A method for extracting lily polysaccharide

Abstract

The invention provides a method for extracting lily polysaccharide, belonging to the technical field of polysaccharide extraction. The extraction method comprises lily pretreatment, crude polysaccharide extraction, preparation of protein adsorbent and preparation of refined polysaccharide. The method for preparing refined polysaccharide comprises dissolving crude polysaccharide in ultrapure water, heating in a water bath to 30-50 DEG C, adding protein adsorbent, stirring and adsorbing for 45-55 minutes, centrifuging for 2.5-3.5 minutes, standing, taking supernatant, adding ethanol solution with a volume of 2.5-3.5 times that of the supernatant, standing for 16-20 hours, filtering, washing with anhydrous ethanol, drying to obtain refined polysaccharide, i.e. lily polysaccharide. In the method for extracting lily polysaccharide of the invention, the yield of crude lily polysaccharide reaches 21.97-22.69%; the loss rate of lily polysaccharide is 4.63-5.04%; the protein removal rate reaches 63.48-67.53% and the purity of refined polysaccharide reaches 95.73-96.18%.

Description

Technical Field

[0001] The invention belongs to the technical field of polysaccharide extraction, and particularly relates to a method for extracting lily polysaccharide. Background Art

[0002] Lily, also known as lily, mountain dan, and heavy lily, belongs to the Liliaceae family. It is a perennial herbaceous bulbous plant that likes a cool, moist semi-shady environment, is relatively cold-resistant, and is a long-day plant. Lily bulbs are spherical, mostly white or lavender in color, and are fleshy and thick. They are the main organs for storing nutrients. At the same time, lily bulbs are often used to treat symptoms such as yin deficiency and chronic cough, blood in sputum, restlessness and palpitations, insomnia and dreaminess, and mental confusion. They have high edible and medicinal value.

[0003] Lily polysaccharide is a mixed polysaccharide extracted from lily bulbs. Pharmacological studies have shown that lily polysaccharide has multiple effects such as anti-inflammatory, antioxidant, anti-tumor, hypoglycemic, and immunity enhancement. The extraction steps of lily polysaccharide are mainly divided into raw material pretreatment, crude polysaccharide extraction, and polysaccharide refining (deproteinization). Among them, the Sevage method for polysaccharide refining (deproteinization) has mild conditions and wide applicability, but the chloroform used in the Sevage method is a toxic substance, and the residual solvent is likely to cause the polysaccharide activity to decrease.

[0004] Patent CN114835831A discloses a method for extracting lily polysaccharides. The patent adopts ultrasonic method instead of water extraction method. The polysaccharide extraction efficiency is high and the polysaccharide activity is well maintained. However, the equipment cost is high, the energy utilization rate is low, and ultrasonic treatment will lead to degradation of the polysaccharide structure. The deproteinization adopts Sevage method, which easily causes the polysaccharide activity to decrease.

[0005] Patent CN116970097A discloses a method for extracting plant polysaccharides and plant polysaccharides. In the patented technical solution, the method for removing protein is to add Fe 3+ The modified organic porous polymer microspheres are added, stirred, filtered, and polysaccharide filtrate is obtained. The polysaccharide filtrate is decolorized, concentrated, dried, washed, and dried to obtain plant polysaccharides. This method is mild and will not reduce the activity of polysaccharides. However, Fe 3+ While citric acid is complexed with proteins, it also combines with the hydroxyl groups in polysaccharides, resulting in increased loss of polysaccharides while removing proteins. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides a method for extracting lily polysaccharides to achieve the following invention objectives: while improving the protein removal rate, reducing the polysaccharide loss rate and improving the polysaccharide yield.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0008] A method for extracting lily polysaccharides comprises the following steps:

[0009] 1. Lily pretreatment

[0010] After washing the lily bulbs with anhydrous ethanol for 9 to 11 minutes, the residual anhydrous ethanol on the surface is washed away with deionized water, and then dried at 30 to 40° C. and ground into 0.20 to 0.25 mm powder to obtain lily bulb powder, which is stored at 4 to 6° C. for later use.

[0011] 2. Extraction of crude polysaccharides

[0012] The lily bulb powder is quickly frozen at -25 to -35°C for 28 to 32 minutes, taken out, thawed at room temperature, then quickly frozen at -45 to -55°C for 12 to 17 minutes, taken out, thawed at room temperature, then quickly frozen at -70 to -90°C for 7 to 15 minutes, taken out, and quickly thawed under a microwave to obtain the thawed lily bulb powder, the thawed lily bulb powder is added to ultrapure water, extracted in a water bath at 50 to 65°C for 40 to 50 minutes, and then transferred to a centrifuge, The mixture was centrifuged at 4000-5000 r / min for 5-10 min. After the centrifugation, the supernatant was extracted and subjected to vacuum distillation. When the volume of the supernatant was concentrated to 1 / 4-1 / 2 of the original volume, the vacuum distillation was stopped to obtain a concentrated supernatant. An ethanol solution 2.5-3.5 times the volume of the concentrated supernatant was added to the concentrated supernatant. The mixture was allowed to stand at 4-6°C for 16-20 h, filtered, the filter cake was cleaned with anhydrous ethanol, and dried at 30-40°C to constant weight to obtain crude polysaccharide.

[0013] The rapid thawing under microwave, the microwave temperature is 40-60°C, the microwave time is 5-10min;

[0014] The mass ratio of the thawed lily bulb powder to ultrapure water is 0.8-1.2:50-60;

[0015] The mass fraction of the ethanol solution is 70-80%.

[0016] 3. Preparation of protein adsorbent

[0017] Tween 60 is added to liquid paraffin, heated in a constant temperature water bath at 50-65°C until completely dissolved, and then starch solution is added dropwise, while controlling the stirring rate to be 500-700 r / min. When all the starch solution is added, stirring is stopped, and citric acid and modified chitosan are added, and cross-linking reaction is carried out at 35-45°C for 2.5-4h. After the reaction is completed, centrifuge at 2500-3500 r / min for 8-12min, and the upper solution is discarded. The lower precipitate is fully washed with anhydrous ethanol for 2-4 times, and then dried in an oven at 55-65°C to constant weight to obtain starch / modified chitosan composite adsorption microspheres, i.e., protein adsorbents;

[0018] The mass fraction of the starch solution is 6 to 10%;

[0019] The mass ratio of the starch solution, liquid paraffin and Tween 60 is 1.2-1.6:13.3-13.8:1.5-1.9;

[0020] The mass ratio of the starch solution, modified chitosan and citric acid is 90-120:7-9:2.5-3.5;

[0021] The preparation method of the modified chitosan comprises: adjusting the pH value of the chitosan solution to 4.5-5.5 using an HCl solution, then adding 12-hydroxydodecanal, stirring and reacting for 4.5-5.5 hours, then adding sodium borohydride and stirring and reacting for 12-15 hours, then adding periodic acid and stirring and reacting for 35-45 minutes, then filtering, washing the filter cake with anhydrous ethanol, and drying it in an oven at 55-65° C. for 8-12 hours to obtain the modified chitosan;

[0022] The mass fraction of the chitosan solution is 12-18%;

[0023] The mass fraction of the HCI solution is 8 to 12%;

[0024] The mass ratio of chitosan, 12-hydroxydodecanal, sodium borohydride and periodic acid is 65-70:1.1-1.5:0.35-0.4:4.0-4.3;

[0025] The stirring rate is 30-40 r / min.

[0026] 4. Preparation of refined polysaccharides

[0027] The crude polysaccharide is dissolved in ultrapure water, heated in a water bath to 30-50°C, and then a protein adsorbent is added, and adsorbed at a rate of 17-25 r / min for 45-55 min, and then centrifuged at a rate of 600-800 r / min for 2.5-3.5 min, and allowed to stand for 25-35 min, and the supernatant is taken, and an ethanol solution 2.5-3.5 times the volume of the supernatant is added to the supernatant, and allowed to stand for 16-20 h at 4-6°C, and filtered, and the filter cake is cleaned with anhydrous ethanol, and dried at 30-40°C to constant weight to obtain a refined polysaccharide, i.e., lily polysaccharide;

[0028] The mass ratio of the crude polysaccharide, ultrapure water and protein adsorbent is 0.8-1.2: 6-8: 0.04-0.06;

[0029] The mass fraction of the ethanol solution is 80-90%.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1. When extracting crude polysaccharides, the method of quick freezing and slow thawing is adopted to make the water in the cells form ice crystals, thereby piercing the cell membrane and cell wall, while causing less damage to the structure of the polysaccharide. Combined with the water extraction method, the yield of crude polysaccharides can be significantly increased to 21.97-22.69%.

[0032] 2. The protein adsorbent of the present invention is an adsorption microsphere formed by citric acid cross-linked starch and modified chitosan. The raw materials used are mild and will not cause the activity of polysaccharides to decrease. The preparation principle of the modified chitosan is that the amino group of chitosan reacts with the aldehyde group in 12-hydroxydodecanal to undergo Schiff base reaction, and a large number of long-chain structures containing hydroxyl groups at the end are connected to the surface of chitosan. Then, periodic acid oxidizes the hydroxyl groups at the ends of the long chains into aldehyde groups. The aldehyde groups react sensitively with the amino groups on the surface of proteins and can quickly and accurately connect to proteins. Therefore, in the process of preparing refined polysaccharides, the protein removal rate is high (63.48-67.53%), that is, the purity of the refined polysaccharides is high (95.73-96.18%), and the polysaccharide loss rate is small (4.63-5.04%). DETAILED DESCRIPTION

[0033] Example 1

[0034] 1. Lily pretreatment

[0035] After washing the lily bulbs with anhydrous ethanol for 10 minutes, the residual anhydrous ethanol on the surface was washed away with deionized water, and then dried at 35°C and ground into 0.21-0.23 mm powder to obtain lily bulb powder, which was stored at 5°C for later use.

[0036] 2. Extraction of crude polysaccharides

[0037] The lily bulb powder was quick-frozen at -30°C for 30 minutes, taken out, thawed at room temperature, then quick-frozen at -50°C for 15 minutes, taken out, thawed at room temperature, then quick-frozen at -80°C for 10 minutes, taken out, and quickly thawed under a microwave to obtain the thawed lily bulb powder; the thawed lily bulb powder was added into ultrapure water, extracted in a 60°C water bath for 45 minutes, transferred into a centrifuge, and centrifuged at 4500r / min for 8 minutes. After the centrifugation, the supernatant was extracted and the supernatant was subjected to reduced pressure distillation. When the volume of the supernatant was concentrated to 1 / 3 of the original volume, the reduced pressure distillation was stopped to obtain a concentrated supernatant; an ethanol solution 3 times the volume of the concentrated supernatant was added to the concentrated supernatant, and the mixture was allowed to stand at 5°C for 18 hours, filtered, the filter cake was cleaned with anhydrous ethanol, and dried at 35°C to constant weight to obtain crude polysaccharide.

[0038] The rapid thawing under microwave, the microwave temperature is 50°C, the microwave time is 7min;

[0039] The mass ratio of the thawed lily bulb powder to ultrapure water is 1:55;

[0040] The mass fraction of the ethanol solution is 75%.

[0041] 3. Preparation of protein adsorbent

[0042] Tween 60 was added to liquid paraffin, heated in a constant temperature water bath at 60°C until completely dissolved, and then starch solution was added dropwise, while the stirring rate was controlled to be 600 r / min. When all the starch solution was added, stirring was stopped, and citric acid and modified chitosan were added. The mixture was cross-linked at 40°C for 3 h. After the reaction was completed, the mixture was centrifuged at 3000 r / min for 10 min, the upper solution was discarded, and the lower precipitate was fully washed with anhydrous ethanol for 3 times, and then dried in an oven at 60°C to constant weight to obtain starch / modified chitosan composite adsorption microspheres, i.e., protein adsorbents.

[0043] The mass fraction of the starch solution is 8%;

[0044] The mass ratio of the starch solution, liquid paraffin and Tween 60 is 1.4:13.6:1.7;

[0045] The mass ratio of the starch solution, modified chitosan and citric acid is 100:8:3;

[0046] The preparation method of the modified chitosan comprises: adjusting the pH value of the chitosan solution to 5 using an HCl solution, then adding 12-hydroxydodecanal, stirring and reacting for 5 hours, then adding sodium borohydride and stirring and reacting for 14 hours, then adding periodic acid and stirring and reacting for 40 minutes, then filtering, washing the filter cake with anhydrous ethanol, and drying it in a 60° C. oven for 10 hours to obtain the modified chitosan;

[0047] The mass fraction of the chitosan solution is 15%;

[0048] The mass fraction of the HCI solution is 10%;

[0049] The mass ratio of chitosan, 12-hydroxydodecanal, sodium borohydride and periodic acid is 67:1.3:0.38:4.1;

[0050] The stirring rate is 35 r / min.

[0051] 4. Preparation of refined polysaccharides

[0052] The crude polysaccharide was dissolved in ultrapure water, heated to 40°C in a water bath, and then a protein adsorbent was added. After adsorption at a rate of 20 r / min for 50 min, the mixture was centrifuged at a rate of 700 r / min for 3 min, and allowed to stand for 30 min. The supernatant was taken, and an ethanol solution 3 times the volume of the supernatant was added to the supernatant. The mixture was allowed to stand at 5°C for 18 h, and filtered. The filter cake was cleaned with anhydrous ethanol, and dried at 35°C to constant weight to obtain a refined polysaccharide, i.e., lily polysaccharide.

[0053] The mass ratio of the crude polysaccharide, ultrapure water and protein adsorbent is 1:7:0.05;

[0054] The mass fraction of the ethanol solution is 85%.

[0055] Example 2

[0056] 1. Lily pretreatment

[0057] After washing the lily bulbs with anhydrous ethanol for 9 minutes, the residual anhydrous ethanol on the surface was washed away with deionized water, and then dried at 30°C and ground into 0.20-0.21 mm powder to obtain lily bulb powder, which was stored at 4°C for later use.

[0058] 2. Extraction of crude polysaccharides

[0059] The lily bulb powder was quick-frozen at -25°C for 28 minutes, taken out, thawed at room temperature, then quick-frozen at -45°C for 12 minutes, taken out, thawed at room temperature, then quick-frozen at -70°C for 7 minutes, taken out, and quickly thawed under a microwave to obtain the thawed lily bulb powder; the thawed lily bulb powder was added into ultrapure water, extracted in a 50°C water bath for 40 minutes, transferred into a centrifuge, and centrifuged at 4000r / min for 5 minutes. After the centrifugation, the supernatant was extracted and the supernatant was subjected to reduced pressure distillation. When the volume of the supernatant was concentrated to 1 / 4 of the original volume, the reduced pressure distillation was stopped to obtain a concentrated supernatant, an ethanol solution 2.5 times the volume of the concentrated supernatant was added to the concentrated supernatant, and the mixture was allowed to stand at 4°C for 16 hours, filtered, the filter cake was cleaned with anhydrous ethanol, and dried at 30°C to constant weight to obtain crude polysaccharide.

[0060] The rapid thawing under microwave, the microwave temperature is 40°C, the microwave time is 5min;

[0061] The mass ratio of the thawed lily bulb powder to ultrapure water is 0.8:50;

[0062] The mass fraction of the ethanol solution is 70%.

[0063] 3. Preparation of protein adsorbent

[0064] Tween 60 was added to liquid paraffin, heated in a constant temperature water bath at 50°C until completely dissolved, and then starch solution was added dropwise, while the stirring rate was controlled to be 500r / min. When all the starch solution was added, stirring was stopped, and citric acid and modified chitosan were added. The cross-linking reaction was carried out at 35°C for 2.5h. After the reaction was completed, centrifuged at 2500r / min for 8min, the upper solution was discarded, and the lower precipitate was fully washed twice with anhydrous ethanol, and then dried in an oven at 55°C to constant weight to obtain starch / modified chitosan composite adsorption microspheres, i.e., protein adsorbents.

[0065] The mass fraction of the starch solution is 6%;

[0066] The mass ratio of the starch solution, liquid paraffin and Tween 60 is 1.2:13.3:1.5;

[0067] The mass ratio of the starch solution, modified chitosan and citric acid is 90:7:2.5;

[0068] The preparation method of the modified chitosan comprises: adjusting the pH value of the chitosan solution to 4.5 using an HCl solution, then adding 12-hydroxydodecanal, stirring and reacting for 4.5 hours, then adding sodium borohydride and stirring and reacting for 12 hours, then adding periodic acid and stirring and reacting for 35 minutes, then filtering, washing the filter cake with anhydrous ethanol, and drying it in a 55° C. oven for 8 hours to obtain the modified chitosan;

[0069] The mass fraction of the chitosan solution is 12%;

[0070] The mass fraction of the HCI solution is 8%;

[0071] The mass ratio of chitosan, 12-hydroxydodecanal, sodium borohydride and periodic acid is 65:1.1:0.35:4.0;

[0072] The stirring rate is 30 r / min.

[0073] 4. Preparation of refined polysaccharides

[0074] The crude polysaccharide was dissolved in ultrapure water, heated to 30°C in a water bath, and then a protein adsorbent was added. After stirring and adsorbing at a rate of 17 r / min for 45 min, the mixture was centrifuged at a rate of 600 r / min for 2.5 min, and allowed to stand for 25 min. The supernatant was taken, and an ethanol solution 2.5 times the volume of the supernatant was added to the supernatant. The mixture was allowed to stand at 4°C for 16 h, and filtered. The filter cake was cleaned with anhydrous ethanol, and dried at 30°C to constant weight to obtain a refined polysaccharide, i.e., lily polysaccharide.

[0075] The mass ratio of the crude polysaccharide, ultrapure water and protein adsorbent is 0.8:6:0.04;

[0076] The mass fraction of the ethanol solution is 80%.

[0077] Example 3

[0078] 1. Lily pretreatment

[0079] After washing the lily bulbs with anhydrous ethanol for 11 minutes, the residual anhydrous ethanol on the surface was washed away with deionized water, and then dried at 40°C and ground into 0.23-0.25 mm powder to obtain lily bulb powder, which was stored at 6°C for later use.

[0080] 2. Extraction of crude polysaccharides

[0081] The lily bulb powder was quickly frozen at -35°C for 32 minutes, taken out, thawed at room temperature, then quickly frozen at -55°C for 17 minutes, taken out, thawed at room temperature, then quickly frozen at -90°C for 15 minutes, taken out, and quickly thawed under a microwave to obtain the thawed lily bulb powder. The thawed lily bulb powder was added to ultrapure water, extracted in a 65°C water bath for 50 minutes, transferred to a centrifuge, and centrifuged at 5000r / min for 10 minutes. After the centrifugation, the supernatant was extracted and the supernatant was subjected to reduced pressure distillation. When the volume of the supernatant was concentrated to 1 / 2 of the original volume, the reduced pressure distillation was stopped to obtain a concentrated supernatant. An ethanol solution 3.5 times the volume of the concentrated supernatant was added to the concentrated supernatant, and the mixture was allowed to stand at 6°C for 20 hours, filtered, and the filter cake was cleaned with anhydrous ethanol, and dried at 40°C to constant weight to obtain crude polysaccharides.

[0082] The rapid thawing under microwave, the microwave temperature is 60°C, the microwave time is 10min;

[0083] The mass ratio of the thawed lily bulb powder to ultrapure water is 1.2:60;

[0084] The mass fraction of the ethanol solution is 80%.

[0085] 3. Preparation of protein adsorbent

[0086] Tween 60 was added to liquid paraffin, heated in a constant temperature water bath at 65°C until completely dissolved, and then starch solution was added dropwise, while the stirring rate was controlled to be 700 r / min. When all the starch solution was added, stirring was stopped, and citric acid and modified chitosan were added. The mixture was cross-linked at 45°C for 4 h. After the reaction was completed, the mixture was centrifuged at 3500 r / min for 12 min, the upper solution was discarded, and the lower precipitate was fully washed with anhydrous ethanol for 4 times, and then dried in an oven at 65°C to constant weight to obtain starch / modified chitosan composite adsorption microspheres, i.e., protein adsorbents.

[0087] The mass fraction of the starch solution is 10%;

[0088] The mass ratio of the starch solution, liquid paraffin and Tween 60 is 1.6:13.8:1.9;

[0089] The mass ratio of the starch solution, modified chitosan and citric acid is 120:9:3.5;

[0090] The preparation method of the modified chitosan comprises: adjusting the pH value of the chitosan solution to 5.5 using an HCl solution, then adding 12-hydroxydodecanal, stirring and reacting for 5.5 hours, then adding sodium borohydride and stirring and reacting for 15 hours, then adding periodic acid and stirring and reacting for 45 minutes, then filtering, washing the filter cake with anhydrous ethanol, and drying it in a 65° C. oven for 12 hours to obtain the modified chitosan;

[0091] The mass fraction of the chitosan solution is 18%;

[0092] The mass fraction of the HCI solution is 12%;

[0093] The mass ratio of chitosan, 12-hydroxydodecanal, sodium borohydride and periodic acid is 70:1.5:0.4:4.3;

[0094] The stirring rate is 40 r / min.

[0095] 4. Preparation of refined polysaccharides

[0096] The crude polysaccharide was dissolved in ultrapure water, heated to 50°C in a water bath, and then a protein adsorbent was added. After adsorption at a rate of 25 r / min for 55 min, the mixture was centrifuged at a rate of 800 r / min for 3.5 min, and allowed to stand for 35 min. The supernatant was taken, and an ethanol solution 3.5 times the volume of the supernatant was added to the supernatant. The mixture was allowed to stand for 20 h at 6°C, and filtered. The filter cake was cleaned with anhydrous ethanol, and dried at 40°C to constant weight to obtain a refined polysaccharide, i.e., lily polysaccharide.

[0097] The mass ratio of the crude polysaccharide, ultrapure water and protein adsorbent is 1.2:8:0.06;

[0098] The mass fraction of the ethanol solution is 90%.

[0099] Comparative Example 1

[0100] On the basis of Example 1, the quick freezing and thawing steps in step 2 are omitted. Specifically, the lily bulb powder is extracted in a 65°C water bath for 50 minutes, and then transferred to a centrifuge and centrifuged at 5000 r / min for 10 minutes. After the centrifugation, the supernatant is extracted and the supernatant is subjected to vacuum distillation. When the volume of the supernatant is concentrated to 1 / 2 of the original volume, the vacuum distillation is stopped to obtain a concentrated supernatant. An ethanol solution 3.5 times the volume of the concentrated supernatant is added to the concentrated supernatant, and the mixture is allowed to stand at 6°C for 20 hours. The mixture is filtered, the filter cake is cleaned with anhydrous ethanol, and dried at 40°C to constant weight to obtain crude polysaccharides.

[0101] Comparative Example 2

[0102] On the basis of Example 1, the preparation of modified chitosan in step 3 was omitted, and the modified chitosan was replaced by chitosan.

[0103] Test example

[0104] The lily polysaccharides prepared in Examples 1 to 3 and Comparative Examples 1 to 2 were tested, and the test items included the yield of crude lily polysaccharides, the loss rate of lily polysaccharides, the protein removal rate, and the purity of refined polysaccharides;

[0105] The test method for the yield of lily crude polysaccharides is as follows:

[0106] Lily crude polysaccharide yield = lily crude polysaccharide mass / lily bulb powder mass;

[0107] The test method for lily polysaccharide loss rate is as follows:

[0108] The phenol-sulfuric acid method was used to determine the polysaccharide content m1 in the crude polysaccharide solution before deproteinization and the polysaccharide content m2 in the refined polysaccharide solution after deproteinization. The lily polysaccharide loss rate = (m1-m2) / m1×100%;

[0109] The protein removal rate test method is as follows:

[0110] The protein content q1 in the crude polysaccharide solution before deproteinization and the protein content q2 in the refined polysaccharide solution after deproteinization were determined by the Coomassie Brilliant Blue method. The protein removal rate = (q1-q2) / q1×100%;

[0111] The calculation method of the purity of refined polysaccharides is as follows:

[0112] Purity of refined polysaccharide = content of polysaccharide in refined polysaccharide solution after deproteinization m2 / mass of refined polysaccharide m×100%;

[0113] The test results of lily polysaccharide yield, lily polysaccharide loss rate, protein removal rate and refined polysaccharide purity are shown in Table 1.

[0114] Table 1

[0115]

[0116] It can be seen from the above results that the yield of crude lily polysaccharide in Comparative Example 1 is lower than that in Example 1 due to the lack of quick freezing and thawing steps during polysaccharide extraction, but the protein adsorbent is not changed. Therefore, during polysaccharide purification, the lily polysaccharide loss rate and protein removal rate are not much different from those in Example 1, and the purified polysaccharide has a higher purity.

[0117] In Comparative Example 2, the crude polysaccharide extraction method was unchanged, and the yield of crude lily polysaccharides was high. However, the prepared protein adsorbent did not modify the chitosan, resulting in the protein adsorbent adsorbing part of the polysaccharides while adsorbing the protein. Therefore, the lily polysaccharide loss rate increased, the protein removal rate decreased, and the purity of the refined polysaccharide decreased.

Claims

1. A method for extracting lily polysaccharides, characterized in that: The extraction method includes lily pretreatment, crude polysaccharide extraction, protein adsorbent preparation, and refined polysaccharide preparation; The method for preparing the protein adsorbent comprises: adding Tween 60 to liquid paraffin, heating in a water bath until completely dissolved, adding starch solution dropwise, and controlling the stirring rate to be 500-700 r / min; when all the starch solution is added, stopping stirring, adding citric acid and modified chitosan, and performing cross-linking reaction at 35-45° C. for 2.5-4 hours; after the reaction is completed, centrifuging, removing the lower precipitate, washing it with anhydrous ethanol, and drying it to obtain the protein adsorbent; The preparation method of the modified chitosan comprises: adjusting the pH value of the chitosan solution to 4.5-5.5 using an HCl solution, then adding 12-hydroxydodecanal, stirring and reacting for 4.5-5.5 hours, then adding sodium borohydride and stirring and reacting for 12-15 hours, then adding periodic acid and stirring and reacting for 35-45 minutes, then filtering, washing with anhydrous ethanol, and drying to obtain the modified chitosan; The method for extracting crude polysaccharides comprises the following steps: quick-freezing lily bulb powder at -25 to -35°C for 28 to 32 minutes, taking it out, thawing it at room temperature, then quick-freezing it at -45 to -55°C for 12 to 17 minutes, taking it out, thawing it at room temperature, then quick-freezing it at -70 to -90°C for 7 to 15 minutes, taking it out, and quickly thawing it under microwave; adding the thawed lily bulb powder into ultrapure water, extracting it in a water bath at 50 to 65°C for 40 to 50 minutes, transferring it into a centrifuge, centrifuging it at 4000 to 5000 r / min for 5 to 10 minutes, and then performing reduced pressure distillation on the supernatant to a volume of 1 / 4 to 1 / 2 of the original volume, adding an ethanol solution 2.5 to 3.5 times the volume of the concentrated supernatant to the obtained concentrated supernatant, and performing standing, filtering, washing with anhydrous ethanol, and drying to obtain crude polysaccharides.

2. The method for extracting lily polysaccharides according to claim 1, characterized in that: In the method for preparing a protein adsorbent, the mass fraction of the starch solution is 6-10%; the mass ratio of the starch solution, liquid paraffin and Tween 60 is 1.2-1.6:13.3-13.8:1.5-1.9; and the mass ratio of the starch solution, modified chitosan and citric acid is 90-120:7-9:2.5-3.

5.

3. The method for extracting lily polysaccharides according to claim 1, characterized in that: In the preparation method of the modified chitosan, the mass fraction of the chitosan solution is 12-18%; the mass fraction of the HCl solution is 8-12%; the mass ratio of chitosan, 12-hydroxydodecanal, sodium borohydride and periodic acid is 65-70:1.1-1.5:0.35-0.4:4.0-4.

3.

4. The method for extracting lily polysaccharides according to claim 1, characterized in that: In the preparation method of the modified chitosan, the drying temperature is 55 to 65° C. and the drying time is 8 to 12 hours.

5. The method for extracting lily polysaccharides according to claim 1, characterized in that: In the crude polysaccharide extraction method, the rapid thawing under microwave, the microwave temperature is 40-60° C., the microwave time is 5-10 min; the mass ratio of the thawed lily bulb powder to ultrapure water is 0.8-1.2:50-60; the mass fraction of the ethanol solution is 70-80%.

6. The method for extracting lily polysaccharides according to claim 1, characterized in that: In the crude polysaccharide extraction method, the standing temperature is 4-6°C, and the drying temperature is 30-40°C.

7. The method for extracting lily polysaccharides according to claim 1, characterized in that: The lily pretreatment method comprises the following steps: washing the lily bulbs with anhydrous ethanol for 9 to 11 minutes, washing away the anhydrous ethanol remaining on the surface with deionized water, drying at 30 to 40° C., grinding into 0.20 to 0.25 mm powder, obtaining lily bulb powder, and storing at 4 to 6° C. for later use.

8. The method for extracting lily polysaccharides according to claim 1, characterized in that: The method for preparing refined polysaccharide comprises the following steps: dissolving crude polysaccharide in ultrapure water, heating the solution in a water bath to 30-50° C., adding a protein adsorbent, stirring and adsorbing the solution for 45-55 minutes, centrifuging the solution at a rate of 600-800 r / min for 2.5-3.5 minutes, standing the solution for 25-35 minutes, taking the supernatant, adding an ethanol solution 2.5-3.5 times the volume of the supernatant, standing the solution for 16-20 hours at 4-6° C., filtering, washing with anhydrous ethanol, and drying the solution to obtain refined polysaccharide, i.e., lily polysaccharide.

9. The method for extracting lily polysaccharides according to claim 8, characterized in that: In the method for preparing refined polysaccharide, the mass ratio of crude polysaccharide, ultrapure water and protein adsorbent is 0.8-1.2:6-8:0.04-0.06; and the mass fraction of ethanol solution is 80-90%.

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