centrifugation, the centrifugal force of the centrifugation is 9000-11000 g, and the centrifugation time is 20-30 min.
Preferably, the sulfate mixture in step 4) comprises ammonium sulfate and sodium sulfate; the mass ratio of the ammonium sulfate to the sodium sulfate is 1 (2-4).
Preferably, the mass ratio of the sulfate mixture to the tatarian aster root is 1 (90-110).
Preferably, the temperature of the second enzymolysis in the step 5) is 45-65 ℃, the time of the second enzymolysis is 1-3 h, and the pH value of the second enzymolysis is 7-10.
Preferably, the alkaline protease is used in the step 5) in an amount of 0.5% to 1.5% by mass of the aster tataricus.
Preferably, the ultrafiltration in step 5) has a molecular weight cut-off of 3000D and the nanofiltration has a molecular weight cut-off of 300D.
The invention has the beneficial effects that: according to the extraction method of the aster polysaccharide and the aster polypeptide, small-molecular peptides and small-molecular polysaccharides in aster are effectively separated and extracted through enzymolysis of cellulase, heating treatment after enzymolysis, salting out of a sulfate mixture and second enzymolysis of alkaline protease; realizes the extraction of micromolecular peptide and micromolecular polysaccharide from the aster at one time, simplifies the production link, saves time and improves the full fruit utilization of the aster. In addition, the aster small-molecule peptide and the aster small-molecule polysaccharide extracted by the method can be used as raw materials of health-care food and functional food. According to the description of the embodiment, the yield of the small-molecule peptide and the small-molecule polysaccharide is remarkably higher than that of the comparative example.
Drawings
FIG. 1 is a flow chart of the method of the present invention.
Detailed Description
The invention provides a method for extracting aster polysaccharide and aster polypeptide, which comprises the following steps: 1) mixing radix Asteris with water, pulping, and ultrasonically treating to obtain radix Asteris slurry; 2) mixing the aster slurry and cellulase, and carrying out first enzymolysis to obtain a first enzymolysis liquid; 3) treating the first enzymolysis liquid at 30-50 ℃ for 30-50 min, then carrying out solid-liquid separation, and collecting liquid-phase components to obtain a first clear liquid; 4) adjusting the pH value of the first clear liquid to 4-7, mixing the first clear liquid with a sulfate mixture, and salting out and layering to obtain an upper-layer second clear liquid and a lower-layer precipitation liquid; 5) mixing the lower-layer precipitation solution with alkaline protease to perform second enzymolysis to obtain a second enzymolysis solution, and performing ultrafiltration, nanofiltration and drying on the second enzymolysis solution to obtain the aster polypeptide; 6) drying the second supernatant in the step 4) to obtain the aster polysaccharide.
In the invention, aster is mixed with water, pulped and ultrasonically treated to obtain aster slurry. In the present invention, the aster is a dried root and rhizome of aster tataricus belonging to the genus aster of the family asteraceae, and the aster is preferably aster tataricus produced in the provinces of anhua and anhui china, and in the practice of the present invention, the aster is preferably aster tataricus produced from constant water. In the present invention, before the aster is mixed with water and pulped, the aster is preferably washed, and the washing is not particularly limited in the present invention, and the aster can be washed clean with conventional clean water. In the present invention, the mass of the water is preferably 5 to 15 times, more preferably 8 to 12 times, and most preferably 10 times the mass of the aster tataricus. In the invention, the pulping is preferably carried out by a mechanical method, and in the specific implementation process of the invention, the conventional pulping equipment is adopted. In the invention, the power of ultrasonic treatment is preferably 20-40 KHz, more preferably 25-35 KHz, and most preferably 30 KHz; the time of ultrasonic treatment is preferably 15-35 min, more preferably 20-30 min, and most preferably 25 min. In the present invention, the sonication serves to disrupt the cell wall of Aster tataricus, facilitating the extraction of proteins and polysaccharides.
After obtaining the aster slurry, the aster slurry is mixed with cellulase, and first enzymolysis is carried out to obtain a first enzymolysis liquid. In the invention, the mass ratio of the cellulase to the aster is preferably 1 (3000-5000), more preferably 1 (3500-4500), and most preferably 1: 4000. In the invention, the temperature of the first enzymolysis is preferably 30-50 ℃, and the time of the first enzymolysis is preferably 2-4 h, specifically 2h, 3h and 4 h; the pH value of the first enzymolysis is preferably 4.5-6.5, more preferably 5.0-6.0, and specifically 4.5, 5.5 and 6.5; in the present invention, the pH value is preferably adjusted by using a citric acid solution, and the citric acid solution is preferably a citric acid solution with a mass percentage of 4% to 6%, and more preferably 5%. The source of the cellulase in the present invention is not particularly limited, and any commercially available product that is conventional in the art may be used.
After the first enzymolysis liquid is obtained, the first enzymolysis liquid is treated at 30-50 ℃ for 30-50 min and then subjected to solid-liquid separation, and collected liquid-phase components are first clear liquid. In the invention, the treatment temperature is preferably 35-45 ℃, more preferably 40 ℃, and particularly can be selected to be 30, 40 and 50 ℃; the treatment time is preferably 35-45 min, and more preferably 40 min; specifically 30, 40 and 50min can be selected. In the invention, the first enzymolysis liquid is treated at 30-50 ℃ to destroy the cell wall of the aster, so that the polypeptide and the polysaccharide are easier to extract. In the invention, after the first enzymolysis liquid is treated at 30-50 ℃, the method preferably further comprises a cooling step, wherein the cooling time is preferably 10-14 h, and more preferably 12 h; the cooling mode is not particularly limited, and the cooling can be realized by adopting conventional natural cooling. In the invention, the solid-liquid separation mode is preferably centrifugation, and the centrifugal force of the centrifugation is preferably 9000-11000 g, and more preferably 10000 g; the time for centrifugation is preferably 20-30 min, and more preferably 25 min.
After the first clear liquid is obtained, adjusting the pH value of the first clear liquid to be 4-7, mixing the clear liquid with a sulfate mixture, and salting out and layering to obtain an upper-layer second clear liquid and a lower-layer precipitation liquid. In the present invention, the pH adjustment of the first clear liquid is preferably adjusted to 4, 5, 6 or 7; the sulfate mixture preferably comprises ammonium sulfate and sodium sulfate; the mass ratio of the ammonium sulfate to the sodium sulfate is preferably 1 (2-4), and specifically 1:2, 1:3 or 1: 4; the mass ratio of the sulfate mixture to the tatarian aster root is preferably 1 (90-110), and more preferably 1: 100. In the specific implementation process of the invention, the sulfate mixture is preferably used in the form of a solution, and the mass concentration of the ammonium sulfate solution is preferably 15% to 25%, and more preferably 20%; the mass concentration of the sodium sulfate is preferably 15-25%, and more preferably 20%; the addition amount of the solution is calculated according to the concentration of the solution and the mass of the required sulfate mixture. In the invention, the environmental temperature in the salting-out and layering process is preferably 20-40 ℃, and can be specifically 20 ℃, 30 ℃ or 40 ℃. The mixture of sulfates can cause the protein to precipitate, with different effects at different temperatures. After the salting-out layering, obtaining an upper second clear solution and a lower precipitation solution; the second supernatant is rich in micromolecular polysaccharide; the lower layer precipitation liquid is rich in protein.
After the lower-layer precipitation solution is obtained, the lower-layer precipitation solution is mixed with alkaline protease for second enzymolysis to obtain a second enzymolysis solution, and the second enzymolysis solution is subjected to ultrafiltration, nanofiltration and drying to obtain the aster polypeptide. In the invention, the temperature of the second enzymolysis is preferably 45-65 ℃, more preferably 50-60 ℃, and specifically can be 45 ℃, 55 ℃ or 60 ℃; the time of the second enzymolysis is preferably 1-3 h, more preferably 1.5-2.5 h, and particularly 1h, 2h or 3 h. In the invention, the pH value of the second enzymolysis is preferably 7-10, and can be specifically 7, 8, 9 or 10; the reagent used for adjusting the pH value is not particularly limited, and the conventional acid-base adjusting agent in the field can be adopted. In the present invention, the alkaline protease is used in an amount of 0.5% to 1.5% by mass of Aster tataricus. In the present invention, the alkaline protease functions to better decompose protein into polypeptide; the source of the alkaline protease is not particularly limited in the present invention, and any commercially available product that is conventional in the art may be used. In the invention, the ultrafiltration preferably has a cut-off molecular weight of 3000D, the ultrafiltration is preferably realized by using an ultrafiltration membrane, the nanofiltration preferably has a cut-off molecular weight of 300D, and the nanofiltration is preferably realized by using a nanofiltration membrane. The molecular weight of the polypeptide in the filtrate collected by ultrafiltration and nanofiltration is very small, so that the enrichment and extraction of the small molecular peptide are really realized. In the invention, the drying temperature of the filtered solution is preferably 0-4 ℃, and more preferably 2 ℃.
In the invention, the second supernatant is dried to obtain the aster polysaccharide. In the invention, the drying is preferably spray drying, and the spray drying condition is preferably that the set temperature is 240 ℃, the frequency of a fan is 40Hz, and the rotating speed of a peristaltic pump is 5 rpm.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The preparation method for simultaneously extracting the small molecular peptide and the small molecular polysaccharide from the aster comprises the following steps:
(1) pretreatment of aster: cleaning radix Asteris with clear water, taking radix Asteris with a mass of 50g, adding water with a mass of 5 times of that of radix Asteris, and pulping by adopting a mechanical method; then, the mixture is subjected to ultrasonic treatment for 15min under the condition of 20 kHz.
(2) Enzymolysis: adding cellulase into the slurry, wherein the mass ratio of the cellulase to the aster is 1:3000, simultaneously adjusting the pH to 5.5, and the enzymolysis time is 2h to obtain a first enzymolysis liquid.
(3) Heating treatment: pouring the first enzymolysis liquid into a container, heating for 30min at 40 deg.C, and cooling for 12 h.
(4) And (3) centrifugal treatment: and putting the heated first enzymolysis liquid into a high-speed freezing centrifugal machine, and centrifuging for 20min at the centrifugal force of 10000 g.
(5) Salting out: taking out the centrifuged supernatant as the first supernatant, adjusting pH to 5 at 20 deg.C, and adding NH4SO4With Na2SO4Mixture of (2), NH4SO4With Na2SO4The mass ratio of the mixture to the aster is 1: 100; NH (NH)4SO4With Na2SO4Is added in the form of a solution, in particular 20% strength NH4SO4With 20% concentration of Na2SO4The solution is mixed according to the volume ratio of 1:2, then added into the first clear liquid, stirred evenly, placed for 12 hours and layered into an upper layer of second clear liquid and a lower layer of precipitation liquid.
(6) Enzymolysis: and (3) adding alkaline protease into the middle-lower layer precipitation liquid in the step (5), wherein the enzyme amount is 0.5 percent of the mass of the aster, adjusting the environmental temperature to 45 ℃, adjusting the pH to 8, and stirring for 1 hour to obtain a second enzymolysis liquid.
(7) Extracting small molecule peptide: putting the second enzymolysis liquid in the step (6) into an ultrafiltration membrane with the relative analytical mass of 3000D for ultrafiltration, then putting the ultrafiltration membrane into a nanofiltration membrane with the relative molecular mass of 300D for interception, and freeze-drying at 2 ℃ to obtain the aster small-molecular peptide;
(8) extracting small molecular polysaccharide: and (5) spray-drying the second supernatant of the middle-upper layer to obtain the aster small-molecular polysaccharide.
Example 2
The preparation method for simultaneously extracting small-molecule peptide and small-molecule polysaccharide from aster comprises the following steps:
(1) pretreatment of aster: cleaning radix Asteris with clear water, taking radix Asteris with a mass of 50g, adding water with a mass of 10 times of that of radix Asteris, and pulping by adopting a mechanical method; then, the mixture is subjected to ultrasonic treatment for 35min under the condition of 30 kHz.
(2) Enzymolysis: adding cellulase into the slurry, wherein the mass ratio of the cellulase to the aster is 1:4000, simultaneously adjusting the pH to 5.5, and the enzymolysis time is 3h to obtain a first enzymolysis liquid.
(3) Heating treatment: pouring the first enzymolysis liquid into a container, heating for 40min at 30 deg.C, and cooling for 12 h.
(4) And (3) centrifugal treatment: and putting the heated first enzymolysis liquid into a high-speed freezing centrifugal machine, and centrifuging for 20min at the centrifugal force of 10000 g.
(5) Salting out: taking out the centrifuged first clear liquid, adjusting pH to 5 at an ambient temperature of 30 deg.C, and adding NH4SO4With Na2SO4Mixture of (2), NH4SO4With Na2SO4The mass ratio of the mixture to the aster is 1: 100; NH (NH)4SO4With Na2SO4Is added in the form of a solution, in particular 20% strength NH4SO4With 20% concentration of Na2SO4The solution is mixed according to the volume ratio of 1:3, then added into the first clear liquid, stirred evenly, placed for 12 hours and layered into an upper layer of second clear liquid and a lower layer of precipitation liquid.
(6) Enzymolysis: and (3) adding alkaline protease into the lower middle layer precipitation liquid in the step (5), wherein the enzyme amount is 1% of the mass of the aster, adjusting the environmental temperature to 55 ℃, adjusting the pH to 9, and stirring for 2 hours to obtain a second enzymolysis liquid.
(7) Extracting small molecule peptide: putting the second enzymolysis liquid in the step (6) into an ultrafiltration membrane with the relative analytical mass of 3000D for ultrafiltration, then putting the ultrafiltration membrane into a nanofiltration membrane with the relative molecular mass of 300D for interception, and freeze-drying to obtain the aster small-molecular peptide;
(8) extracting small molecular polysaccharide: and (5) spray-drying the second supernatant of the middle-upper layer to obtain the aster small-molecular polysaccharide.
Example 3
The preparation method for simultaneously extracting small-molecule peptide and small-molecule polysaccharide from aster comprises the following steps:
(1) pretreatment of aster: cleaning radix Asteris with clear water, adding water 15 times of radix Asteris, and pulping by mechanical method; then, the mixture is subjected to ultrasonic treatment for 35min under the condition of 20 kHz.
(2) Enzymolysis: adding cellulase into the slurry, wherein the mass ratio of the cellulase to the aster is 1:5000, simultaneously adjusting the pH to 6.5, and the enzymolysis time is 3h to obtain a first enzymolysis liquid.
(3) Heating treatment: pouring the first enzymolysis liquid into a container, heating for 50min at 40 deg.C, and cooling for 12 h.
(4) And (3) centrifugal treatment: and putting the heated first enzymolysis liquid into a high-speed freezing centrifugal machine, and centrifuging for 25min at the centrifugal force of 10000 g.
(5) Salting out: taking out the centrifuged first clear liquid, adjusting pH to 6 at 40 deg.C, and adding NH4SO4With Na2SO4Mixture of (2), NH4SO4With Na2SO4The mass ratio of the mixture to the aster is 1: 100; NH (NH)4SO4With Na2SO4Is added in the form of a solution, in particular 20% strength NH4SO4With 20% concentration of Na2SO4The solution is mixed according to the volume ratio of 1:3, then added into the first clear liquid, placed for 12 hours and separated into an upper layer second clear liquid and a lower layer precipitation liquid.
(6) Enzymolysis: and (3) adding alkaline protease into the lower-middle layer precipitation liquid in the step (5), wherein the enzyme adding amount is 1.5% of the mass of the aster, adjusting the environmental temperature to 55 ℃, adjusting the pH to 9, and stirring for 2 hours to obtain a second enzymolysis liquid.
(7) Extracting small molecule peptide: putting the second enzymolysis liquid in the step (6) into an ultrafiltration membrane with the relative analytical mass of 3000D for ultrafiltration, then putting the ultrafiltration membrane into a nanofiltration membrane with the relative molecular mass of 300D for interception, and freeze-drying to obtain the aster small-molecular peptide;
(8) extracting small molecular polysaccharide: and (5) spray-drying the second supernatant of the middle-upper layer to obtain the aster small-molecular polysaccharide.
Example 4
The preparation method for simultaneously extracting small-molecule peptide and small-molecule polysaccharide from aster comprises the following steps:
(1) pretreatment of aster: cleaning radix Asteris with clear water, adding water 10 times of radix Asteris, and pulping by mechanical method; then, the mixture is subjected to ultrasonic treatment for 25min under the condition of 40 kHz.
(2) Enzymolysis: adding cellulase into the slurry, wherein the mass ratio of the cellulase to the aster is 1:4000, simultaneously adjusting the pH to 4.5, and the enzymolysis time is 4h to obtain a first enzymolysis liquid.
(3) Heating treatment: pouring the first enzymolysis liquid into a container, heating for 30min at 50 deg.C, and cooling for 12 h.
(4) And (3) centrifugal treatment: and putting the heated first enzymolysis liquid into a high-speed freezing centrifugal machine, and centrifuging for 30min at the centrifugal force of 10000 g.
(5) Salting out: taking out the centrifuged first clear liquid, adjusting pH to 4 at 20 deg.C, and adding NH4SO4With Na2SO4Mixture of (2), NH4SO4With Na2SO4The mass ratio of the mixture to the aster is 1: 100; NH (NH)4SO4With Na2SO4Is added in the form of a solution, in particular 20% strength NH4SO4With 20% concentration of Na2SO4The solution is mixed according to the volume ratio of 1:4, then added into the first clear liquid, placed for 12 hours and separated into an upper layer second clear liquid and a lower layer precipitation liquid.
(6) Enzymolysis: adding alkaline protease into the lower middle layer precipitation liquid in the step (5), wherein the addition amount of the alkaline protease is 1% of the mass of the aster, adjusting the environmental temperature to 65 ℃, adjusting the pH to 10, and stirring for 3 hours to obtain a second enzymolysis liquid;
(7) extracting small molecule peptide: putting the second enzymolysis liquid in the step (6) into an ultrafiltration membrane with the relative analytical mass of 3000D for ultrafiltration, then putting the ultrafiltration membrane into a nanofiltration membrane with the relative molecular mass of 300D for interception, and freeze-drying to obtain the aster small-molecular peptide;
(8) extracting small molecular polysaccharide: and (5) spray-drying the second supernatant of the middle-upper layer to obtain the aster small-molecular polysaccharide.
Comparative example 1
The preparation method for simultaneously extracting the small-molecule peptide and the small-molecule polysaccharide from the aster comprises the following steps:
(1) pretreatment of aster: cleaning radix Asteris with clear water, adding water 10 times of radix Asteris, and pulping by mechanical method; then, the mixture is subjected to ultrasonic treatment for 25min under the condition of 30 kHz.
(2) Heating treatment: pouring the aster slurry into a container, heating for 30min at 40 deg.C, and cooling for 12 h.
(3) And (3) centrifugal treatment: the slurry mixture of radix Asteris after heat treatment was centrifuged in a high-speed refrigerated centrifuge at a centrifugal force of 10000g for 25 min.
(4) Salting out: taking out the centrifuged supernatant, adjusting pH to 6 at an ambient temperature of 30 deg.C, and adding NH4SO4With Na2SO4Mixture of (2), NH4SO4With Na2SO4The mass ratio of the mixture to the aster is 1: 100; NH (NH)4SO4With Na2SO4Is added in the form of a solution, in particular 20% strength NH4SO4With 20% concentration of Na2SO4The solution is mixed according to the volume ratio of 1:2, then added into the first clear liquid, stirred evenly, placed for 12 hours and layered into supernatant and lower precipitate.
(5) Enzymolysis: adding alkaline protease into the precipitation solution in the step (4), wherein the enzyme adding amount is 1% of the mass of the aster, adjusting the environmental temperature to 45 ℃, adjusting the pH to 8, and stirring for enzymolysis for 1 h;
(6) extracting small molecule peptide: putting the liquid obtained in the step (5) into an ultrafiltration membrane with the relative analytical mass of 3000D for ultrafiltration, then putting the ultrafiltration membrane into a nanofiltration membrane with the relative molecular mass of 300D for interception, and freeze-drying to obtain the aster small-molecule peptide;
(7) extracting small molecular polysaccharide: and (4) spray-drying the supernatant in the step (4) to obtain the aster small-molecular polysaccharide.
Comparative example 2
The preparation method for simultaneously extracting the small-molecule peptide and the small-molecule polysaccharide from the aster comprises the following steps:
(1) pretreatment of aster: cleaning radix Asteris with clear water, adding water 15 times of radix Asteris, and pulping by mechanical method; then, the mixture is subjected to ultrasonic treatment for 25min under the condition of 20 kHz.
(2) Enzymolysis: adding cellulase with the mass ratio of the cellulase to the aster tataricus being 1:4000 into the slurry, adjusting the pH to 5.5 and the enzymolysis time to 3 h.
(3) Heating treatment: pouring the enzymolyzed radix Asteris slurry into a container, heating for 40min at 40 deg.C, and cooling for 12 hr.
(4) And (3) centrifugal treatment: the slurry mixture of radix Asteris after heat treatment was centrifuged in a high-speed refrigerated centrifuge at a centrifugal force of 10000g for 20 min.
(5) Salting out: taking out the centrifuged supernatant, adjusting pH to 5 at 20 deg.C, and adding 20% NH4SO4Solution of said NH4SO4NH in solution4SO4The mass ratio of the radix asteris to the radix asteris is 1:100, the mixture is stirred evenly, placed for 12 hours and layered into supernatant and lower-layer precipitation.
(6) Enzymolysis: adding alkaline protease into the precipitation solution in the step (5), wherein the enzyme amount is 1.5% of the mass of the aster, adjusting the environmental temperature to 45 ℃, adjusting the pH to 8, and stirring for 2 h.
(7) Extracting small molecule peptide: putting the liquid obtained in the step (6) into an ultrafiltration membrane with the relative analytical mass of 3000D for ultrafiltration, then putting the ultrafiltration membrane into a nanofiltration membrane with the relative molecular mass of 300D for interception, and freeze-drying to obtain the aster small-molecule peptide;
(8) extracting small molecular polysaccharide: and (5) spray-drying the supernatant to obtain the aster small-molecular polysaccharide.
TABLE 1 extraction rates of small-molecule peptides and small-molecule polysaccharides from Aster tataricus
Figure BDA0002345276830000101
As can be seen from Table 1, the yield of the aster small-molecular peptide and the aster small-molecular polysaccharide extracted by the extraction method of the embodiments 1 to 4 is obviously higher than that of the comparative examples 1 and 2.
The aster small molecular peptide is identified by high performance liquid chromatography, the molecular weight is between 180D and 260D, and the aster small molecular polysaccharide is identified by high performance gel permeation chromatography, and the molecular weight is between 1000 and 1800D.
From the above embodiments, the method provided by the invention realizes the extraction of the small molecule peptide and the small molecule polysaccharide from the aster tataricus at one time, simplifies the production link, saves the time, and improves the full fruit utilization of the aster tataricus. In addition, the aster small-molecule peptide and the aster small-molecule polysaccharide extracted by the method can be used as raw materials of health-care food and functional food.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (10)

1. A method for extracting aster polysaccharide and aster polypeptide comprises the following steps:
1) mixing radix Asteris with water, pulping, and ultrasonically treating to obtain radix Asteris slurry;
2) mixing the aster slurry and cellulase, and carrying out first enzymolysis to obtain a first enzymolysis liquid;
3) treating the first enzymolysis liquid at 30-50 ℃ for 30-50 min, then carrying out solid-liquid separation, and collecting liquid-phase components to obtain a first clear liquid;
4) adjusting the pH value of the first clear liquid to 4-7, mixing the first clear liquid with a sulfate mixture, and salting out and layering to obtain an upper-layer second clear liquid and a lower-layer precipitation liquid;
5) mixing the lower-layer precipitation solution with alkaline protease to perform second enzymolysis to obtain a second enzymolysis solution, and performing ultrafiltration, nanofiltration and drying on the second enzymolysis solution to obtain the aster polypeptide;
6) drying the second supernatant in the step 4) to obtain the aster polysaccharide.
2. The extraction method according to claim 1, wherein the power of the ultrasonic treatment in the step 1) is 20-40 KHz, and the time of the ultrasonic treatment is 15-35 min.
3. The extraction method according to claim 1 or 2, wherein the mass ratio of the cellulase to the aster in the step 2) is 1 (3000-5000).
4. The extraction method according to claim 3, wherein the temperature of the first enzymolysis is 30-50 ℃, the time of the first enzymolysis is 2-4 h, and the pH value of the first enzymolysis is 4.5-6.5.
5. The extraction method according to claim 1, wherein the solid-liquid separation in step 3) is centrifugation, the centrifugal force is 9000-11000 g, and the centrifugation time is 20-30 min.
6. The extraction process according to claim 1, wherein the sulfate mixture in step 4) comprises ammonium sulfate and sodium sulfate; the mass ratio of the ammonium sulfate to the sodium sulfate is 1 (2-4).
7. The extraction method according to claim 1 or 6, wherein the mass ratio of the sulfate mixture to the tatarian aster root is 1 (90-110).
8. The extraction method according to claim 1, wherein the temperature of the second enzymolysis in the step 5) is 45-65 ℃, the time of the second enzymolysis is 1-3 h, and the pH value of the second enzymolysis is 7-10.
9. The extraction method according to claim 1, wherein the alkaline protease is used in an amount of 0.5% to 1.5% by mass of the aster tataricus in step 5).
10. The extraction process according to claim 1, 8 or 9, characterized in that the ultrafiltration in step 5) has a molecular weight cut-off of 3000D and the nanofiltration has a molecular weight cut-off of 300D.
CN201911392161.9A 2019-12-30 2019-12-30 Method for extracting aster polysaccharide and aster polypeptide Pending CN110964767A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201911392161.9A CN110964767A (en) 2019-12-30 2019-12-30 Method for extracting aster polysaccharide and aster polypeptide

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201911392161.9A CN110964767A (en) 2019-12-30 2019-12-30 Method for extracting aster polysaccharide and aster polypeptide

Publications (1)

Publication Number Publication Date
CN110964767A true CN110964767A (en) 2020-04-07

Family

ID=70037274

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201911392161.9A Pending CN110964767A (en) 2019-12-30 2019-12-30 Method for extracting aster polysaccharide and aster polypeptide

Country Status (1)

Country Link
CN (1) CN110964767A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114250261A (en) * 2021-12-28 2022-03-29 河南省国德科果蔬研究院有限公司 Extraction method of red date kernel polypeptide

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104762356A (en) * 2015-04-08 2015-07-08 西北农林科技大学 Pumpkin leaf polysaccharide and polypeptide and preparation method thereof
CN106480143A (en) * 2015-08-31 2017-03-08 四川科伦新光健康药业有限公司 A kind of Lepidinm meyenii Walp oligosaccharide polypeptide and preparation method thereof
CN106619796A (en) * 2017-01-19 2017-05-10 大连豪翔生物酶工程有限公司 Technology for integrated extraction of active components of Semen Cassiae
CN107083413A (en) * 2017-03-16 2017-08-22 华侨大学 A kind of method that sea cucumber internal organ prepare sea cucumber peptide and sea cucumber polysaccharide
CN109608514A (en) * 2018-11-29 2019-04-12 河北中科汉禧生物科技有限公司 The method of protein and polysaccharide is extracted from turnip
CN110240626A (en) * 2019-06-25 2019-09-17 中藜农业科技(江苏)有限公司 A kind of quinoa polysaccharide polypeptide production method

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104762356A (en) * 2015-04-08 2015-07-08 西北农林科技大学 Pumpkin leaf polysaccharide and polypeptide and preparation method thereof
CN106480143A (en) * 2015-08-31 2017-03-08 四川科伦新光健康药业有限公司 A kind of Lepidinm meyenii Walp oligosaccharide polypeptide and preparation method thereof
CN106619796A (en) * 2017-01-19 2017-05-10 大连豪翔生物酶工程有限公司 Technology for integrated extraction of active components of Semen Cassiae
CN107083413A (en) * 2017-03-16 2017-08-22 华侨大学 A kind of method that sea cucumber internal organ prepare sea cucumber peptide and sea cucumber polysaccharide
CN109608514A (en) * 2018-11-29 2019-04-12 河北中科汉禧生物科技有限公司 The method of protein and polysaccharide is extracted from turnip
CN110240626A (en) * 2019-06-25 2019-09-17 中藜农业科技(江苏)有限公司 A kind of quinoa polysaccharide polypeptide production method

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DONGLIANG等: ""Oligopeptides from Aster tataricus"", 《PHYTOCHEMISTRY》 *
彭文静等: ""紫菀化学成分及药理作用研究进展"", 《动物医学进展》 *
房慧勇等: ""紫菀的化学成分及其药理活性研究进展"", 《医学研究与教育》 *
范玲等: ""紫菀化学成分及药理作用研究进展"", 《吉林中医药》 *
黄方言: "《现代实用中药新剂型新技术》", 30 April 2001, 北京：人民卫生出版社 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114250261A (en) * 2021-12-28 2022-03-29 河南省国德科果蔬研究院有限公司 Extraction method of red date kernel polypeptide
CN114250261B (en) * 2021-12-28 2023-08-22 河南省国德科果蔬研究院有限公司 Method for extracting red date pit polypeptide

Similar Documents

Publication Publication Date Title
CN103598544B (en) Extraction process for carrying out comprehensive utilization on garlic as raw material
CN104789629A (en) Chickpea oligopeptide and preparation method thereof
CN110016086B (en) Refining process of ganoderma lucidum polysaccharide
CN106282282A (en) A kind of extract oils and fats and albumen and/or the method for glucosides in moringa seeds simultaneously
CN104788511A (en) Method for preparing persimmon tannins with high micromolecular tannin content from persimmons
CN107446978A (en) A kind of stem of noble dendrobium small-molecular peptides and preparation method thereof
CN105167064A (en) Method of processing clear wolfberry juice by comprehensive utilization of wolfberry residues
CN103860631A (en) Method for preparing liquorice active substances
CN106832027A (en) The extracting method of gumbo polysaccharide
CN103689158A (en) Preparation method of chrysanthemum morifolium extract
CN110964767A (en) Method for extracting aster polysaccharide and aster polypeptide
CN106046189A (en) Extraction and purification method of pumpkin polysaccharide
CN103923050B (en) Utilize rutin to prepare the method for Quercetin
CN105326049A (en) Processing method of rubus rosaefolius smith-pomegranate seed nutritional powder
CN112707852B (en) Combined preparation method of garlic extract
CN1927857A (en) Extraction and purification process of buckwheat sprout total flavone
CN110777173B (en) Method for preparing momordica grosvenori amino acid by using momordica grosvenori centrifugal waste residues
CN107349239A (en) A kind of extracting method of mango leaf polyphenol
CN114874284B (en) Method for synchronously extracting and separating matrimony vine protein and polysaccharide
CN112442136A (en) Method for extracting functional components from tremella
CN109021142A (en) One grows tea the extracting method that milli extracts tea polyphenols
CN105639657A (en) Method for extracting mineral elements in boxthorn leaves through enzymolysis
CN115463181B (en) Preparation method of agilawood leaf extract
CN108813026A (en) A kind of preparation process of folium cortex eucommiae wild chrysanthemum tea powder
CN109468358B (en) Method for extracting cistanche chelating peptide

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
RJ01 Rejection of invention patent application after publication

Application publication date: 20200407

RJ01 Rejection of invention patent application after publication