CN106381319B - Efficient extraction and separation method for grape seed procyanidin oligomers - Google Patents
Efficient extraction and separation method for grape seed procyanidin oligomers Download PDFInfo
- Publication number
- CN106381319B CN106381319B CN201610792583.5A CN201610792583A CN106381319B CN 106381319 B CN106381319 B CN 106381319B CN 201610792583 A CN201610792583 A CN 201610792583A CN 106381319 B CN106381319 B CN 106381319B
- Authority
- CN
- China
- Prior art keywords
- procyanidin
- grape seed
- ethanol
- extracting
- oligomers
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- 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
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
- C12P17/162—Heterorings having oxygen atoms as the only ring heteroatoms, e.g. Lasalocid
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
- C07D311/60—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2
- C07D311/62—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2 with oxygen atoms directly attached in position 3, e.g. anthocyanidins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07G—COMPOUNDS OF UNKNOWN CONSTITUTION
- C07G99/00—Subject matter not provided for in other groups of this subclass
-
- 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
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
Abstract
The invention belongs to the technical field of comprehensive utilization of grape seeds, and particularly relates to a high-efficiency extraction and separation method of grape seed procyanidin oligomers. The method for efficiently extracting and separating the grape seed procyanidin oligomer comprises the following steps: (1) drying and crushing at low temperature; (2) degreasing; (3) enzyme treatment; (4) extracting procyanidine; (5) concentrating at low temperature; (6) degrading procyanidine polymer; (7) purifying; (8) concentrating at low temperature, and drying. The extraction and separation method obviously improves the yield of the grape seed procyanidin and the content of the oligomeric procyanidin in the extracting solution, simultaneously well maintains the biological activity of the procyanidin by low-temperature extraction and separation, and obtains the high-purity procyanidin oligomer by column chromatography fractional purification.
Description
Technical Field
The invention belongs to the technical field of comprehensive utilization of grape seeds, and particularly relates to a high-efficiency extraction and separation method of grape seed procyanidin oligomers.
Background
The grape seed procyanidin has a structure of C6-C3-C6, and is a polyphenol compound formed by condensation of C4-C8 or C4-C6 by taking flavan-3-alcohol as a basic composition unit, wherein n is 2-5 for oligomer procyanidin, and n is 5 for polymer procyanidin.
The procyanidin has biochemical activity in resisting oxidation, scavenging free radicals such as superoxide anion and hydroxyl radical, protecting cardiovascular system, reducing blood lipid, reducing blood glucose, and resisting radiation. Free radicals are harmful compounds generated in the oxidation reaction of the body, have strong oxidizing property, and can damage tissues and cells of the body, thereby causing chronic diseases and aging effects. The research shows that the procyanidin is an effective free radical scavenger, particularly the oligomeric procyanidin has far higher scavenging capacity on free radicals and active oxygen than other antioxidants (such as VE and VC), and can prevent arteriosclerosis caused by the oxidation of low-density lipoprotein in human blood. The antioxidant activity of procyanidin is influenced by polymerization degree, the oligomer activity is high, and the higher the polymerization degree is, the lower the antioxidant activity is. With the intensive knowledge of procyanidin and the discovery of the biological activity of procyanidin with different polymerization degrees, the research and separation of oligomeric procyanidin are started.
In recent years, many methods for separating the procyanidin oligomers are available, for example, the grape seed procyanidin oligomers are purified by a high-pressure microfiltration membrane technology, the efficiency is high, the process is simple, but the industrial production is difficult and the production cost is high, and the concentrated high-polymer procyanidin is easy to reduce the permeability of the microfiltration membrane; or by supercritical CO2The extraction technology adds entrainers such as methanol or ethanol and the like to separate the grape seed procyanidin oligomers, the methanol effect is good, the monomer and dimer content in the extract is high, but the total extraction rate is low, and a part of the oligomer procyanidin and the high polymer are still remained in the grape seeds, so that the resource waste is caused, and the utilization rate is not high. The prior art focuses on the research of procyanidine extraction or polymer degradation process, and has no clear expression or even strong conclusion on the preparation, separation and purification method of oligomers.
Therefore, no very effective method for preliminarily separating and identifying the oligomeric procyanidin in the grape seeds exists so far. Grape seeds account for about 5% of the weight of wine grapes, are a large amount of resource byproducts generated in the grape industry, the yield of the wine grapes is nearly 100 ten thousand tons, and only the grape seeds generated by wine production can reach tens of thousands tons every year. Grape seeds contain rich polyphenols, wherein the procyanidin accounts for about 5% of the grape seeds, so that the research on the effective separation and purification of procyanidin oligomers not only has important theoretical value, but also has important significance on prolonging the grape industry chain, protecting the environment and effectively utilizing resources.
Disclosure of Invention
The invention aims to provide a method for efficiently extracting and separating grape seed procyanidin oligomers aiming at the defects, the method for extracting and separating the grape seed procyanidin oligomers obviously improves the yield of the grape seed procyanidin and the content of low-polymer procyanidin in an extracting solution, simultaneously, the low-temperature extraction and separation well keeps the biological activity of the procyanidin, and the high-purity procyanidin oligomers are obtained through column chromatography fractional purification.
The technical scheme of the invention is as follows: a method for efficiently extracting and separating grape seed procyanidin oligomers comprises the following steps:
(1) drying and crushing at low temperature: firstly, grape seeds are dried at low temperature; then crushing to obtain 40-60 mesh grape seed powder;
(2) degreasing: extracting grape seed oil from the obtained grape seed powder by supercritical carbon dioxide to remove oil at low temperature to obtain degreased grape seed powder;
(3) performing enzyme treatment, namely performing biological enzyme treatment on the obtained defatted grape seed powder by adopting cellulase and β -glucosidase, namely adding distilled water into the defatted grape seed powder according to the solid-to-liquid ratio of 1:20, taking the defatted grape seed powder as a substrate, and respectively adding 20U/m L cellulase and 10U/m Lβ -glucosidase under the enzymolysis conditions of pH value of 4.4, enzymolysis temperature of 47.8 ℃ and enzymolysis time of 2 hours;
(4) extracting procyanidine: extracting the degreased grape seed powder treated by the biological enzyme by using ethanol with the volume fraction of 70% to obtain procyanidine extracting solution; the average polymerization degree of procyanidine in the extracting solution is 3-10, and the content of oligomers is 40-60%;
(5) low-temperature concentration: concentrating the procyanidin extractive solution at 40 deg.C and vacuum degree of 0.08Mpa, centrifuging, and collecting supernatant to obtain procyanidin water solution;
(6) degrading the procyanidin polymer: degrading the obtained procyanidin aqueous solution by using composite acid to obtain procyanidin oligomer mixture solution, wherein the composite acid is prepared by compounding oxalic acid, glacial acetic acid and sulfurous acid;
(7) and (3) purification: firstly, macroporous resin adsorption and washing with water are carried out on the degraded procyanidine oligomer mixture solution to remove impurities; then, gradient elution is carried out by adopting ethanol, and oligomer procyanidine ethanol solutions with different polymerization degrees are sequentially obtained;
(8) low-temperature concentration and drying: concentrating the ethanol solution of the oligomeric proanthocyanidins with different polymerization degrees at low temperature to obtain aqueous solution of oligomeric proanthocyanidins; and (5) obtaining a finished product by freeze drying.
And (2) drying the grape seeds at low temperature in the step (1) under the conditions that the temperature is 30-50 ℃ and the vacuum degree is 0.06-0.09 MPa.
The extraction pressure of the supercritical carbon dioxide extraction in the step (2) is 32.30MPa, the extraction temperature is 41.5 ℃, and the extraction time is 75 min; the final oil yield is 16.0 percent, and the residual oil yield is 0.2 to 0.5 percent.
The step (3) adopts a pH buffer NaH2PO4。
The solid-liquid ratio of the extraction in the step (4) is 1:10, the temperature is 70 ℃, hydrochloric acid is adopted to adjust the pH value to 3.0, and the extraction time is 2 hours for 3 times.
The step (6) degradation is carried out for 30 minutes at the temperature of 50 ℃, wherein the oxalic acid concentration is 20 g/L;
the composite acid is oxalic acid: glacial acetic acid: the ratio of sulfurous acid to sulfurous acid is 1:1: 1; the volume ratio of the composite acid to the procyanidine aqueous solution is 5: 1.
The step (7) of purification adopts macroporous resin HPD100 for adsorption, wherein the adsorption sample concentration is 8 g/L, the sample injection speed is 2bv/h, the water washing speed is 2bv/h, and the adsorption time is 2h, the ethanol gradient elution is specifically that when the ethanol volume fraction is 0-10%, the procyanidin oligomer with the polymerization degree of 1.47 is eluted, when the ethanol volume fraction is 10-20%, the procyanidin oligomer with the polymerization degree of 1.78 is eluted, when the ethanol volume fraction is 20-30%, the procyanidin oligomer with the polymerization degree of 2.53 is eluted, when the ethanol volume fraction is 30-40%, the procyanidin oligomer with the polymerization degree of 2.78 is eluted, when the ethanol volume fraction is 40-50%, the procyanidin oligomer with the polymerization degree of 3.545 is eluted, and when the ethanol volume fraction is 50-60%, the procyanidin oligomer with the polymerization degree of 4.40 is eluted.
In the step (1), unfermented white grape seeds are adopted as the grape seeds.
The step (8) of low-temperature concentration and drying is carried out at 40 ℃ and under the condition of 0.09Mpa of vacuum degree; the freeze drying condition is vacuum degree of 1.1pa, pre-freezing initial temperature is-36 deg.C, and heating temperature is-10 deg.C.
The invention has the beneficial effects that: the method for efficiently extracting and separating the grape seed procyanidin oligomer takes unfermented white grape seeds as raw materials, and the raw materials are dried, crushed and subjected to supercritical CO2Removing oil by a fluid extraction method, treating grape seed powder by a biological enzyme method, extracting a target substance by an acid solvent method, hydrolyzing an extracting solution by an acid reagent to obtain high-content procyanidin oligomers, separating the procyanidin oligomers by macroporous resin column chromatography, and drying to obtain a sample.
The method is characterized in that the procyanidin oligomer is separated and extracted by combining the synergy of a solvent extraction method, an acid degradation method and a column chromatography fractionation method, the content of procyanidin, particularly the procyanidin oligomer in the extracting solution can be greatly improved, the nature of procyanidin in an acidic condition is stable, the content of procyanidin in the extracting solution is increased by 10-15% on the same scale, and the average polymerization degree in the procyanidin extracting solution is reduced by 2-3 units.
The method comprises the steps of adopting cellulose to destroy cell walls and promote the dissolution of procyanidine in grape seeds, adopting β -glucosidase to treat grape seed powder, not only destroying plant cell walls, but also decomposing anthocyanin (combined with polysaccharide) into free procyanidine, enabling the cellulose to act on β -1, 4-glucoside bonds of cell wall cellulose to the maximum extent, destroying cell walls, reducing mass transfer resistance and accelerating the dissolution efficiency of procyanidine, β -glucosidase is few in free states under natural conditions, mainly exists in a glucoside form, procyanidine usually forms anthocyanin with one or more of glucose, rhamnose, galactose, arabinose and the like through the glucoside bonds, and the extraction rate of procyanidine can be increased after enzymolysis by adding β -glucosidase, wherein the enzyme is endoprotease which has the specific action of β -1, 3 and 1, 4-glucoside bonds of glucan.
The following experimental tests were performed on the amount matching of the synergistic combination of cellulase and β -glucosidase during the use, and the results are shown in table 1.
TABLE 1
Table 1 shows that the proanthocyanidins can be effectively dissolved by increasing the addition of the cellulase and β -glucosidase, and when the addition of the cellulase exceeds 20U/m L and the addition of the β -glucosidase exceeds 10U/m L, the extraction amount of the proanthocyanidins is not increased continuously, which indicates that the enzymolysis efficiency is highest in the enzyme concentration range, so that the addition of the cellulase is 20U/m L-glucosidase is 10U/m L.
The following experiment compares the single acid degradation with the complex acid degradation, and see table 2 below in detail, wherein the acid hydrolysis temperature is 50 ℃ and the degradation time is 30 min.
TABLE 2
The experiment of degrading high polymer in the procyanidin extracting solution by different acid reagents in the table 2 proves that the addition of the composite acid compounded by oxalic acid, glacial acetic acid and sulfurous acid can effectively degrade the high polymer, the sulfurous acid is added to degrade the procyanidin into oligomer procyanidin, the oxidation of the procyanidin in the reaction process can be greatly reduced, and the antioxidant activity of the oligomer procyanidin is improved.
Detailed Description
The present invention will be described in detail below with reference to examples.
Example 1
The method for efficiently extracting and separating the grape seed procyanidin oligomer comprises the following steps:
(1) drying and crushing at low temperature: firstly, grape seeds are dried at low temperature of 30 ℃ and under the vacuum degree of 0.06 Mpa; then crushing to obtain 40-mesh grape seed powder, wherein the grape seeds adopt unfermented white grape seeds;
(2) degreasing: extracting grape seed oil with supercritical carbon dioxide to remove oil at low temperature to obtain defatted grape seed powder, wherein the supercritical carbon dioxide extraction pressure is 32.30MPa, and the extraction temperature is 41.5 deg.C; the extraction time is 75 min; the final oil yield is 16.0 percent, and the residual oil yield is 0.2 to 0.5 percent.
(3) Performing enzyme treatment by using cellulase and β -glucosidase to obtain defatted grape seed powder, adding distilled water into the defatted grape seed powder at a solid-to-liquid ratio of 1:20, adding 20U/m L cellulase and 10U/m Lβ -glucosidase into the defatted grape seed powder as a substrate respectively, and performing enzymolysis by using pH buffer NaH2PO4Adjusting pH to 4.4, enzymolysis temperature to 47.8 deg.C, and enzymolysis time to 2 hr;
(4) extracting procyanidine: extracting the degreased grape seed powder treated by the biological enzyme by using ethanol with the volume fraction of 70% to obtain procyanidine extracting solution; the average polymerization degree of procyanidine in the extracting solution is 3-10, and the content of oligomers is 40-60%; wherein the solid-liquid ratio of extraction is 1:10, the temperature is 70 ℃, hydrochloric acid is adopted to adjust the pH value to 3.0, and the extraction time is 2 hours for 3 times;
(5) low-temperature concentration: concentrating the procyanidin extractive solution at 40 deg.C and vacuum degree of 0.08Mpa, centrifuging, and collecting supernatant to obtain procyanidin water solution;
(6) degrading procyanidin high polymer, namely performing degradation reaction on the obtained procyanidin aqueous solution for 30 minutes at 50 ℃ by adopting composite acid to obtain procyanidin oligomer mixture solution, wherein the composite acid is prepared by compounding oxalic acid, glacial acetic acid and sulfurous acid according to the volume ratio of 1:1:1, the concentration of the oxalic acid is 20 g/L, and the volume ratio of the composite acid to the procyanidin aqueous solution is 5: 1;
(7) purifying, namely firstly adsorbing and washing a degraded proanthocyanidin oligomer mixture solution by using macroporous resin HPD100 for impurity removal, wherein the adsorbed sample concentration is 8 g/L, the sample injection speed is 2bv/h, the water washing speed is 2bv/h, and the adsorption time is 2h, then carrying out gradient elution by using ethanol, and sequentially and respectively obtaining proanthocyanidin ethanol solutions with different polymerization degrees, wherein specifically, when the volume fraction of the ethanol is 0-10%, proanthocyanidin oligomers with the polymerization degree of 1.47 are eluted, when the volume fraction of the ethanol is 10-20%, proanthocyanidin oligomers with the polymerization degree of 1.78 are eluted, when the volume fraction of the ethanol is 20-30%, proanthocyanidin oligomers with the polymerization degree of 2.53 are eluted, when the volume fraction of the ethanol is 30-40%, proanthocyanidin oligomers with the polymerization degree of 2.78 are eluted, when the volume fraction of the ethanol is 40-50%, proanthocyanidin oligomers with the polymerization degree of 3.545 are eluted, and when the volume fraction of the ethanol is 50-60%, proanthocyanidin oligomers with the polymerization degree of 4.40 are eluted;
(8) low-temperature concentration and drying: concentrating the ethanol solution of oligomeric proanthocyanidins with different polymerization degrees at 40 deg.C under vacuum degree of 0.09Mpa to obtain oligomeric proanthocyanidins water solution; the finished product is obtained by freeze drying, wherein the freeze drying condition is that the vacuum degree is 1.1pa, the pre-freezing initial temperature is-36 ℃, and the heating temperature is-10 ℃.
Example 2
The method for efficiently extracting and separating the grape seed procyanidin oligomer comprises the following steps:
(1) drying and crushing at low temperature: firstly, grape seeds are dried at low temperature of 50 ℃ and under the vacuum degree of 0.09 MPa; then crushing to obtain 60-mesh grape seed powder, wherein the grape seeds adopt unfermented white grape seeds;
(2) degreasing: extracting grape seed oil with supercritical carbon dioxide to remove oil at low temperature to obtain defatted grape seed powder, wherein the supercritical carbon dioxide extraction pressure is 32.30MPa, and the extraction temperature is 41.5 deg.C; the extraction time is 75 min; the final oil yield is 16.0 percent, and the residual oil yield is 0.2 to 0.5 percent.
(3) Enzyme treatment with cellulase β -glucosePerforming biological enzyme treatment on the defatted grape seed powder with glycosidase, adding distilled water into defatted grape seed powder at a solid-to-liquid ratio of 1:20, adding 20U/m L cellulase and 10U/m Lβ -glucosidase into defatted grape seed powder as substrate, respectively, and performing enzymolysis with pH buffer NaH2PO4Adjusting pH to 4.4, enzymolysis temperature to 47.8 deg.C, and enzymolysis time to 2 hr;
(4) extracting procyanidine: extracting the degreased grape seed powder treated by the biological enzyme by using ethanol with the volume fraction of 70% to obtain procyanidine extracting solution; the average polymerization degree of procyanidine in the extracting solution is 3-10, and the content of oligomers is 40-60%; wherein the solid-liquid ratio of extraction is 1:10, the temperature is 70 ℃, hydrochloric acid is adopted to adjust the pH value to 3.0, and the extraction time is 2 hours for 3 times;
(5) low-temperature concentration: concentrating the procyanidin extractive solution at 40 deg.C and vacuum degree of 0.08Mpa, centrifuging, and collecting supernatant to obtain procyanidin water solution;
(6) degrading procyanidin high polymer, namely performing degradation reaction on the obtained procyanidin aqueous solution for 30 minutes at 50 ℃ by adopting composite acid to obtain procyanidin oligomer mixture solution, wherein the composite acid is prepared by compounding oxalic acid, glacial acetic acid and sulfurous acid according to the volume ratio of 1:1:1, the concentration of the oxalic acid is 20 g/L, and the volume ratio of the composite acid to the procyanidin aqueous solution is 5: 1;
(7) purifying, namely firstly adsorbing and washing a degraded proanthocyanidin oligomer mixture solution by using macroporous resin HPD100 for impurity removal, wherein the adsorbed sample concentration is 8 g/L, the sample injection speed is 2bv/h, the water washing speed is 2bv/h, and the adsorption time is 2h, then carrying out gradient elution by using ethanol, and sequentially and respectively obtaining proanthocyanidin ethanol solutions with different polymerization degrees, wherein specifically, when the volume fraction of the ethanol is 0-10%, proanthocyanidin oligomers with the polymerization degree of 1.47 are eluted, when the volume fraction of the ethanol is 10-20%, proanthocyanidin oligomers with the polymerization degree of 1.78 are eluted, when the volume fraction of the ethanol is 20-30%, proanthocyanidin oligomers with the polymerization degree of 2.53 are eluted, when the volume fraction of the ethanol is 30-40%, proanthocyanidin oligomers with the polymerization degree of 2.78 are eluted, when the volume fraction of the ethanol is 40-50%, proanthocyanidin oligomers with the polymerization degree of 3.545 are eluted, and when the volume fraction of the ethanol is 50-60%, proanthocyanidin oligomers with the polymerization degree of 4.40 are eluted;
(8) low-temperature concentration and drying: concentrating the ethanol solution of oligomeric proanthocyanidins with different polymerization degrees at 40 deg.C under vacuum degree of 0.09Mpa to obtain oligomeric proanthocyanidins water solution; the finished product is obtained by freeze drying, wherein the freeze drying condition is that the vacuum degree is 1.1pa, the pre-freezing initial temperature is-36 ℃, and the heating temperature is-10 ℃.
Example 3
The method for efficiently extracting and separating the grape seed procyanidin oligomer comprises the following steps:
(1) drying and crushing at low temperature: firstly, grape seeds are dried at low temperature of 45 ℃ and under the vacuum degree of 0.07 MPa; then crushing to obtain 40-60 mesh grape seed powder, wherein the grape seeds are unfermented white grape seeds;
(2) degreasing: extracting grape seed oil with supercritical carbon dioxide to remove oil at low temperature to obtain defatted grape seed powder, wherein the supercritical carbon dioxide extraction pressure is 32.30MPa, and the extraction temperature is 41.5 deg.C; the extraction time is 75 min; the final oil yield is 16.0 percent, and the residual oil yield is 0.2 to 0.5 percent.
(3) Performing enzyme treatment by using cellulase and β -glucosidase to obtain defatted grape seed powder, adding distilled water into the defatted grape seed powder at a solid-to-liquid ratio of 1:20, adding 20U/m L cellulase and 10U/m Lβ -glucosidase into the defatted grape seed powder as a substrate respectively, and performing enzymolysis by using pH buffer NaH2PO4Adjusting pH to 4.4, enzymolysis temperature to 47.8 deg.C, and enzymolysis time to 2 hr;
(4) extracting procyanidine: extracting the degreased grape seed powder treated by the biological enzyme by using ethanol with the volume fraction of 70% to obtain procyanidine extracting solution; the average polymerization degree of procyanidine in the extracting solution is 3-10, and the content of oligomers is 40-60%; wherein the solid-liquid ratio of extraction is 1:10, the temperature is 70 ℃, hydrochloric acid is adopted to adjust the pH value to 3.0, and the extraction time is 2 hours for 3 times;
(5) low-temperature concentration: concentrating the procyanidin extractive solution at 40 deg.C and vacuum degree of 0.08Mpa, centrifuging, and collecting supernatant to obtain procyanidin water solution;
(6) degrading procyanidin high polymer, namely performing degradation reaction on the obtained procyanidin aqueous solution for 30 minutes at 50 ℃ by adopting composite acid to obtain procyanidin oligomer mixture solution, wherein the composite acid is prepared by compounding oxalic acid, glacial acetic acid and sulfurous acid according to the volume ratio of 1:1:1, the concentration of the oxalic acid is 20 g/L, and the volume ratio of the composite acid to the procyanidin aqueous solution is 5: 1;
(7) purifying, namely firstly adsorbing and washing a degraded proanthocyanidin oligomer mixture solution by using macroporous resin HPD100 for impurity removal, wherein the adsorbed sample concentration is 8 g/L, the sample injection speed is 2bv/h, the water washing speed is 2bv/h, and the adsorption time is 2h, then carrying out gradient elution by using ethanol, and sequentially and respectively obtaining proanthocyanidin ethanol solutions with different polymerization degrees, wherein specifically, when the volume fraction of the ethanol is 0-10%, proanthocyanidin oligomers with the polymerization degree of 1.47 are eluted, when the volume fraction of the ethanol is 10-20%, proanthocyanidin oligomers with the polymerization degree of 1.78 are eluted, when the volume fraction of the ethanol is 20-30%, proanthocyanidin oligomers with the polymerization degree of 2.53 are eluted, when the volume fraction of the ethanol is 30-40%, proanthocyanidin oligomers with the polymerization degree of 2.78 are eluted, when the volume fraction of the ethanol is 40-50%, proanthocyanidin oligomers with the polymerization degree of 3.545 are eluted, and when the volume fraction of the ethanol is 50-60%, proanthocyanidin oligomers with the polymerization degree of 4.40 are eluted;
(8) low-temperature concentration and drying: concentrating the ethanol solution of oligomeric proanthocyanidins with different polymerization degrees at 40 deg.C under vacuum degree of 0.09Mpa to obtain oligomeric proanthocyanidins water solution; the finished product is obtained by freeze drying, wherein the freeze drying condition is that the vacuum degree is 1.1pa, the pre-freezing initial temperature is-36 ℃, and the heating temperature is-10 ℃.
Claims (9)
1. A method for efficiently extracting and separating grape seed procyanidin oligomers comprises the following steps:
(1) drying and crushing at low temperature: firstly, grape seeds are dried at low temperature; then crushing to obtain 40-60 mesh grape seed powder;
(2) degreasing: extracting grape seed oil from the obtained grape seed powder by supercritical carbon dioxide to remove oil at low temperature to obtain degreased grape seed powder;
(3) performing enzyme treatment, namely performing biological enzyme treatment on the obtained defatted grape seed powder by adopting cellulase and β -glucosidase, namely adding distilled water into the defatted grape seed powder according to the solid-to-liquid ratio of 1:20, taking the defatted grape seed powder as a substrate, and respectively adding 20U/m L cellulase and 10U/m Lβ -glucosidase under the enzymolysis conditions of pH value of 4.4, enzymolysis temperature of 47.8 ℃ and enzymolysis time of 2 hours;
(4) extracting procyanidine: extracting the degreased grape seed powder treated by the biological enzyme by using ethanol with the volume fraction of 70% to obtain procyanidine extracting solution; the average polymerization degree of procyanidine in the extracting solution is 3-10, and the content of oligomers is 40-60%;
(5) low-temperature concentration: concentrating the procyanidin extractive solution at 40 deg.C and vacuum degree of 0.08Mpa, centrifuging, and collecting supernatant to obtain procyanidin water solution;
(6) degrading the procyanidin polymer: degrading the obtained procyanidin aqueous solution by adopting composite acid to obtain procyanidin oligomer mixture solution, wherein the composite acid is prepared by compounding oxalic acid, glacial acetic acid and sulfurous acid, and the composite acid is prepared from the following components in percentage by volume: glacial acetic acid: the ratio of sulfurous acid to sulfurous acid is 1:1: 1;
(7) and (3) purification: firstly, macroporous resin adsorption and washing with water are carried out on the degraded procyanidine oligomer mixture solution to remove impurities; then, gradient elution is carried out by adopting ethanol, and oligomer procyanidine ethanol solutions with different polymerization degrees are sequentially obtained;
(8) low-temperature concentration and drying: concentrating the ethanol solution of the oligomeric proanthocyanidins with different polymerization degrees at low temperature to obtain aqueous solution of oligomeric proanthocyanidins; and (5) obtaining a finished product by freeze drying.
2. The method for efficiently extracting and separating the grape seed procyanidin oligomers as claimed in claim 1, wherein the grape seeds are dried at a low temperature of 30-50 ℃ and a vacuum degree of 0.06-0.09 Mpa in the step (1).
3. The method for efficiently extracting and separating the grape seed procyanidin oligomers as claimed in claim 1, wherein the supercritical carbon dioxide extraction in the step (2) is performed at an extraction pressure of 32.30MPa, an extraction temperature of 41.5 ℃ and an extraction time of 75 min; the final oil yield is 16.0 percent, and the residual oil yield is 0.2 to 0.5 percent.
4. The method for efficiently extracting and separating the grape seed procyanidin oligomers as claimed in claim 1, wherein the pH buffer NaH is adopted in the step (3)2PO4。
5. The method for efficiently extracting and separating the grape seed procyanidin oligomers as claimed in claim 1, wherein the extraction solid-to-liquid ratio in the step (4) is 1:10, the temperature is 70 ℃, hydrochloric acid is adopted to adjust the pH value to 3.0, and the extraction time is 2 hours for 3 times.
6. The method for efficiently extracting and separating the grape seed procyanidin oligomers as claimed in claim 1, wherein the degradation in the step (6) is carried out for 30 minutes at 50 ℃, wherein the oxalic acid concentration is 20 g/L, and the volume ratio of the composite acid to the procyanidin aqueous solution is 5: 1.
7. The method for efficiently extracting and separating the grape seed procyanidin oligomers as claimed in claim 1, wherein macroporous resin HPD100 is adopted for adsorption in the purification in the step (7), wherein the adsorption sample concentration is 8 g/L, the sample injection speed is 2bv/h, the water washing speed is 2bv/h, and the adsorption time is 2h, the ethanol gradient elution is specifically that the procyanidin oligomers with the polymerization degree of 1.47 are eluted when the ethanol volume fraction is 0-10%, the procyanidin oligomers with the polymerization degree of 1.78 are eluted when the ethanol volume fraction is 10-20%, the procyanidin oligomers with the polymerization degree of 2.53 are eluted when the ethanol volume fraction is 20-30%, the procyanidin oligomers with the polymerization degree of 2.78 are eluted when the ethanol volume fraction is 30-40%, the procyanidin oligomers with the polymerization degree of 3.545 are eluted when the ethanol volume fraction is 40-50%, and the procyanidin oligomers with the polymerization degree of 4.40 are eluted when the ethanol volume fraction is 50-60%.
8. The method for efficiently extracting and separating the grape seed procyanidin oligomers as claimed in claim 1, wherein unfermented white grape seeds are adopted as the grape seeds in the step (1).
9. The method for efficiently extracting and separating the grape seed procyanidin oligomers of claim 1, wherein the step (8) of low-temperature concentration and drying is carried out at 40 ℃ and under the vacuum degree of 0.09 Mpa; the freeze drying condition is vacuum degree of 1.1pa, pre-freezing initial temperature is-36 deg.C, and heating temperature is-10 deg.C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610792583.5A CN106381319B (en) | 2016-08-31 | 2016-08-31 | Efficient extraction and separation method for grape seed procyanidin oligomers |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610792583.5A CN106381319B (en) | 2016-08-31 | 2016-08-31 | Efficient extraction and separation method for grape seed procyanidin oligomers |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106381319A CN106381319A (en) | 2017-02-08 |
| CN106381319B true CN106381319B (en) | 2020-07-14 |
Family
ID=57938188
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610792583.5A Active CN106381319B (en) | 2016-08-31 | 2016-08-31 | Efficient extraction and separation method for grape seed procyanidin oligomers |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106381319B (en) |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106923136B (en) * | 2017-03-02 | 2020-08-11 | 吉林农业大学 | Anthocyanin complex acid stabilizer and ultrahigh pressure auxiliary stabilizing method |
| CN107467571B (en) * | 2017-08-23 | 2021-04-02 | 湖南胖哥食品有限责任公司 | Areca nut additive and preparation method thereof, and areca nut product and preparation method thereof |
| CN107556283A (en) * | 2017-10-19 | 2018-01-09 | 阜康市天鼎生物科技有限公司 | A kind of process of the extraction of the efficient low-consume from grape pip OPC |
| CN107556282A (en) * | 2017-10-19 | 2018-01-09 | 阜康市天鼎生物科技有限公司 | A kind of overcritical method from extracting proanthocyanidin from grape seeds |
| CN108003128B (en) * | 2017-12-25 | 2020-09-15 | 北京颐方生物科技有限公司 | Method for extracting grape seed procyanidin |
| CN109705080A (en) * | 2018-12-20 | 2019-05-03 | 宁波天鼎生物科技有限公司 | The extracting method of Proanthocyanidins from Grape Seeds high polymer cracking and procyanidine B4 |
| CN109651322A (en) * | 2018-12-20 | 2019-04-19 | 浙江大学宁波理工学院 | The extracting method of Proanthocyanidins from Grape Seeds high polymer cracking and procyanidin B 2 |
| CN110878075A (en) * | 2019-12-06 | 2020-03-13 | 怀化学院 | Method for extracting procyanidine from grape seeds and procyanidine |
| CN113951512A (en) * | 2021-11-11 | 2022-01-21 | 郑州轻工业大学 | Grape seed procyanidin oligomer micro powder and preparation method thereof |
| CN114031590A (en) * | 2021-11-17 | 2022-02-11 | 中北大学 | Method for efficiently preparing high-purity oligomeric proanthocyanidins from sea buckthorn seed meal |
| CN114890973B (en) * | 2022-04-21 | 2023-09-19 | 陕西嘉禾药业有限公司 | Method for extracting and separating water-soluble procyanidine in grape seeds |
| CN117160270B (en) * | 2023-11-03 | 2024-03-29 | 宁德九展农业有限公司 | Emulsifying device for camellia oil milk and production method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2920987A1 (en) * | 2007-09-14 | 2009-03-20 | Pascal Marionnet | Cosmetic composition made of a plant extract, preferably strawberry roots, useful in dermatological cosmetic creams and lotions |
| CN103193751A (en) * | 2013-03-27 | 2013-07-10 | 吉首大学 | Method for continuously extracting active ingredients from grape seeds |
| CN103923052A (en) * | 2014-04-09 | 2014-07-16 | 完美（中国）有限公司 | Method for preparing oligomeric proanthocyanidins |
-
2016
- 2016-08-31 CN CN201610792583.5A patent/CN106381319B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2920987A1 (en) * | 2007-09-14 | 2009-03-20 | Pascal Marionnet | Cosmetic composition made of a plant extract, preferably strawberry roots, useful in dermatological cosmetic creams and lotions |
| CN103193751A (en) * | 2013-03-27 | 2013-07-10 | 吉首大学 | Method for continuously extracting active ingredients from grape seeds |
| CN103923052A (en) * | 2014-04-09 | 2014-07-16 | 完美（中国）有限公司 | Method for preparing oligomeric proanthocyanidins |
Non-Patent Citations (1)
| Title |
|---|
| 树莓籽原花青素的降解及抗氧化研究;吴迪;《中国优秀硕士学位论文全文数据库》;20141015;第8，11-18页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106381319A (en) | 2017-02-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106381319B (en) | Efficient extraction and separation method for grape seed procyanidin oligomers | |
| US20100119651A1 (en) | Bio-Enzyme-Assisted Extraction Method for Inulin | |
| CN106632205A (en) | Method for extracting proanthocyanidins from Yunnan pine bark | |
| CN112521523B (en) | Method for extracting and purifying inonotus obliquus polysaccharide | |
| RU2011124540A (en) | PHYTOCOMPLEX FROM FRUITS OF BERGAMOTA, METHOD OF PRODUCTION AND APPLICATION AS A FOOD ADDITIVE AND IN THE FIELD OF PHARMACOLOGY | |
| CN107468738A (en) | A kind of extracting method of chrysanthemum total flavone | |
| CN104045724A (en) | Method for extracting and preparing polysaccharide from inonotus obliquus | |
| CN104886595A (en) | Method for extracting flavonoids from peanut hulls with aqueous enzymatic method and alcohol and water solvent assisted by microwave | |
| CN111574570B (en) | Comprehensive utilization method of cordyceps militaris culture residues | |
| CN1303095C (en) | Ginseng saponin superhigh pressure extraction process | |
| CN1248992C (en) | Preparation of oligomer and monomer from tannin by catalytic hydrogen degradation | |
| CN108822305B (en) | Method for extracting gutta-percha from eucommia bark based on imidazole ionic liquid pretreatment | |
| CN1488364A (en) | Method for treating aloe gel, aloe leaf gel and aloe hide glue and aloe polyaccharide obtained thereof | |
| CN108252143B (en) | Method for extracting plant salt and cellulose from suaeda salsa | |
| CN1266160C (en) | Process for extracting rubusoside of fruit of Gorsvenor Momordica | |
| CN110194810A (en) | A kind of grading purification method for the external glue polysaccharide of water shield improving hypoglycemic activity | |
| CN110669035A (en) | Method for preparing anthocyanin from indigo fruit | |
| CN105566507A (en) | Preparation method of tea polysaccharides | |
| CN108033874B (en) | Biological extraction method of resveratrol | |
| CN114042092A (en) | Cordycepin mixture and effective separation method and application thereof | |
| CN110054709B (en) | Method for efficiently extracting and purifying inonotus obliquus polysaccharide | |
| CN113897406A (en) | Method for extracting and purifying salidroside from rhodiola rosea powder | |
| CN1788822A (en) | Low-temperature differential pressure type extractive technique | |
| CN105420293A (en) | Method for separating and purifying resveratrol from traditional Chinese medicine polygonum cuspidatum extraction solution | |
| CN110590526A (en) | Method for extracting perillaldehyde from perilla frutescens |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |