CN115820778A - Method for industrially producing sea cucumber polypeptide rich in sea cucumber polysaccharide - Google Patents
Method for industrially producing sea cucumber polypeptide rich in sea cucumber polysaccharide Download PDFInfo
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Abstract
The invention discloses a method for industrially producing sea cucumber polypeptide rich in sea cucumber polysaccharide, belonging to the technical field of polypeptide production. The method comprises the following steps: simply cooking the sea cucumber with viscera and sand removed, treating the sea cucumber at 121 ℃ and 0.1MPa for 10-15min, crushing, carrying out enzymolysis for 150-180min by using alkaline protease and neutral protease and carrying out enzymolysis for 30-60min by using flavourzyme, inactivating, filtering, centrifuging, carrying out filter pressing, passing through an ultrafiltration membrane with the molecular weight cutoff of 10-20ku, decoloring the permeation solution by using a 3nm activated carbon fiber membrane, treating the retention solution at 121 ℃ and 0.1MPa for 10-20min, carrying out filter pressing, mixing the retention solution and the permeation solution, concentrating the three sections, and drying to form dry powder. The invention has the advantages that: the polypeptide and the polysaccharide are separated by an ultrafiltration membrane, and then are independently treated and mixed to finally obtain the sea cucumber polypeptide rich in the sea cucumber polysaccharide, which has higher activity and is more suitable for industrial production.
Description
Technical Field
The invention relates to a method for industrially producing sea cucumber polypeptide rich in sea cucumber polysaccharide, belonging to the technical field of polypeptide production.
Background
The sea cucumber polysaccharide is one of important active ingredients of the body wall of the sea cucumber, and the content of the sea cucumber polysaccharide accounts for 4-10% of the total organic matters of the dried sea cucumber. The sea cucumber polysaccharide comprises sea cucumber chondroitin sulfate and sea cucumber fucosan sulfate. Wherein the sea cucumber chondroitin sulfate is acidic mucopolysaccharide with fucose branched chains, is mainly formed by connecting D-N-acetylgalactosamine, D-glucuronic acid and L-fucose by glycosidic bonds, and is branched heteropolysaccharide; the sea cucumber fucoidan sulfate is formed by connecting L-fucose by glycosidic bonds, and is a straight-chain polysaccharide. The chemical structures of the sea cucumber chondroitin sulfate and the sea cucumber fucosan sulfate are special and are peculiar to the sea cucumber, and the sulfation degrees of the sea cucumber chondroitin sulfate and the sea cucumber fucosan sulfate are both as high as more than 30%.
Because the molecular weight of the sea cucumber chondroitin sulfate and the sea cucumber fucosan sulfate is especially large (the relative molecular weight of the sea cucumber chondroitin sulfate is 40-50ku, and the relative molecular weight of the sea cucumber fucosan sulfate is 80-100 ku), and the glycosidic bond of the sea cucumber chondroitin sulfate and the sea cucumber fucosan sulfate cannot be destroyed (the viscosity is high) in the protein enzymolysis process and under the conditions of high temperature, high pressure and neutral pH, in addition, the sea cucumber chondroitin sulfate and the sea cucumber fucosan sulfate both contain sulfate groups (with negative charges), the sea cucumber chondroitin sulfate and the sea cucumber fucosan sulfate are basically removed in the process flows of purification, fishy smell removal, decoloration and the like of sea cucumber polypeptide, the content of sea cucumber polysaccharide in the sea cucumber polypeptide product on the market is basically lower than 0.5%, and the sea cucumber polysaccharide is seriously wasted.
Chinese patent CN101191139a discloses a comprehensive extraction process of sea cucumber polypeptide and polysaccharide, which is not suitable for industrial production, and more suitable for laboratory small-scale preparation of high-purity sea cucumber polypeptide and high-purity complete sea cucumber polysaccharide for academic research, and specifically:
(1) The comprehensive extraction process separates sea cucumber polypeptide and sea cucumber polysaccharide by two enzymolysis and alcohol precipitation methods, the supernatant mainly comprises free amino acids (with average molecular weight of 128 u), dipeptides and tripeptides, the average molecular weight is less than 350u (one peptide bond is formed to remove one molecule of water, and the relative molecular weight of the water is 18), and the activity of the product is low, so the comprehensive extraction process is not suitable for industrial production;
(2) Although the comprehensive extraction process carries out enzymolysis for two times, only free amino acid, dipeptide and tripeptide can be dissolved in 60 percent ethanol solution, the product yield is only 68.2 percent, and the product yield is lower, so the comprehensive extraction process is not suitable for industrial production;
(3) The comprehensive extraction process needs two times of alcohol precipitation, and the consumption of ethanol is large, so the comprehensive extraction process is not suitable for industrial production;
(4) The comprehensive extraction process needs to add potassium acetate in the refining process of the crude sea cucumber polysaccharide, obtains the sea cucumber polysaccharide with higher purity by salting out, and is not suitable for industrial production due to the introduction of a foreign substance potassium acetate, the need of removing residues in the later period and the like;
(5) The comprehensive extraction process needs dialysis treatment of the sea cucumber polysaccharide solution for 48 hours, and is not suitable for industrial production because the industrial production is not carried out in an aseptic environment and the feed liquid is easy to deteriorate due to overlong dialysis time.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a method for industrially producing sea cucumber polypeptide rich in sea cucumber polysaccharide.
In order to achieve the above object, the present invention adopts the following technical solutions:
a method for industrially producing sea cucumber polypeptide rich in sea cucumber polysaccharide comprises the following steps:
(1) Soaking dried sea cucumber, cutting open, removing viscera and sand mouth, and cleaning with pure water;
(2) Putting the cleaned sea cucumber into a cooking pot, adding water, simply cooking the sea cucumber, fishing out the sea cucumber, and removing soup;
(3) Placing the simply cooked sea cucumber into a high-pressure kettle, adding water, and treating at 121 deg.C and 0.1MPa for 10-15min;
(4) Crushing the sea cucumbers subjected to high-temperature and high-pressure treatment by using a cutting and mixing machine;
(5) Pouring the crushed sea cucumber into an enzymolysis tank, pumping the rest cooking liquor in the high-temperature high-pressure treatment step into the enzymolysis tank, adding pure water, adjusting the pH value of the feed liquid in the enzymolysis tank to 7-9, heating to 50-55 ℃, sequentially adding alkaline protease accounting for 0.2% of the weight of the dried sea cucumber and neutral protease accounting for 0.2% of the weight of the dried sea cucumber, carrying out enzymolysis for 150-180min, finally adding flavourzyme accounting for 0.1% of the weight of the dried sea cucumber, carrying out enzymolysis for 30-60min, and continuously stirring in the enzymolysis process;
(6) Heating the enzymolysis feed liquid to 85-90 deg.C, and maintaining for 10min;
(7) Cooling the inactivated feed liquid to 50-55 ℃, filtering with a filter screen of 200-250 meshes, pouring the filtrate into a horizontal spiral sedimentation centrifuge for centrifugation, and finally pumping the centrifugal liquid back to the enzymolysis tank;
(8) Introducing the feed liquid in the enzymolysis tank into a plate-and-frame filter press for filter pressing;
(9) Passing the feed liquid after filter pressing through a roll type ultrafiltration membrane with the molecular weight cutoff of 10-20ku, and respectively collecting the permeate and the retentate;
(10) Heating the permeate to 35 deg.C, introducing into a decolorization device with stainless steel skeleton and activated carbon fiber membrane for deodorization and decolorization, wherein the diameter of micropores of the activated carbon fiber is 3nm, and collecting filtrate;
(11) Pumping the trapped fluid into an autoclave, adjusting the pH value of the trapped fluid to 5 by using acetic acid, then carrying out constant-temperature and constant-pressure treatment for 10-20min at the temperature of 121 ℃ and under the pressure of 0.1MPa, adjusting the pH value of the trapped fluid to 6.5-7 by using a sodium hydroxide solution when the trapped fluid is cooled to 50-60 ℃, then introducing into a plate and frame filter press for filter pressing, and collecting filtrate;
(12) Mixing the trapped liquid subjected to high-temperature and high-pressure treatment and plate-and-frame filter pressing treatment with the permeate subjected to deodorization and decoloration treatment, and then sequentially carrying out primary nanofiltration, secondary nanofiltration and tertiary nanofiltration on the mixed liquid by using a rolled nanofiltration membrane;
(13) And (3) carrying out instantaneous spray drying on the concentrated solution obtained after the three-stage concentration to form dry powder, thus obtaining the sea cucumber polypeptide.
Preferably, in the step (1), the method for foaming the dried sea cucumber comprises the following steps: soaking dried sea cucumber in water of 4-10 deg.C for 48 hr, and changing water for 2 times.
Preferably, in the step (2), the ratio of the materials to the liquid is 1:4-5, adding water; taking out the sea cucumber when the water is heated to 80-90 ℃.
Preferably, in the step (3), the ratio of the feed liquid to the feed liquid is 1:2-3, adding water.
Preferably, in the step (5), after adding pure water, the final solid-to-liquid ratio is 1:4-5.
Preferably, in step (7), the centrifugation speed is 4800r/min and the productivity is 0.8m 3 /h。
Preferably, in step (8), the plate and frame filter press contains AG-3000 # Diatomaceous earth.
Preferably, in step (11), the plate and frame filter press is internally provided with SD 307 # Diatomite special for sugar production.
Preferably, in step (13), the method of instantaneous spray drying is: heating the concentrated solution to 90 ℃, and pumping the concentrated solution into a drying tower for instantaneous spray drying, wherein the drying temperature is 150-160 ℃.
The invention has the advantages that:
(1) According to the invention, micromolecular sea cucumber polypeptide and macromolecular sea cucumber polysaccharide (sea cucumber chondroitin sulfate and sea cucumber fucosan sulfate) are separated by using an ultrafiltration membrane, then the micromolecular sea cucumber polypeptide and the macromolecular sea cucumber polysaccharide are separately treated, and finally the micromolecular sea cucumber polypeptide and the macromolecular sea cucumber polysaccharide are mixed, so that the sea cucumber polypeptide rich in the sea cucumber polysaccharide (the content of the sea cucumber polysaccharide reaches 6.02-6.07%) is finally obtained, and the waste of the sea cucumber polysaccharide is avoided;
(2) The sea cucumber polysaccharide is treated for 10-20min at constant temperature and pressure under the conditions of pH =5, 121 ℃ and 0.1MPa, so that part of glucoside bonds of the sea cucumber chondroitin sulfate and the sea cucumber fucosan sulfate are broken, the molecular weight of the sea cucumber polysaccharide subjected to moderate degradation is reduced, the water solubility is improved, and the viscosity is reduced;
(3) According to the inventionThe method has the advantages that the yield of the sea cucumber polypeptide can reach 85 percent, the total nitrogen content (calculated by dry basis) in the sea cucumber polypeptide is 14.8-14.9 percent, the protein content (calculated by dry basis) is 92.5-93.1 percent, the proportion of the small molecular peptides with relative molecular mass less than 1000u in the sea cucumber polypeptide is 76.42-78.30 percent, the average relative molecular mass of the sea cucumber polypeptide is 742u-798u (molecular weight standard regression linear equation: Y = -0.24742X +7.23795, R 2 = 0.9917), moderate molecular weight and high activity;
(4) The method of the invention does not need to consume a large amount of ethanol, does not introduce foreign substances, does not need long-time dialysis, and is more suitable for industrial production.
Drawings
FIG. 1 is a diagram showing the molecular weight distribution of a sea cucumber polypeptide prepared in example 1 of the present invention;
FIG. 2 is a graph showing the molecular weight distribution of the sea cucumber polypeptide prepared in example 2 of the present invention.
Detailed Description
The invention is described in detail below with reference to the figures and the embodiments.
Example 1
1. Pretreatment of
Soaking dried sea cucumber in 4 deg.C water for 48 hr, and changing water for 2 times. Then cutting open the sea cucumber, removing internal organs and sand mouths, and cleaning with pure water.
2. Removing holothurin
Sea cucumber saponin is a secondary metabolite of sea cucumber, when the sea cucumber is attacked by natural enemies, the Vickers organ extends out of a body cavity and releases substances with fish toxicity and hemolysis to serve as a defense tool, the saponin is contained in the sea cucumber saponin, and meanwhile, toxin is secreted from the surface of the body wall of the sea cucumber, and the main component of the toxin is the saponin.
Stichopus japonicus saponins have various physiological activities, but excessive amounts of Stichopus japonicus saponins have strong irritation to mucous membranes. In addition, the sea cucumber saponin also has strong hygroscopicity, is easy to absorb moisture and is not easy to store. Therefore, most of holothurin needs to be removed when preparing a holothurin polypeptide product.
Since the aqueous solution of holothurian saponin can generate persistent foam after being strongly shaken and can not disappear after being heated, if the holothurian saponin is not removed in advance, a large amount of persistent foam can be generated in the subsequent enzymolysis process (continuous stirring is needed), which is very unfavorable for industrial production. Therefore, most of the holothurin in the feed liquid is removed in advance before enzymolysis.
The method for removing the holothurian saponins is arranged after pretreatment, and comprises the following steps:
putting the cleaned sea cucumbers into a cooking pot, and mixing the materials according to a material-liquid ratio of 1:4 adding water, simply cooking the sea cucumber, fishing out the sea cucumber when the water is heated to 80 ℃, and discarding the soup.
The sea cucumber saponin is soluble in water, and particularly easily soluble in hot water, so that when the sea cucumber is simply cooked, the sea cucumber saponin can be dissolved in the soup, and a large amount of saponin can be removed by discarding the soup, and simultaneously, bad flavors such as astringent taste, spicy taste and the like can be removed.
About 70% of protein in the body wall of the sea cucumber is collagen, the sea cucumber collagen is type I collagen formed by three alpha polypeptide chains, the type I collagen is rich in alanine and sugar-combined hydroxylysine, the terminal peptides of the alpha polypeptide chains form a spherical structural region of the collagen, and the three alpha polypeptide chains are crosslinked and polymerized into a triple helix structure (the diameter is less than 50 nm) by virtue of Van der Waals force, hydrogen bonds and covalent bonds to form insoluble collagen, so that the loss of the collagen is very small when the sea cucumber is steamed and boiled simply.
The sea cucumber polysaccharide molecules are connected with the core protein of the proteoglycan through carbohydrate peptide bonds, and in addition, the sea cucumber polysaccharide molecules and the protein chains form macromolecular aggregates with space conformations of spiral, folding, coiling and rolling through non-covalent bonds (secondary bonds such as hydrogen bonds) so that the loss of the sea cucumber polysaccharide is very small when the sea cucumber is simply cooked.
3. High temperature high pressure treatment
The sea cucumber is reduced in volume and more elastic after being simply cooked in the previous step, which is very unfavorable for the subsequent crushing and enzymolysis. The high temperature and high pressure treatment can solve the problem well.
Therefore, after the sea cucumbers are simply cooked, the sea cucumbers are subjected to high-temperature high-pressure treatment, and the high-temperature high-pressure treatment method comprises the following steps:
placing the simply cooked sea cucumbers into an autoclave, and mixing the materials according to a material-liquid ratio of 1:2 adding water, and then treating for 10min at constant temperature and pressure of 0.1MPa at 121 ℃.
The sea cucumber processed at high temperature and high pressure has larger volume and smaller elasticity.
4. Pulverizing
Crushing the sea cucumbers treated at high temperature and high pressure by using a cutting and mixing machine.
5. Enzymolysis
Pouring the crushed sea cucumbers into an enzymolysis tank, pumping the cooking liquor left in the high-temperature high-pressure treatment step into the enzymolysis tank, and adding pure water, wherein the final solid-to-liquid ratio is 1: and 4, adjusting the pH value of the feed liquid in the enzymolysis tank to 7, heating to 50 ℃, sequentially adding alkaline protease (Angel AP-200A series) accounting for 0.2 percent of the weight of the dry sea cucumber and neutral protease accounting for 0.2 percent of the weight of the dry sea cucumber, carrying out enzymolysis for 150min, finally adding flavourzyme accounting for 0.1 percent of the weight of the dry sea cucumber, carrying out enzymolysis for 30min, and continuously stirring in the enzymolysis process.
6. Inactivating
Heating the enzymolysis feed liquid to 85 deg.C, and maintaining for 10min.
7. Centrifugation
Cooling the inactivated feed liquid to 50 ℃, filtering with a 200-mesh filter screen, pouring the filtrate into a horizontal spiral sedimentation centrifuge, wherein the centrifugal speed is 4800r/min, and the centrifugal production capacity is 0.8m 3 And h, finally pumping the centrifugal liquid back to the enzymolysis tank.
8. Plate frame filter pressing
Introducing the feed liquid (centrifugate) in the enzymolysis tank into a tank filled with AG-3000 # Filter pressing is carried out in a plate-and-frame filter press of diatomite (filter aid).
AG-3000 # The diatomite has a medium-coarse microporous structure, so that the sea cucumber polypeptide liquid can obtain a better flow speed ratio, and fine suspended matters in the sea cucumber polypeptide liquid can be filtered.
9. Membrane filtration
And (3) passing the feed liquid after filter pressing through a roll-type ultrafiltration membrane with the molecular weight cutoff of 10ku, and respectively collecting the permeation liquid and the trapped liquid.
The relative molecular mass of the sea cucumber chondroitin sulfate is 40-50ku, the relative molecular mass of the sea cucumber fucosan sulfate is 80-100ku, and the relative molecular mass of the small molecular peptide is less than 5ku. Therefore, the permeate liquid is mainly composed of small molecular peptides, and the retentate liquid is mainly composed of macromolecular sea cucumber polysaccharide (sea cucumber chondroitin sulfate and sea cucumber fucosan sulfate).
10. Treatment permeate (deodorization and decoloration)
The temperature of the permeate is raised to 35 ℃, then the permeate is led into a decoloring device of activated carbon fiber membranes with stainless steel frameworks (the pore diameter of micropores of the activated carbon fibers is 3 nm) for deodorization and decoloring, and the filtrate is collected.
11. Treatment of the retentate
Pumping the trapped fluid into autoclave, adjusting pH value of the trapped fluid to 5 with acetic acid, and treating at 121 deg.C and 0.1MPa for 10min. When the trapped fluid is cooled to 50 ℃, the pH value of the trapped fluid is adjusted to 6.5 by using sodium hydroxide solution with the mass concentration of 5 percent, and then the trapped fluid is led into a device filled with SD 307 # Filter pressing is carried out in a plate-and-frame filter press of diatomite (filter aid) special for sugar production, and filtrate is collected.
When the trapped fluid is treated at high temperature and high pressure, the glycosidic bond of the sea cucumber polysaccharide can be destroyed and degraded only when the pH value is less than 7. In addition, when the sea cucumber polysaccharide is heated within the range of 100-121 ℃, the lower the pH value, the higher the temperature and the longer the heating time are, the more obvious the molecular weight reduction is. But the content of sulfate radicals is an extremely important index for determining the activity of the sea cucumber polysaccharide, and the loss of the sulfate radicals is increased along with the reduction of the pH value and the increase of the heating time. Comprehensively considering the factors of corrosion of acid to equipment, loss of sulfate radicals, energy conservation and the like, the method finally determines to adjust the pH value of the trapped fluid to 5, and treats the trapped fluid for 10-20min at constant temperature and constant pressure at 121 ℃ and 0.1 MPa. The sea cucumber polysaccharide which is properly degraded has the advantages of reduced molecular weight, better water solubility and reduced viscosity.
12. Mixing
Mixing the trapped liquid after high-temperature and high-pressure treatment and plate-and-frame filter pressing treatment with the permeate liquid after deodorization and decoloration treatment, and then sequentially carrying out primary nanofiltration, secondary nanofiltration and tertiary nanofiltration on the mixed liquid by using a rolled nanofiltration membrane.
After the mixed liquid is concentrated in three stages, the concentration is gradually improved, and meanwhile, ash content is also removed.
13. Drying
Heating the concentrated solution obtained after the three-stage concentration to 90 ℃, pumping the concentrated solution into a drying tower for instantaneous spray drying at the drying temperature of 150 ℃ to form dry powder, and obtaining the sea cucumber polypeptide with the yield of 85%.
And pouring the obtained sea cucumber polypeptide into water to prepare a sample solution with the mass concentration of 2%, wherein the sample solution is clear and transparent.
Through detection, the content of the sea cucumber polysaccharide in the sample is 6.07%, the total nitrogen content (calculated on a dry basis) is 14.8%, and the protein content (calculated on a dry basis) is 92.5%.
In addition, the molecular weight distribution of the sample is shown in fig. 1, and the specific data are shown in table 1 below:
TABLE 1 molecular weight distribution of samples
| Size of molecular weight | Ratio of occupation of |
| <180u | 13.49% |
| 180-500u | 37.89% |
| 500-1000u | 25.04% |
| 1000-2000u | 14.56% |
| 2000-5000u | 8.08% |
| >5000u | 0.93% |
As can be seen from the table above, the proportion of the molecular weight of the sea cucumber polypeptide prepared by the invention which is less than 1000u is 76.42%.
In addition, according to the regression linear equation of the molecular weight standard: y = -0.24742X +7.23795 2 =0.9917, and the average relative molecular mass of the sea cucumber polypeptide prepared by the invention is 798u through calculation, the molecular weight is moderate, and the activity is high.
Example 2
1. Pretreatment of
Soaking dried sea cucumber in 10 deg.C water for 48 hr, and changing water for 2 times. Then cutting open the sea cucumber, removing viscera and sand mouth, and cleaning with pure water.
2. Removing holothurin
Putting the cleaned sea cucumbers into a cooking pot, and mixing the materials according to a material-liquid ratio of 1:5 adding water, simply cooking the sea cucumber, fishing out the sea cucumber when the water is heated to 90 ℃, and discarding the soup.
3. High temperature high pressure treatment
Placing the simply cooked sea cucumbers into an autoclave, and mixing according to a feed-liquid ratio of 1:3 adding water, and then treating for 15min at constant temperature and pressure of 0.1MPa at 121 ℃.
4. Pulverizing
Crushing the sea cucumbers treated at high temperature and high pressure by using a cutting and mixing machine.
5. Enzymolysis
Pouring the crushed sea cucumbers into an enzymolysis tank, pumping the cooking liquor left in the high-temperature high-pressure treatment step into the enzymolysis tank, and adding pure water, wherein the final solid-to-liquid ratio is 1:5, adjusting the pH value of the feed liquid in the enzymolysis tank to 8, heating to 55 ℃, sequentially adding alkaline protease (Angel AP-200A series) accounting for 0.2% of the weight of the dry sea cucumber and neutral protease accounting for 0.2% of the weight of the dry sea cucumber, carrying out enzymolysis for 180min, finally adding flavor protease accounting for 0.1% of the weight of the dry sea cucumber, carrying out enzymolysis for 60min, and continuously stirring in the enzymolysis process.
6. Inactivating
Heating the enzymolysis feed liquid to 90 deg.C, and maintaining for 10min.
7. Centrifugation
Cooling the inactivated feed liquid to 55 ℃, filtering with a 250-mesh filter screen, pouring the filtrate into a horizontal spiral sedimentation centrifuge, wherein the centrifugal speed is 4800r/min, and the centrifugal production capacity is 0.8m 3 And h, finally pumping the centrifugal liquid back to the enzymolysis tank.
8. Plate frame filter pressing
Introducing the feed liquid (centrifugate) in the enzymolysis tank into a tank filled with AG-3000 # Filter pressing is carried out in a plate-and-frame filter press of diatomite (filter aid).
9. Membrane filtration
And (3) passing the feed liquid after filter pressing through a roll-type ultrafiltration membrane with the molecular weight cutoff of 20ku, and respectively collecting the permeate and the retentate.
10. Treating the permeate (deodorization and decoloration)
The temperature of the permeate is raised to 35 ℃, then the permeate is led into a decoloring device of activated carbon fiber membranes with stainless steel frameworks (the pore diameter of micropores of the activated carbon fibers is 3 nm) for deodorization and decoloring, and the filtrate is collected.
11. Treatment of trapped fluid
Pumping the trapped fluid into autoclave, adjusting pH value of the trapped fluid to 5 with acetic acid, and treating at 121 deg.C and 0.1MPa for 20min. When the trapped fluid is cooled to 60 ℃, the pH value of the trapped fluid is adjusted to 7 by using a sodium hydroxide solution with the mass concentration of 5 percent, and then the trapped fluid is led into a container filled with SD 307 # Filter pressing is carried out in a plate-and-frame filter press of diatomite (filter aid) special for sugar production, and filtrate is collected.
12. Mixing
Mixing the trapped liquid after high-temperature and high-pressure treatment and plate-and-frame filter pressing treatment with the permeate liquid after deodorization and decoloration treatment, and then sequentially carrying out primary nanofiltration, secondary nanofiltration and tertiary nanofiltration on the mixed liquid by using a rolled nanofiltration membrane.
13. Drying
Heating the concentrated solution obtained after the three-stage concentration to 90 ℃, pumping the concentrated solution into a drying tower for instantaneous spray drying at the drying temperature of 150 ℃ to form dry powder, and obtaining the sea cucumber polypeptide with the yield of 85%.
And pouring the obtained sea cucumber polypeptide into water to prepare a sample solution with the mass concentration of 2%, wherein the sample solution is clear and transparent.
Through detection, the content of the sea cucumber polysaccharide in the sample is 6.02%, the total nitrogen content (calculated on a dry basis) is 14.9%, and the protein content (calculated on a dry basis) is 93.1%.
In addition, the molecular weight distribution of the sample is shown in fig. 2, and the specific data are shown in table 2 below:
TABLE 2 molecular weight distribution of samples
| Size of molecular weight | Ratio of the ingredients |
| <180u | 13.92% |
| 180-500u | 39.67% |
| 500-1000u | 24.71% |
| 1000-2000u | 14.25% |
| 2000-5000u | 6.71% |
| >5000u | 0.74% |
As can be seen from the above table, the proportion of the sea cucumber polypeptide with the molecular weight of less than 1000u is 78.30%.
In addition, the linear equation is regressed according to molecular weight standards: y = -0.24742X +7.23795 2 =0.9917, and the average relative molecular mass of the sea cucumber polypeptide prepared by the invention is 742u, the molecular weight is moderate, and the activity is high.
It should be noted that the above-mentioned embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. It is not exhaustive here for all embodiments. All obvious changes and modifications which are obvious and belong to the technical scheme of the invention are still in the protection scope of the invention.
Claims (10)
1. A method for industrially producing sea cucumber polypeptide rich in sea cucumber polysaccharide is characterized by comprising the following steps:
(1) Soaking dried sea cucumber, cutting open, removing viscera and sand mouth, and cleaning with pure water;
(2) Putting the cleaned sea cucumber into a cooking pot, adding water, simply cooking the sea cucumber, fishing out the sea cucumber, and removing soup;
(3) Placing the simply cooked sea cucumber into a high-pressure kettle, adding water, and treating at 121 deg.C and 0.1MPa for 10-15min;
(4) Crushing the sea cucumbers subjected to high-temperature and high-pressure treatment by using a cutting and mixing machine;
(5) Pouring the crushed sea cucumber into an enzymolysis tank, pumping the rest cooking liquor obtained in the high-temperature high-pressure treatment step into the enzymolysis tank, adding pure water, adjusting the pH value of the liquor in the enzymolysis tank to 7-9, heating to 50-55 ℃, sequentially adding alkaline protease accounting for 0.2% of the weight of the dry sea cucumber and neutral protease accounting for 0.2% of the weight of the dry sea cucumber, carrying out enzymolysis for 150-180min, finally adding flavor protease accounting for 0.1% of the weight of the dry sea cucumber, carrying out enzymolysis for 30-60min, and continuously stirring in the enzymolysis process;
(6) Heating the enzymolysis feed liquid to 85-90 deg.C, and maintaining for 10min;
(7) Cooling the inactivated feed liquid to 50-55 ℃, filtering with a 200-250 mesh filter screen, pouring the filtrate into a horizontal spiral sedimentation centrifuge for centrifugation, and finally pumping the centrifugal liquid back to the enzymolysis tank;
(8) Introducing the feed liquid in the enzymolysis tank into a plate-and-frame filter press for filter pressing;
(9) Passing the feed liquid after filter pressing through a roll type ultrafiltration membrane with the molecular weight cutoff of 10-20ku, and respectively collecting the permeate and the retentate;
(10) Heating the permeate to 35 deg.C, introducing into a stainless steel skeleton activated carbon fiber membrane decolorizer for deodorizing and decolorizing, wherein the diameter of micropores of the activated carbon fiber is 3nm, and collecting filtrate;
(11) Pumping the trapped fluid into an autoclave, adjusting the pH value of the trapped fluid to 5 by using acetic acid, then carrying out constant-temperature and constant-pressure treatment for 10-20min at the temperature of 121 ℃ and under the pressure of 0.1MPa, adjusting the pH value of the trapped fluid to 6.5-7 by using a sodium hydroxide solution when the trapped fluid is cooled to 50-60 ℃, then introducing into a plate and frame filter press for filter pressing, and collecting filtrate;
(12) Mixing the trapped liquid subjected to high-temperature and high-pressure treatment and plate-and-frame filter pressing treatment with the permeate subjected to deodorization and decoloration treatment, and then sequentially performing primary nanofiltration, secondary nanofiltration and tertiary nanofiltration on the mixed liquid by using a rolled nanofiltration membrane;
(13) And (3) carrying out instantaneous spray drying on the concentrated solution obtained after the three-stage concentration to form dry powder, thus obtaining the sea cucumber polypeptide.
2. The method for industrially producing sea cucumber polypeptide rich in sea cucumber polysaccharide according to claim 1, wherein in the step (1), the method for soaking and expanding the dried sea cucumber comprises the following steps: soaking dried sea cucumber in water of 4-10 deg.C for 48 hr, and changing water for 2 times.
3. The method for industrially producing the sea cucumber polypeptide rich in the sea cucumber polysaccharide as claimed in claim 1, wherein, in the step (2), the ratio of the feed to the liquid is 1:4-5, adding water.
4. The method for industrially producing sea cucumber polypeptide rich in sea cucumber polysaccharide as claimed in claim 1, wherein in the step (2), the sea cucumber is fished out when the water is heated to 80-90 ℃.
5. The method for industrially producing the sea cucumber polypeptide rich in the sea cucumber polysaccharide as claimed in claim 1, wherein, in the step (3), the ratio of the feed to the liquid is 1:2-3, adding water.
6. The method for industrially producing the sea cucumber polypeptide rich in the sea cucumber polysaccharide as claimed in claim 1, wherein in the step (5), after pure water is added, the final solid-to-liquid ratio is 1:4-5.
7. The method for industrially producing sea cucumber polypeptide rich in sea cucumber polysaccharide as claimed in claim 1, wherein in the step (7), the centrifugal speed is 4800r/min, and the centrifugal productivity is 0.8m 3 /h。
8. The method for industrially producing sea cucumber polypeptide rich in sea cucumber polysaccharide as claimed in claim 1, wherein in the step (8), AG-3000 is installed in a plate and frame filter press # Diatomaceous earth.
9. The method for industrially producing the sea cucumber polypeptide rich in the sea cucumber polysaccharide as claimed in claim 1, wherein in the step (11), the plate-and-frame filter press is filled with SD 307 # Diatomite special for sugar production.
10. The method for industrially producing sea cucumber polypeptide rich in sea cucumber polysaccharide as claimed in claim 1, wherein in the step (13), the instantaneous spray drying method comprises: heating the concentrated solution to 90 deg.C, pumping into drying tower for instantaneous spray drying at 150-160 deg.C.
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