CN110627863B - Proteoglycan extraction method, proteoglycan extract, application of proteoglycan extract and cosmetic - Google Patents
Proteoglycan extraction method, proteoglycan extract, application of proteoglycan extract and cosmetic Download PDFInfo
- Publication number
- CN110627863B CN110627863B CN201910954373.5A CN201910954373A CN110627863B CN 110627863 B CN110627863 B CN 110627863B CN 201910954373 A CN201910954373 A CN 201910954373A CN 110627863 B CN110627863 B CN 110627863B
- Authority
- CN
- China
- Prior art keywords
- proteoglycan
- solution
- red algae
- wall
- leaching
- 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
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/73—Polysaccharides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/82—Preparation or application process involves sonication or ultrasonication
Abstract
The invention provides a proteoglycan extraction method, a proteoglycan extract, application of the proteoglycan extract and cosmetics, and relates to the technical field of proteoglycan extraction. The extraction method of the proteoglycan comprises the following steps of carrying out ultrasonic leaching on red algae palm leaf trees in the presence of a wall-breaking enzyme to obtain a leaching solution, then using ammonium sulfate to precipitate the leaching solution to obtain crude proteoglycan, and then using ethanol to purify the crude proteoglycan to obtain proteoglycan; wherein the wall-breaking enzyme comprises cellulase, hemicellulase and pectinase. The protein extraction method has the advantages of wide raw material source, low extraction cost, simple operation and easy industrial production.
Description
Technical Field
The invention relates to the technical field of proteoglycan extraction, and particularly relates to a proteoglycan extraction method, a proteoglycan extract, application of the proteoglycan extract and cosmetics.
Background
Proteoglycan is a kind of complex formed by connecting carbohydrate and protein through one or more covalent bonds, and is one of important biological macromolecular substances in organisms. Due to the special properties of proteoglycan, the value of proteoglycan is more and more recognized, and proteoglycan is widely applied to the fields of biochemistry, medicine, food, cosmetics and the like. Has effects in enhancing immunity, resisting oxidation, relieving fatigue, resisting radiation and virus, improving cell membrane ductility, and protecting cell.
In the past, natural marine proteoglycan is mainly derived from marine animals, such as whale, shark and other cartilages, but due to the difficulty and the limited capture amount of such marine organisms, it is difficult to obtain marine proteoglycan in large quantities. Therefore, a process is needed that allows for easier access to proteoglycans of marine origin.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide a proteoglycan extraction method, which alleviates the problem of lacking a method for easily obtaining proteoglycan of marine origin in the prior art.
The second purpose of the invention is to provide an extract prepared by the extraction method of the rhodophyta palm leaf tree-derived proteoglycan.
The third purpose of the invention is to provide a method for extracting proteoglycan from the rhodophyta palm leaf tree or application of the extract in preparing cosmetics.
The fourth object of the present invention is to provide a cosmetic composition comprising the above extract.
In order to solve the technical problems, the invention adopts the following technical scheme:
according to one aspect of the present invention, there is provided a proteoglycan extraction method, comprising: ultrasonically leaching red algae palm leaf trees in the presence of a wall-breaking enzyme to obtain a leaching solution, then precipitating the leaching solution by using ammonium sulfate to obtain crude proteoglycan, and purifying the crude proteoglycan by using ethanol to obtain proteoglycan; the wall-breaking enzyme comprises cellulase, hemicellulase and pectinase.
Preferably, the ultrasonic leaching time is 6-8 h, preferably 6.5-7.5 h, and more preferably 7 h;
preferably, the method comprises the steps of firstly crushing the anthurium andraeanum leaf trees, sieving to obtain anthurium andraeanum leaf tree powder, and then ultrasonically leaching a mixture of the powder of the anthurium andraeanum leaf trees, water and wall breaking enzyme;
preferably, the amount of water is 20-25 times of the weight of the powder of the red algae palm leaf tree;
preferably, the mass percent of cellulase in the wall-breaking enzyme is 25-50%, the mass percent of hemicellulase is 30-45%, and the balance is pectinase;
preferably, the dosage of the wall-breaking enzyme is 0.03-0.07 time of the weight of the red algae palm leaf tree, and more preferably 0.04 time;
preferably, the water comprises deionized water;
preferably, the crushed anthurium andraeanum leaf trees comprise crushed and dried anthurium andraeanum leaf trees;
preferably, the drying comprises freeze drying.
Preferably, after the red algae palm leaf tree is subjected to ultrasonic leaching, the leaching solution is obtained after the solid-liquid separation of the product of the ultrasonic leaching, and then ammonium sulfate is used for precipitating the leaching solution;
preferably, ammonium sulfate precipitation of the leachate comprises: the concentration of ammonium sulfate in the leaching liquor is 70-90%, and crude proteoglycan is obtained after precipitation;
preferably, adding saturated ammonium sulfate solution into the leaching liquor until the concentration of ammonium sulfate in the leaching liquor is 70-90%;
preferably, ammonium sulfate is used for precipitating the leaching solution for 12-18 h;
preferably, before the leaching liquor is precipitated by using ammonium sulfate, the leaching liquor is subjected to decolorization treatment;
preferably, the leachate is decolorized using activated carbon.
Preferably, removing sulfate ions in crude proteoglycan, and purifying the crude proteoglycan by using ethanol;
preferably, the sulfate ions in the crude proteoglycan are removed by a dialysis method;
preferably, the buffer used for dialysis comprises a phosphate buffer; the concentration of the phosphate buffer solution is preferably 0.005-0.015 mol/L, and the pH is preferably 6.5-7.5;
preferably, the buffer solution is replaced 5-7 times in the dialysis process.
Preferably, the ethanol purification of the crude proteoglycan comprises: the final volume concentration of ethanol in the solution containing crude proteoglycan is 40-50%, and proteoglycan is obtained after precipitation;
preferably, ethanol with a volume concentration of 85-95% is added to the solution containing crude proteoglycan until the final volume concentration of ethanol in the solution containing crude proteoglycan is 40-50%;
preferably, the ethanol in the solution containing the crude proteoglycan is kept still for 10-15 hours under the temperature condition of 0-5 ℃ after the final volume concentration of 40-50 percent, so as to obtain the precipitate.
Preferably, the ethanol purified product is dried to obtain the proteoglycan;
preferably, the drying comprises drying at 40-45 ℃.
Preferably, the extraction method comprises the following steps:
(a) freeze drying the washed red algae palm leaf tree;
(b) crushing the freeze-dried anthurium andraeanum leaf trees by using a crusher, and sieving to obtain anthurium andraeanum leaf tree powder;
(c) adding the powder of the red algae palm leaf tree into deionized water with the weight of 20-25 times that of the red algae palm leaf tree powder, adding a wall breaking enzyme, and performing ultrasonic extraction for 7 hours;
(d) centrifuging and filtering the sample treated in the step (c) to obtain a leaching solution;
(e) adding active carbon into the leaching liquor obtained in the step (d) for decoloring;
(f) adding a saturated ammonium sulfate solution into the decolorized leaching liquor in the step (e) until the concentration of the saturated ammonium sulfate solution is 70% -90%, precipitating for 12-18 h, and centrifuging to obtain a crude proteoglycan precipitate;
(g) dialyzing the precipitate obtained in the step (f) by using a phosphate buffer solution with the concentration of 0.01mol/L, pH of 7, and changing the dialyzate for 5-7 times until sulfate ions do not exist in the solution;
(h) adding 85-95% ethanol into the solution to remove sulfate ions until the final concentration is 40-50%; standing for 10-15 h at 0-5 ℃, and centrifuging to obtain a precipitate which is a purified proteoglycan;
(i) and (5) drying the precipitate obtained in the step (h) at 40-45 ℃ to obtain the proteoglycan.
According to one aspect of the invention, the invention also provides an extract prepared by the proteoglycan extraction method.
According to one aspect of the invention, the invention also provides the proteoglycan extraction method or the application of the extract in preparing cosmetics.
According to one aspect of the present invention, there is also provided a cosmetic comprising the above extract.
Compared with the prior art, the invention has the following beneficial effects:
the method for extracting proteoglycan provided by the invention takes the red algae palmaria palmata as a raw material, and the red algae palmaria palmata is also known as palmate red skin algae, and is widely applied to the fields of food, health care products and cosmetic skin care, so the source of the raw material is safe. The red algae palm leaf trees grow vigorously, are low in cost and easy to circulate, and can effectively increase the marine bioavailability.
The method for extracting proteoglycan provided by the invention adopts ultrasonic extraction of the red algae palm leaf tree in the presence of wall-breaking enzyme. The wall-breaking enzyme used in the invention comprises cellulase, hemicellulase and pectinase, and can break the cell wall of the red algae palm leaf tree and promote the cell sap to flow out. The cell lysate of the red algae palm leaf tree can be efficiently obtained by combining the wall-breaking enzyme and the ultrasound. Ammonium sulfate and ethanol are sequentially adopted for fractional precipitation, and proteoglycan with higher purity can be effectively obtained. And the wall-breaking enzyme, ammonium sulfate and ethanol are used as main extraction reagents, so that the method is safe and easy to obtain, the process flow is simple, the process is green and environment-friendly, no pollution is caused to the environment, and the product extracted by the process flow has high extraction rate, is simple and convenient to operate and is easy for industrial production.
The extract prepared by the proteoglycan extraction method provided by the invention has the main component of proteoglycan of marine origin, has good effects of anti-inflammation, moisture retention, antioxidation, melanin inhibition and the like, and can be used for preparing cosmetics.
The proteoglycan extraction method or the application of the extract in preparing cosmetics can endow the cosmetics with good anti-inflammatory, moisturizing, antioxidant, melanin inhibiting and the like. And because the proteoglycan extraction method has wide raw material sources and low cost, the proteoglycan extraction method or the extract can be applied to the preparation of cosmetics, so that the production cost of the cosmetics can be reduced.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a graph showing a standard curve of a protein in an effect example of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that:
in the present invention, all embodiments and preferred methods mentioned herein may be combined with each other to form a new technical solution, if not specifically stated; all the technical features mentioned herein, as well as preferred features, can be combined with each other to form new solutions; the components concerned or their preferred components can be combined with one another to form new solutions.
In the present invention, unless otherwise stated, the numerical range "a-b" represents a shorthand representation of any combination of real numbers between a and b, where a and b are both real numbers. For example, a numerical range of "1 to 10" means that all real numbers between "1 to 10" have been listed herein, and "1 to 10" is only a shorthand representation of the combination of these values. The "ranges" disclosed herein may have one or more lower limits and one or more upper limits, respectively, in the form of lower limits and upper limits.
Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.
According to one aspect of the present invention, there is provided a method for extracting proteoglycan, comprising: ultrasonically leaching red algae palm leaf trees in the presence of a wall-breaking enzyme to obtain a leaching solution, then precipitating the leaching solution by using ammonium sulfate to obtain crude proteoglycan, and purifying the crude proteoglycan by using ethanol to obtain proteoglycan, wherein the wall-breaking enzyme comprises cellulase, hemicellulase and pectinase.
The method for extracting proteoglycan provided by the invention takes the red algae palmaria palmata as a raw material, and the red algae palmaria palmata, also called palmate red skin algae, is rich in abundant bioactive substances, has excellent effects of anti-inflammation, antioxidation, melanin inhibition and the like, and is widely applied to the field of food, health care products and cosmetic skin care. The red algae palm leaf trees are mainly distributed on the coastlines of the Atlantic ocean and the Pacific ocean, the utilization rate of the red algae palm leaf trees in China is low at present, the red algae palm leaf trees are often hidden troubles due to excessive growth in re-cultivation areas, the red algae palm leaf trees which grow vigorously are used as extraction raw materials of proteoglycan, the cost is low, the circulation is easy, and the marine bioavailability can be effectively increased.
The wall-breaking enzyme used in the invention comprises cellulase, hemicellulase and pectinase, and can break the cell wall of the red algae palm leaf tree and promote the cell sap to flow out. And the wall-breaking enzyme, the ammonium sulfate and the ethanol are used as main extraction reagents, so that the method is safe and easy to obtain, the process flow is simple, the process is green and environment-friendly, no pollution is caused to the environment, the extraction rate of the product extracted by the process flow is high, the operation is simple and convenient, and the industrial production is easy to realize.
In some preferred embodiments, the time for the ultrasonic leaching is 6 to 8 hours, such as but not limited to 6 hours, 6.5 hours, 7 hours, 7.5 hours or 8 hours, preferably 6.5 to 7.5 hours, and more preferably 7 hours.
In some preferred embodiments, the red algae palm leaf tree is first crushed, and preferably dried red algae palm leaf tree, more preferably freeze-dried red algae palm leaf tree is used as the red algae palm leaf tree; sieving the crushed anthurium andraeanum leaves to obtain anthurium andraeanum leaves powder, and then ultrasonically leaching a mixture of the powder of the anthurium andraeanum leaves, water and wall-breaking enzyme, wherein the using amount of the water is preferably 20-25 times of the weight of the powder of the anthurium andraeanum leaves, for example, but not limited to, 20 times, 21 times, 22 times, 23 times, 24 times or 25 times, and the water is preferably deionized water with less impurities. The dosage of each component in the wall-breaking enzyme is preferably 25-50% by mass of the cellulase, for example, but not limited to, 25%, 30%, 35%, 40%, 45% or 50%; the mass percentage of the hemicellulase is 30-45%, for example, but not limited to 30%, 35%, 40% or 45%, and the balance is pectinase, for example, but not limited to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45%. The amount of the wall-breaking enzyme is preferably 0.03 to 0.07 times of the weight of the red algae palm leaf tree, and may be, but is not limited to, 0.03 times, 0.04 times, 0.05 times, 0.06 times, or 0.07 times, and more preferably 0.04 times.
In some preferred embodiments, after the red algae palm leaf tree is subjected to ultrasonic leaching, the product of the ultrasonic leaching is subjected to solid-liquid separation to remove solid impurities in the leaching liquor, the solid-liquid separation is preferably performed by centrifugation with simple operation and good separation effect and filtration, the obtained filtrate is palm leaf tree cell lysate, the filtrate is used as the leaching liquor, and then ammonium sulfate is used for precipitating the leaching liquor.
In some preferred embodiments, the leachate is decolorized prior to precipitation with ammonium sulfate to remove colored species from the leachate, thereby reducing impurities in the proteoglycans. The leaching liquor is preferably decolorized by using activated carbon with high decolorization efficiency and no side effect.
In some preferred embodiments, the step of precipitating the leachate using ammonium sulphate comprises adjusting the concentration of ammonium sulphate in the leachate to between 70% and 90%, for example but not limited to between 70%, 72%, 75%, 78%, 80%, 83%, 85%, 88% or 90%, preferably precipitating the leachate by slowly adding a saturated solution of ammonium sulphate to the leachate to a concentration of 70% to 90% ammonium sulphate in said leachate. When the concentration of the ammonium sulfate in the leaching liquor is 70-90%, the proteoglycan in the leaching liquor can be effectively precipitated. Taking the precipitate obtained from the leaching liquor treated by ammonium sulfate as crude proteoglycan, preferably adding ammonium sulfate into the leaching liquor and precipitating for 12-18 h to obtain crude proteoglycan precipitate which is taken as the material for ethanol purification.
In some preferred embodiments, the crude proteoglycan is purified by ethanol, and the sulfate ion in the crude proteoglycan is removed to prevent the residue of the sulfate ion from affecting the purification of proteoglycan by ethanol. In some preferred embodiments, the sulfate ion is removed from the crude proteoglycan by dialysis. The dialysate is preferably phosphate buffer solution during dialysis, wherein the concentration of the phosphate buffer solution is preferably 0.005-0.015 mol/L, and the pH is preferably 6.5-7.5. In order to remove the sulfate ions in the crude proteoglycan more completely, the buffer solution is preferably replaced 5 to 7 times during the dialysis process.
In some preferred embodiments, purifying the crude proteoglycan using ethanol comprises bringing the final volume concentration of ethanol in the solution containing the crude proteoglycan to 40% to 50%, for example, but not limited to, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49% or 50%, and obtaining a precipitate after solid-liquid separation, i.e., the precipitate is the proteoglycan. When the solution containing crude proteoglycan contains ethanol with the final volume concentration of 40-50%, proteoglycan can be effectively precipitated, and the purity of non-proteoglycan substances is reduced, so that the purity of proteoglycan is improved.
In some preferred embodiments, it is preferred to add ethanol to the solution containing crude proteoglycan at a volume concentration of 85% to 95%, for example, but not limited to, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or 95%, to a final volume concentration of ethanol in the solution containing crude proteoglycan of 40% to 50%.
In some preferred embodiments, after the final volume concentration of ethanol in the solution containing crude proteoglycan is 40% to 50%, the solution is allowed to stand at 0 to 5 ℃, for example, but not limited to, 0 ℃, 1 ℃, 2 ℃, 3 ℃, 4 ℃ or 5 ℃ for 10 to 15 hours, for example, but not limited to, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours or 15 hours, under which the purity of proteoglycan in the precipitate obtained is high.
In some preferred embodiments, the ethanol purified product is dried to obtain the proteoglycan, preferably at 40-45 ℃, such as but not limited to 41 ℃, 42 ℃, 43 ℃, 44 ℃ or 45 ℃.
In some preferred embodiments, the following method is adopted to extract proteoglycan from the red algae palm leaf tree, and the effect is better:
(a) freeze drying the washed red algae palm leaf tree;
(b) crushing the freeze-dried anthurium andraeanum leaf trees by using a crusher, and sieving to obtain anthurium andraeanum leaf tree powder;
(c) adding the powder of the red algae palm leaf tree into deionized water with the weight of 20-25 times that of the red algae palm leaf tree powder, adding a wall breaking enzyme, and performing ultrasonic extraction for 7 hours;
(d) centrifuging and filtering the sample treated in the step (c) to obtain a leaching solution;
(e) adding active carbon into the leaching liquor obtained in the step (d) for decoloring;
(f) adding saturated ammonium sulfate solution into the decolorized leaching liquor in the step (e) until the concentration of the saturated ammonium sulfate solution is 70% -90%, precipitating overnight, and centrifuging to obtain crude proteoglycan precipitate;
(g) dialyzing the precipitate obtained in the step (f) by using a phosphate buffer solution with the concentration of 0.01mol/L, pH of 7, and changing the dialyzate for 5-7 times until sulfate ions do not exist in the solution;
(h) adding 85-95% ethanol into the solution to remove sulfate ions until the final concentration is 40-50%; standing for 10-15 h at 0-5 ℃, and centrifuging to obtain a precipitate which is a purified proteoglycan;
(i) and (5) drying the precipitate obtained in the step (h) at 40-45 ℃ to obtain the proteoglycan.
According to one aspect of the invention, the invention also provides an extract prepared by the proteoglycan extraction method. The extract prepared by the method has marine proteoglycan as main ingredient, has good antiinflammatory, moisturizing, antioxidant, and melanin inhibiting effects, and can be used for preparing cosmetics.
According to one aspect of the invention, the invention also provides the proteoglycan extraction method or the application of the extract in preparing cosmetics, which can endow the cosmetics with good anti-inflammatory, moisturizing, antioxidant, melanin inhibiting and the like. And because the proteoglycan extraction method has wide raw material sources and low cost, the proteoglycan extraction method or the extract can be applied to the preparation of cosmetics, so that the production cost of the cosmetics can be reduced.
According to one aspect of the present invention, there is also provided a cosmetic comprising the above extract. The extract can be used as main active ingredient in cosmetic, or used as auxiliary active ingredient of cosmetic to cooperate with other active ingredients. The cosmetic containing the extract has good effects of whitening, repairing, delaying oxidation, brightening skin color, tightening, moistening, moisturizing and moisturizing. The cosmetic can be, but not limited to, various skin care products, color cosmetics or cosmeceuticals for cleaning, maintaining, beautifying, decorating, changing appearance and the like; the cosmetic can be, but not limited to, moisturizer, skin lotion, skin essence, skin cream, facial cleanser, facial mask, etc.
The technical solution and the advantages of the present invention will be further explained with reference to the preferred embodiments.
Example 1
The embodiment provides a method for extracting proteoglycan, which comprises the following steps:
(a) collecting appropriate amount of red algae palm leaf tree from sea area, washing with tap water, removing attachments, and freeze drying;
(b) crushing the freeze-dried anthurium andraeanum leaf trees by using a crusher, and sieving to obtain anthurium andraeanum leaf tree powder;
(c) weighing red algae palm leaf tree powder, adding the red algae palm leaf tree powder into deionized water with the weight of 25 times, adding wall breaking enzyme with the weight of 0.04 time of the red algae palm leaf tree powder, wherein the wall breaking enzyme contains 40% of cellulase, 40% of hemicellulase and 20% of pectinase according to mass percentage, and carrying out ultrasonic extraction for 7 hours;
(d) centrifuging and filtering the sample treated in the step (c) to obtain a leaching solution;
(e) adding active carbon into the leaching liquor obtained in the step (d) for decoloring;
(f) adding a saturated ammonium sulfate solution into the decolorized leaching liquor in the step (e) until the concentration of the saturated ammonium sulfate solution is 80%, precipitating for 12 hours, and centrifuging to obtain a crude proteoglycan precipitate;
(g) dialyzing the precipitate obtained in the step (f) by using a phosphate buffer solution with the concentration of 0.01mol/L, pH of 7, and replacing the dialyzate for 6 times until sulfate ions are not contained in the solution;
(h) adding 90% ethanol to the solution from which sulfate ions are to be removed until the final concentration is 45%; standing at 4 ℃ for 12h, and centrifuging to obtain a precipitate which is a purified proteoglycan;
(i) drying the precipitate obtained in step (h) at 45 ℃ to obtain proteoglycan.
Example 2
The embodiment provides a method for extracting proteoglycan, which comprises the following steps:
(a) collecting appropriate amount of red algae palm leaf tree from sea area, washing with tap water, removing attachments, and freeze drying;
(b) crushing the freeze-dried anthurium andraeanum leaf trees by using a crusher, and sieving to obtain anthurium andraeanum leaf tree powder;
(c) weighing the powder of the red algae palm leaf trees, adding the powder into deionized water with the weight being 20 times that of the red algae palm leaf trees, adding wall breaking enzyme with the weight being 0.07 time that of the powder of the red algae palm leaf trees, wherein the wall breaking enzyme contains 25% of cellulase, 45% of hemicellulase and 30% of pectinase according to mass percentage, and carrying out ultrasonic extraction for 6 hours;
(d) centrifuging and filtering the sample treated in the step (c) to obtain a leaching solution;
(e) adding active carbon into the leaching liquor obtained in the step (d) for decoloring;
(f) adding a saturated ammonium sulfate solution into the decolorized leaching liquor in the step (e) until the concentration of the saturated ammonium sulfate solution is 90%, precipitating for 18h, and centrifuging to obtain a crude proteoglycan precipitate;
(g) dialyzing the precipitate obtained in the step (f) by using a phosphate buffer solution with the concentration of 0.01mol/L, pH of 7, and replacing the dialyzate for 7 times until sulfate ions are not contained in the solution;
(h) adding 85% ethanol to the solution from which sulfate ions are to be removed until the final concentration is 50%; standing at 4 ℃ for 15h, and centrifuging to obtain a precipitate which is a purified proteoglycan;
(i) drying the precipitate obtained in step (h) at 40 ℃ to obtain proteoglycan.
Example 3
The embodiment provides a method for extracting proteoglycan, which comprises the following steps:
(a) collecting appropriate amount of red algae palm leaf tree from sea area, washing with tap water, removing attachments, and freeze drying;
(b) crushing the freeze-dried anthurium andraeanum leaf trees by using a crusher, and sieving to obtain anthurium andraeanum leaf tree powder;
(c) weighing the powder of the red algae palm leaf trees, adding the powder into deionized water with the weight being 20 times that of the red algae palm leaf trees, adding wall breaking enzyme with the weight being 0.03 time that of the powder of the red algae palm leaf trees, wherein the wall breaking enzyme contains 50% of cellulase, 30% of hemicellulase and 20% of pectinase according to mass percentage, and carrying out ultrasonic extraction for 8 hours;
(d) centrifuging and filtering the sample treated in the step (c) to obtain a leaching solution;
(e) adding active carbon into the leaching liquor obtained in the step (d) for decoloring;
(f) adding a saturated ammonium sulfate solution into the decolorized leaching liquor in the step (e) until the concentration of the saturated ammonium sulfate solution is 70%, precipitating for 12 hours, and centrifuging to obtain a crude proteoglycan precipitate;
(g) dialyzing the precipitate obtained in the step (f) by using a phosphate buffer solution with the concentration of 0.01mol/L, pH of 7, and replacing the dialyzate for 5 times until sulfate ions are not contained in the solution;
(h) adding 95% ethanol to the solution from which sulfate ions are to be removed until the final concentration is 40%; standing at 0 ℃ for 15h, and centrifuging to obtain a precipitate which is a purified proteoglycan;
(i) drying the precipitate obtained in step (h) at 40 ℃ to obtain proteoglycan.
Example 4
The embodiment provides a method for extracting proteoglycan, which comprises the following steps:
(a) collecting appropriate amount of red algae palm leaf tree from sea area, washing with tap water, removing attachments, and freeze drying;
(b) crushing the freeze-dried anthurium andraeanum leaf trees by using a crusher, and sieving to obtain anthurium andraeanum leaf tree powder;
(c) weighing red algae palm leaf tree powder, adding the red algae palm leaf tree powder into deionized water with the weight of 30 times, adding wall breaking enzyme with the weight of 0.05 time of the red algae palm leaf tree powder, wherein the wall breaking enzyme contains 20% of cellulase, 50% of hemicellulase and 30% of pectinase according to mass percentage, and carrying out ultrasonic extraction for 5 hours;
(d) centrifuging and filtering the sample treated in the step (c) to obtain a leaching solution;
(e) adding active carbon into the leaching liquor obtained in the step (d) for decoloring;
(f) adding a saturated ammonium sulfate solution into the decolorized leaching liquor in the step (e) until the concentration of the saturated ammonium sulfate solution is 60%, precipitating for 12 hours, and centrifuging to obtain a crude proteoglycan precipitate;
(g) dialyzing the precipitate obtained in the step (f) by using a phosphate buffer solution with the concentration of 0.01mol/L, pH of 7, and replacing the dialyzate for 5 times until sulfate ions are not contained in the solution;
(h) adding 95% ethanol to the solution from which sulfate ions are to be removed until the final concentration is 60%; standing at 0 ℃ for 5h, and centrifuging to obtain a precipitate which is a purified proteoglycan;
(i) drying the precipitate obtained in step (h) at 45 ℃ to obtain proteoglycan.
Example 5
The embodiment provides a method for extracting proteoglycan, which comprises the following steps:
(a) collecting appropriate amount of red algae palm leaf tree from sea area, washing with tap water, removing attachments, and freeze drying;
(b) crushing the freeze-dried anthurium andraeanum leaf trees by using a crusher, and sieving to obtain anthurium andraeanum leaf tree powder;
(c) weighing red algae palm leaf tree powder, adding the red algae palm leaf tree powder into deionized water with the weight 15 times that of the red algae palm leaf tree powder, adding wall breaking enzyme with the weight 0.02 time that of the red algae palm leaf tree powder, wherein the wall breaking enzyme contains 20% of cellulase, 50% of hemicellulase and 30% of pectinase according to mass percentage, and performing ultrasonic extraction for 10 hours;
(d) centrifuging and filtering the sample treated in the step (c) to obtain a leaching solution;
(e) adding active carbon into the leaching liquor obtained in the step (d) for decoloring;
(f) adding a saturated ammonium sulfate solution into the decolorized leaching liquor in the step (e) until the concentration of the saturated ammonium sulfate solution is 90%, precipitating for 5 hours, and centrifuging to obtain a crude proteoglycan precipitate;
(g) dialyzing the precipitate obtained in the step (f) by using a phosphate buffer solution with the concentration of 0.01mol/L, pH of 7, and replacing the dialyzate for 5 times until sulfate ions are not contained in the solution;
(h) adding 95% ethanol to the solution from which sulfate ions are to be removed until the final concentration is 30%; standing at 0 ℃ for 20h, and centrifuging to obtain a precipitate which is a purified proteoglycan;
(i) drying the precipitate obtained in step (h) at 45 ℃ to obtain proteoglycan.
Example 6
This example provides a proteoglycan extraction method, which is different from example 1 in that the leaching solution is not decolored using activated carbon.
Example 7
This example provides a method for extracting proteoglycan, which is different from example 1 in that sulfate ions are removed without dialysis and ethanol precipitation is directly used.
Example 8
This example provides a method for extracting proteoglycan, which is different from example 1 in that the undried red algae palm leaf tree is directly crushed.
Comparative example 1
This comparative example provides a proteoglycan extraction method, which is different from example 1 in that a wall-breaking enzyme is not used in the ultrasonic extraction process.
Comparative example 2
This comparative example provides a proteoglycan extraction method, which is different from example 1 in that the extraction method does not include a step of ethanol purification, i.e., the precipitate obtained is directly dried after performing step (f).
Comparative example 3
This comparative example provides a proteoglycan extraction method, which is different from example 1 in that the decolorized leaching solution is directly precipitated using ethanol, that is, step (e) is performed and then step (h) is directly performed.
Examples of effects
Qualitative analysis of proteoglycans prepared in examples 1 to 7 and comparative examples 1 to 3 was performed by biuret reaction, CTAB reaction, feilin reagent detection, and iodine-potassium iodide reaction, and the results are shown in table 1:
TABLE 1
In the above experiment, the biuret reaction was positive, indicating that the product contained protein; the positive CTAB reaction indicates that the product contains acidic polysaccharide; the detection result of the Fehling reagent is negative, which indicates that the obtained product does not contain free polysaccharide substances; the negative iodine-potassium iodide reaction indicates that the obtained product does not contain starch substances.
As can be seen from the comparison between the above examples 1-3 and examples 4-5, the optimization of the process parameters can improve the extraction effect of proteoglycan; as can be seen from the comparison between example 1 and example 6, the effect of extracting proteoglycan can be improved by carrying out the decoloring treatment on the leaching solution; as can be seen from the comparison between example 1 and example 7, after the leaching liquor is precipitated by ammonium sulfate, the residual sulfate ions are removed, so that the extraction efficiency of proteoglycan can be further improved; as can be seen from the comparison of example 1 and comparative example 1, the absence of the use of the wall-breaking enzyme results in a significant reduction in the extraction effect of proteoglycan; as can be seen from the comparison between example 1 and comparative examples 2-3, the proteoglycan with higher purity can be effectively obtained by fractional precipitation using ammonium sulfate and ethanol.
The related quantitative detection data calculation formula is as follows:
(1) quantitative correlation of proteins
a. Protein standard curves are plotted, and the protein standard curves are shown in figure 1.
Wherein, the regression equation y is 0.6119x +0.075 (R)2＝0.9994)。
b. Purity determination formula:
the mass of the glycoprotein is (cxV × M)/M
Wherein, c: calculating the concentration of mg/mL by using a standard curve equation; v: sample volume, mL; m: total crude glycoprotein mass; m: the quality of the powder of the red algae palm leaf tree is improved.
Glycoprotein purity:
the purity of glycoprotein is the mass of the extracted glycoprotein/the mass of the powder of the red alga palm leaf tree is multiplied by 100 percent;
(2) the extraction rate (%). ratio of extracted proteoglycan mass/extracted powder of red algae palm leaf tree powder mass x 100%.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (7)
1. A method for extracting proteoglycan, comprising: ultrasonically leaching red algae palm leaf trees in the presence of a wall-breaking enzyme to obtain a leaching solution, then precipitating the leaching solution by using ammonium sulfate to obtain crude proteoglycan, and purifying the crude proteoglycan by using ethanol to obtain proteoglycan; the wall-breaking enzyme comprises cellulase, hemicellulase and pectinase;
the method specifically comprises the following steps:
(a) freeze drying the washed red algae palm leaf tree;
(b) crushing the freeze-dried anthurium andraeanum leaf trees by using a crusher, and sieving to obtain anthurium andraeanum leaf tree powder;
(c) adding the powder of the red algae palm leaf tree into deionized water with the weight of 20-25 times that of the red algae palm leaf tree powder, adding a wall breaking enzyme, and performing ultrasonic extraction for 7 hours;
(d) centrifuging and filtering the sample treated in the step (c) to obtain a leaching solution;
(e) adding active carbon into the leaching liquor obtained in the step (d) for decoloring;
(f) adding a saturated ammonium sulfate solution into the decolorized leaching liquor in the step (e) until the concentration of the saturated ammonium sulfate solution is 70% -90%, precipitating for 12-18 h, and centrifuging to obtain a crude proteoglycan precipitate;
(g) dialyzing the precipitate obtained in the step (f) by using a phosphate buffer solution with the concentration of 0.01mol/L, pH of 7, and changing the dialyzate for 5-7 times until sulfate ions do not exist in the solution;
(h) adding 85-95% ethanol into the solution from which the sulfate ions are removed until the final concentration is 40-50%; standing for 10-15 h at 0-5 ℃, and centrifuging to obtain a precipitate which is a purified proteoglycan;
(i) and (5) drying the precipitate obtained in the step (h) at 40-45 ℃ to obtain the proteoglycan.
2. The extraction method according to claim 1,
the mass percent of cellulase in the wall-breaking enzyme is 25-50%, the mass percent of hemicellulase is 30-45%, and the balance is pectinase.
3. The extraction method according to claim 1, wherein the amount of the wall-breaking enzyme is 0.03-0.07 times of the weight of the red algae palm leaf tree.
4. The extraction method according to claim 1, wherein the amount of the wall-breaking enzyme is 0.04 times of the weight of the red algae palm leaf tree.
5. An extract prepared by the proteoglycan extraction method of any one of claims 1 to 4.
6. Use of the proteoglycan extraction method of any one of claims 1 to 4 or the extract of claim 5 for preparing cosmetics.
7. A cosmetic comprising the extract according to claim 5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910954373.5A CN110627863B (en) | 2019-10-09 | 2019-10-09 | Proteoglycan extraction method, proteoglycan extract, application of proteoglycan extract and cosmetic |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910954373.5A CN110627863B (en) | 2019-10-09 | 2019-10-09 | Proteoglycan extraction method, proteoglycan extract, application of proteoglycan extract and cosmetic |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110627863A CN110627863A (en) | 2019-12-31 |
| CN110627863B true CN110627863B (en) | 2021-07-06 |
Family
ID=68975907
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910954373.5A Active CN110627863B (en) | 2019-10-09 | 2019-10-09 | Proteoglycan extraction method, proteoglycan extract, application of proteoglycan extract and cosmetic |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110627863B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112341548B (en) * | 2020-11-05 | 2021-09-24 | 江南大学 | Processing method and application of grain-derived polysaccharide with high moisturizing effect |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1206587A (en) * | 1997-07-29 | 1999-02-03 | 张许昌 | Natural spiroalgae cosmetic for skin and hair cleaning and protection |
| CN1660897A (en) * | 2004-12-16 | 2005-08-31 | 集美大学 | Method for extracting and purifying glycoprotein shuck |
| CN1844259A (en) * | 2006-04-29 | 2006-10-11 | 大连大学 | Method for integrated extraction of active substances in Platymonas |
| CN101760492A (en) * | 2008-12-24 | 2010-06-30 | 徐宝贞 | Method for extracting algae glycoproteins |
| CN103951737A (en) * | 2014-04-22 | 2014-07-30 | 钦州市山海生物科技有限公司 | Method for extracting glycotropic protein from seaweed |
| WO2015004255A1 (en) * | 2013-07-12 | 2015-01-15 | Danmarks Tekniske Universitet | Isolation of glycoproteins from brown algae |
| CN109608559A (en) * | 2019-01-15 | 2019-04-12 | 广东海洋大学 | A method of extracting active polysaccharide from seaweed |
| CN109953905A (en) * | 2019-05-14 | 2019-07-02 | 深圳市仙迪化妆品有限公司 | A kind of preparation method of the cosmetics of chitosan containing algae |
| CN110256542A (en) * | 2019-06-03 | 2019-09-20 | 中国海洋大学 | A kind of preparation method of red algae phycoerythrin |
-
2019
- 2019-10-09 CN CN201910954373.5A patent/CN110627863B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1206587A (en) * | 1997-07-29 | 1999-02-03 | 张许昌 | Natural spiroalgae cosmetic for skin and hair cleaning and protection |
| CN1660897A (en) * | 2004-12-16 | 2005-08-31 | 集美大学 | Method for extracting and purifying glycoprotein shuck |
| CN1844259A (en) * | 2006-04-29 | 2006-10-11 | 大连大学 | Method for integrated extraction of active substances in Platymonas |
| CN101760492A (en) * | 2008-12-24 | 2010-06-30 | 徐宝贞 | Method for extracting algae glycoproteins |
| WO2015004255A1 (en) * | 2013-07-12 | 2015-01-15 | Danmarks Tekniske Universitet | Isolation of glycoproteins from brown algae |
| CN103951737A (en) * | 2014-04-22 | 2014-07-30 | 钦州市山海生物科技有限公司 | Method for extracting glycotropic protein from seaweed |
| CN109608559A (en) * | 2019-01-15 | 2019-04-12 | 广东海洋大学 | A method of extracting active polysaccharide from seaweed |
| CN109953905A (en) * | 2019-05-14 | 2019-07-02 | 深圳市仙迪化妆品有限公司 | A kind of preparation method of the cosmetics of chitosan containing algae |
| CN110256542A (en) * | 2019-06-03 | 2019-09-20 | 中国海洋大学 | A kind of preparation method of red algae phycoerythrin |
Non-Patent Citations (3)
| Title |
|---|
| Nutritional value of proteins from edible seaweed Palmaria palmata(dulse);Anne Valerie Galland Irmouli等;《The Journal of Nutritional Biochemistry》;19990607;第10卷(第6期);353-359 * |
| 海洋动物源性糖蛋白研究进展;于志鹏等;《食品工业科技》;20160323;第37卷(第13期);第3.5节、第379页左栏第4段 * |
| 食源性植物糖蛋白研究进展;赵文竹等;《食品工业科技》;20160426;第37卷(第16期);389-395 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110627863A (en) | 2019-12-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103141895B (en) | Natural marine animal peptide beautifying drink | |
| CN103757084B (en) | A kind of rice wine vinasse extract and its preparation method and application | |
| KR100594342B1 (en) | Cosmetics | |
| CN104726527A (en) | Preparation technology for producing collagen through enzymolysis of fish scales | |
| CN103445159A (en) | Enzymatic preparation method of all-component ginseng juice | |
| JP6016878B2 (en) | Proteoglycan production method | |
| CN105663021B (en) | A kind of glutathione fermentation facial mask and preparation method thereof | |
| CN102961297A (en) | Novel moisturizing and ageing resisting skin care product containing natural tremella polysaccharide | |
| EP1074262A1 (en) | Laminaria algae extract, method of preparation and cosmetic or pharmaceutical compositions containing the same | |
| CN104862366B (en) | A kind of technique from black sharkskin extraction collagen peptide, dermatan sulfate, hydroxyapatite and melanin | |
| CN114557927B (en) | Yeast essence water composition, yeast essence water, and preparation method and application thereof | |
| KR101025292B1 (en) | Fermentation Condiment of Sea-Tangle Extract by Yeast and Preparation Method Thereof | |
| CN104558115A (en) | Antioxidant polypeptide with Raja porosa meat protein as well as preparation method and application of antioxidant polypeptide | |
| CN110627863B (en) | Proteoglycan extraction method, proteoglycan extract, application of proteoglycan extract and cosmetic | |
| CN101225421A (en) | Novel process for extracting golden fungus polysaccharides by step enzyme method | |
| WO2018105919A2 (en) | Pomegranate beverage containing marine collagen and method for manufacturing same | |
| CN104055702B (en) | A kind of skin antiallergic wetting agent | |
| CN101427819A (en) | Holothurian essence and method of producing the same | |
| CN104725466A (en) | Method for extracting earthworm active protein for cosmetics | |
| CN101302251B (en) | Cyanea-originated glucoprotein having immunopotentiation activity, preparation and use thereof | |
| KR102099573B1 (en) | Manufacturing method of fermented product of fucoidan for functional cosmetics having good whitening function and anti-wrinkling function | |
| KR101989934B1 (en) | Method of preparing Cocoon Sericin extract, and composition including the same | |
| CN111437242A (en) | Crystal mask prepared from jellyfish | |
| KR101880056B1 (en) | Cosmetic composition containing Chojeong carbonated water for hair damage prevention | |
| CN105061623A (en) | Mulberry polysaccharide preparation method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |