CN112314770A - Method for simultaneously preparing polysaccharide and protein from sparassis crispa - Google Patents
Method for simultaneously preparing polysaccharide and protein from sparassis crispa Download PDFInfo
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- CN112314770A CN112314770A CN202011071580.5A CN202011071580A CN112314770A CN 112314770 A CN112314770 A CN 112314770A CN 202011071580 A CN202011071580 A CN 202011071580A CN 112314770 A CN112314770 A CN 112314770A
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- sparassis crispa
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/006—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from vegetable materials
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
Abstract
The invention discloses a method for simultaneously preparing polysaccharide and protein from sparassis crispa, which comprises the following steps: s1, crushing dry sparassis crispa sporocarp, adding an ammonium sulfate solution and tert-butyl alcohol, and mixing; s2, uniformly mixing the mixture, placing the mixture in a bipolar square wave high-voltage pulse electric field for extraction, and collecting an extracting solution; s3, after the pulsed electric field extraction, carrying out microwave extraction on the mixed solution; s4, after the microwave extraction is finished, centrifuging the mixed solution; s5, after the centrifugation is finished, placing the supernatant in a shaking table for oscillation; s6, standing after oscillation is finished, dividing the mixed solution into three layers, adding tert-butyl alcohol on the uppermost layer, evaporating and recycling, adding dichloromethane into the protein layer on the middle layer for extraction, then carrying out rotary drying to remove dichloromethane to obtain the sparassis crispa protein, and dialyzing the lowermost layer to obtain the sparassis crispa polysaccharide. The method can simultaneously and efficiently prepare the Sparassis crispa polysaccharide and protein.
Description
Technical Field
The invention relates to the field of sparassis crispa processing, and particularly relates to a method for simultaneously preparing polysaccharide and protein from sparassis crispa.
Background
Sparassis Crispa (Sparassis Crispa) is a precious fungus used as both medicine and food, and shares the reputation of "king of Wangu mushroom", modern researches show that Sparassis Crispa fruiting bodies are rich in proteins, polysaccharides, ergosterol, glucosylceramide, adenosine and other small molecular compounds and the like; the Sparassis crispa contains a large amount of beta-glucan, antioxidant substances, vitamins and minerals, contains superoxide dismutase and vitamin E, is used for treating prostate diseases, and has various pharmacological activities of improving immunity, resisting tumor, resisting hypertension, resisting diabetes and the like.
Research shows that the protein content in the sparassis crispa fruiting body is 13.4%, and the crude fat content is lower than that of common edible fungi. In the deep processing process of the sparassis crispa, polysaccharide extraction is generally used as a main raw material, and protein is generally treated as waste, so that the extraction cost is increased, and a large amount of sparassis crispa resources are wasted.
For example, chinese patent document CN110776582A discloses a method for extracting β -glucan from sparassis crispa, which comprises the following steps: s1: drying, crushing and grinding Sparassis crispa to obtain superfine powder; s2: adding a wall breaking enzyme agent into the superfine powder, and performing ultrasonic enzymolysis to obtain a free liquid; s3: adding the free liquid into a high-pressure microwave tank, performing high-pressure microwave extraction, and filtering to obtain an extracting solution; s4: adding an enzyme removing agent into the extracting solution, stirring, standing, and centrifuging to obtain a supernatant; s5: adding ammonium sulfate into the supernatant, dissolving, adding ethanol, centrifuging, and collecting precipitate to obtain crude extract; s6: dissolving the crude extract, and drying to obtain the beta-glucan. To the best of the inventors, the patent document adds ammonium sulfate and ethanol to the precipitate collected by centrifugation, and then dissolves and dries the precipitate to obtain beta-glucan. This patent document describes: the method comprises the steps of adding an amylase and a protease into a raw material, and carrying out enzymolysis on the raw material to remove starch, protein and other polysaccharides. Then precipitating and centrifuging by using ethanol to obtain the beta-glucan.
Chinese patent document CN110835380A discloses a microwave-assisted process for extracting sparassis crispa polysaccharide, which comprises the following steps: dehydrating fresh sparassis crispa in a physical extrusion mode; crushing the dehydrated sparassis crispa; adding cellulase and pure water into pulverized Sparassis crispa to obtain mixed solution, adding pH regulator to adjust pH to 4.5-5.5, and performing microwave treatment to obtain Sparassis crispa crude polysaccharide extract; and (3) inactivating enzyme, concentrating, precipitating with ethanol, and drying the Sparassis crispa crude polysaccharide extract to obtain Sparassis crispa crude polysaccharide. The invention adopts a mode of enzymolysis and microwave assistance to extract polysaccharide.
Chinese patent document CN110606899A discloses a method for extracting Sparassis crispa polysaccharide by enzymolysis, which comprises the following steps: 1) raw material degreasing pretreatment: taking a proper amount of dried sparassis crispa, and crushing the sparassis crispa into 300 meshes; soaking the powder in 80-100% ethanol solution (v/w) with the volume 5 times that of the powder; distilling, condensing, refluxing and filtering the soak solution to obtain residues, and placing the residues in a shade place for air drying; 2) hot water enzymolysis and leaching: adding 1-15% of enzyme (w/w) and 20-60 times of distilled water (v/w) into the residue obtained in the step (1), uniformly stirring to obtain a mixed solution, standing the mixed solution at the temperature of 20-60 ℃ for 2-10 h for enzymolysis, performing centrifugal treatment on an enzymolysis solution to separate solid from liquid, and filtering solid residue to obtain a filtrate; repeatedly leaching for 2-3 times, and mixing the above filtrates to obtain Sparassis crispa extractive solution; 3) ethanol precipitation and centrifugation: concentrating, inactivating and cooling the sparassis crispa extracting solution obtained in the step 2; adding a 95% ethanol solution with the volume 5 times that of the mixture, uniformly stirring, precipitating at the temperature of-4 ℃ for 8-12 hours, centrifuging, removing supernate, and collecting precipitate; 4) vacuum freeze drying: and (4) freeze-drying the precipitate collected in the step (3) in a vacuum environment to obtain the crude polysaccharide of the sparassis crispa. Wherein the enzyme in the step 2) comprises one or more of amylase, pectinase, glucoamylase and papain. The invention is that after the sparassis crispa is extracted and degreased by ethanol, the sparassis crispa crude polysaccharide is obtained by water enzymolysis extraction, ethanol precipitation and vacuum freeze-drying.
Chinese patent document CN110981987A discloses a process for extracting sparassis crispa polysaccharide by an ultrasonic-assisted method, which comprises the following steps: dehydrating fresh sparassis crispa in a physical extrusion mode; crushing the dehydrated sparassis crispa; adding cellulase, pure water and pH regulator into pulverized Sparassis crispa, adjusting pH to 4.5-5.5, and performing enzymolysis to obtain enzymolysis solution; carrying out ultrasonic treatment on the enzymolysis liquid to obtain a crude polysaccharide extracting solution of the sparassis crispa; and (3) inactivating enzyme, concentrating, precipitating with ethanol, and drying the Sparassis crispa crude polysaccharide extract to obtain Sparassis crispa crude polysaccharide. The invention adopts a mode of enzymolysis matched with ultrasonic assistance to extract polysaccharide.
Chinese patent document CN104448014A discloses a method for extracting alkali-soluble polysaccharides from sparassis crispa, which comprises the following steps: (1) alkali-soluble crude polysaccharide extraction is carried out by adding water into sparassis crispa sporophore powder for swelling overnight, extracting for 3 times in water bath for 2 hours, centrifuging, breaking cell wall of obtained residue by a cell crusher for 10min, extracting for 3 times by 0.1mol/mL sodium hydroxide for 2 hours, centrifuging again, mixing supernate, neutralizing by 0.1mol/mL hydrochloric acid, concentrating to 10mL, carrying out alcohol precipitation on concentrated solution by 3 times volume of absolute ethyl alcohol for overnight centrifugation, adding absolute ethyl alcohol and ethyl ether into precipitate for washing, centrifuging, and drying the precipitate to obtain the alkali-soluble crude polysaccharide. (2) Purifying and accurately weighing the alkali-soluble crude polysaccharide, preparing 5% sugar solution by using distilled water, adding 10% trichloroacetic acid to adjust the pH value to 3, standing overnight, centrifuging for 10min at 5000r/min, adding 95% ethanol with 4 times of volume into the supernatant, standing overnight, centrifuging, adding absolute ethyl alcohol and diethyl ether into the precipitate, washing, and drying in a vacuum drier to obtain the deproteinized alkali-soluble polysaccharide.
In the existing method for extracting the effective components from the sparassis crispa, polysaccharide is basically extracted, and protein is directly removed in the extraction preparation process, for example, the protein is directly subjected to enzymolysis by using enzyme. No method for simultaneously preparing polysaccharide and protein from Sparassis crispa was found.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for simultaneously preparing polysaccharides and proteins from Sparassis crispa, and simultaneously, efficiently preparing Sparassis crispa polysaccharides and proteins.
The adopted technical scheme is as follows:
a method for simultaneously preparing polysaccharide and protein from sparassis crispa comprises the following steps:
s1, crushing dry sparassis crispa sporocarp, adding an ammonium sulfate solution and tert-butyl alcohol, and mixing;
s2, uniformly mixing the mixture, placing the mixture in a bipolar square wave high-voltage pulse electric field for extraction, and collecting an extracting solution;
s3, after the pulsed electric field extraction, carrying out microwave extraction on the mixed solution;
s4, after the microwave extraction is finished, centrifuging the mixed solution;
s5, after the centrifugation is finished, placing the supernatant in a shaking table for oscillation;
s6, standing after oscillation is finished, dividing the mixed solution into three layers, adding tert-butyl alcohol on the uppermost layer, evaporating and recycling, adding dichloromethane into the protein layer on the middle layer for extraction, then carrying out rotary drying to remove dichloromethane to obtain the sparassis crispa protein, and dialyzing the lowermost layer to obtain the sparassis crispa polysaccharide.
Further, in S1, a 10 to 20 times by weight ammonium sulfate solution having a mass concentration of 20% was added.
Further, in S1, adding a mixture of the S1 and the Sparassis crispa in a mass-volume ratio of 1: 15-20 of tert-butanol.
Further, in S1, the pH of the mixture was adjusted to 6 with a pH adjuster.
Further, the pH regulator is a citric acid solution or a formic acid solution.
Further, in S2, the intensity of the pulse electric field extracted by the pulse electric field is 10-50Kv/cm, and the frequency is 200-1000 Hz.
Further, in S3, the power of microwave extraction is 200-500W, and the time is 5-10 min.
Further, in S5, the supernatant was put on a shaker at 20-30 ℃ for 50-70 minutes with shaking at 100 rpm.
Compared with the prior art, the extraction and separation process combines a pulse electric field, microwaves and a two-aqueous-phase system to prepare the Sparassis crispa polysaccharide and protein at one time, and has the following beneficial effects:
1. the function of the ammonium sulfate is mainly to interact with protein in sparassis crispa and promote salting out and collection of the protein.
2. The tertiary butanol is used as a synergist of the invention to make the protein layer of the aqueous two-phase more stable.
3. The method combines a pulse electric field, microwaves and a two-aqueous-phase system, simultaneously efficiently prepares the Sparassis crispa polysaccharide and protein, and has simple process and high extraction rate.
Detailed Description
The present invention is further illustrated by the following examples and comparative examples, but it should not be construed that the scope of the above subject matter is limited to the following examples, and all the technologies realized based on the above contents of the present invention are within the scope of the present invention.
Example 1
A method for simultaneously preparing polysaccharide and protein from sparassis crispa comprises the following steps:
(1) pulverizing 10g of dried Sparassis crispa fruiting body, adding 10 weight times of (NH) with mass concentration of 20%4)2SO4Adding a solution into the mixture, wherein the mass volume ratio of the solution to the sparassis crispa is 1: 15, tert-butanol, pH 6 adjusted with formic acid solution;
(2) mixing the mixture uniformly, placing the mixture in a bipolar square wave high-voltage pulse electric field for extraction, wherein the intensity of the pulse electric field is 10Kv/cm, the frequency is 200Hz, and collecting an extracting solution;
(3) performing microwave extraction on the mixed solution after the pulsed electric field extraction for 5min at the power of 200W;
(4) after extraction, centrifuging the mixed solution;
(5) after the centrifugation is finished, putting the supernatant into a shaking table, and oscillating at 28 ℃ and 100rpm for 60 minutes;
(6) after the oscillation is finished, standing, dividing the mixed solution into three layers, wherein the uppermost layer is tert-butyl alcohol, evaporating and recycling, the middle layer is a protein layer, adding dichloromethane for extraction, then carrying out rotary drying to remove dichloromethane, so as to obtain 0.85g of sparassis crispa protein, and the lowermost layer is dialyzed and freeze-dried to obtain 1.67g of sparassis crispa polysaccharide.
Example 2
A method for simultaneously preparing polysaccharide and protein from sparassis crispa comprises the following steps:
(1) pulverizing 10g of dried Sparassis crispa fruiting body, adding 15 weight times of (NH) with mass concentration of 20%4)2SO4Adding a solution into the mixture, wherein the mass volume ratio of the solution to the sparassis crispa is 1: 17, tert-butanol, adjusting the pH to 6 with a proper amount of formic acid solution;
(2) mixing the mixture uniformly, placing the mixture in a bipolar square wave high-voltage pulse electric field for extraction, wherein the intensity of the pulse electric field is 30Kv/cm, the frequency is 400Hz, and collecting an extracting solution;
(3) performing microwave extraction on the mixed solution after the pulsed electric field extraction, wherein the power is 300W, and extracting for 6 min;
(4) after extraction, centrifuging the mixed solution;
(5) after the centrifugation is finished, putting the supernatant into a shaking table, and oscillating at 28 ℃ and 100rpm for 60 minutes;
(6) after the oscillation is finished, standing, dividing the mixed solution into three layers, wherein the uppermost layer is tert-butyl alcohol, evaporating and recycling, the middle layer is a protein layer, adding dichloromethane for extraction, then carrying out rotary drying to remove dichloromethane, so as to obtain 0.99g of sparassis crispa protein, and the lowermost layer is dialyzed and freeze-dried to obtain 2.23g of sparassis crispa polysaccharide.
Example 3
A method for simultaneously preparing polysaccharide and protein from sparassis crispa comprises the following steps:
(1) pulverizing 10g of dry Sparassis crispa fruiting body, adding 18 weight times of (NH) with mass concentration of 20%4)2SO4Adding a solution into the mixture, wherein the mass volume ratio of the solution to the sparassis crispa is 1: 19, adjusting the pH value to 6 by using a proper amount of formic acid solution;
(2) mixing the mixture uniformly, placing the mixture in a bipolar square wave high-voltage pulse electric field for extraction, wherein the intensity of the pulse electric field is 40Kv/cm, the frequency is 600Hz, and collecting an extracting solution;
(3) performing microwave extraction on the mixed solution after the pulsed electric field extraction, wherein the power is 400W, and extracting for 8 min;
(4) after extraction, centrifuging the mixed solution;
(5) after the centrifugation is finished, putting the supernatant into a shaking table, and oscillating at 28 ℃ and 100rpm for 60 minutes;
(6) after the oscillation is finished, standing, dividing the mixed solution into three layers, wherein the uppermost layer is tert-butyl alcohol, evaporating and recycling, the middle layer is a protein layer, adding dichloromethane for extraction, then carrying out rotary drying to remove dichloromethane, so as to obtain 1.02g of sparassis crispa protein, and the lowermost layer is dialyzed and freeze-dried to obtain 2.47g of sparassis crispa polysaccharide.
Example 4
A method for simultaneously preparing polysaccharide and protein from sparassis crispa comprises the following steps:
(1) pulverizing 10g of dry Sparassis crispa fruiting body, adding 20 weight times of (NH) with mass concentration of 20%4)2SO4Adding a solution into the mixture, wherein the mass volume ratio of the solution to the sparassis crispa is 1: 20 of tert-butanol, the pH of which is adjusted to 6 by using a proper amount of formic acid solution;
(2) mixing the mixture uniformly, placing the mixture in a bipolar square wave high-voltage pulse electric field for extraction, wherein the intensity of the pulse electric field is 50Kv/cm, the frequency is 900Hz, and collecting an extracting solution;
(3) performing microwave extraction on the mixed solution after the pulsed electric field extraction, wherein the power is 500W, and extracting for 10 min;
(4) after extraction, centrifuging the mixed solution;
(5) after the centrifugation is finished, putting the supernatant into a shaking table, and oscillating at 28 ℃ and 100rpm for 60 minutes;
(6) after the oscillation is finished, standing, dividing the mixed solution into three layers, wherein the uppermost layer is tert-butyl alcohol, evaporating and recycling, the middle layer is a protein layer, adding dichloromethane for extraction, then carrying out rotary drying to remove dichloromethane, so as to obtain 1.39g of sparassis crispa protein, and the lowermost layer is dialyzed and freeze-dried to obtain 2.88g of sparassis crispa polysaccharide.
Comparative example 1
Referring to example 1, in contrast to example 1, No (NH) was added in an amount of 18 times by weight to give a mass concentration of 20%4)2SO4Solution, and as a result, an intermediate protein layer was not effectively obtained. The effect of ammonium sulfate is mainly to interact with protein in sparassis crispa and promote salting out and collection of protein. If ammonium sulfate is not added, no protein is obtained.
Comparative example 2
Referring to example 1, in contrast to example 1, no reaction mixture was added to the reaction mixture in a mass to volume ratio of 1: 15, the intermediate protein layer was not apparent. The tertiary butanol is used as a synergist of the invention to make the protein layer of the aqueous two-phase more stable.
The above-listed detailed description is only a specific description of a possible embodiment of the present invention, and they are not intended to limit the scope of the present invention, and equivalent embodiments or modifications made without departing from the technical spirit of the present invention should be included in the scope of the present invention.
Claims (8)
1. A method for simultaneously preparing polysaccharide and protein from sparassis crispa is characterized by comprising the following steps:
s1, crushing dry sparassis crispa sporocarp, adding an ammonium sulfate solution and tert-butyl alcohol, and mixing;
s2, uniformly mixing the mixture, placing the mixture in a bipolar square wave high-voltage pulse electric field for extraction, and collecting an extracting solution;
s3, after the pulsed electric field extraction, carrying out microwave extraction on the mixed solution;
s4, after the microwave extraction is finished, centrifuging the mixed solution;
s5, after the centrifugation is finished, placing the supernatant in a shaking table for oscillation;
s6, standing after oscillation is finished, dividing the mixed solution into three layers, adding tert-butyl alcohol on the uppermost layer, evaporating and recycling, adding dichloromethane into the protein layer on the middle layer for extraction, then carrying out rotary drying to remove dichloromethane to obtain the sparassis crispa protein, and dialyzing the lowermost layer to obtain the sparassis crispa polysaccharide.
2. The method for simultaneously preparing polysaccharide and protein from sparassis crispa according to claim 1, wherein ammonium sulfate solution with mass concentration of 20% is added in an amount of 10-20 times by weight to S1.
3. The method for simultaneously preparing polysaccharide and protein from sparassis crispa according to claim 1, wherein S1 is added with the mass-to-volume ratio of sparassis crispa to S1 of 1: 15-20 of tert-butanol.
4. The method for simultaneously preparing polysaccharide and protein from Sparassis crispa as claimed in claim 1, wherein in S1, pH of the mixture is adjusted to 6 with pH regulator.
5. The method for simultaneously preparing polysaccharide and protein from Sparassis crispa as claimed in claim 4, wherein the pH regulator is citric acid solution or formic acid solution.
6. The method as claimed in claim 1, wherein the pulsed electric field is extracted at a frequency of 200-1000Hz and a field intensity of 10-50Kv/cm in S2.
7. The method as claimed in claim 1, wherein the power of the microwave extraction in S3 is 200-500W and the time is 5-10 min.
8. The method for simultaneously preparing polysaccharide and protein from Sparassis crispa as claimed in claim 1, wherein in S5, the supernatant is placed in a shaker at 20-30 deg.C and shaking at 100rpm for 50-70 min.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113951367A (en) * | 2021-11-16 | 2022-01-21 | 安徽中志科技有限公司 | Edible fungus protein powder with high organic selenium content, preparation method and application |
| CN115028755A (en) * | 2022-07-01 | 2022-09-09 | 福建省农业科学院食用菌研究所 | Preparation method of high molecular weight Sparassis crispa beta-glucan |
| CN116284465A (en) * | 2022-08-05 | 2023-06-23 | 西北农林科技大学 | Preparation method of eucommia ulmoides polysaccharide and application of eucommia ulmoides polysaccharide prepared by preparation method in anti-depression |
-
2020
- 2020-10-09 CN CN202011071580.5A patent/CN112314770A/en not_active Withdrawn
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113951367A (en) * | 2021-11-16 | 2022-01-21 | 安徽中志科技有限公司 | Edible fungus protein powder with high organic selenium content, preparation method and application |
| CN115028755A (en) * | 2022-07-01 | 2022-09-09 | 福建省农业科学院食用菌研究所 | Preparation method of high molecular weight Sparassis crispa beta-glucan |
| CN115028755B (en) * | 2022-07-01 | 2023-08-11 | 福建省农业科学院食用菌研究所 | Preparation method of high molecular weight sparassis crispa beta-glucan |
| CN116284465A (en) * | 2022-08-05 | 2023-06-23 | 西北农林科技大学 | Preparation method of eucommia ulmoides polysaccharide and application of eucommia ulmoides polysaccharide prepared by preparation method in anti-depression |
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