CN106749507B - Method for extracting nostoc proteins - Google Patents
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Abstract
The invention relates to a nostoc protein extraction method, which comprises the steps of extracting nostoc phycobiliprotein by pure water, sequentially extracting alkali-soluble protein from filter residues by using a sodium hydroxide solution, extracting alcohol-soluble protein by using an ethanol solution, extracting salt-soluble protein by using a sodium chloride solution, mixing the obtained nostoc phycobiliprotein crude extract, the alkali-soluble protein crude extract, the alcohol-soluble protein crude extract and the salt-soluble protein crude extract, performing ultrafiltration impurity removal by using a ceramic membrane, performing ultrafiltration concentration by using an organic membrane, and performing freeze drying to obtain nostoc protein. Compared with the existing repeated freeze-thawing method, the method greatly simplifies the operation difficulty, overcomes the harsh conditions, has the extraction rate far superior to that of the prior art, and can extract about 70 percent of protein in the nostoc sphaeroides. Compared with the prior art that phycoerythrin and phycocyanin are extracted singly or phycobiliprotein with more components is extracted, the comprehensive nostoc sphaeroides protein extracted by the method can be used as a comprehensive nutrient to be added into industrial food and medicines, and has wide application prospect.
Description
Technical Field
The invention relates to a method for extracting nostoc sphaeroids kutz protein.
Background
Nostoc sphaeroides Kutzing is a famous Nostoc sphaerotheca, belongs to Nostoc of Nostocaceae, belongs to Nostoc, belongs to the same genus as Nostoc, is called Nostoc sphaerotheca and Nostoc commune, is called Nostoc commune Vauch, is called Nostoc commune, and is a precious dual-purpose nitrogen-fixing blue algae for medicine and food in the traditional export of China. The plant is a multicellular filamentous plant, the cellular structure of the plant is simple, an individual is an unbranched single-row filament consisting of spherical cells, the filament is in the shape of a moniliform, a group is in the shape of colloid, sphere or other irregular shapes, and the filament is blue-green or yellow-brown, can be seen by naked eyes and has the nitrogen fixation capacity. The nostoc sphaeroides is called by Ben Cao gang mu, medicine property examination, national Chinese herbal medicine compilation and the like, and has the effects of improving eyesight, tonifying qi, bringing people with children, clearing heat, benefiting intestines and stomach, treating phlegm fire, prolonging life after long-term eating, eliminating fatigue, astringing, treating night blindness, burning fire and the like.
Nostoc sphaeroids Kutz is rich in protein, and can be divided into two major types, namely simple protein and complex protein. The simple protein mainly comprises albumin (mainly phycobiliprotein), salt-soluble protein, alcohol-soluble protein and alkali-soluble protein, and the complex protein is composite protein. Wherein the simple protein accounts for more than 50% of the nostoc proteins. Phycobiliproteins are light harvesting chromophorin proteins widely present in Phycobilisomes (PBS) from red, blue and green algae, including the four classes Phycocyanin (PC), Phycoerythrin (PE), Phycoerythrin (PEC) and Allophycocyanin (APC). Researches show that the nostoc protein, polysaccharide and amino acid have high content, reasonable composition and balanced proportion, and the variety of the necessary amino acid is more than 16. Wherein, the protein content is 30.8g/100g, the total content of amino acid is 31.06g/100g, even exceeding the FAO standard. The nostoc proteins are complete in species and rich in content, and are superior to other algae. Therefore, nostoc is a good protein extraction source.
At present, the extraction of nostoc proteins is mainly limited to the extraction of phycobiliproteins, and the extraction of total nostoc proteins is reported fresh, and the extraction method of the phycobiliproteins mainly comprises a repeated freeze-thaw method, a chemical reagent treatment method, a swelling method, an ultrasonic method and a tissue mashing method, although the methods can extract nostoc proteins, the methods are complex and harsh, and require repeated freezing at the temperature of-20 ℃, so that a large amount of manpower, material resources and financial resources are consumed; or the extraction yield is low, and is only 7 percent at most; or introduce chemical reagents which are difficult to remove, and the purity of the product is difficult to ensure. In addition, most of documents extract phycobiliprotein or single components thereof, such as phycoerythrin and phycocyanin, and do not consider that nostoc sphaeroides protein rich in various components is a good nutritional product, so that the nostoc sphaeroides protein has a wide development prospect. Therefore, there is a need to develop a method for efficiently extracting various proteins from nostoc sphaeroides for industrial production and application.
Disclosure of Invention
The invention aims to provide a method for efficiently extracting nostoc sphaeroides protein.
In order to achieve the above object, the present invention provides a nostoc proteins extraction method, comprising the following steps:
1) crushing dried nostoc sphaeroides to 10-200 meshes, adding water which is 50-70 times of the weight of the nostoc sphaeroides powder, extracting for 3 times, extracting for 4-8 hours each time, and performing ultrasonic treatment for 10-30 min by 400-900W every 1 hour; centrifuging and filtering to obtain crude extract of Nostoc sphaeroids Kutz bile protein; filtering residues for later use;
2) adding 60-90 times of sodium hydroxide solution by weight into the nostoc sphaeroides filter residue obtained in the step 1), extracting for 3 times, extracting for 3-7 h each time, performing ultrasonic treatment for 10-30 min by using ultrasonic waves of 400-900W every 1h, centrifuging, and filtering to obtain nostoc sphaeroides alkali-soluble protein crude extract; filtering residues for later use;
3) adding 50-70 times of ethanol solution by weight into the nostoc sphaeroides filter residue obtained in the step 2), extracting for 3 times with the concentration of the ethanol solution being 75-100% for 0-6 h every time, performing ultrasonic treatment for 10-30 min by using 400-900W of ultrasonic waves every 1h, centrifuging, and filtering to obtain a nostoc sphaeroides prolamin crude extract; filtering residues for later use;
4) adding 50-90 times of NaCl solution by weight into the nostoc sphaeroides filter residue obtained in the step 3), wherein the concentration of the NaCl solution is 0.1-0.5 mol/L, extracting for 3 times, extracting for 0-6 h each time, performing ultrasonic treatment for 10-30 min by using ultrasonic waves of 400-900W every 1h, centrifuging, filtering to obtain crude extract of nostoc sphaeroides salt-soluble protein, and discarding filter residue;
5) mixing the obtained crude extract of the nostoc sphaeroides gallbladder protein, the crude extract of the nostoc sphaeroides alkali-soluble protein, the crude extract of the nostoc sphaeroides alcohol-soluble protein and the crude extract of the nostoc sphaeroides salt-soluble protein, performing ultrafiltration twice for removing impurities by using a ceramic membrane with the interception aperture of 5000-300000D, performing ultrafiltration concentration by using an organic membrane with the interception aperture of 5,000-10,000D, and performing freeze drying to obtain the nostoc sphaeroides protein.
The extraction method of nostoc proteins comprises the following steps of 1):
preferably, the water content of the dried nostoc sphaeroids kutz is 11-15%, and more preferably 14.6%.
Preferably, the grain diameter of the nostoc sphaeroides is 80-200 meshes, and more preferably 120-200 meshes.
Preferably, the weight ratio of the nostoc sphaeroids kutz to the water is 1: 60.
Preferably, purified water is used.
Preferably, each extraction time is 6 h.
Preferably, the extraction temperature is between room temperature and 45 ℃, and the room temperature is generally 20-25 ℃.
Preferably, the ultrasonic power is 500-800W, and more preferably 700W.
Preferably, the sonication time is 20 min.
Specifically, the step 1) comprises pulverizing dried nostoc sphaeroides (with a water content of about 14.6%) to 120-200 meshes, adding 50-70 times of purified water, extracting at room temperature to 45 ℃ for 6h, performing ultrasonic treatment at intervals of 1h with 700W for 20min, extracting for 3 times, centrifuging, and filtering. And filtering the filter residue for later use.
The extraction method of nostoc proteins comprises the following steps of 2):
preferably, the weight ratio of the nostoc sphaeroides to the sodium hydroxide solution is 1: 80.
Preferably, the concentration of the NaOH solution is 0.2 mol/L.
Preferably, each extraction time is 5 h.
Preferably, the extraction temperature is between room temperature and 45 ℃, and the room temperature is generally 20-25 ℃.
Preferably, the ultrasonic power is 500-800W, and more preferably 700W.
Preferably, the sonication time is 20 min.
Specifically, the step 2) comprises adding the nostoc sphaeroides filter residue obtained in the step 1) into 0.2mol/L NaOH solution 70-90 times the weight of the nostoc sphaeroides filter residue, extracting for 5 hours at 45 ℃, performing ultrasonic treatment for 20 minutes at intervals of 1 hour by using 700W ultrasonic waves, extracting for 3 times, centrifuging, and filtering.
The method for extracting nostoc proteins comprises the step 3)
Preferably, the weight ratio of the nostoc sphaeroides to the ethanol solution is 1: 60.
Preferably, the concentration of the ethanol solution is 95%.
Preferably, each extraction time is 3 h.
Preferably, the extraction temperature is between room temperature and 45 ℃, and the room temperature is generally 20-25 ℃.
Preferably, the ultrasonic power is 500-800W, and more preferably 700W.
Preferably, the sonication time is 20 min.
Specifically, the step 3) comprises adding 50-70 times of 95% ethanol solution by weight of the nostoc sphaeroides filter residue in the step 2), extracting for 3 hours at 45 ℃, performing ultrasonic treatment for 20 minutes at intervals of 1 hour by using 700W ultrasonic waves, extracting for 3 times, centrifuging, and filtering. And filtering the filter residue for later use.
The method for extracting nostoc proteins comprises the step 4)
Preferably, the weight ratio of the nostoc sphaeroids kutz filter residue to the NaCl solution is 1: 70.
Preferably, the concentration of the NaCl solution is 0.3 mol/L.
Preferably, each extraction time is 3 h.
Preferably, the extraction temperature is between room temperature and 45 ℃, and the room temperature is generally 20-25 ℃.
Preferably, the ultrasonic power is 500-800W, and more preferably 700W.
Preferably, the sonication time is 20 min.
Specifically, the step 4) comprises adding the nostoc sphaeroides filter residue obtained in the step 3) into 0.3mol/L NaCl solution with the weight 50-90 times of that of the nostoc sphaeroides filter residue, extracting for 3 hours at 45 ℃, performing ultrasonic treatment for 20 minutes at intervals of 1 hour by using 700W ultrasonic waves, extracting for 3 times, centrifuging, and filtering. And filtering the filter residue for later use.
The method for extracting nostoc proteins comprises the step 5)
Preferably, mixing the four crude protein extracting solutions, performing primary ultrafiltration by using a ceramic membrane with the interception aperture of 150,000-450,000D, stopping ultrafiltration until liquid in a barrel becomes viscous and bluish purple is not obvious, and taking a filtrate; and (3) performing secondary ultrafiltration on the filtrate by using a ceramic membrane with the interception aperture of 5,000-15,000D, continuously supplementing pure water into an ultrafiltration barrel, taking liquid in the barrel after 4 hours, concentrating by 4-5 times by using a plate type organic membrane with the interception aperture of 5,000-10,000D, and freeze-drying the filtrate to obtain the nostoc protein extract.
Preferably, the pore size of the first ultrafiltration ceramic membrane is 300,000D.
Preferably, the pore size of the second ultrafiltration ceramic membrane is 5,000D.
Preferably, the pore size of the organic membrane is 8,000D.
More specifically, the method for extracting nostoc proteins comprises the following steps:
1) crushing dried nostoc sphaeroides to 120-200 meshes, adding water 60 times the weight of nostoc sphaeroides, extracting for 3 times, extracting for 6 hours each time, and performing ultrasonic treatment for 20min by 700W every 1 hour; centrifuging and filtering to obtain crude extract of Nostoc sphaeroids Kutz bile protein; filtering residues for later use;
2) adding 80 times of sodium hydroxide solution by weight into the nostoc sphaeroides filter residue obtained in the step 1), extracting for 3 times with the concentration of 0.2mol/L for 5h each time, performing ultrasonic treatment for 20min by using 700W ultrasonic waves every 1h, centrifuging, and filtering to obtain crude nostoc sphaeroides alkali-soluble protein extract; filtering residues for later use;
3) adding 60 times of ethanol solution with the concentration of 95% into the nostoc sphaeroides filter residue obtained in the step 2), extracting for 3 times, each time for 3 hours, performing ultrasonic treatment for 20min at intervals of 1 hour by using 700W ultrasonic waves, centrifuging, and filtering to obtain a nostoc sphaeroides gliadin crude extract; filtering residues for later use;
4) adding 70 times of NaCl solution by weight into the nostoc sphaeroides filter residue obtained in the step 3), extracting for 3 times with the concentration of the NaCl solution being 0.3mol/L, performing ultrasonic treatment for 20min by 700W ultrasonic wave every 1h, centrifuging, and filtering to obtain nostoc sphaeroides salt-soluble protein crude extract; filtering residues for later use;
5) mixing the obtained crude extractive solution of Nostoc sphaeroids Kutz bile protein, crude extractive solution of Nostoc sphaeroids Kutz salt-soluble protein, crude extractive solution of Nostoc sphaeroids Kutz alcohol-soluble protein and crude extractive solution of Nostoc sphaeroids Kutz alkali-soluble protein, sequentially ultrafiltering with ceramic membrane with retention pore size of 300,000D and 5,000D for twice removing impurities, ultrafiltering with organic membrane with retention pore size of 8,000D for concentration, and freeze drying to obtain Nostoc sphaeroids Kutz protein.
Compared with the prior art, the invention has the following advantages:
1) the ultrasonic-assisted extraction method is combined with the swelling method, compared with the existing repeated freezing and thawing method, the operation difficulty is greatly simplified, the harsh conditions are overcome, the extraction rate is far better than that of the prior art, and about 70 percent of protein in the nostoc sphaeroides can be extracted.
2) The method adopts a ceramic membrane ultrafiltration method to remove nostoc polysaccharide, NaCl, NaOH, ethanol, pigment and other impurities dissolved out of the crude extract, and purifies nostoc protein, compared with the methods such as an ammonium sulfate multistage precipitation method and a dialysis method, the method adopts a physical method, has mild conditions, no phase change, low energy consumption, no need of introducing inorganic salt ions, simple and convenient operation, short time consumption, easy storage and large-scale production;
3) the dried nostoc sphaeroids kutz powder is adopted as a raw material for extraction, so that the nostoc sphaeroids kutz powder is easy to realize and store industrially, and the problems that fresh nostoc sphaeroids kutz products are easy to deteriorate after being stored for a long time and the like are solved;
4) compared with the method for storing the protein in the dialyzate, the dried protein product obtained by adopting the freeze-drying method is easier to be actually stored, and a convenient, quick and easy-to-store method is provided for industrially applying the nostoc sphaeroides protein;
5) compared with the prior art that phycoerythrin and phycocyanin are extracted singly or phycobiliprotein with more components is extracted, the method extracts comprehensive nostoc proteins including phycobiliprotein (containing phycocyanin, phycoerythrin, phycoerythrocyanin and allophycocyanin), salt-soluble protein, alcohol-soluble protein and alkali-soluble protein, and the product can be used as a comprehensive nutrient to be added into industrial food, medicines and cosmetics and skin care products, and has wide application prospect;
6) the method adopts the extraction from room temperature to 45 ℃, has wide extraction temperature range, avoids low-temperature operation and is easy to realize industrially.
Drawings
FIG. 1 shows the effect of the number of grain sizes of Nostoc sphaeroides rice flour on the extraction rate of Nostoc sphaeroides biliprotein in Experimental example 1;
FIG. 2 shows the effect of the ultrasonic time on the extraction rate of the gall protein of Nostoc sphaeroids Kutz in Experimental example 2;
FIG. 3 shows the effect of ultrasonic power on the extraction rate of the gall protein of Nostoc sphaeroides in Experimental example 3;
FIG. 4 shows the effect of the treatment method of Experimental example 4 on the extraction rate of the gall protein of Nostoc sphaeroides;
FIG. 5 shows the effect of the feed liquid ratio of Experimental example 5 on the extraction rate of phycobiliprotein from nostoc sphaeroids kutz;
FIG. 6 shows the effect of extraction time on the extraction rate of C.nostoc biliary protein in Experimental example 6;
FIG. 7 shows the effect of the number of extractions on the extraction rate of C.nostoc biliary protein in Experimental example 7;
FIG. 8 shows the effect of extraction temperature on the extraction rate of C.nostoc biliary protein in Experimental example 8;
FIG. 9 shows the effect of alkali concentration on the extraction rate of alkali-soluble protein from nostoc sphaeroides in Experimental example 9;
FIG. 10 shows the effect of the feed liquid ratio of Experimental example 10 on the extraction rate of soluble protein from nostoc sphaeroides;
FIG. 11 shows the effect of the extraction time of Experimental example 11 on the extraction rate of alkali-soluble protein from nostoc;
FIG. 12 shows the effect of alcohol concentration on the gliadin extraction rate of nostoc 12;
FIG. 13 shows the effect of feed liquid ratio on the extraction rate of gliadin from Nostoc commune in Experimental example 13;
FIG. 14 shows the effect of extraction time on the prolamin extraction rate of nostoc sphaeroides in Experimental example 14;
FIG. 15 shows the effect of the salt concentration in Experimental example 15 on the extraction rate of the salt-soluble protein of nostoc;
FIG. 16 shows the effect of the feed liquid ratio of Experimental example 16 on the extraction rate of the salt-soluble protein of nostoc;
FIG. 17 shows the effect of the extraction time of Experimental example 17 on the extraction rate of the salt-soluble protein of nostoc sphaeroides.
Detailed Description
The following examples and experimental examples are provided to illustrate the present invention, but are not intended to limit the scope of the present invention. The following does not specify particular techniques or conditions, according to the techniques or conditions described in the literature of the art, or according to the product specifications. The reagents or instruments used are not indicated by manufacturers, and are all conventional products which can be purchased by normal distributors.
The following phycobiliprotein content detection method is Coomassie brilliant blue G250 staining method, and the absorbance at 595nm is measured, and the content is calculated according to the recording method of Liheng's plant physiological and biochemical experiment principle and technology'.
The following method for calculating the extraction rate:
the extraction rate%.
The best mode of the invention is as follows, and other modes can adjust the relevant parameters according to actual conditions (specifically referring to the summary of the invention and the following experimental examples), and the invention can be realized.
A method for extracting nostoc proteins comprises the following steps:
1) crushing dried nostoc sphaeroides to 120-200 meshes, adding water 60 times the weight of nostoc sphaeroides, extracting for 3 times, extracting for 6 hours each time, and performing ultrasonic treatment for 20min by 700W every 1 hour; centrifuging and filtering to obtain crude extract of Nostoc sphaeroids Kutz bile protein; filtering residues for later use;
2) adding 80 times of sodium hydroxide solution by weight into the nostoc sphaeroides filter residue obtained in the step 1), extracting for 3 times with the concentration of 0.2mol/L for 5h each time, performing ultrasonic treatment for 20min by using 700W ultrasonic waves every 1h, centrifuging, and filtering to obtain crude nostoc sphaeroides alkali-soluble protein extract; filtering residues for later use;
3) adding 60 times of ethanol solution with the concentration of 95% into the nostoc sphaeroides filter residue obtained in the step 2), extracting for 3 times, each time for 3 hours, performing ultrasonic treatment for 20min at intervals of 1 hour by using 700W ultrasonic waves, centrifuging, and filtering to obtain a nostoc sphaeroides gliadin crude extract; filtering residues for later use;
4) adding 70 times of NaCl solution by weight into the nostoc sphaeroides filter residue obtained in the step 3), extracting for 3 times with the concentration of the NaCl solution being 0.3mol/L, performing ultrasonic treatment for 20min by 700W ultrasonic wave every 1h, centrifuging, and filtering to obtain nostoc sphaeroides salt-soluble protein crude extract; filtering residues for later use;
5) mixing the obtained crude extractive solution of Nostoc sphaeroids Kutz bile protein, crude extractive solution of Nostoc sphaeroids Kutz salt-soluble protein, crude extractive solution of Nostoc sphaeroids Kutz alcohol-soluble protein and crude extractive solution of Nostoc sphaeroids Kutz alkali-soluble protein, sequentially ultrafiltering with ceramic membrane with retention pore size of 300,000D and 5,000D for twice removing impurities, ultrafiltering with organic membrane with retention pore size of 8,000D for concentration, and freeze drying to obtain Nostoc sphaeroids Kutz protein.
Experimental example 1 extraction of phycobiliprotein in laboratory bench scale
Crushing the dried nostoc sphaeroides kutz product (with the water content of 14.6%) which is dried or baked by using a universal crusher to be more than 10-200 meshes, sieving the crushed product by using a sieve to obtain particles with the number of 10-24 meshes, 24-65 meshes, 65-80 meshes, 80-120 meshes, 120-200 meshes and more than 200 meshes, respectively taking 0.5g of the particles, adding 25mL of purified water, extracting the mixture at 30 ℃ for 6 hours, centrifuging the mixture at 4000r/min for 10min, and filtering the mixture to obtain a crude extracting solution. The results are shown in Table 1 and FIG. 1, and it can be seen from FIG. 1 that the optimum particle size of Nostoc sphaeroids kutz is 120-200 mesh.
TABLE 1
Experimental example 2 extraction of phycobiliprotein in laboratory bench scale
Crushing dried nostoc sphaeroides (with the water content of 14.6%) dried nostoc sphaeroides in a drying or baking mode into 120-200 meshes by using a universal crusher, respectively taking 0.5g of the dried nostoc sphaeroides, adding 25mL of purified water, adding water into nostoc sphaeroides powder, dissolving the powder by ultrasonic treatment for 30min, extracting the powder for 1h, then performing ultrasonic treatment for 10min, 20min, 30min, 40min, 50min and 60min, centrifuging the powder for 10min at 4000r/min after ultrasonic treatment, and filtering the solution to obtain a crude extracting solution. The results are shown in the following table 2 and fig. 2, and it can be seen from fig. 2 that the extraction rate of the nostoc sphaeroides bile protein is basically kept flat along with the extension of the ultrasonic time, and the optimal ultrasonic time is 20min in comprehensive consideration.
TABLE 2
Experimental example 3 extraction of phycobiliprotein in laboratory bench scale
Crushing the dried nostoc sphaeroides kutz product (with the water content of 14.6%) which is dried or baked by using a universal crusher to 120-200 meshes, respectively taking 0.5g of the dried nostoc sphaeroides kutz, adding 25mL of purified water, extracting for 1h, then carrying out ultrasonic treatment for 20min at the ultrasonic power of 400W, 500W, 600W, 700W, 800W and 900W, centrifuging for 10min at the speed of 4000r/min after ultrasonic treatment, and filtering to obtain a crude extract. The results are shown in Table 3 below and FIG. 3. from FIG. 3, it can be seen that the optimum ultrasonic power is 700W.
TABLE 3
Experimental example 4 extraction of phycobiliprotein in laboratory bench scale
The dried nostoc sphaeroides kutz product (with the water content of 14.6%) which is dried or baked is crushed to be more than 10-200 meshes by a universal crusher and is sieved to obtain particles with the number of the particles being respectively 10-24 meshes, 24-65 meshes, 65-80 meshes, 80-120 meshes, 120-200 meshes and more than 200 meshes. Carrying out different treatment modes on the nostoc sphaeroids kutz powder, setting the nostoc sphaeroids kutz powder into three groups, wherein the first group is a direct water extraction group, taking 0.5g of a sample of the direct water extraction group, directly adding 25mL of pure water, stirring for dissolving, and extracting at room temperature for 21 hours; the second group is an ultrasonic-assisted extraction group, 0.5g of a sample of the group is added into 25mL of pure water, ultrasonic treatment is carried out at 700W power for 20min for dissolution, extraction is carried out at room temperature, ultrasonic treatment is carried out once every 1h, and extraction is carried out for 21 h; and the third group is a freeze-thaw group, 0.5g of samples of the freeze-thaw group are added into 25mL of pure water and rehydrated for 20h, and the samples are placed in a refrigerator for refrigeration for 21h at the temperature of 20 ℃ below zero, taken out and thawed for 5h at room temperature. Centrifuging at 4000r/min for 10min, and filtering to obtain crude extractive solution. The results are shown in Table 4 below and FIG. 4. from FIG. 4, it can be seen that the most preferred treatment method is ultrasonic assisted extraction.
TABLE 4
Experimental example 5 extraction of phycobiliprotein in laboratory bench scale
Crushing the dried nostoc sphaeroides kutz product (with the water content of 14.6%) which is dried or baked by using a universal crusher to 120-200 meshes, respectively taking 0.5g of the dried nostoc sphaeroides kutz, adding 15mL, 20mL, 25mL, 30mL, 35mL, 40mL and 45mL of purified water, carrying out ultrasonic treatment at 700W for 20min every 1h, extracting at the temperature of 30 ℃ for 6h, centrifuging at 4000r/min for 10min, and filtering to obtain a crude extract. The results are shown in the following table 5 and fig. 5, and it can be seen from fig. 5 that the optimum ratio of the guxian rice flour to the pure water is 1: 60.
TABLE 5
Experimental example 6 extraction of phycobiliprotein in laboratory A dried product of nostoc sphaeroides (water content 14.6%) dried in the sun or oven dried is pulverized to 120-200 mesh by a universal pulverizer, 0.5g of the dried product is taken respectively, 30mL of purified water is added, ultrasonic treatment is carried out for 20min every 1h 700W, extraction is carried out at 30 ℃, and the protein content is measured after 0h, 0.5h, 1h, 2h, 3h, 4h, 5h and 6 h. As shown in Table 6 and FIG. 6, it can be seen from FIG. 6 that the extraction rate of the phycobiliprotein from Nostoc sphaeroids Kutz gradually increases with the increase of the extraction time, the rate of the increase is very fast, then gradually becomes gentle, and the extraction time reaches 24.15% after 6 h.
TABLE 6
Experimental example 7 extraction of phycobiliprotein in laboratory bench scale
The dried nostoc sphaeroides kutz product (with the water content of 14.6%) which is dried or baked is crushed to be more than 10-200 meshes by a universal crusher and is sieved to obtain particles with the number of the particles being respectively 10-24 meshes, 24-65 meshes, 65-80 meshes, 80-120 meshes, 120-200 meshes and more than 200 meshes. Respectively taking 0.5g, adding 30mL of purified water, performing ultrasonic treatment at 700W every 1h for 20min, extracting at 30 ℃ for 6h, centrifuging at 4000r/min for 10min, and filtering to obtain crude extract. Extracting for 1 time, 2 times and 3 times. As shown in table 7 and fig. 7, it can be seen from fig. 7 that the extraction more than 3 times has no significant effect on the extraction rate of phycobiliprotein, and thus the optimal extraction number is 3 times.
TABLE 7
Experimental example 8 extraction of phycobiliprotein in laboratory bench scale
Crushing dried nostoc sphaeroides kutz (with the water content of 14.6%) which is dried or baked by using a universal crusher into 120-200 meshes, respectively taking 0.5g of the dried nostoc sphaeroides kutz, adding 30mL of purified water, carrying out ultrasonic treatment for 20min at intervals of 1h 700W, extracting for 6h at 15 ℃, 30 ℃, 45 ℃ and 60 ℃, centrifuging for 10min at 4000r/min, and filtering to obtain a crude extract. As shown in Table 8 and FIG. 8, it is understood from FIG. 8 that the optimum extraction temperature is 45 ℃ and the extraction rate at room temperature is 20% or more.
TABLE 8
Experimental example 9 extraction of alkali-soluble protein in laboratory bench scale
Pulverizing dried Nostoc sphaeroids Kutz (water content 14.6%) with universal pulverizer to 120-200 mesh, respectively taking 0.5g, adding 30mL of purified water, ultrasonic extracting at intervals of 1h 700W for 20min, extracting at 45 deg.C for 6h, centrifuging at 4000r/min for 10min, and filtering. Adding 30mL of NaOH solution into filter residue, wherein the concentration is respectively 0.05mol/L, 0.1mol/L, 0.15mol/L, 0.2mol/L, 0.25mol/L and 0.3mol/L, performing ultrasonic-assisted extraction at 45 ℃ for 6h, centrifuging at 4000r/min for 10min, and filtering to obtain a crude extract. The results are shown in Table 9 below and FIG. 9, and it can be seen from FIG. 9 that the optimum alkali concentration is 0.2 mol/L.
TABLE 9
Experimental example 10 extraction of alkali-soluble protein in laboratory bench scale
Pulverizing dried Nostoc sphaeroids Kutz (water content 14.6%) with universal pulverizer to 120-200 mesh, respectively taking 0.5g, adding 30mL of purified water, ultrasonic extracting at intervals of 1h 700W for 20min, extracting at 45 deg.C for 6h, centrifuging at 4000r/min for 10min, and filtering. Adding 0.2mol/L NaOH solution 15mL, 20mL, 25mL, 30mL, 35mL, 40mL, 45mL into the filter residue, performing ultrasonic-assisted extraction at 45 deg.C for 6h, centrifuging at 4000r/min for 10min, and filtering to obtain crude extract. The results are shown in Table 10 and FIG. 10. from FIG. 10, it can be seen that the optimum feed-to-liquid ratio of Gexian rice flour to alkali solution is 1: 80.
Experimental example 11 extraction of alkali-soluble protein in laboratory bench scale
Pulverizing dried Nostoc sphaeroids Kutz (water content 14.6%) with universal pulverizer to 120-200 mesh, respectively taking 0.5g, adding 30mL of purified water, ultrasonic extracting at intervals of 1h 700W for 20min, extracting at 45 deg.C for 6h, centrifuging at 4000r/min for 10min, and filtering. Adding the filter residue into 40mL of 0.2mol/L NaOH solution, performing ultrasonic-assisted extraction at 45 ℃ for 0h, 1h, 2h, 3h, 4h, 5h and 6h, centrifuging at 4000r/min for 10min, and filtering to obtain a crude extract. The results are shown in Table 11 and FIG. 11, and it can be seen from FIG. 11 that the optimum extraction time for alkali-soluble protein was 5 hours.
TABLE 11
Experimental example 12 laboratory bench scale extraction of prolamins
Pulverizing dried Nostoc sphaeroids Kutz (water content 14.6%) with universal pulverizer to 120-200 mesh, respectively taking 0.5g, adding 30mL of purified water, ultrasonic extracting at intervals of 1h 700W for 20min, extracting at 45 deg.C for 6h, centrifuging at 4000r/min for 10min, and filtering. Adding filter residue into 40mL of 0.2mol/L NaOH solution, performing ultrasonic treatment at intervals of 1h and 700W for 20min, extracting at 45 ℃ for 5h, centrifuging at 4000r/min for 10min, and filtering. Adding the filter residue into 30mL of 45%, 55%, 65%, 75%, 85% and 95% alcoholic solution, performing ultrasonic treatment at intervals of 1h and 700W for 20min, extracting at 45 ℃ for 6h, centrifuging at 4000r/min for 10min, and filtering to obtain a crude extract. The results are shown in Table 12 below and FIG. 12, and it can be seen from FIG. 12 that the optimum alcohol concentration is 95%.
TABLE 12
Experimental example 13 laboratory bench scale extraction of prolamins
Pulverizing dried Nostoc sphaeroids Kutz (water content 14.6%) with universal pulverizer to 120-200 mesh, respectively taking 0.5g, adding 30mL of purified water, ultrasonic extracting at intervals of 1h 700W for 20min, extracting at 45 deg.C for 6h, centrifuging at 4000r/min for 10min, and filtering. Adding filter residue into 40mL of 0.2mol/L NaOH solution, performing ultrasonic treatment at intervals of 1h and 700W for 20min, extracting at 45 ℃ for 5h, centrifuging at 4000r/min for 10min, and filtering. Adding 15mL, 20mL, 25mL, 30mL, 35mL, 40mL and 45mL of 95% alcoholic solution into the filter residue, performing ultrasonic treatment at 700W every 1h for 20min, extracting at 45 ℃ for 6h, centrifuging at 4000r/min for 10min, and filtering to obtain a crude extract. The results are shown in the following table 13 and fig. 13, and it can be seen from fig. 13 that the optimum ratio of the griffia root rice flour to the alcohol solution is 1: 60.
Watch 13
Experimental example 14 laboratory bench scale extraction of prolamins
Pulverizing dried Nostoc sphaeroids Kutz (water content 14.6%) with universal pulverizer to 120-200 mesh, respectively taking 0.5g, adding 30mL of purified water, ultrasonic extracting at intervals of 1h 700W for 20min, extracting at 45 deg.C for 6h, centrifuging at 4000r/min for 10min, and filtering. Adding filter residue into 40mL of 0.2mol/L NaOH solution, performing ultrasonic treatment at intervals of 1h and 700W for 20min, extracting at 45 ℃ for 5h, centrifuging at 4000r/min for 10min, and filtering. Adding 95% alcohol solution 30mL into the filter residue, performing ultrasonic treatment at intervals of 1h 700W for 20min, extracting at 45 ℃ for 0h, 1h, 2h, 3h, 4h, 5h and 6h, centrifuging at 4000r/min for 10min, and filtering to obtain a crude extract. The results are shown in table 14 and fig. 14, and it can be seen from fig. 14 that the optimum extraction time of prolamin is 3 h.
TABLE 14
Experimental example 15 extraction of salt-soluble protein in laboratory bench scale
Pulverizing dried Nostoc sphaeroids Kutz (water content 14.6%) with universal pulverizer to 120-200 mesh, respectively taking 0.5g, adding 30mL of purified water, ultrasonic extracting at intervals of 1h 700W for 20min, extracting at 45 deg.C for 6h, centrifuging at 4000r/min for 10min, and filtering. Adding filter residue into 40mL of 0.2mol/L NaOH solution, performing ultrasonic treatment at intervals of 1h and 700W for 20min, extracting at 45 ℃ for 5h, centrifuging at 4000r/min for 10min, and filtering. Adding 95% alcohol solution 30mL into the filter residue, performing ultrasonic treatment at intervals of 1h 700W for 20min, extracting at 45 deg.C for 3h, centrifuging at 4000r/min for 10min, and filtering. Adding the filter residue into 30mL of NaCl solution with the concentration of 0.1mol/L, 0.2mol/L, 0.3mol/L, 0.4mol/L, 0.5mol/L and 0.6mol/L respectively, performing ultrasonic treatment at intervals of 1h and 700W for 20min, extracting at 45 ℃ for 6h, centrifuging at 4000r/min for 10min, and filtering to obtain a crude extract. The results are shown in Table 15 below and FIG. 15, and it can be seen from FIG. 15 that the optimum salt concentration is 0.3 mol/L.
Experimental example 16 extraction of salt-soluble protein in laboratory bench scale
Pulverizing dried Nostoc sphaeroids Kutz (water content 14.6%) with universal pulverizer to 120-200 mesh, respectively taking 0.5g, adding 30mL of purified water, ultrasonic extracting at intervals of 1h 700W for 20min, extracting at 45 deg.C for 6h, centrifuging at 4000r/min for 10min, and filtering. Adding filter residue into 40mL of 0.2mol/L NaOH solution, performing ultrasonic treatment at intervals of 1h and 700W for 20min, extracting at 45 ℃ for 5h, centrifuging at 4000r/min for 10min, and filtering. Adding 95% alcohol solution 30mL into the filter residue, performing ultrasonic treatment at intervals of 1h 700W for 20min, extracting at 45 deg.C for 3h, centrifuging at 4000r/min for 10min, and filtering. Adding the filter residue into 0.3mol/L NaCl solution 15mL, 20mL, 25mL, 30mL, 35mL, 40mL, 45mL, performing ultrasonic treatment at intervals of 1h and 700W for 20min, extracting at 45 ℃ for 6h, centrifuging at 4000r/min for 10min, and filtering to obtain a crude extract. The results are shown in Table 16 and FIG. 16, and it can be seen from FIG. 16 that the optimum feed-to-liquid ratio of Guxian rice flour to salt solution is 1: 70.
TABLE 16
Experimental example 17 extraction of salt-soluble protein in laboratory bench scale
Pulverizing dried Nostoc sphaeroids Kutz (water content 14.6%) with universal pulverizer to 120-200 mesh, respectively taking 0.5g, adding 30mL of purified water, ultrasonic extracting at intervals of 1h 700W for 20min, extracting at 45 deg.C for 6h, centrifuging at 4000r/min for 10min, and filtering. Adding filter residue into 40mL of 0.2mol/L NaOH solution, performing ultrasonic treatment at intervals of 1h and 700W for 20min, extracting at 45 ℃ for 5h, centrifuging at 4000r/min for 10min, and filtering. Adding 95% alcohol solution 30mL into the filter residue, performing ultrasonic treatment at intervals of 1h 700W for 20min, extracting at 45 deg.C for 3h, centrifuging at 4000r/min for 10min, and filtering. Adding 35mL of 0.3mol/L NaCl solution into the filter residue, performing ultrasonic treatment at intervals of 1h 700W for 20min, extracting at 45 ℃ for 0h, 1h, 2h, 3h, 4h, 5h and 6h, centrifuging at 4000r/min for 10min, and filtering to obtain a crude extract. The results are shown in Table 17 below and FIG. 17, and it can be seen from FIG. 17 that the optimum extraction time for the salt-solubilized protein was 3 hours.
TABLE 17
Experimental example 18 Effect of different extraction methods on the Total protein content of Nostoc sphaeroides
The dried nostoc sphaeroides product (with the water content of 14.6%) which is dried or baked is crushed into 120-200 meshes by a universal crusher, 0.5g of the dried nostoc sphaeroides product is respectively taken and divided into 6 groups, different proteins are extracted from each group according to different sequences, phycobiliprotein is extracted in the first step, and salt-soluble protein, alcohol-soluble protein and alkali-soluble protein are extracted according to different sequences in the second step, the fourth step and the fifth step.
The experimental conditions are as follows: and (3) extraction of phycobiliprotein: extracting with pure water at a ratio of 1:60 for 6h at 45 deg.C for 700W every 1h for 20 min; extracting the salt-soluble protein: NaCl concentration of 0.3mol/L, material-liquid ratio of 1:70, extraction time of 3h, extraction temperature of 45 ℃, and ultrasonic treatment for 20min at intervals of 1h and 700W; extracting alkali-soluble protein: the concentration of NaOH is 0.2mol/L, the ratio of material to liquid is 1:80, the extraction time is 5h, the extraction temperature is 45 ℃, and the ultrasonic treatment is carried out for 20min at intervals of 1h and 700W; alcohol soluble protein extraction: the ethanol concentration is 95%, the feed-liquid ratio is 1:70, the extraction time is 3h, the extraction temperature is 45 ℃, and the ultrasonic treatment is carried out for 20min at intervals of 1h and 700W. The results are given in Table 18 below.
As can be seen from table 18, the total extraction rate of the first step of phycobiliprotein extraction, the second step of alkali-soluble protein extraction, the third step of prolamin extraction, and the fourth step of salt-soluble protein extraction is the highest, and is significantly higher than that of the other groups.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (3)
1. The nostoc sphaeroids kutz protein extraction method is characterized by comprising the following steps of:
1) crushing dried nostoc sphaeroides with the water content of 11-15% to 120-200 meshes, adding water 60 times the weight of nostoc sphaeroides, extracting for 3 times, extracting for 6 hours each time, extracting at the temperature of 45 ℃, and performing ultrasonic treatment for 20min by 700W every 1 hour; centrifuging and filtering to obtain crude extract of Nostoc sphaeroids Kutz bile protein; filtering residues for later use;
2) adding 80 times of sodium hydroxide solution by weight into the nostoc sphaeroides filter residue obtained in the step 1), extracting for 3 times, wherein the concentration of the sodium hydroxide solution is 0.2mol/L, the extraction time is 5h each time, the extraction temperature is 45 ℃, ultrasonic treatment is carried out for 20min by using 700W ultrasonic waves every 1h, and centrifugation and filtration are carried out to obtain nostoc sphaeroides alkali-soluble protein crude extract; filtering residues for later use;
3) adding 70 times of ethanol solution with the concentration of 95% into the nostoc sphaeroides filter residue obtained in the step 2), extracting for 3 times, each time for 3 hours, at the extraction temperature of 45 ℃, performing ultrasonic treatment for 20 minutes by using 700W ultrasonic waves every 1 hour, centrifuging, and filtering to obtain a nostoc sphaeroides alcohol soluble protein crude extract; filtering residues for later use;
4) adding 70 times of NaCl solution by weight into the nostoc sphaeroides filter residue obtained in the step 3), extracting for 3 times, wherein the concentration of the NaCl solution is 0.3mol/L, the extraction temperature is 45 ℃, ultrasonic processing is performed for 20min by 700W ultrasonic waves every 1h, centrifuging and filtering are performed, so as to obtain crude nostoc sphaeroides salt-soluble protein extract, and the filter residue is discarded;
5) mixing the obtained crude extract of the nostoc sphaeroides gallbladder protein, the crude extract of the nostoc sphaeroides alkali-soluble protein, the crude extract of the nostoc sphaeroides alcohol-soluble protein and the crude extract of the nostoc sphaeroides salt-soluble protein, performing ultrafiltration twice for removing impurities by using a ceramic membrane with the interception aperture of 5000-300000D, performing ultrafiltration concentration by using an organic membrane with the interception aperture of 5,000-10,000D, and performing freeze drying to obtain the nostoc sphaeroides protein.
2. The method according to claim 1, characterized in that in step 1): the water content of the dried nostoc sphaeroides is 14.6 percent.
3. The extraction method as claimed in claim 1, wherein step 5) comprises mixing the crude extractive solution of nostoc phycobiliprotein, crude extractive solution of nostoc salt-soluble protein, crude extractive solution of nostoc alcohol-soluble protein and crude extractive solution of nostoc alkali-soluble protein, sequentially ultrafiltering with ceramic membrane with cut-off pore size of 300,000D and 5,000D for two times to remove impurities, ultrafiltering with organic membrane with cut-off pore size of 8,000D for concentration, and freeze drying to obtain nostoc protein.
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