CN108191952B - Method for extracting and separating proteins from stems and leaves of ginseng - Google Patents
Method for extracting and separating proteins from stems and leaves of ginseng Download PDFInfo
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- CN108191952B CN108191952B CN201810238528.0A CN201810238528A CN108191952B CN 108191952 B CN108191952 B CN 108191952B CN 201810238528 A CN201810238528 A CN 201810238528A CN 108191952 B CN108191952 B CN 108191952B
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- 241000208340 Araliaceae Species 0.000 title claims abstract description 95
- 235000008434 ginseng Nutrition 0.000 title claims abstract description 95
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 title claims abstract description 94
- 235000003140 Panax quinquefolius Nutrition 0.000 title claims abstract description 94
- 238000000034 method Methods 0.000 title claims abstract description 27
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 25
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 25
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims abstract description 46
- 101710138460 Leaf protein Proteins 0.000 claims abstract description 30
- 239000006228 supernatant Substances 0.000 claims abstract description 24
- 239000000843 powder Substances 0.000 claims abstract description 19
- 239000012153 distilled water Substances 0.000 claims abstract description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 17
- 238000000605 extraction Methods 0.000 claims abstract description 8
- 239000012535 impurity Substances 0.000 claims abstract description 8
- 239000003960 organic solvent Substances 0.000 claims abstract description 8
- 238000002156 mixing Methods 0.000 claims abstract description 3
- 239000012460 protein solution Substances 0.000 claims description 34
- 239000002244 precipitate Substances 0.000 claims description 21
- 238000002386 leaching Methods 0.000 claims description 15
- 239000008055 phosphate buffer solution Substances 0.000 claims description 12
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 10
- 238000001914 filtration Methods 0.000 claims description 7
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 6
- 238000000751 protein extraction Methods 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 2
- 240000004371 Panax ginseng Species 0.000 claims 1
- 235000002789 Panax ginseng Nutrition 0.000 claims 1
- 239000007853 buffer solution Substances 0.000 abstract description 11
- 238000000926 separation method Methods 0.000 abstract description 4
- 238000000746 purification Methods 0.000 abstract description 3
- 235000013376 functional food Nutrition 0.000 abstract description 2
- 238000009776 industrial production Methods 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 238000002360 preparation method Methods 0.000 abstract description 2
- 239000000047 product Substances 0.000 abstract description 2
- 238000004108 freeze drying Methods 0.000 abstract 1
- 238000009010 Bradford assay Methods 0.000 description 4
- 238000010298 pulverizing process Methods 0.000 description 4
- 239000000706 filtrate Substances 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 244000025254 Cannabis sativa Species 0.000 description 1
- 241001295925 Gegenes Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 235000002791 Panax Nutrition 0.000 description 1
- 241000208343 Panax Species 0.000 description 1
- 241000037831 Polygonatum sibiricum Species 0.000 description 1
- 229930182558 Sterol Natural products 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000036449 good health Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 229930182490 saponin Natural products 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
- 235000017709 saponins Nutrition 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 150000003432 sterols Chemical class 0.000 description 1
- 235000003702 sterols Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 239000000341 volatile oil Substances 0.000 description 1
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- 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
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Plant Substances (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses an extraction and separation method of ginseng stem leaf protein, and belongs to the technical field of protein separation and purification. Extracting ginseng protein from ginseng stems and leaves, removing impurities with an organic solvent, extracting with a near-neutral buffer solution twice, mixing the supernatants, standing with cold acetone, dissolving with distilled water again, and freeze drying to obtain ginseng protein powder. The method of the invention has simple preparation method and high extraction rate; the ginseng stem leaves are low in price, and the production cost can be reduced by extracting ginseng protein from the ginseng stem leaves, so that higher economic benefit is achieved; is suitable for industrial production, and the product can be used for development of functional food.
Description
Technical Field
The invention belongs to the technical field of protein separation and purification, and relates to a method for extracting ginseng stem leaf protein.
Background
Ginseng belongs to the genus Panax of Araliaceae, is a perennial root herbaceous plant, and ancient Ginseng is elegantly called Polygonatum sibiricum Red, Gegen and Shencao. Ginseng is mainly produced in northeast China, is one of three treasures in the east China, is a famous and precious medicinal material and a good health care product which are known by China, abroad and old and young, and is called 'king of all grass'.
Ginseng, including the root, stem and leaf, is a Chinese herb medicine for the whole body. Ginseng contains many effective medicinal components including ginseng polysaccharide, saponin, fatty acid, sterol, vitamins, volatile oil, protein, polypeptide, etc. In recent years, many studies have been made on the separation and purification of ginseng components, and much progress has been made, but there is little concern about the study of proteins in ginseng.
Proteins play a very important role in the course of life activities and are present in all animals and plants. At present, people mainly extract ginseng protein from ginseng roots, and research on extracting ginseng protein from ginseng stems and leaves is less.
Disclosure of Invention
The invention aims to develop a novel, simple and high-extraction-rate ginseng protein extraction method: the protein of the ginseng stem leaves is extracted by taking the ginseng stem leaves as a raw material through the methods of removing impurities by an organic solvent, leaching by a buffer solution, standing by cold acetone, re-dissolving by distilled water, freezing and drying in vacuum and the like.
The specific technical scheme for extracting the ginseng stem leaf protein is as follows.
A method for extracting protein from caulis Et folium Ginseng comprises extracting protein, standing with cold acetone, re-dissolving with distilled water, and post-treating;
the protein extraction process comprises the steps of adding ginseng stem and leaf powder into a phosphate buffer solution with the pH value of 7.4-7.8, leaching for 5-10 hours, and centrifuging to obtain a supernatant and a precipitate, wherein the mass-to-volume ratio (g/mL) of the ginseng stem and leaf powder to the phosphate buffer solution is 1: 10-20; adding the precipitate into a phosphate buffer solution with the pH value of 7.4-7.8, leaching for 5-10 hours, centrifuging and collecting a supernatant, wherein the mass-to-volume ratio (g/mL) of the precipitate to the phosphate buffer solution is 1: 10-20; mixing the two supernatants to obtain ginseng stem and leaf protein solution;
the cold acetone standing and distilled water redissolving process comprises the steps of adding cold acetone with the volume of 5-10 times of that of the protein solution of the ginseng stem leaves into the protein solution of the ginseng stem leaves, standing for 3-10 hours at the temperature of minus 20 ℃, centrifuging, removing supernatant, adding the precipitate into distilled water, and redissolving to obtain the total protein solution of the ginseng stem leaves, wherein the using amount of the distilled water is 0.2-0.5 time of the volume of the protein solution of the ginseng stem leaves;
the post-treatment process comprises pre-cooling the total protein solution of the ginseng stem and leaf at-20 ℃ for 5 hours, and then carrying out vacuum freeze drying to obtain the ginseng stem and leaf protein powder.
The phosphate buffer solution may be a commercial 1 XPBS buffer solution or may be prepared by itself.
Preferably, an impurity removal process is carried out before the protein extraction process, wherein the impurity removal process comprises the steps of adding ginseng stem leaf powder into an organic solvent, stirring for 3-5 hours, filtering and retaining filter residue, and then adding the filter residue into a phosphate buffer solution with the pH value of 7.4-7.8 for leaching; the organic solvent is acetone, absolute ethyl alcohol or n-hexane.
In the impurity removal process, the mass-to-volume ratio (g/mL) of the ginseng stem and leaf powder to the organic solvent is 1: 10. The preferred organic solvent is n-hexane.
During the standing of cold acetone and the re-dissolving of distilled water, the cold acetone may be acetone at-20 deg.c.
In the protein extraction process, the preferable leaching time is 10 hours each time, and the preferable mass volume ratio (g/mL) of the ginseng stem leaf powder, the precipitate and the phosphate buffer solution is 1: 10; in the cold acetone standing and distilled water redissolving process, cold acetone with the volume 5 times that of the ginseng stem and leaf protein solution is preferably added and is kept stand for 3 hours at the temperature of minus 20 ℃.
The method can obtain the ginseng stem leaf protein powder; the preparation method is simple and convenient, has high extraction rate, is suitable for industrial production, and can be used for developing functional foods; the ginseng stem leaves are low in price, and the production cost can be reduced by extracting ginseng protein from the ginseng stem leaves, so that higher economic benefit is achieved.
Drawings
FIG. 1 is an SDS-PAGE pattern of ginseng proteins prepared according to various embodiments of the present invention.
In FIG. 1, M is Marker; 1 is the map of example 1; 2 is the map of example 2; 3 is the map of example 3; 4 is the map of example 4.
Detailed Description
Example 1
1) Pulverizing dried caulis Et folium Ginseng with pulverizer to obtain radix Ginseng powder;
2) adding acetone according to the mass-to-volume ratio (g/mL) of 1:10, stirring for 5 hours at room temperature, and filtering to obtain filtrate and filter residue;
3) collecting filter residues, adding 1 XPBS buffer solution with the pH value of 7.4-7.8 according to the mass-volume ratio (g/mL) of 1:10, leaching for 10 hours at room temperature, and centrifuging to obtain supernate and sediment; collecting the precipitate, adding 1 XPBS buffer solution with the pH value of 7.4-7.8 according to the mass-to-volume ratio (g/mL) of 1:20, carrying out secondary leaching for 5 hours, centrifuging to collect supernatant, and combining the supernatants to obtain the ginseng stem leaf protein solution;
4) and adding 5 times of cold acetone with the volume of-20 ℃ into the obtained ginseng stem and leaf protein solution, standing for 3 hours at the temperature of-20 ℃, centrifuging at a high speed, removing supernatant, adding a proper amount of distilled water (the volume of which is 0.2-0.5 times of that of the ginseng stem and leaf protein solution) into the precipitate, and dissolving again to obtain the ginseng stem and leaf protein solution.
5) Precooling the total protein solution of the ginseng stem and leaf for 5 hours at the temperature of minus 20 ℃, and then carrying out vacuum freeze drying to obtain the protein powder of the ginseng stem and leaf.
The extraction rate of the stem and leaf protein of ginseng was found to be 0.236% by Bradford method.
Example 2
This embodiment is the best embodiment of the present invention.
1) Pulverizing dried caulis Et folium Ginseng with pulverizer to obtain radix Ginseng powder;
2) adding n-hexane according to the mass-volume ratio (g/mL) of 1:10, stirring for 3 hours at room temperature, and filtering to obtain filtrate and filter residue;
3) collecting filter residues, adding 1 XPBS buffer solution with the pH value of 7.4-7.8 according to the mass-volume ratio (g/mL) of 1:10, leaching for 10 hours at room temperature, and filtering to obtain supernatant and precipitate; collecting the precipitate, adding 1 XPBS buffer solution with the pH value of 7.4-7.8 according to the mass-to-volume ratio (g/mL) of 1:10, carrying out secondary leaching for 10 hours, centrifuging to collect supernatant, and combining the supernatants to obtain the ginseng stem leaf protein solution;
4) and adding 5 times of cold acetone with the volume of-20 ℃ into the obtained ginseng stem and leaf protein solution, standing for 3 hours at the temperature of-20 ℃, centrifuging at a high speed, removing supernatant, adding a proper amount of distilled water (the volume of which is 0.2-0.5 times of that of the ginseng stem and leaf protein solution) into the precipitate, and dissolving again to obtain the ginseng stem and leaf protein solution.
5) Precooling the total protein solution of the ginseng stem and leaf for 5 hours at the temperature of minus 20 ℃, and then carrying out vacuum freeze drying to obtain the protein powder of the ginseng stem and leaf.
The extraction rate of the ginseng stem and leaf protein can be measured to be 1.068% by using the Bradford method.
Example 3
1) Pulverizing dried caulis Et folium Ginseng with pulverizer to obtain radix Ginseng powder;
2) adding absolute ethyl alcohol according to the mass-to-volume ratio (g/mL) of 1:10, stirring for 5 hours at room temperature, and filtering to obtain filtrate and filter residue;
3) collecting filter residues, adding 1 XPBS buffer solution with the pH value of 7.4-7.8 according to the mass-to-volume ratio (g/mL) of 1:20, leaching for 5 hours at room temperature, and filtering to obtain supernatant and precipitate; collecting the precipitate, adding 1 XPBS buffer solution with the pH value of 7.4-7.8 according to the mass-to-volume ratio (g/mL) of 1:10, carrying out secondary leaching for 10 hours, centrifuging to collect supernatant, and combining the supernatants to obtain the ginseng stem leaf protein solution;
4) and adding 5 times of cold acetone with the volume of-20 ℃ into the obtained ginseng stem and leaf protein solution, standing for 10 hours at-20 ℃, centrifuging at a high speed, removing supernatant, adding a proper amount of distilled water (the volume of which is 0.2-0.5 times of that of the ginseng stem and leaf protein solution) into the precipitate, and dissolving again to obtain the ginseng stem and leaf protein solution.
5) The obtained ginseng stem and leaf total protein solution is pre-cooled for 5 hours at the temperature of minus 20 ℃, and then is subjected to vacuum freeze drying to obtain the ginseng stem and leaf protein powder.
The extraction rate of the stem and leaf protein of ginseng was determined to be 0.248% by Bradford method.
Example 4
1) Pulverizing dried caulis Et folium Ginseng with pulverizer to obtain radix Ginseng powder;
2) adding 1 XPBS buffer solution with the pH value of 7.4-7.8 according to the mass-to-volume ratio (g/mL) of 1:10, leaching for 10 hours at room temperature, and centrifuging to obtain supernatant and precipitate; collecting the precipitate, adding 1 XPBS buffer solution with the pH value of 7.4-7.8 according to the mass-to-volume ratio (g/mL) of 1:10, carrying out secondary leaching for 10 hours, centrifuging to collect supernatant, and combining the supernatants to obtain the ginseng stem leaf protein solution;
3) and adding 5 times of cold acetone with the volume of-20 ℃ into the obtained ginseng stem and leaf protein solution, standing for 10 hours at-20 ℃, centrifuging at a high speed, removing supernatant, adding a proper amount of distilled water (the volume of which is 0.2-0.5 times of that of the ginseng stem and leaf protein solution) into the precipitate, and dissolving again to obtain the ginseng stem and leaf protein solution.
4) Precooling the total protein solution of the ginseng stem and leaf for 5 hours at the temperature of minus 20 ℃, and then carrying out vacuum freeze drying to obtain the protein powder of the ginseng stem and leaf.
The extraction rate of the stem and leaf protein of ginseng was found to be 0.836% by Bradford method.
Claims (4)
1. A method for extracting and separating protein from stem and leaf of Ginseng radix comprises removing impurities, extracting protein, standing with cold acetone, re-dissolving with distilled water, and post-treating;
the impurity removal process comprises the steps of adding ginseng stem leaf powder into an organic solvent n-hexane, stirring for 3 hours, filtering and retaining filter residues;
adding filter residues into a phosphate buffer solution with the pH value of 7.4-7.8, leaching for 5-10 hours, and centrifuging to obtain a supernatant and a precipitate, wherein the mass volume ratio of the filter residues to the phosphate buffer solution is 1: 10-20; adding the precipitate into a phosphate buffer solution with the pH value of 7.4-7.8, leaching for 5-10 hours, centrifuging and collecting a supernatant, wherein the mass volume ratio of the precipitate to the phosphate buffer solution is 1: 10-20; mixing the two supernatants to obtain ginseng stem and leaf protein solution;
the cold acetone standing and distilled water redissolving process comprises the steps of adding cold acetone with the volume of 5-10 times of that of the protein solution of the ginseng stem leaves into the protein solution of the ginseng stem leaves, standing for 3-10 hours at the temperature of minus 20 ℃, centrifuging, removing supernatant, adding the precipitate into distilled water, and redissolving to obtain the total protein solution of the ginseng stem leaves, wherein the using amount of the distilled water is 0.2-0.5 time of the volume of the protein solution of the ginseng stem leaves;
the post-treatment process comprises pre-cooling the total protein solution of the ginseng stem and leaf at-20 ℃ for 5 hours, and then carrying out vacuum freeze drying to obtain the ginseng stem and leaf protein powder.
2. The method for extracting and separating the protein of the ginseng stem and leaf according to claim 1, wherein the mass volume ratio of the ginseng stem and leaf powder to the organic solvent n-hexane is 1:10 in the impurity removal process.
3. The method for extracting and separating proteins from stems and leaves of Panax ginseng as claimed in claim 1 or 2, wherein the cold acetone is acetone at-20 ℃.
4. The method for extracting and separating the proteins of the ginseng stems and leaves according to the claim 1 or 2, wherein in the protein extraction process, the extraction time is 10 hours each time, and the mass-volume ratio of filter residues, precipitates and a phosphate buffer solution is 1: 10; the cold acetone standing and distilled water re-dissolving process is to add cold acetone of 5 times volume into ginseng protein solution and to stand at-20 deg.c for 3 hr.
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| CN109517870A (en) * | 2019-01-24 | 2019-03-26 | 吉林大学 | A kind of method of double enzyme stepwise discretization preparation ginseng polypeptides |
| CN114276408A (en) * | 2022-01-18 | 2022-04-05 | 长春中医药大学 | Extraction method and application of acanthopanax senticosus glycoprotein |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101906130A (en) * | 2010-07-07 | 2010-12-08 | 北华大学 | Wild ginseng protein extracting method suitable for dielectrophoresis |
| CN105154509A (en) * | 2015-09-28 | 2015-12-16 | 宗树伟 | Extraction method of ginseng peptide |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101906130A (en) * | 2010-07-07 | 2010-12-08 | 北华大学 | Wild ginseng protein extracting method suitable for dielectrophoresis |
| CN105154509A (en) * | 2015-09-28 | 2015-12-16 | 宗树伟 | Extraction method of ginseng peptide |
Non-Patent Citations (3)
| Title |
|---|
| Protein extraction from the stem of Panax ginseng C. A. Meyer: A tissue of lower protein extraction efficiency for proteomic analysis;Sun, LW et al.;《AFRICAN JOURNAL OF BIOTECHNOLOGY》;20110523;图1 * |
| 人参茎双向电泳图谱的建立及不同生长人参茎的比较蛋合质组学研;马朋涛;《中国优秀硕士学位论文全文数据库》;20111015;第8页第2段，第25页倒数第1段和第24页倒数第3段 * |
| 人参蛋白四种提取方法的比较研究;王伟楠等;《食品工业科技》;20100525(第5期);第1.2.1和1.2.3部分 * |
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