CN114316077A - Preparation method and application of sea cucumber polysaccharide - Google Patents
Preparation method and application of sea cucumber polysaccharide Download PDFInfo
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Abstract
The invention discloses a preparation method of sea cucumber polysaccharide, which comprises the following steps: 1) pulverizing Stichopus japonicus, soaking in water, sequentially performing enzymolysis with compound protease and flavourzyme, boiling to inactivate enzyme after enzymolysis, centrifuging, and collecting supernatant; ultrafiltering the supernatant with hollow fiber column, collecting ultrafiltration retentate to obtain crude product solution of Stichopus japonicus polysaccharide; 2) putting the sea cucumber polysaccharide crude product solution obtained in the step 1) on an anion exchange chromatographic column, sequentially eluting with water and NaCl solution, and collecting NaCl eluent; 3) desalting the NaCl eluate with nanofiltration membrane, collecting nanofiltration trapped fluid, concentrating under reduced pressure, and freeze drying to obtain sea cucumber polysaccharide. The sea cucumber polysaccharide can be used for preparing anticoagulant drugs or health care products, has the effect of preventing thrombus, and has good auxiliary prevention effect on the old and people with high risk of thrombus.
Description
Technical Field
The invention relates to a sea cucumber polysaccharide and a preparation method and application thereof.
Technical Field
Sea cucumber is a superior tonic good medicine and is popular as a functional food. In recent years, with the rapid development of the health care product industry, more and more health care products using sea cucumbers as main raw materials are provided, and meanwhile, the research on the medicinal value and the health care function of the sea cucumbers is further advanced. Sea cucumber contains a plurality of important chemical components, mainly comprising sea cucumber polysaccharide, sea cucumber saponin, sea cucumber collagen, sea cucumber polypeptide, lipid substances and the like, and the active components have biological activities of resisting tumor, oxidation, immunity regulation, bacteria, viruses, blood sugar and blood coagulation and the like, and can be used for preventing and assisting in treating certain diseases (Wahao and the like, research progress of chemical components and biological activity of sea cucumber [ J ], food safety quality detection report, 2017, 8(06): 2054-.
Relevant research at home and abroad shows that the sea cucumber polysaccharide mainly comprises two types: one is holothurian glycosaminoglycan or mucopolysaccharide (HG), which is a branched heteropolysaccharide composed of D-N-acetylgalactosamine, D-glucuronic acid and L-fucose, and the relative molecular mass is 40000-50000; the other is Holothurian Fucosan (HF), which is a straight-chain homogeneous polysaccharide composed of L-fucose and has a relative molecular mass of 80000-.
According to the reports of related documents, the current common extraction methods of sea cucumber polysaccharide mainly comprise a solvent extraction method and a protease hydrolysis method, and the protease hydrolysis method is an ideal method for extracting the sea cucumber polysaccharide (Zhang hongling et al, the research progress on the extraction and separation and the biological activity of the sea cucumber polysaccharide [ J ], the report on the quality detection of food safety, 2017, 8(06):2062 and 2067). The commonly used separation and purification methods mainly include fractional precipitation, salting-out, metal complexation, organic salt precipitation, quaternary ammonium salt precipitation, column chromatography, etc. (Zea Binxin, etc., separation and purification method of sea cucumber polysaccharide and its main biological activity [ J ], Fujian aquatic product, 2008(03): 70-74). The strong alkaline anion exchanger in the column chromatography can be combined with the sea cucumber acidic mucopolysaccharide, and the sea cucumber acidic mucopolysaccharide is eluted and separated by the high-concentration salt solution, so that the high-purity sea cucumber polysaccharide is obtained.
Disclosure of Invention
The first purpose of the invention is to provide a preparation method of sea cucumber polysaccharide. The preparation method can improve the yield of the sea cucumber polysaccharide, is suitable for industrial production and reduces energy consumption.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for preparing sea cucumber polysaccharide comprises the following steps:
1) pulverizing Stichopus japonicus, soaking in water, sequentially performing enzymolysis with compound protease and flavourzyme, boiling to inactivate enzyme after enzymolysis, centrifuging, and collecting supernatant; ultrafiltering the supernatant with hollow fiber column, collecting ultrafiltration retentate to obtain crude product solution of Stichopus japonicus polysaccharide;
2) putting the sea cucumber polysaccharide crude product solution obtained in the step 1) on an anion exchange chromatographic column, sequentially eluting with water and NaCl solution, and collecting NaCl eluent;
3) desalting NaCl eluate with nanofiltration membrane, collecting nanofiltration trapped fluid, concentrating under reduced pressure, and freeze drying to obtain sea cucumber polysaccharide.
The sea cucumber in the present invention is typically a dried sea cucumber body wall. The sea cucumber may be any of a variety of sea cucumbers known in the art, including but not limited to: thelenota ananas, Apostichopus japonicus (Apostichopus japonicus), Pseudobulbus japonicas (Holothuria fuscophylla Jaeger), Pantoea polyphylla (Stichopus naga), Holothuria scabra (Holothuria scabra), Rhamnus alba (Actinophyta mauritiana), Actinophyta canaliculata (Actinophyta mitis), Holothuria scabra (Acaudina molpadioides), Holothuria atrata (Bohadschia argus), Holothuria aurantia (Holothuria edulis), Holothuria nobilis selenka (Holothuria nobilis), Holothuria nobilis selenka (Holothuria), Holothuria america (Isostichopus japonicus), Thelephora japonica (Thelephorax), and Holothuria gracilis (Lucilia japonica), and the like.
Preferably, in the step 1), the sea cucumber is crushed into coarse particles and then soaked, so that the reaction is facilitated. Soaking sea cucumber in 8-10 times of water for preferably 0.5 hr or more, such as 1 hr.
Preferably, in the step 1), the enzymolysis conditions of the compound protease are as follows: the enzyme adding amount is 1-2% of the weight of the sea cucumber, the pH value is 6.0-7.5, the temperature is 50-60 ℃, and the time is 1-2 h; the flavor protease enzymolysis conditions are as follows: the enzyme is added in an amount of 0.1-0.3% of the weight of the sea cucumber, the pH value is 6.0-7.0, the temperature is 50-60 ℃, and the time is 3-4 h.
Preferably, in the step 1), the centrifugation speed is 4000-15000 rpm. Preferably, the supernatant after centrifugation is ultrafiltered by a hollow fiber column with the molecular weight cut-off of 5KD, and the ultrafiltration conditions are as follows: the reflux pressure is less than or equal to 0.15 MPa. More preferably, when the volume of the ultrafiltration retentate is half of the volume before ultrafiltration, the deionized water is added to the volume of the liquid before ultrafiltration; and (3) repeating the water replenishing for 2 times, and stopping ultrafiltration to obtain ultrafiltration retentate, namely the sea cucumber polysaccharide crude product solution.
Preferably, in the above step 2), the anion exchange chromatography column is packed with a strongly basic anion exchange chromatography resin (e.g., D204 anion exchange resin) or a weakly basic anion exchange resin (e.g., DEAE-52 cellulose), more preferably a strongly basic anion exchange chromatography resin.
Preferably, in the step 2), the anion exchange chromatography column is sequentially and respectively eluted by 2-3 times of column volume of deionized water and 0.5-2.5 mol/L NaCl solution (such as 0.5, 2.0 and 3.0mol/L NaCl solution) in a gradient manner, and the elution flow rate is 2 BV/h. Preferably, in the step 2), the anion exchange chromatography column is sequentially and respectively eluted by deionized water with 2-3 times of column volume, NaCl solution with 2-3 times of column volume and 0.3-0.7mol/L and NaCl solution with 2-3 times of column volume and 1.8-2.4mol/L in a gradient manner, the elution flow rate is 2BV/h, and the eluent when the eluent is eluted by 1.8-2.4mol/L NaCl solution is collected; the column was then washed by elution with about 3mol/L NaCl solution. More preferably, the anion exchange chromatography column is eluted by 3 times of column volume of deionized water, 3 times of column volume of 0.5mol/L NaCl solution and 3 times of column volume of 2.0mol/L NaCl solution in sequence, and the eluent when being eluted by 2mol/L NaCl solution is collected, wherein the elution flow rate is 2 BV/h; then eluted with 3mol/L NaCl solution to wash the column.
Preferably, in the step 2), the collected eluent is desalted by a nanofiltration membrane with the molecular weight cut-off of 100-.
Preferably, in each of the above steps, the water used is deionized water.
Another object of the present invention is to provide a sea cucumber polysaccharide prepared according to any one of the above preparation methods.
The invention also aims to provide the application of the sea cucumber polysaccharide prepared by the preparation method in preparing medicines or health products or foods. The use may be various uses of sea cucumber polysaccharides known in the art, including but not limited to: hyperlipemia, antioxidation, anticoagulation, antitumor, immunity improving, etc.
The invention has the beneficial effects that: 1) in the preparation process of the crude product of the sea cucumber polysaccharide, the ultrafiltration step of the hollow fiber column can enrich the crude product of the sea cucumber polysaccharide with molecular weight of more than 5 KD; 2) in the purification process of the crude polysaccharide, the nanofiltration desalination process is adopted, compared with the existing ultrafiltration desalination process, the nanofiltration separation effect is finer, the accurate removal of soluble inorganic salt can be ensured during desalination, and the loss of part of polysaccharide samples with smaller molecular weight can be avoided, so that the yield of the sea cucumber polysaccharide is higher and can reach 4% -6%, and the content of the sea cucumber polysaccharide reaches more than 65% and even 75%; 3) the vacuum concentration step of ultrafiltration trapped fluid is reduced, the purification and decoloration treatment can be directly carried out by using a column chromatography method, the pollution to the environment caused by using an organic reagent in the large-scale production is avoided, the industrial production is convenient, and the energy consumption is reduced.
Detailed Description
The present invention is described in further detail by examples. The following examples are for illustrative purposes only and should not be construed as arbitrarily limiting the scope of the present invention. Unless otherwise specified, materials, reagents and apparatuses used in the examples are generally commercially available or conventional products. Unless otherwise specified, the methods of operation in the examples are conventional, such as: the total polysaccharide content of sea cucumber is determined by using phenol-sulfuric acid method, which is a conventional method well known to those skilled in the art.
Example 1 preparation of sea cucumber polysaccharide
1) Extracting a sea cucumber polysaccharide crude product: taking 3kg of dried thelenota ananas, crushing into coarse particles, adding deionized water with the weight 8 times that of the thelenota ananas, soaking for 1h, and sequentially carrying out enzymolysis by using compound protease and flavourzyme, wherein the enzymolysis conditions of the compound protease are as follows: the enzyme addition amount is 1% of the weight of thelenota ananas, the pH value is 6.0, the temperature is 50 ℃, and the time is 1 h; the flavor protease enzymolysis conditions are as follows: the enzyme is added in an amount of 0.1% of the weight of thelenota ananas, and has a pH of 6.0, a temperature of 50 deg.C, and a time of 4 h. Boiling for 15min to inactivate enzyme after enzymolysis, centrifuging at 6000rpm, and collecting supernatant; the supernatant is ultrafiltered by a 5KD hollow fiber column under the ultrafiltration conditions that: the reflux pressure is less than or equal to 0.15MPa, and when the volume of the ultrafiltration trapped fluid reaches one half of the volume before ultrafiltration, the deionized water is supplemented until the volume of the fluid before ultrafiltration; and (3) repeating the water replenishing for 2 times, and stopping ultrafiltration to obtain ultrafiltration retentate, namely the sea cucumber polysaccharide crude product solution.
2) Separation and purification of sea cucumber polysaccharide: dividing the crude sea cucumber polysaccharide solution into two parts, which are named as a sample 1 and a sample 2 respectively;
sample 1 was treated as follows: adding the sample 1 into a pretreated D204 type strong-base anion resin column (specification)
Resin wet volume is 100ml), eluting with 2 times of column volume of deionized water, 2 times of column volume of 0.6mol/L NaCl solution, 2 times of column volume of 2.2mol/L NaCl solution and 2 times of column volume of 3.1mol/L NaCl solution (for cleaning the column) in sequence, wherein the elution flow rate is 2BV/h, and collecting 2.2mol/L NaCl eluent;
sample 2 was treated as follows: sample 2 was added to a pretreated DEAE-52 cellulose chromatographic column (Specification)
Wet volume of resin 100 ml); eluting with deionized water 2 times the column volume, eluting with 0.6mol/L NaCl solution 2 times the column volume, eluting with 2.2mol/L NaCl solution 2 times the column volume, eluting with 3.1mol/L NaCl solution 2 times the column volume (for cleaning the column) at an elution flow rate of 2BV/h, and collecting 2.2mol/L NaCl eluate;
3) performing nanofiltration desalination treatment of 100D on NaCl eluent of the two chromatographic columns respectively, collecting nanofiltration trapped fluid, concentrating under reduced pressure, and freeze-drying to obtain sea cucumber polysaccharide; 63.452g of sea cucumber polysaccharide of sample 1 purified by a D204 type strongly basic anion resin column has the yield of 4.23%; 57.158g of sea cucumber polysaccharide of sample 2 purified by DEAE-52 cellulose chromatographic column was obtained, the yield was 3.81%.
Example 2 preparation of sea cucumber polysaccharide
1) Extracting a sea cucumber polysaccharide crude product: taking 3kg of dried thelenota ananas, crushing into coarse particles, adding deionized water with the weight 9 times that of the thelenota ananas, soaking for 1h, and sequentially carrying out enzymolysis by using compound protease and flavourzyme, wherein the enzymolysis conditions of the compound protease are as follows: the enzyme addition amount is 1.5 percent of the weight of thelenota ananas, the pH value is 6.5, the temperature is 55 ℃, and the time is 1.5 h; the flavor protease enzymolysis conditions are as follows: the enzyme is added in an amount of 0.2% of the weight of thelenota ananas, and has a pH of 6.5, a temperature of 55 deg.C, and a time of 3.5 h. Boiling for 15min to inactivate enzyme after enzymolysis, centrifuging at 10000rpm, and collecting supernatant; the supernatant is ultrafiltered by a 5KD hollow fiber column under the ultrafiltration conditions that: the reflux pressure is less than or equal to 0.15MPa, and when the volume of the ultrafiltration trapped fluid reaches one half of the volume before ultrafiltration, the deionized water is supplemented until the volume of the fluid before ultrafiltration; and (3) repeating the water replenishing for 2 times, and stopping ultrafiltration to obtain ultrafiltration retentate, namely the sea cucumber polysaccharide crude product solution.
2) Separation and purification of sea cucumber polysaccharide: dividing the crude sea cucumber polysaccharide solution into two parts, which are named as a sample 3 and a sample 4 respectively;
sample 3 was treated as follows: adding the sample 3 into a pretreated D204 type strong-base anion resin column (specification)
The wet volume of the resin is 100ml), eluting by using deionized water with 3 times of column volume, 0.4mol/L NaCl solution with 3 times of column volume, 1.9mol/L NaCl solution with 3 times of column volume and 3mol/L NaCl solution with 3 times of column volume in sequence, wherein the elution flow rate is 2BV/h, and collecting 1.9mol/L NaCl eluent;
sample 4 was treated as follows: sample 4 was added to a pretreated DEAE-52 cellulose chromatographic column (Specification)
Wet volume of resin 100 ml); eluting with deionized water of 3 times of column volume, eluting with 0.4mol/L NaCl solution of 3 times of column volume, eluting with 1.9mol/L NaCl solution of 3 times of column volume, eluting with 3mol/L NaCl solution of 3 times of column volume in sequence at an elution flow rate of 2BV/h, and collecting 1.9mol/L NaCl eluate;
3) respectively carrying out 200D nanofiltration desalination treatment on NaCl eluent of the two chromatographic columns, collecting nanofiltration trapped fluid, carrying out reduced pressure concentration and freeze drying to obtain the sea cucumber polysaccharide; 71.104g of sea cucumber polysaccharide of sample 3 purified by a D204 type strongly basic anion resin column has the yield of 4.74 percent; 64.582g of sea cucumber polysaccharide of sample 4 purified by DEAE-52 cellulose chromatographic column was obtained, and the yield was 4.31%.
Example 3 preparation of sea cucumber polysaccharide
1) Extracting a sea cucumber polysaccharide crude product: taking 3kg of dried thelenota ananas, crushing into coarse particles, adding deionized water with the weight being 10 times that of the thelenota ananas, soaking for 1h, and sequentially carrying out enzymolysis by using compound protease and flavourzyme, wherein the enzymolysis conditions of the compound protease are as follows: the enzyme adding amount is 2% of the weight of thelenota ananas, the pH value is 7.0, the temperature is 60 ℃, and the time is 2 hours; the flavor protease enzymolysis conditions are as follows: the enzyme is added in an amount of 0.3% of the weight of thelenota ananas, has a pH of 7.0, and is heated at 60 deg.C for 3 h. After enzymolysis, boiling for 15min to inactivate enzyme, centrifuging at 15000rpm, and ultrafiltering the supernatant with 5KD hollow fiber column under the ultrafiltration conditions: the reflux pressure is less than or equal to 0.15MPa, and when the volume of the ultrafiltration trapped fluid reaches one half of the volume before ultrafiltration, the deionized water is supplemented until the volume of the fluid before ultrafiltration; and (3) repeating the water replenishing for 2 times, and stopping ultrafiltration to obtain ultrafiltration retentate, namely the sea cucumber polysaccharide crude product solution.
2) Separation and purification of sea cucumber polysaccharide: dividing the crude sea cucumber polysaccharide solution into two parts, which are named as sample 5 and sample 6 respectively;
sample 5 was treated as follows: sample 5 was added to a pretreated D204 type strongly basic anion resin column (specification)
Resin wet volume is 100ml), eluting with 3 times of column volume of deionized water, 3 times of column volume of 0.5mol/L NaCl solution, 3 times of column volume of 2mol/L NaCl solution and 3 times of column volume of 3mol/L NaCl solution in sequence, wherein the elution flow rate is 2BV/h, and collecting 2mol/L NaCl eluate;
sample 6 was treated as follows: sample 6 was added to a pretreated DEAE-52 cellulose chromatographic column (Specification)
Wet volume of resin 100 ml); eluting with deionized water 3 times the column volume, 0.5mol/L NaCl 3 times the column volume, and 3 times the column volumeEluting with 2.0mol/L NaCl solution, eluting with 3mol/L NaCl solution with 3 times of column volume at the flow rate of 2BV/h, and collecting 2.0mol/L NaCl eluate;
3) respectively carrying out 500D nanofiltration desalination treatment on NaCl eluent of the two chromatographic columns, collecting nanofiltration trapped fluid, carrying out reduced pressure concentration and freeze drying to obtain the sea cucumber polysaccharide; 76.813g of Stichopus japonicus polysaccharide of sample 5 purified by D204 type strongly basic anion resin column, yield of 5.12%, and 71.726g of Stichopus japonicus polysaccharide of sample 6 purified by DEAE-52 cellulose chromatographic column, yield of 4.78%.
The three embodiments are combined to obtain: the sea cucumber polysaccharide yield of the embodiment 3 is higher, and is a preferable scheme; in order to examine the sea cucumber polysaccharide yield of different sea cucumbers in the scheme, the same amount of polygonatum odoratum and ivory is taken and operated according to the same method of the embodiment 3.
Example 4 preparation of sea cucumber polysaccharide
1) Extracting a sea cucumber polysaccharide crude product: taking 3kg of dried polygonatum odoratum, crushing into coarse particles, adding deionized water with the weight being 10 times that of the polygonatum odoratum, soaking for 1 hour, and sequentially carrying out enzymolysis by using compound protease and flavourzyme, wherein the enzymolysis conditions of the compound protease are as follows: the enzyme addition amount is 2% of the weight of the polygonatum odoratum, the pH value is 7.0, the temperature is 60 ℃, and the time is 2 hours; the flavor protease enzymolysis conditions are as follows: the enzyme is added at a pH of 7.0 and at a temperature of 60 deg.C for 3 hr, and accounts for 0.3% of the weight of radix polygonati officinalis. After enzymolysis, boiling for 15min to inactivate enzyme, centrifuging at 15000rpm, and ultrafiltering the supernatant with 5KD hollow fiber column under the ultrafiltration conditions: the reflux pressure is less than or equal to 0.15MPa, and when the volume of the ultrafiltration trapped fluid reaches one half of the volume before ultrafiltration, the deionized water is supplemented until the volume of the fluid before ultrafiltration; and (3) repeating the water replenishing for 2 times, and stopping ultrafiltration to obtain ultrafiltration retentate, namely the sea cucumber polysaccharide crude product solution.
2) Separation and purification of sea cucumber polysaccharide: dividing the crude sea cucumber polysaccharide solution into two parts, which are named as a sample 7 and a sample 8 respectively;
sample 7 was treated as follows: sample 7 was added to a pretreated D204 type strongly basic anion resin column (specification)
Wet volume of resin is 100ml), 3 times of columns are used in sequenceEluting with deionized water, eluting with 0.5mol/L NaCl solution 3 times the column volume, eluting with 2mol/L NaCl solution 3 times the column volume, eluting with 3mol/L NaCl solution 3 times the column volume at an elution flow rate of 2BV/h, and collecting 2mol/L NaCl eluate;
sample 8 was treated as follows: sample 8 was added to a pretreated DEAE-52 cellulose chromatographic column (Specification)
Wet volume of resin 100 ml); eluting with deionized water of 3 times of column volume, eluting with 0.5mol/L NaCl solution of 3 times of column volume, eluting with 2.0mol/L NaCl solution of 3 times of column volume, eluting with 3mol/L NaCl solution of 3 times of column volume in sequence at an elution flow rate of 2BV/h, and collecting 2.0mol/L NaCl eluate;
3) respectively carrying out 500D nanofiltration desalination treatment on NaCl eluent of the two chromatographic columns, collecting nanofiltration trapped fluid, carrying out reduced pressure concentration and freeze drying to obtain the sea cucumber polysaccharide; 61.353g of sea cucumber polysaccharide of sample 7 purified by a D204 type strongly basic anion resin column has the yield of 4.09%; 57.316g of sea cucumber polysaccharide of sample 8 purified by DEAE-52 cellulose chromatographic column was obtained, and the yield was 3.82%.
Example 5 preparation of sea cucumber polysaccharide
1) Extracting a sea cucumber polysaccharide crude product: taking 3kg of dried ivy, crushing into coarse particles, adding deionized water with the weight being 10 times that of the ivy, soaking for 1h, and sequentially carrying out enzymolysis by using compound protease and flavourzyme, wherein the enzymolysis conditions of the compound protease are as follows: the enzyme addition amount is 2% of the weight of the ivory, the pH value is 7.0, the temperature is 60 ℃, and the time is 2 hours; the flavor protease enzymolysis conditions are as follows: the enzyme is added at a pH of 7.0 and a temperature of 60 deg.C for 3 hr, wherein the weight of the enzyme is 0.3% of that of the ivory. After enzymolysis, boiling for 15min to inactivate enzyme, centrifuging at 15000rpm, and ultrafiltering the supernatant with 5KD hollow fiber column under the ultrafiltration conditions: the reflux pressure is less than or equal to 0.15MPa, and when the volume of the ultrafiltration trapped fluid reaches one half of the volume before ultrafiltration, the deionized water is supplemented until the volume of the fluid before ultrafiltration; and (3) repeating the water replenishing for 2 times, and stopping ultrafiltration to obtain ultrafiltration retentate, namely the sea cucumber polysaccharide crude product solution.
2) Separation and purification of sea cucumber polysaccharide: dividing the crude sea cucumber polysaccharide solution into two parts, which are named as a sample 9 and a sample 10 respectively;
sample 9 was treated as follows: sample 9 was added to a pretreated D204 type strongly basic anion resin column (specification)
Resin wet volume is 100ml), eluting with 3 times of column volume of deionized water, 3 times of column volume of 0.5mol/L NaCl solution, 3 times of column volume of 2mol/L NaCl solution and 3 times of column volume of 3mol/L NaCl solution in sequence, wherein the elution flow rate is 2BV/h, and collecting 2mol/L NaCl eluate;
sample 10 was treated as follows: the sample 10 was added to a pretreated DEAE-52 cellulose chromatographic column (Specification)
Wet volume of resin 100 ml); eluting with deionized water of 3 times of column volume, eluting with 0.5mol/L NaCl solution of 3 times of column volume, eluting with 2.0mol/L NaCl solution of 3 times of column volume, eluting with 3mol/L NaCl solution of 3 times of column volume in sequence at an elution flow rate of 2BV/h, and collecting 2.0mol/L NaCl eluate;
3) respectively carrying out 500D nanofiltration desalination treatment on NaCl eluent of the two chromatographic columns, collecting nanofiltration trapped fluid, carrying out reduced pressure concentration and freeze drying to obtain the sea cucumber polysaccharide; 53.742g of sea cucumber polysaccharide of sample 9 purified by a D204 type strongly basic anion resin column has the yield of 3.58%; 50.259g of sea cucumber polysaccharide of sample 10 purified by DEAE-52 cellulose chromatographic column was obtained, and the yield was 3.35%.
Comparative example 1 preparation of sea cucumber polysaccharide
1) Extracting a sea cucumber polysaccharide crude product: weighing 2kg of dried thelenota ananas (same as example 3 in batches), crushing into coarse particles, adding pure water with the weight 10 times of that of the sea cucumbers, and carrying out enzymolysis by using papain under the conditions as follows: the enzyme addition amount is 2 percent of the weight of the sea cucumber, the pH value is 7.0, the temperature is 55 ℃, and the time is 5 hours; boiling to inactivate enzyme after enzymolysis, centrifuging at 12000rpm in tubular centrifuge, collecting supernatant, concentrating under reduced pressure (temperature 50 deg.C, vacuum degree less than or equal to-0.085 MPa) to 22% of original volume, centrifuging at 14000rpm in tubular centrifuge again for 15min, and collecting supernatant; salting out and precipitating supernatant by alcohol for the first time: adding potassium acetate (to make potassium acetate final concentration be 0.5M) and ethanol (to make ethanol final concentration be 60%), centrifuging with tubular centrifuge, and collecting precipitate; dissolving the precipitate with pure water, and performing salting out and alcohol precipitation for the second time: adding potassium acetate (to make potassium acetate concentration 1.0M) and ethanol (to make ethanol concentration 40%), centrifuging in tubular centrifuge, and collecting precipitate; dissolving the precipitate with pure water, and performing salting out and alcohol precipitation for the third time: adding potassium acetate (to make final concentration of potassium acetate 1.0M) and ethanol (to make final concentration of ethanol 40%), centrifuging with tubular centrifuge to obtain precipitate, washing the precipitate with anhydrous ethanol twice, and volatilizing ethanol to obtain 227.4g of crude product of Stichopus japonicus polysaccharide with yield of 11.37%.
2) Dissolving the crude product with pure water, and purifying with D204 type anion chromatographic column (OH)-) The elution is sequentially gradient-eluted by pure water with 3 times of the volume of the column bed and NaCl solutions with the concentration of 0.5mol/L, 1.0 mol/L, 2.0mol/L and 3.0mol/L, and the elution solution eluted by the NaCl solution with the concentration of 2.0mol/L is collected.
3) And (3) carrying out ultrafiltration and desalination treatment on the collected eluent by using a hollow fiber column with 5KD, collecting ultrafiltration retentate, carrying out reduced pressure concentration (the temperature is 50 ℃, and the vacuum degree is less than or equal to-0.085 MPa), and freeze-drying to obtain 72.4g of the sea cucumber polysaccharide of the sample 11, wherein the yield of the sea cucumber polysaccharide is 3.62%.
Comparative example 2 preparation of sea cucumber polysaccharide
Extracting a sea cucumber polysaccharide crude product: weighing 2kg of dried thelenota ananas (same as example 3 in batches), crushing into coarse particles, adding pure water with the weight 10 times of that of the sea cucumbers, and carrying out enzymolysis by using papain under the conditions as follows: the enzyme adding amount is 3 percent of the weight of the sea cucumber, the pH value is 6.5, the temperature is 60 ℃, and the time is 2 hours; the subsequent operation is carried out in the same pair 3; the sea cucumber polysaccharide of the sample 12 purified by the D204 type strongly basic anion resin column is 32.17g, and the yield is 3.22%; the sample 13 purified by DEAE-52 cellulose chromatographic column had 30.26g of sea cucumber polysaccharide, and the yield was 3.03%.
Taking 10 sea cucumber polysaccharide samples of the 5 examples and samples of the comparative examples 1 and 2, and simultaneously measuring the total polysaccharide content of the sea cucumber by using a conventional phenol-sulfuric acid method; the yield and total polysaccharide content of the sea cucumber are shown in table 1.
TABLE 1 sea cucumber polysaccharide yield and total polysaccharide content in sea cucumber
As can be seen from the examples, 1) the sea cucumber polysaccharide obtained in example 3 has higher yield, which is a preferable scheme, and the yield and the content of the sea cucumber polysaccharide obtained by adopting the D204 type strong-base anion resin column for purification are both obtained by the DEAE-52 cellulose chromatographic column for purification; 2) from the comparison research of different sea cucumber raw materials, the yield and the content of the sea cucumber polysaccharide obtained by thelenota ananas are slightly higher than those of the holothuria leucospilota and the holothuria ivory; 3) in the purification process of the crude polysaccharide, compared with the existing ultrafiltration desalination process, the method adopts the nanofiltration membrane for desalination, which can ensure that soluble inorganic salt can be removed accurately, and can also avoid the loss of part of polysaccharide samples with smaller molecular weight, so that the yield of the sea cucumber polysaccharide is higher and can reach 3.35-5.12%, the content of the sea cucumber polysaccharide reaches more than 65%, even 75%, and a good effect is achieved.
The above examples are merely illustrative of the present invention. Modifications and improvements may be made by those skilled in the art without departing from the spirit of the invention.
Claims (10)
1. A method for preparing sea cucumber polysaccharide comprises the following steps:
1) pulverizing Stichopus japonicus, soaking in water, sequentially performing enzymolysis with compound protease and flavourzyme, boiling to inactivate enzyme after enzymolysis, centrifuging, and collecting supernatant; ultrafiltering the supernatant with hollow fiber column, collecting ultrafiltration retentate to obtain crude product solution of Stichopus japonicus polysaccharide;
2) putting the sea cucumber polysaccharide crude product solution obtained in the step 1) on an anion exchange chromatographic column, sequentially eluting with water and NaCl solution, and collecting NaCl eluent;
3) desalting the NaCl eluate with nanofiltration membrane, collecting nanofiltration trapped fluid, concentrating under reduced pressure, and freeze drying to obtain sea cucumber polysaccharide.
2. The method according to claim 1, wherein in step 1), the sea cucumber is soaked in water 8-10 times its weight for 0.5h or more.
3. The method according to claim 1, wherein in step 1), the compound protease enzymolysis conditions are as follows: the enzyme adding amount is 1-2% of the weight of the sea cucumber, the pH value is 6.0-7.5, the temperature is 50-60 ℃, and the time is 1-2 h; the flavor protease enzymolysis conditions are as follows: the enzyme is added in an amount of 0.1-0.3% of the weight of the sea cucumber, the pH value is 6.0-7.0, the temperature is 50-60 ℃, and the time is 3-4 h.
4. The method as claimed in claim 1, wherein in step 1), the centrifugation speed is 4000-15000 rpm; and (3) ultrafiltering the centrifuged supernatant by a hollow fiber column with the molecular weight cut-off of 5KD, wherein the ultrafiltration conditions are as follows: the reflux pressure is less than or equal to 0.15 MPa; when the volume of the ultrafiltration trapped fluid is half of the volume before ultrafiltration, the deionized water is added to the volume of the fluid before ultrafiltration; and (3) repeating the water replenishing for 2 times, and stopping ultrafiltration to obtain ultrafiltration retentate, namely the sea cucumber polysaccharide crude product solution.
5. The method according to claim 1, wherein in step 2), the packing of the anion exchange chromatography column is a strongly basic anion exchange chromatography resin or a weakly basic anion exchange resin.
6. The method according to claim 1, wherein in step 2), the anion exchange chromatography column is sequentially and respectively eluted with deionized water with 2-3 times of column volume and NaCl solution with 0.5-2.5 mol/L in a gradient manner, and the elution flow rate is 2 BV/h.
7. The method according to claim 1, wherein in step 2), the anion exchange chromatography column is sequentially and respectively eluted with 2-3 times of column volume of deionized water, 2-3 times of column volume of 0.3-0.7mol/L NaCl solution and 2-3 times of column volume of 1.8-2.4mol/L NaCl solution in a gradient manner at an elution flow rate of 2BV/h, and the eluent eluted with 1.8-2.4mol/L NaCl solution is collected.
8. The method as claimed in claim 1, wherein in step 3), the collected eluate is desalted by using a nanofiltration membrane with molecular weight cut-off of 100-500 Da.
9. A sea cucumber polysaccharide prepared according to the method of any one of claims 1 to 8.
10. The application of the sea cucumber polysaccharide of claim 9 in preparing medicines or health products or foods for resisting hyperlipidemia, oxidation, coagulation, tumors and immunity.
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| CN115894734A (en) * | 2022-12-02 | 2023-04-04 | 大连深蓝肽科技研发有限公司 | Method for extracting sea cucumber polysaccharide from sea cucumber cooking liquor with high efficiency and low pollution |
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| CN115894734A (en) * | 2022-12-02 | 2023-04-04 | 大连深蓝肽科技研发有限公司 | Method for extracting sea cucumber polysaccharide from sea cucumber cooking liquor with high efficiency and low pollution |
| CN115894734B (en) * | 2022-12-02 | 2024-02-23 | 大连深蓝肽科技研发有限公司 | Method for extracting sea cucumber polysaccharide from sea cucumber cooking liquor with high efficiency and low pollution |
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