CN115197339A - Method for combined extraction of protein and polysaccharide - Google Patents
Method for combined extraction of protein and polysaccharide Download PDFInfo
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- CN115197339A CN115197339A CN202210821396.0A CN202210821396A CN115197339A CN 115197339 A CN115197339 A CN 115197339A CN 202210821396 A CN202210821396 A CN 202210821396A CN 115197339 A CN115197339 A CN 115197339A
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- 150000004676 glycans Chemical class 0.000 title claims abstract description 60
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 60
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 60
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 50
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 50
- 238000000034 method Methods 0.000 title claims abstract description 45
- 238000000605 extraction Methods 0.000 title claims description 22
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 86
- 241000195620 Euglena Species 0.000 claims abstract description 67
- 239000002244 precipitate Substances 0.000 claims abstract description 43
- 239000006228 supernatant Substances 0.000 claims abstract description 36
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 35
- 238000000926 separation method Methods 0.000 claims abstract description 31
- 239000000843 powder Substances 0.000 claims abstract description 29
- 239000000243 solution Substances 0.000 claims abstract description 27
- 239000007787 solid Substances 0.000 claims abstract description 25
- 239000000047 product Substances 0.000 claims abstract description 18
- 239000011259 mixed solution Substances 0.000 claims abstract description 10
- 239000000203 mixture Substances 0.000 claims abstract description 10
- 238000001035 drying Methods 0.000 claims abstract description 9
- 239000012670 alkaline solution Substances 0.000 claims abstract description 8
- 238000002156 mixing Methods 0.000 claims abstract description 8
- 238000005303 weighing Methods 0.000 claims abstract description 8
- 238000001816 cooling Methods 0.000 claims description 10
- 238000003756 stirring Methods 0.000 claims description 7
- 230000007935 neutral effect Effects 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims 1
- 235000019441 ethanol Nutrition 0.000 abstract description 11
- 235000015097 nutrients Nutrition 0.000 abstract description 5
- 239000000126 substance Substances 0.000 abstract description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 15
- 238000007696 Kjeldahl method Methods 0.000 description 8
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 description 8
- 238000000643 oven drying Methods 0.000 description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 239000012141 concentrate Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000001704 evaporation Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 230000018044 dehydration Effects 0.000 description 2
- 238000006297 dehydration reaction Methods 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 241000206602 Eukaryota Species 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241001248610 Ophiocordyceps sinensis Species 0.000 description 1
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 1
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 1
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010364 biochemical engineering Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000003467 diminishing effect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000013376 functional food Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 210000004994 reproductive system Anatomy 0.000 description 1
- 230000027756 respiratory electron transport chain Effects 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 229940116269 uric acid Drugs 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 238000003809 water extraction Methods 0.000 description 1
Classifications
-
- 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
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
-
- 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
-
- 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 provides a method for extracting protein and polysaccharide in a combined manner, which comprises the following steps: weighing a certain amount of euglena dry powder, adding an alkaline solution, and extracting in a water bath; centrifuging the product, collecting supernatant, adding appropriate amount of water into the separated solid, and extracting in water bath; centrifuging the product, collecting supernatant, mixing with the supernatant, adjusting pH of the mixed solution to neutrality, and concentrating the volume of the mixed solution to 1/15-1/30 of the initial volume; adding anhydrous ethanol into the concentrated solution to make the volume fraction of ethanol be 30-50%, standing the solution for a period of time, performing centrifugal separation on the mixture, collecting precipitate, and dehydrating and drying the precipitate in sequence to obtain euglena protein; adding absolute ethyl alcohol into the supernatant, and performing centrifugal separation to obtain a second product with the main component of euglena polysaccharide. The method can quickly realize the separation of protein and polysaccharide in the euglena and improve the utilization efficiency of nutrient substances in the euglena.
Description
Technical Field
The invention relates to the technical field of biochemical engineering, in particular to a method for combined extraction of protein and polysaccharide.
Background
Euglena is a unicellular eukaryote, which is very rich in nutrients and is called "Cordyceps sinensis" in water. The Euglena contains abundant nutrients such as polysaccharide, protein, unsaturated fatty acid, vitamins, amino acids, etc. Wherein the total content of protein and polysaccharide accounts for more than 50% of the dry weight. The euglena polysaccharide is the most important product in the euglena, mainly comprises beta-1, 3-glucose, and has the functions of resisting virus, enhancing immunity, diminishing inflammation, reducing uric acid, reducing blood fat, reducing blood sugar, reducing blood pressure, protecting reproductive system and the like; the Euglena protein is also called as Euglena active protein, can promote energy synthesis and electron transfer in Euglena, has effects of regulating cell activity and immunity, and has killing effect on tumor cells and some harmful cells. Therefore, the euglena polysaccharide and the euglena protein have wide research prospects and application prospects in the fields of functional foods, biomedicines, clinics, aquatic products and the like. However, domestic research on euglena products and development and utilization thereof is still in the beginning stage at present, and most of the existing technologies focus on the extraction process of euglena polysaccharide which is a single substance and have low extraction rate.
Disclosure of Invention
In view of this, the invention provides a method for combined extraction of protein and polysaccharide, and aims to solve the problems that multiple nutrients in euglena are difficult to extract simultaneously and the extraction rate is low in the prior art.
The invention provides a method for extracting protein and polysaccharide in a combined manner, which comprises the following steps: the method comprises the following steps:
step 1, weighing a certain amount of euglena dry powder, adding an alkaline solution into the euglena dry powder according to a preset mass ratio, carrying out water bath extraction at a first preset temperature for a first preset time, and cooling to room temperature;
step 2, performing centrifugal separation on the mixture obtained in the step 1, collecting first supernatant, adding a proper amount of water into the separated solid, performing water bath extraction at a second preset temperature for a second preset time, and cooling to room temperature;
step 3, performing centrifugal separation on the product obtained in the step 2, collecting a second supernatant, mixing the second supernatant with the first supernatant, adjusting the pH of the mixed solution to be neutral, and concentrating the volume of the mixed solution to be 1/15-1/30 of the initial volume;
step 4, adding absolute ethyl alcohol into the concentrated solution under the stirring state at room temperature until the proportion of the absolute ethyl alcohol in the solution reaches a first integral number, standing for a period of time, carrying out centrifugal separation on the mixture, collecting first precipitates, and sequentially dehydrating and drying the first precipitates to obtain a first product with the main component of euglena protein;
and 5, adding absolute ethyl alcohol into the supernatant obtained after centrifugal separation in the step 4 until the proportion of the absolute ethyl alcohol in the solution reaches a second volume number, standing for a period of time, carrying out centrifugal separation on the mixture, collecting second precipitates, and sequentially dehydrating and drying the second precipitates to obtain a second product with the main component of euglena polysaccharide.
Further, in the above method, the mass ratio in step 1 is 1.
Further, in the above method, in the step 2, the volume of the added water is 10 to 30 times of the volume of the separated solid.
Further, in the above method, in the step 2, ultrasonic wave is adopted for auxiliary extraction while water bath extraction is performed.
Further, in the above method, the first preset temperature is 60 to 90 ℃; and/or the second preset temperature is 55-85 ℃.
Further, in the above method, the first volume fraction is 30 to 50%.
Further, in the above method, the second volume fraction is 50 to 70%.
Further, in the above method, in the step 4, the first precipitate is dehydrated by using absolute ethyl alcohol.
Further, in the above method, in the step 5, the second precipitate is dehydrated by using absolute ethyl alcohol.
Further, in the above method, the rotation speed of the centrifugal separation in the step 2 is 3000-5000rpm; the rotation speed of the centrifugal separation in the step 3 is 3000-5000rpm; the rotation speed of the centrifugal separation in the step 4 is 3000-5000rpm; the rotation speed of the centrifugal separation in the step 5 is 3000-5000rpm.
According to the method for combined extraction of protein and polysaccharide, provided by the invention, the water extract in the euglena dry powder is separated for multiple times by selecting safe and nontoxic absolute ethyl alcohol, so that on one hand, the separation of protein and polysaccharide in the euglena can be quickly realized, and the utilization efficiency of nutrient substances in the euglena is improved; on the other hand, the extracted protein and polysaccharide can be safely applied to the fields related to food and health care products; the method has the advantages of simple process route, short flow, small equipment investment and low production cost, and can quickly realize industrial production.
Detailed Description
While the preferred embodiments of the present invention are described below, it should be understood that various changes and modifications can be made by one skilled in the art without departing from the principles of the invention, and such changes and modifications are also considered to be within the scope of the invention.
The invention provides a method for extracting protein and polysaccharide in a combined manner, which comprises the following steps:
step 1, weighing a certain amount of euglena dry powder, adding an alkaline solution into the euglena dry powder according to a preset mass ratio, carrying out water bath extraction at a first preset temperature for a first preset time, and cooling to room temperature.
Specifically, the alkaline solution may be a sodium hydroxide solution, a potassium hydroxide solution, a sodium carbonate solution, a sodium bicarbonate solution, a potassium carbonate solution, or the like. The mass ratio of the euglena dry powder to the alkaline solution is 1. The mass concentration of the alkaline solution is 0.5-2.5%. In the step, the polysaccharide and the protein in the euglena are quickly dissolved out by adopting an alkaline solution.
The first predetermined temperature is preferably 60-90 deg.c. For example, the first predetermined temperature may be 60 ℃,70 ℃,80 ℃,90 ℃. The first preset time period may be 1-3 hours.
Preferably, ultrasonic agitation is used for auxiliary extraction during the first water bath extraction to promote dissolution of protein and polysaccharide.
And 2, performing centrifugal separation on the mixture obtained in the step 1, collecting first supernatant, adding a proper amount of water into the separated solid, performing water bath extraction at a second preset temperature for a second preset time, and cooling to room temperature.
Specifically, the volume of water added is 10 to 30 times the volume of the separated solid.
The second predetermined temperature is preferably 55-85 deg.c. For example, the second predetermined temperature may be 55 ℃, 65 ℃,70 ℃,80 ℃, 85 ℃. The second preset time period may be 1-3 hours.
Preferably, ultrasonic agitation is used for auxiliary extraction during the second water bath extraction to promote dissolution of protein and polysaccharide in the remaining solids.
In this step, the rotational speed of the centrifugal separation is 3000 to 5000rpm, preferably 4000rpm.
And 3, performing centrifugal separation on the product obtained in the step 2, collecting a second supernatant, mixing the second supernatant with the first supernatant, adjusting the pH of the mixed solution to be neutral, and concentrating the volume of the mixed solution to be 1/15-1/30 of the initial volume. Wherein, the function of adjusting pH is to make the whole solution in neutral, thus avoiding the denaturation of protein and polysaccharide caused by long-term alkaline environment, and the neutral is also beneficial to the direct utilization of subsequent products. The concentration aims at improving the solid content, reducing the ethanol consumption in the subsequent treatment process and improving the product yield.
Specifically, the pH of the mixed solution can be adjusted by using phosphoric acid, boric acid, or the like.
In this step, the rotational speed of the centrifugal separation is 3000 to 5000rpm, preferably 4000rpm. In this step, a rotary evaporator may be selected for the concentration of the mixed solution.
And 4, adding absolute ethyl alcohol into the concentrated solution under the stirring state at room temperature until the proportion of the absolute ethyl alcohol in the solution reaches a first integral number, standing for a period of time, carrying out centrifugal separation on the mixture, collecting first precipitates, and sequentially dehydrating and drying the first precipitates to obtain a first product with the main component of euglena protein.
In particular, the first volume fraction is 30-50%. Adding anhydrous ethanol into the concentrated solution, and standing for 30-60min.
The principle is as follows: in water, most polysaccharides have much higher solubility than most proteins, and after ethanol is added, ethanol will bind with water molecules competitively with proteins and polysaccharides, and because ethanol has stronger competitive power, water molecules will bind with ethanol preferentially, while water molecules binding with proteins and polysaccharides will decrease correspondingly, so with the addition of ethanol, proteins with lower solubility will precipitate first due to the decrease of water binding with them, and the precipitation of polysaccharides will require ethanol with higher concentration. Since the protein and polysaccharide are uniformly mixed with each other throughout the solution, when the protein is precipitated first, a small amount of polysaccharide is also entrained by the protein precipitate and precipitated together. At the same time, a small amount of less soluble polysaccharide will precipitate at this ethanol concentration, so the first precipitation will contain a small amount of polysaccharide, but most of the polysaccharide will remain in solution.
In this embodiment, preferably, the first precipitate is dehydrated by using absolute ethyl alcohol; in order to ensure the dehydration effect, the first precipitate can be dehydrated for a plurality of times by adopting absolute ethyl alcohol. For example, the first precipitate is dehydrated three times with absolute ethanol.
In this step, the rotational speed of the centrifugal separation is 3000 to 5000rpm, preferably 4000rpm.
And 5, adding absolute ethyl alcohol into the supernatant obtained after centrifugal separation in the step 4 until the proportion of the absolute ethyl alcohol in the solution reaches a second volume fraction, standing for a period of time, carrying out centrifugal separation on the mixture, collecting a second precipitate, and sequentially dehydrating and drying the second precipitate to obtain a second product with the main component of euglena polysaccharide.
In particular, the second volume fraction is 50-70%. Adding anhydrous ethanol into the supernatant, and standing for 30-60min.
In this step, the rotational speed of the centrifugal separation is 3000 to 5000rpm, preferably 4000rpm.
Preferably, the second precipitate is dehydrated by absolute ethyl alcohol; in order to ensure the dehydration effect, the second precipitate can be dehydrated for a plurality of times by adopting absolute ethyl alcohol. For example, the second precipitate is removed by using absolute ethyl alcohol
Water was used three times.
The invention is described in detail below in terms of several specific examples.
Example 1
Weighing 15g of euglena dry powder, adding a 0.5% sodium hydroxide solution according to the mass ratio of 1;
centrifuging at 4000rpm for 5min, collecting supernatant, adding 15 times of water to the rest solid, extracting in water bath at 80 deg.C for 1 hr under the assistance of ultrasonic wave, and cooling to room temperature;
centrifuging at 4000rpm for 5min, collecting supernatant, mixing with the supernatant in step 2, adjusting pH to neutrality, and rotary evaporating to concentrate to 1/15 of original volume;
adding anhydrous ethanol into the concentrated solution twice, adding anhydrous ethanol under stirring at room temperature for the first time until the volume fraction of the anhydrous ethanol reaches 45%, standing for 30min, centrifuging at 4000rpm for 5min, collecting precipitate, dehydrating the precipitate with anhydrous ethanol for three times, drying to obtain euglena protein, and collecting 0.83g of euglena protein solid powder. The obtained euglena protein solid powder is detected by adopting a Kjeldahl method and a phenol-sulfuric acid method, the protein content is 78.8 percent, and the polysaccharide content is 18.6 percent.
Adding anhydrous ethanol into the supernatant for the second time until the volume fraction of the anhydrous ethanol reaches 65%, standing for 30min, centrifuging at 4000rpm for 5min, collecting precipitate, dehydrating the precipitate with anhydrous ethanol for three times, and oven drying to obtain crude polysaccharide of Euglena, and collecting Euglena polysaccharide powder 3.76g. The obtained euglena polysaccharide solid powder is detected by adopting a Kjeldahl method and a phenol-sulfuric acid method, the protein content is 19.4 percent, and the polysaccharide content is 76.3 percent.
Example 2
Weighing 15g of euglena dry powder, adding a 1.0% sodium hydroxide solution according to the mass ratio of 1;
adjusting the pH value of the solution to 7.0 by using phosphoric acid, centrifuging for 5min at 4000rpm, collecting supernatant, adding 20 times of water into the residual solid, extracting for 2 hours in a water bath at 80 ℃ under the assistance of ultrasonic waves, and cooling to room temperature;
centrifuging at 4000rpm for 5min, collecting supernatant, mixing with the supernatant in step 2, adjusting pH to neutrality, and rotary evaporating to concentrate to 1/15 of original volume;
adding anhydrous ethanol into the concentrated solution twice, adding anhydrous ethanol under stirring at room temperature for the first time until the volume fraction of the anhydrous ethanol reaches 50%, standing for 30min, centrifuging at 4000rpm for 5min, collecting precipitate, dehydrating the precipitate with anhydrous ethanol for three times, oven drying to obtain euglena protein, and collecting 0.96g of euglena protein solid powder. The obtained euglena protein solid powder is detected by adopting a Kjeldahl method and a phenol-sulfuric acid method, the protein content is 73.4 percent, and the polysaccharide content is 21.6 percent.
Adding anhydrous ethanol into the supernatant for the second time until the volume fraction of the anhydrous ethanol reaches 70%, standing for 30min, centrifuging at 4000rpm for 5min, collecting precipitate, dehydrating the precipitate with anhydrous ethanol for three times, and oven drying to obtain Euglena polysaccharide, and collecting Euglena polysaccharide powder 4.15g. The obtained euglena polysaccharide solid powder is detected by adopting a Kjeldahl method and a phenol-sulfuric acid method, the protein content is 14.9 percent, and the polysaccharide content is 81.53 percent.
Example 3
Weighing 10g of euglena dry powder, adding a 2.0% sodium hydroxide solution according to a mass ratio of 1;
centrifuging at 4000rpm for 5min, collecting supernatant, adding 30 times of water into the rest solid, extracting in 80 deg.C water bath for 3 hr under the assistance of ultrasonic wave, and cooling to room temperature;
centrifuging at 4000rpm for 5min, collecting supernatant, mixing with the supernatant obtained in step 2, adjusting pH to neutrality, and rotary evaporating to concentrate to 1/25 of original volume;
adding absolute ethanol into the concentrated solution twice, stirring at room temperature for the first time, adding absolute ethanol until the volume fraction of the absolute ethanol reaches 45%, standing for 30min, centrifuging at 4000rpm for 5min, collecting precipitate, dehydrating the precipitate with absolute ethanol for three times, and oven drying to obtain euglena protein, wherein 0.98g of euglena protein solid powder is collected. The obtained euglena protein solid powder is detected by adopting a Kjeldahl method and a phenol-sulfuric acid method, the protein content is 78.4 percent, and the polysaccharide content is 18.7 percent.
Adding absolute ethanol into the supernatant for the second time until the volume fraction of the absolute ethanol reaches 65%, standing for 30min, centrifuging at 4000rpm for 5min, collecting precipitate, dehydrating the precipitate with absolute ethanol for three times, and oven drying to obtain Euglena polysaccharide, wherein the Euglena polysaccharide powder is collected in an amount of 4.36g. The obtained euglena polysaccharide solid powder is detected by adopting a Kjeldahl method and a phenol-sulfuric acid method, the protein content is 8.9 percent, and the polysaccharide content is 82.33 percent.
Example 4
Weighing 10g of euglena dry powder, adding a 2.5% sodium hydroxide solution according to the mass ratio of 1;
centrifuging at 4000rpm for 5min, collecting supernatant, adding 30 times of water to the rest solid, extracting in 80 deg.C water bath for 3 hr under the assistance of ultrasonic wave, and cooling to room temperature;
centrifuging at 4000rpm for 5min, collecting supernatant, mixing with the supernatant obtained in step 2, adjusting pH to neutrality, and rotary evaporating to concentrate to 1/30 of original volume;
adding anhydrous ethanol into the concentrated solution twice, adding anhydrous ethanol under stirring at room temperature for the first time until the volume fraction of the anhydrous ethanol reaches 35%, standing for 30min, centrifuging at 4000rpm for 5min, collecting precipitate, dehydrating the precipitate with anhydrous ethanol for three times, drying to obtain euglena protein, and collecting 1.15g of euglena protein solid powder. The obtained euglena protein solid powder is detected by adopting a Kjeldahl method and a phenol-sulfuric acid method, the protein content is 73.4 percent, and the polysaccharide content is 22.7 percent.
Adding anhydrous ethanol into the supernatant for the second time until the volume fraction of the anhydrous ethanol reaches 60%, standing for 30min, centrifuging at 4000rpm for 5min, collecting precipitate, dehydrating the precipitate with anhydrous ethanol for three times, and oven drying to obtain Euglena polysaccharide, wherein the Euglena polysaccharide powder is collected in total by 4.29g. The obtained euglena polysaccharide solid powder is detected by adopting a Kjeldahl method and a phenol-sulfuric acid method, the protein content is 7.6 percent, and the polysaccharide content is 85.61 percent.
In conclusion, the method for extracting the protein and the polysaccharide in a combined manner provided by the invention has the advantages that the polysaccharide and the protein in the euglena are quickly dissolved out under the alkaline condition, and then the polysaccharide and the protein in the euglena are extracted in a combined manner by adopting a water extraction and alcohol precipitation method; the method adopts ethanol with different concentrations to precipitate and separate protein and polysaccharide, simultaneously obtains the euglena protein and the euglena polysaccharide by a one-step method, quickly realizes the separation of the protein and the polysaccharide in the euglena, has simple process route, short flow, small equipment investment and low production cost, and can quickly realize industrial production.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent should be subject to the appended claims.
Claims (10)
1. A method for extracting protein and polysaccharide in a combined way is characterized by comprising the following steps:
step 1, weighing a certain amount of euglena dry powder, adding an alkaline solution into the euglena dry powder according to a preset mass ratio, carrying out water bath extraction at a first preset temperature for a first preset time, and cooling to room temperature;
step 2, performing centrifugal separation on the mixture obtained in the step 1, collecting first supernatant, adding a proper amount of water into the separated solid, performing water bath extraction at a second preset temperature for a second preset time, and cooling to room temperature;
step 3, performing centrifugal separation on the product obtained in the step 2, collecting a second supernatant, mixing the second supernatant with the first supernatant, adjusting the pH of the mixed solution to be neutral, and concentrating the volume of the mixed solution to be 1/15-1/30 of the initial volume;
step 4, adding absolute ethyl alcohol into the concentrated solution under the stirring state at room temperature until the proportion of the absolute ethyl alcohol in the solution reaches a first integral number, standing for a period of time, carrying out centrifugal separation on the mixture, collecting first precipitate, and sequentially dehydrating and drying the first precipitate to obtain a first product with the main component of euglena protein;
and 5, adding absolute ethyl alcohol into the supernatant obtained after centrifugal separation in the step 4 until the proportion of the absolute ethyl alcohol in the solution reaches a second volume fraction, standing for a period of time, carrying out centrifugal separation on the mixture, collecting a second precipitate, and sequentially dehydrating and drying the second precipitate to obtain a second product with the main component of euglena polysaccharide.
2. The method according to claim 1, wherein the mass ratio in step 1 is 1.
3. The method of claim 1, wherein in step 2, the volume of water added is 10 to 30 times the volume of the separated solid.
4. The method as claimed in claim 1, wherein in step 2, ultrasonic wave is used for auxiliary extraction while the water bath extraction is performed.
5. The method of claim 1, wherein the first predetermined temperature is 60-90 ℃; and/or the second preset temperature is 55-85 ℃.
6. The method of claim 1, wherein the first volume fraction is 30-50%.
7. The method of claim 1, wherein the second volume fraction is 50-70%.
8. The method according to claim 1, wherein in the step 4, the first precipitate is dehydrated by using absolute ethyl alcohol.
9. The method according to claim 1, wherein in the step 5, the second precipitate is dehydrated by using absolute ethanol.
10. The method according to claim 1, wherein the rotational speed of the centrifugation in the step 2 is 3000-5000rpm; the rotation speed of the centrifugal separation in the step 3 is 3000-5000rpm; the rotation speed of the centrifugal separation in the step 4 is 3000-5000rpm; the rotation speed of the centrifugal separation in the step 5 is 3000-5000rpm.
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