CN101451157B - Method for preparing low molecular weight sea cucumber polysaccharide - Google Patents
Method for preparing low molecular weight sea cucumber polysaccharide Download PDFInfo
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Abstract
The invention discloses a method for preparing low molecular weight holothurian polysaccharide. Sea cucumbers are cleaned, minced, heat-gelatinated, freeze-dried, coarse ground, airflow crushed and nanometer crushed to produce nanometer sea cucumber powder, then prolease is used to perform 1 to 3 times of enzymolysis, and then a low molecular weight holothurian polysaccharide solution is obtained through the centrifugation; and then a crude low molecular weight holothurian total polysaccharide product is obtained through salt-adding alcohol precipitation and centrifugation, and finally is subjected to hydrogen peroxide decolorization, salt-adding alcohol precipitation and centrifugal drying to obtain the low molecular weight holothurian polysaccharide. The obtained crude low molecular weight holothurian total polysaccharide product is dissolved, is added with potassium salt and precipitates a holothurian glycosaminoglyan precipitate at low temperature, a supernatant fluid is a holothurian fucoidin solution, and the holothurian glycosaminoglyan precipitate and the holothurian fucoidin solution are separated through the centrifugation, and are decolored respectively. Low molecular weight holothurian fucoidin is obtained through salt-adding alcohol precipitation, separation and drying; and the potassium salt is added to precipitate at low temperature, and low molecular weight holothurian glycosaminoglyan is obtained through separation and drying. The method utilizes gelatinated nanometer sea cucumbers to directly perform the low molecular weight polysaccharide, and the low molecular weight polysaccharide has low molecular weight, has good absorption of human bodies, greatly reduces the dosage, and obviously strengthens the effect.
Description
Technical field
The invention belongs to and derive from food of sea cucumber, the invention belongs to the sea cucumbers field of food, adopt the working method of heating, lyophilize, comminution by gas stream, ball mill pulverizing; Simultaneously, also relate to the pharmaceutical product of sea cucumber, particularly prepare the method for polysaccharide with zymolysis technique.
Background technology
Sea cucumber is one of China's famous " seafood delights eight delicacies ", and ancestor's beche-de-mer nourishing and health preserving of the Chinese nation has long history, and the staple in the imitative stichopus japonicus is a collagen protein; With directly carrying out lyophilize after the fresh and alive sea cucumbers cleaning; Its protein contnt is about 45%, and lipid accounts for about 20%, and polysaccharide accounts for about 8-10%; Inorganic salt account for about 20%, wherein also comprise compositions such as saponin in the lipid.Show through the analysis of experiments result: if the collagen protein in the sea cucumber without any processing, like heating etc., effective specific absorption of human body is extremely low, less than 1%.This result has bibliographical information.In " Chinese marine drug " magazine calendar year 2001 the 4th interim " sea cucumber: marine genseng " literary composition; For the composition of holothurian collagen with do in order to through detailed introduction has been arranged, the donkey-hide gelatin, Colla Plastri Testudinis, Deerhorn Glue that prompting sea cucumber albumin glue can be mended glue with traditional Chinese medicine is at composition and use and compare favourably.The NCP composition in the sea cucumber has simultaneously improved the nutritive value of single collagen protein, this be again above-mentioned traditional drugs glue can't be obtained.Lipid in the sea cucumber is main with phosphatide, and it is a kind of low-cholesterol animal food, contains a large amount of compositions such as pufas to the human body beneficial, like DHA, EPA, AA etc.The saponin that is contained in the sea cucumber simultaneously has multiple physiologically active, can resist multiple persistent ailment.
Polysaccharide is the another kind of important component of sea cucumber, has the reported in literature sea cucumber polysaccharide to mainly contain two kinds, and one is sea cucumber ammonia polyose of candy or mucopolysaccharide (Holothurian Glycosaminoglycan; HG), by branch's mixed polysaccharide that D-N-acetylamino galactosamine, D-glucuronic acid and L-Fucose are formed, relative molecular mass is at 4-5 about ten thousand; Another kind of sea cucumber fucoidin (Holothurian Fucan; HF), be the even polysaccharide of side chain that constitutes by the L-Fucose, relative molecular mass is at 8-10 about ten thousand.Definite at present, the structure of two kinds of sea cucumber polysaccharides is special, all is that sea cucumber institute is peculiar.Simultaneously, sea cucumber polysaccharide is big with the degree of the height of its content in tissue and polysaccharide, and is very rare in animal based food resource.In recent years; Both at home and abroad to sea cucumber polysaccharide; Particularly to HG and change the structure thing (like depolymerizing substance; DHG) extensive pharmacological research confirms that it has multiple drug effect: 1, the growth of HG antagonism kinds of experiments animal tumor, and respectively up to more than 79% and 60%, artificial lung transfer and Lewis lung cancer that the while can also be suppressed M737 mammary cancer shift naturally to MA-737 mammary cancer and T795 lung cancer growth inhibition ratio; 2, HG improves the body cell immunologic function, improves and strengthens because of lotus knurl or the animal body immunologic hypofunction situation of using cancer therapy drug to cause; 3, HG antagonism new vessel generates, and comprises that the new vessel that transplanted tumor brings out generates; 4, HG is a kind of novel antithrombotics, in the different links demonstration multiple actions of coagulation process.HG is better than heparin at aspects such as anti-freezing mechanism and securities; 5, HG can promote fibrinolysis through plasminogen activation, and HG can suppress polymerization between fibrinous monomer, change the fibrin clot structure simultaneously, thereby helps the medicine fibrinolytic; 6, in animal phlebothrombosis model and acute mouse lung bolt match model, HG can suppress embolism and form and improve the animal survival rate.HG treatment disseminated intravascular coagulation (DIC) and antithrombotic effect have been confirmed in the preliminary clinical trial; 7, HG has the effect that reduces WBV and plasma viscosity, shows that simultaneously the function of regulating blood fat promptly reduces serum cholesterol and triglyceride levels, high density lipoprotein increasing and reduction low-density lipoprotein.
The multiple pharmacological function of sea cucumber it has been seen in that the exploitation of sea cucumber and lower molecular weight ammonia polyose of candy thereof is worth.
Yet sea cucumber polysaccharide forms protein-polysaccharide in sea cucumber and protein binding, under the prerequisite that collagen structure is not destroyed, and especially can't absorbing contacting.The outer prior reason of order is that sea cucumber polysaccharide is big because of its molecular weight, can't effectively receive through human body is oral, and the route of administration in the report of aspects such as some curative effects of the relevant sea cucumber polysaccharide of reporting in the document all is a drug administration by injection.
Preparation about sea cucumber (gluing) polysaccharide has had many patent reports.The method that ZL 99113247 provides is to carry out enzymolysis with bromeline list enzyme or with medium-sized proteolytic enzyme of Bacillus subtilus and the two enzymes of stomach en-after sea cucumber is rubbed pulping, and it contains 2-10 peptide 5～20%, mucopolysaccharide 0.5～5%.ZL 03111925.5 described method is that to utilize the enzyme solution of autolytic enzyme to make molecular weight after the sea cucumber slurrying be 2～50,000 saccharan.And patent documents such as ZL200410054772.X, CN 200410038284.X and CN 200710017839.6 report all is to carry out enzymolysis with fresh sea cucumber basically; Comprise that autolytic enzyme, external application enzyme carry out enzymolysis, then through separation, alcohol precipitation, refining and obtain sea cucumber polysaccharide.Can have sea cucumber polysaccharide molecular weight much, that obtain to have much, as whether to reach HG and FG requirements, human body absorb as for the enzymolysis degree of its preparation method all has no way of understanding.
Summary of the invention
The present invention is raw material and provide a kind of with the nanometer sea cucumber for solving the problem that exists in the prior art, prepares the method for sea cucumber lower molecular weight ammonia polyose of candy.
Technical scheme of the present invention is to utilize sea cucumber (to comprise bright sea cucumber; Salt marsh, half-dried, dry Stichopus japonicus hair products) process the nanometer Stichopus japonicus powder; Make low molecule sea cucumber polysaccharide with nanometer Stichopus japonicus powder enzymolysis: comprising low molecule ammonia polyose of candy (the Depolymrized Holothurian Glycosaminoglycan of sea cucumber; Be called for short DHG) and the low molecule fucoidin of sea cucumber (Depolymrized Holothurian Fucan is called for short DHF).
Preparation process of the present invention is following:
(1) raw material is handled: fresh and alive sea cucumbers being cut off, taken out internal organ, it is fully cleaned up respectively, can only be to use the sea cucumber body wall, also can rub together with the sea cucumber internal organ, places encloses container.Raw material also can adopt various forms of cucumber products, like dry Stichopus japonicus, half-dried sea cucumber, salt marsh sea cucumber etc., it is made to clean up places encloses container.
(2) heating gel: 70～130 ℃ are heated 1min～20h down.Preferred 100～105 ℃, 1h.
(3) lyophilize: the sea cucumber after the gel is carried out lyophilize, and moisture is less than 10%, and preferred moisture is less than 3%.
(4) coarse reduction: the sea cucumber after the lyophilize is carried out coarse reduction, and to obtain fineness be the Stichopus japonicus powder that 10～300 orders do not wait.
(5) micronizing: the Stichopus japonicus powder of coarse reduction is carried out micronizing with micronizer mill, and smashing fineness is 100～3000 orders.
(6) nano-pulverization: the sea cucumber super-fine powder that will pass through comminution by gas stream carries out nanometer with the high-energy ball milling kibbler to be pulverized, and the pulverizing time is 4～20h, preferred 10～12h, and fineness can reach 10～1000nm.Wherein use X-ray detection size-grade distribution, in 0～300nm scope, its median size is 100～200nm.
(7) enzymolysis and separation: nanometer Stichopus japonicus powder and water are dissolved in water with 1: 3～10 mass ratioes; In the nanometer Stichopus japonicus powder: proteolytic enzyme be the ratio of 1g: 0.1～1010mg add proteolytic enzyme under corresponding pH value in 40～70 ℃ of enzymolysis that carry out 1～5h; The enzyme that goes out of then heating; 0～10 ℃ of high speed centrifugation is got supernatant; The proteolytic enzyme that enzymolysis is selected for use can be various enzymes, like bromeline, and papoid; Sumizyme MP, neutral protease, flavor protease; Trypsinase etc. can be simultaneously single enzyme enzymolysis of planting, and also can be to select for use two or more proteolytic enzyme to carry out enzymolysis.Preferred nanometer sea cucumber powder and water carry out mixing according to 1: 7 ratio, carry out double enzymolysis with Sumizyme MP Acalase and pancreatin.Hydrolysis temperature is 40～70 ℃; Earlier pH is adjusted to 7～8, according to the mass ratio of nanometer sea cucumber powder and enzyme (1 gram: 0.1～10mg) adding Sumizyme MP Acalase, carry out enzymolysis 0.1h～5h after; With pH regulator to 8～10; (1 gram: 10mg～1000mg) adding trypsinase carries out enzymolysis 0.1～5h 2 times, and hydrolysis temperature is 40～70 ℃, earlier pH is adjusted to 8～10 according to the mass ratio of nanometer sea cucumber powder and enzyme.After enzyme reaction finishes with enzyme digestion reaction liquid in 90～100 ℃ of heating 1～20min enzyme that goes out.Enzymolysis product is carried out spinning, get the enzymolysis supernatant.
(8) alcohol precipitation with separate: according to the volume of enzymolysis supernatant; To wherein add salt to whole mass concentration and reach 1～8%; The salt that adds can be sodium-chlor, calcium acetate etc., adds ethanolic soln and carries out alcohol precipitation, and the ethanol mass concentration in the final enzymolysis solution is 20～70%; Low temperature (0～10 ℃) is centrifugal, obtains the total Crude polysaccharides of degraded sea cucumber.
(9) the total Crude polysaccharides of the sea cucumber that makes can be made with extra care together, also can the TGSS C3 in the total reducing sugar and Fucose be made with extra care after separately.
(10) the total Crude polysaccharides of sea cucumber is refining:
(i) decolouring: the low total Crude polysaccharides of molecule of the sea cucumber that makes is added water dissolve; The mass ratio of polysaccharide and water is 1: 5～20 water, decolours to yellow or colourless with ydrogen peroxide 50, and pH value of solution is 8～12 during decolouring; The whole mass concentration of the ydrogen peroxide 50 of 40～60 ℃ of addings of temperature is 0.1～5.0%; After the decolouring pH value of solution is adjusted to 2, the centrifuging and taking supernatant is to remove acidic protein.
(ii) purifying: adding salt to whole mass concentration in the total Crude polysaccharides aqueous solution of the sea cucumber after will decolouring is 1～8%; The salt that adds can be sodium-chlor, calcium acetate etc.; Add ethanol then and carry out alcohol precipitation, adding the whole mass concentration of alcoholic acid is 40～80%, centrifugal after; Pure hypostasis is dehydrated, and sub-sea cucumber total polysaccharides promptly makes low score.
(11) refining after thick DHG, the thick DHF separation of polysaccharides:
(i) separate: the low molecule sea cucumber total polysaccharides that will make adds water and dissolves; The mass ratio that adds water is 1: 5～20, adds sylvite such as Potassium ethanoate that whole mass concentration is 1～5M or Repone K according to the volume of Crude polysaccharides solution then, is positioned under 0～10 ℃ of low temperature; To being arranged, deposition separates out; It is centrifugal down in low temperature (0～10 ℃) to take out the back, is precipitated as low molecule sea cucumber ammonia polyose of candy raw product after centrifugal, and supernatant is the aqueous solution raw product of low molecule sea cucumber fucoidin.
(ii) decolouring: the aqueous solution raw product that will be dissolved in water into solution (mass ratio of raw sugar and water is 1: 5～20) and low molecule sea cucumber fucoidin from the low molecule sea cucumber ammonia polyose of candy raw product that step (i) separation obtains all can decolour respectively to yellow or colourless with ydrogen peroxide 50; PH value of solution is 8～12 during decolouring; 40～60 ℃ of temperature, the whole mass concentration of the ydrogen peroxide 50 of adding is 0.1～5.0%, after the decolouring pH value of solution is adjusted to 2; Centrifugal to remove acidic protein, get supernatant.
(iii) DHF is refining: adding salt to whole mass concentration in the DHF solution after will decolouring is 1～8%; The salt that adds can be sodium-chlor, calcium acetate etc.; Add ethanol then and carry out alcohol precipitation, adding the whole mass concentration of alcoholic acid is 40～80%, centrifugal after; Pure hypostasis is dehydrated, promptly get the low molecule fucoidin of sea cucumber.
(iv) DHG is refining: adding whole mass concentration in the DHG solution after will decolouring is the Potassium ethanoate of 1～5M, is positioned under 0～10 ℃ of low temperature, centrifugal under low temperature to there being deposition to separate out, and centrifugal sediment is dehydrated, and promptly obtains the low molecule ammonia polyose of candy of sea cucumber.
Low molecular weight sea cucumber ammonia polyose of candy after purifying, decolour, dewatering is light yellow or white powder, is prone to the moisture absorption, is prone to be dissolved in the water, and the aqueous solution is light yellow or water white.Viscosity-average molecular weight is 20000～40000.
Low molecular weight sea cucumber Fucose after purifying, decolour, dewatering is white or buff powder, is prone to the moisture absorption, is prone to be dissolved in the water, and the aqueous solution is light yellow or water white.Viscosity-average molecular weight is 40000～60000.
The viscosity-average molecular weight of the sea cucumber ammonia polyose of candy that experiment proof makes with method of the present invention (comprising gel sea cucumber system nano powder, enzymolysis and purification) is that the viscosity-average molecular weight (80000) of 20000～40000 sea cucumber ammonia polyose of candy that make than the micronization Stichopus japonicus powder is much little.Therefore, the absorption that people are oral when taking will be easy to many.
Compared with prior art, the advantage that the present invention gives prominence to is: the preparation process of prior art is to extract sea cucumber polysaccharide earlier, and then sea cucumber polysaccharide is degraded, thereby obtains the low molecular weight sea cucumber ammonia polyose of candy.The present invention utilizes gel nanometer sea cucumber directly to hang down molecule ammonia polyose of candy and extraction, and its molecular weight is low, has improved the bioavailability of sea cucumber polysaccharide, can practice thrift the sea cucumber resource.Simultaneously the ammonia polyose of candy of sea cucumber degraded after, be developed to protective foods after, absorption by human body is good, dose reduces greatly, and effect obviously strengthens.Also can directly develop sea cucumber ammonia polyose of candy healthcare products and the medicine that Gong to orally use.
Embodiment
Embodiment 1
(1) raw material is handled: fresh and alive sea cucumbers is cut off, taken out internal organ, the sea cucumber body wall is fully cleaned up respectively, place encloses container.
(2) gel: said vesse in 70～80 ℃, is heated 20h.
(3) vacuum lyophilization: the sea cucumber after the gel is carried out lyophilize, and moisture is 0.1%.
(4) coarse reduction: the sea cucumber after the lyophilize is carried out coarse reduction.Obtaining fineness is the Stichopus japonicus powder that 10～300 orders do not wait.
(5) micronizing: the Stichopus japonicus powder of coarse reduction is carried out micronizing with micronizer mill, and obtaining smashing fineness is 100～3000 purpose sea cucumber super-fine powder.
(6) nano-pulverization: the sea cucumber super-fine powder that will pass through comminution by gas stream carries out nanometer with the high-energy ball milling kibbler to be pulverized, and the pulverizing time is 4h.
(7) enzymolysis: after proportionally 1: 3 ratio adds with nanometer sea cucumber powder and water (weight ratio); Stir; Add the mass ratio 1g of bromeline according to nanometer sea cucumber powder and bromeline enzyme: 10mg, pH6-7, hydrolysis temperature are 40 ℃; After carrying out enzymolysis 5h, after enzyme reaction finishes enzyme digestion reaction liquid is heated 20min in 90 ℃.Enzymolysis product is carried out spinning, get the enzymolysis supernatant.
(8) alcohol precipitation with separate: according to the volume of enzymolysis supernatant; To wherein add sodium-chlor to whole mass concentration and reach 1%; Add ethanolic soln and carry out alcohol precipitation, the ethanol mass concentration in the final enzymolysis solution is 20% back low-temperature centrifugation, obtains the total Crude polysaccharides of degraded sea cucumber.
(9) thick DHG, thick DHF separation of polysaccharides: the low molecule Crude polysaccharides of the sea cucumber that makes is added water dissolve; The ratio of amount of water is that 1 gram Crude polysaccharides adds 5ml water, and the volume according to Crude polysaccharides solution adds the Potassium ethanoate that whole mass concentration is 1M then, is positioned under 0～10 ℃ of low temperature; To being arranged, deposition separates out; It is centrifugal to take out back (0～10 ℃) under low temperature, and being precipitated as after centrifugal promptly obtains low molecule sea cucumber ammonia polyose of candy raw product, and supernatant is the aqueous solution raw product of low molecule sea cucumber fucoidin.
(10) decolouring: will add water by the low molecule sea cucumber ammonia polyose of candy raw product that (9) obtain and dissolve (ratio of amount of water is that 1 gram Crude polysaccharides adds 5g water); Itself and the aqueous solution raw product of low molecule sea cucumber fucoidin are decoloured to yellow or colourless with ydrogen peroxide 50 respectively, and pH value of solution is 8～9 during decolouring, 40 ℃ of temperature; The whole mass concentration of the ydrogen peroxide 50 that adds is 0.1%, after the decolouring pH value of solution is adjusted to 2, removes acidic protein; Centrifugal, get supernatant.
(11) DHF is refining: adding sodium chloride salt to whole mass concentration in the DHF solution after will decolouring is 1%; Add ethanol then and carry out alcohol precipitation, adding the whole mass concentration of alcoholic acid is 40%, centrifugal after; Pure hypostasis is dehydrated, promptly get the low molecule fucoidin of sea cucumber.
(12) DHG is refining: adding whole mass concentration in the DHG solution after will decolouring is the Potassium ethanoate of 1M, is positioned under 0～10 ℃ of low temperature, to there being deposition to separate out; It is centrifugal down in low temperature (0～10 ℃) to take out the back; After centrifugal, centrifugal sediment is dehydrated, promptly obtain the low molecule ammonia polyose of candy of sea cucumber.
(13) the low molecular weight sea cucumber ammonia polyose of candy after making with extra care is a white powder, is prone to the moisture absorption, is prone to be dissolved in the water, and the aqueous solution is water white.Viscosity-average molecular weight is 35000.
Low molecular weight sea cucumber Fucose after refining is a buff powder, is prone to the moisture absorption, is prone to be dissolved in the water, and the aqueous solution is light yellow.Viscosity-average molecular weight is 51000.
Embodiment 2
(1) raw material is handled: fresh and alive sea cucumbers is cut off, taken out internal organ, with the sea cucumber body wall, internal organ fully clean up respectively, place encloses container together.
(2) gel: said vesse in 80～90 ℃, is heated 15h.
(3) vacuum lyophilization: the sea cucumber after the gel is carried out lyophilize, and moisture is 1%.
(4) coarse reduction: the sea cucumber after the lyophilize is carried out coarse reduction.Obtaining fineness is the Stichopus japonicus powder that 10～300 orders do not wait.
(5) micronizing: the Stichopus japonicus powder of coarse reduction is carried out micronizing with micronizer mill, and obtaining smashing fineness is 100～3000 purpose sea cucumber super-fine powder.
(6) nano-pulverization: the sea cucumber super-fine powder that will pass through comminution by gas stream carries out nanometer with the high-energy ball milling kibbler to be pulverized, and the pulverizing time is 8h.
(7) enzymolysis: after proportionally 1: 4 ratio adds with nanometer sea cucumber powder and water (weight ratio), stir,
Add the mass ratio 1g of Sumizyme MP according to nanometer sea cucumber powder and Sumizyme MP: 0.5mg, pH7～8, hydrolysis temperature is 65 ℃, carry out enzymolysis 2h after, after enzyme reaction finishes enzyme digestion reaction liquid is heated 15min in 95 ℃.Enzymolysis product is carried out spinning, get the enzymolysis supernatant.
(8) alcohol precipitation with separate: according to the volume of enzymolysis supernatant, will wherein add calcium acetate and reach 2% to whole mass concentration, and add ethanolic soln and carry out alcohol precipitation, the alcohol concn in the final enzymolysis solution is 30%, and low-temperature centrifugation obtains the total Crude polysaccharides of degraded sea cucumber.
(9) thick DHG, thick DHF separation of polysaccharides: the low molecule Crude polysaccharides of the sea cucumber that makes is added water dissolve; The ratio of amount of water is that 1 gram Crude polysaccharides adds 10ml water, and the volume according to Crude polysaccharides solution adds the Potassium ethanoate that whole mass concentration is 2M then, is positioned under 0～10 ℃ of low temperature; To being arranged, deposition separates out; It is centrifugal under low temperature to take out the back, and being precipitated as after centrifugal promptly obtains low molecule sea cucumber ammonia polyose of candy raw product, and supernatant is the aqueous solution raw product of low molecule sea cucumber fucoidin.
(10) decolouring: will add water by the low molecule sea cucumber ammonia polyose of candy raw product that (9) obtain and dissolve (ratio of amount of water is that 1 gram Crude polysaccharides adds 10g water); Itself and the aqueous solution raw product of low molecule sea cucumber fucoidin are decoloured to yellow or colourless with ydrogen peroxide 50 respectively; PH value of solution is 9～10 during decolouring, 45 ℃ of temperature, and the whole mass concentration of the ydrogen peroxide 50 of adding is 0.5%; After the decolouring pH value of solution is adjusted to 2; Remove acidic protein, centrifugal, get supernatant.
(11) DHF is refining: adding calcium chloride to whole mass concentration in the HF solution after will decolour is 2%, adds ethanol then and carries out alcohol precipitation, and adding the whole mass concentration of alcoholic acid is 50%, centrifugal after, pure hypostasis is dehydrated, promptly get sea cucumber and hang down the molecule fucoidin.
(12) DHG is refining: adding whole mass concentration in the HG solution after will decolouring is the Potassium ethanoate of 2M, is positioned under 0～10 ℃ of low temperature, to there being deposition to separate out; It is centrifugal under low temperature to take out the back; After centrifugal, centrifugal sediment is dehydrated, promptly obtain the low molecule ammonia polyose of candy of sea cucumber.
(13) the low molecular weight sea cucumber ammonia polyose of candy after making with extra care is a white powder, is prone to the moisture absorption, is prone to be dissolved in the water, and the aqueous solution is water white.Viscosity-average molecular weight is 31000.
Low molecular weight sea cucumber Fucose after refining is a white powder, is prone to the moisture absorption, is prone to be dissolved in the water, and the aqueous solution is water white.Viscosity-average molecular weight is 53000.
Embodiment 3
(1) raw material is handled: get dry Stichopus japonicus, in water, soak soft after, body wall is cut off, clean up, place encloses container.
(2) gel: said vesse in 90～100 ℃, is heated 10h.
(3) vacuum lyophilization: the sea cucumber after the gel is carried out lyophilize, and moisture is 3%.
(4) coarse reduction, (5) micronizing and (6) nano-pulverization are all with embodiment 2.
(7) enzymolysis: after proportionally 1: 5 ratio adds with nanometer sea cucumber powder and water (weight ratio); Stir; Add trypsinase according to nanometer sea cucumber powder and tryptic mass ratio 1g: 10mg, pH8～9, hydrolysis temperature is 45 ℃; After carrying out enzymolysis 5h, after enzyme reaction finishes enzyme digestion reaction liquid is heated 10min in 100 ℃.Enzymolysis product is carried out spinning, get the enzymolysis supernatant.
(8) alcohol precipitation with separate: according to the volume of enzymolysis supernatant, will wherein add calcium acetate and reach 3% to whole mass concentration, and add ethanolic soln and carry out alcohol precipitation, the alcohol concn in the final enzymolysis solution is 40% low-temperature centrifugation, obtains the total Crude polysaccharides of degraded sea cucumber.
(9) thick DHG, thick DHF separation of polysaccharides: the low molecule Crude polysaccharides of the sea cucumber that makes is added water dissolve; The ratio of amount of water is that 1 gram Crude polysaccharides adds 15ml water, and the volume according to Crude polysaccharides solution adds the Repone K that whole mass concentration is 2.5M then, is positioned under 0～10 ℃ of low temperature; To being arranged, deposition separates out; It is centrifugal under low temperature to take out the back, and being precipitated as after centrifugal promptly obtains low molecule sea cucumber ammonia polyose of candy raw product, and supernatant is the aqueous solution raw product of low molecule sea cucumber fucoidin.
(10) decolouring: will add water by the low molecule sea cucumber ammonia polyose of candy raw product that (9) obtain and dissolve (ratio of amount of water is that 1 gram Crude polysaccharides adds 15g water); Itself and the aqueous solution raw product of low molecule sea cucumber fucoidin are decoloured to yellow or colourless with ydrogen peroxide 50 respectively, and pH value of solution is 10～11 during decolouring, 50 ℃ of temperature; The whole mass concentration of the ydrogen peroxide 50 that adds is 1%, after the decolouring pH value of solution is adjusted to 2, removes acidic protein; Centrifugal, get supernatant.
(11) DHF is refining: adding calcium acetate to whole mass concentration in the HF solution after will decolour is 3%, adds ethanol then and carries out alcohol precipitation, and adding the whole mass concentration of alcoholic acid is 55%, centrifugal after, pure hypostasis is dehydrated, promptly get sea cucumber and hang down the molecule fucoidin.
(12) DHG is refining: adding whole mass concentration in the HG solution after will decolouring is the Potassium ethanoate of 2.5M, is positioned under 0～10 ℃ of low temperature, to there being deposition to separate out; It is centrifugal under low temperature to take out the back; After centrifugal, centrifugal sediment is dehydrated, promptly obtain the low molecule ammonia polyose of candy of sea cucumber.
(13) the low molecular weight sea cucumber ammonia polyose of candy after making with extra care is a white powder, is prone to the moisture absorption, is prone to be dissolved in the water, and the aqueous solution is water white.Viscosity-average molecular weight is 28000.
Low molecular weight sea cucumber Fucose after refining is a buff powder, is prone to the moisture absorption, is prone to be dissolved in the water, and the aqueous solution is light yellow.Viscosity-average molecular weight is 47000.
Embodiment 4
(1) raw material is handled: get the salt marsh dry Stichopus japonicus, in water, remove the intravital salinity of sea cucumber, soak soft after, body wall is cut off, clean up, place encloses container.
(2) gel: said vesse in 100～105 ℃, is heated 5h.
(3) vacuum lyophilization: the sea cucumber after the gel is carried out lyophilize, and moisture is 5%.
(4) coarse reduction and (5) micronizing are all with embodiment 2.
(6) the nano-pulverization sea cucumber super-fine powder that will pass through comminution by gas stream carries out nanometer with the high-energy ball milling kibbler and pulverizes, and the pulverizing time is 12h.
(7) enzymolysis: after proportionally 1: 6 ratio adds with nanometer sea cucumber powder and water (weight ratio); Stir; Add the mass ratio 1g of neutral protease according to nanometer sea cucumber powder and neutral protease: 1mg, pH6.7～7, hydrolysis temperature is 50 ℃; After carrying out enzymolysis 1h, after enzyme reaction finishes enzyme digestion reaction liquid is heated 5min in 100 ℃.Enzymolysis product is carried out spinning, get the enzymolysis supernatant.
(8) alcohol precipitation with separate: according to the volume of enzymolysis supernatant, will wherein add the whole mass concentration of sodium-chlor and reach 4%, and add ethanolic soln and carry out alcohol precipitation, the alcohol concn in the final enzymolysis solution is 45%, and low-temperature centrifugation obtains the total Crude polysaccharides of degraded sea cucumber.
(9) thick DHG, thick DHF separation of polysaccharides: the low molecule Crude polysaccharides of the sea cucumber that makes is added water dissolve; The ratio of amount of water is that 1 gram Crude polysaccharides adds 20ml water, and the volume according to Crude polysaccharides solution adds the Potassium ethanoate that whole mass concentration is 3M then, is positioned under 0 ℃ of-10 ℃ of low temperature; To being arranged, deposition separates out; It is centrifugal under low temperature to take out the back, and being precipitated as after centrifugal promptly obtains low molecule sea cucumber ammonia polyose of candy raw product, and supernatant is the aqueous solution raw product of low molecule sea cucumber fucoidin.
(10) decolouring: will add water by the low molecule sea cucumber ammonia polyose of candy raw product that (9) obtain and dissolve (ratio of amount of water is that 1 gram Crude polysaccharides adds 20g water); Itself and the aqueous solution raw product of low molecule sea cucumber fucoidin are decoloured to yellow or colourless with ydrogen peroxide 50 respectively, and pH value of solution is 11～12 during decolouring, 55 ℃ of temperature; The whole mass concentration of the ydrogen peroxide 50 that adds is 2%, after the decolouring pH value of solution is adjusted to 2, removes acidic protein; Centrifugal, get supernatant.
(11) DHF is refining: adding sodium-chlor to whole mass concentration in the HF solution after will decolour is 4%, adds ethanol then and carries out alcohol precipitation, and adding the whole mass concentration of alcoholic acid is 60%, centrifugal after, pure hypostasis is dehydrated, promptly get sea cucumber and hang down the molecule fucoidin.
(12) DHG is refining: adding whole mass concentration in the HG solution after will decolouring is the Potassium ethanoate of 3M, is positioned under 0～10 ℃ of low temperature, to there being deposition to separate out; It is centrifugal under low temperature to take out the back; After centrifugal, centrifugal sediment is dehydrated, promptly obtain the low molecule ammonia polyose of candy of sea cucumber.(13) the low molecular weight sea cucumber ammonia polyose of candy after making with extra care is a white powder, is prone to the moisture absorption, is prone to be dissolved in the water, and the aqueous solution is water white.Viscosity-average molecular weight is 24000.
Low molecular weight sea cucumber Fucose after refining is a white powder, is prone to the moisture absorption, is prone to be dissolved in the water, and the aqueous solution is water white.Viscosity-average molecular weight is 43000.
Embodiment 5
(1) raw material is handled: get the salt marsh dry Stichopus japonicus, soaks soft after, 90～100 ℃ at water heating 1h, body wall is cut off, clean up, remove the intravital salinity of sea cucumber with pure water, play the effect of a Stichopus japonicus (processed) simultaneously.Place encloses container.
(2) gel: said vesse in 105～110 ℃, is heated 2h.
(3) vacuum lyophilization: the sea cucumber after the gel is carried out lyophilize, and moisture is 7%.
(4) coarse reduction and (5) micronizing are with embodiment 1.
(6) nano-pulverization: the sea cucumber super-fine powder that will pass through comminution by gas stream carries out nanometer with the high-energy ball milling kibbler to be pulverized, and the pulverizing time is 16h.
(7) enzymolysis: after proportionally 1: 7 ratio adds with nanometer sea cucumber powder and water (weight ratio), stir, add the mass ratio 1g of alkalescence property proteolytic enzyme according to nanometer sea cucumber powder and Sumizyme MP: 0.1mg; PH7～8, hydrolysis temperature are 65 ℃, carry out enzymolysis 3h after; Again temperature is adjusted into 45 ℃; Add trypsinase, add the weight ratio 1g of trypsinase according to nanometer sea cucumber powder and pancreatin: 10mg, pH 8～9; Carry out enzymolysis 3h, after enzyme reaction finishes enzyme digestion reaction liquid is heated 10min in 100 ℃.Enzymolysis product is carried out spinning, get the enzymolysis supernatant.
(8) alcohol precipitation with separate: according to the volume of enzymolysis supernatant, will wherein add calcium chloride and reach 5% to whole mass concentration, and add ethanolic soln and carry out alcohol precipitation, the alcohol concn in the final enzymolysis solution is 50%, and low-temperature centrifugation obtains the total Crude polysaccharides of degraded sea cucumber.
(9) thick DHG, thick DHF separation of polysaccharides: the low molecule Crude polysaccharides of the sea cucumber that makes is added water dissolve; The ratio of amount of water is that 1 gram Crude polysaccharides adds 10ml water, and the volume according to Crude polysaccharides solution adds the Repone K that whole mass concentration is 4M then, is positioned under 0～10 ℃ of low temperature; To being arranged, deposition separates out; It is centrifugal under low temperature to take out the back, and being precipitated as after centrifugal promptly obtains low molecule sea cucumber ammonia polyose of candy raw product, and supernatant is the aqueous solution raw product of low molecule sea cucumber fucoidin.
(10) decolouring: will add water by the low molecule sea cucumber ammonia polyose of candy raw product that (9) obtain and dissolve (ratio of amount of water is that 1 gram Crude polysaccharides adds 10g water); Itself and the aqueous solution raw product of low molecule sea cucumber fucoidin are decoloured to yellow or colourless with ydrogen peroxide 50 respectively, and pH value of solution is 9～10 during decolouring, 50 ℃ of temperature; The whole mass concentration of the ydrogen peroxide 50 that adds is 4%, after the decolouring pH value of solution is adjusted to 2, removes acidic protein; Centrifugal, get supernatant.
(11) DHF is refining: adding calcium chloride to whole mass concentration in the HF solution after will decolour is 5%, adds ethanol then and carries out alcohol precipitation, and adding the whole mass concentration of alcoholic acid is 65%, centrifugal after, pure hypostasis is dehydrated, promptly get sea cucumber and hang down the molecule fucoidin.
(12) DHG is refining: adding whole mass concentration in the HG solution after will decolouring is the Potassium ethanoate of 3.5M, is positioned under 0～10 ℃ of low temperature, to there being deposition to separate out; It is centrifugal under low temperature to take out the back; After centrifugal, centrifugal sediment is dehydrated, promptly obtain the low molecule ammonia polyose of candy of sea cucumber.(13) the low molecular weight sea cucumber ammonia polyose of candy after making with extra care is a white powder, is prone to the moisture absorption, is prone to be dissolved in the water, and the aqueous solution is water white.Viscosity-average molecular weight is 36000.
Low molecular weight sea cucumber Fucose after refining is a buff powder, is prone to the moisture absorption, is prone to be dissolved in the water, and the aqueous solution is light yellow.Viscosity-average molecular weight is 58000.
Embodiment 6
(1) raw material is handled: get the half-dried sea cucumber of salt marsh, soak soft after, body wall is cut off, clean up, remove the intravital salinity of sea cucumber with pure water.Place encloses container.
(2) gel: said vesse in 110～120 ℃, is heated 1h.
(3) vacuum lyophilization: the sea cucumber after the gel is carried out lyophilize, and moisture is 9%.
(4) coarse reduction and (5) micronizing are all with embodiment 1.
(6) nano-pulverization: the sea cucumber super-fine powder that will pass through comminution by gas stream carries out nanometer with the high-energy ball milling kibbler to be pulverized, and the pulverizing time is 18h.
(7) enzymolysis: after proportionally 1: 8 ratio adds with nanometer sea cucumber powder and water (weight ratio), stir, add the mass ratio 1g of alkalescence property proteolytic enzyme according to nanometer sea cucumber powder and Sumizyme MP: 0.1mg; PH7～8, hydrolysis temperature are 65 ℃, carry out enzymolysis 1h after; Add Sumizyme MP again; Add the mass ratio 1g of Sumizyme MP according to nanometer sea cucumber powder and Sumizyme MP: 0.1mg, pH 7～8,65 ℃ of temperature; Carry out enzymolysis 3h, after enzyme reaction finishes enzyme digestion reaction liquid is heated 10min in 100 ℃.Enzymolysis product is carried out spinning, get the enzymolysis supernatant.
(8) alcohol precipitation with separate: according to the volume of enzymolysis supernatant, will wherein add calcium chloride and reach 6% to whole mass concentration, and add ethanolic soln and carry out alcohol precipitation, the alcohol concn in the final enzymolysis solution is 60%, and low-temperature centrifugation obtains the total Crude polysaccharides of degraded sea cucumber.
(9) thick DHG, thick DHF separation of polysaccharides: the low molecule Crude polysaccharides of the sea cucumber that makes is added water dissolve; The ratio of amount of water is that 1 gram Crude polysaccharides adds 10ml water, and the volume according to Crude polysaccharides solution adds the Potassium ethanoate that whole mass concentration is 4M then, is positioned under 0～10 ℃ of low temperature; To being arranged, deposition separates out; It is centrifugal under low temperature to take out the back, and being precipitated as after centrifugal promptly obtains low molecule sea cucumber ammonia polyose of candy raw product, and supernatant is the aqueous solution raw product of low molecule sea cucumber fucoidin.
(10) decolouring: will add water by the low molecule sea cucumber ammonia polyose of candy raw product that (9) obtain and dissolve (ratio of amount of water is that 1 gram Crude polysaccharides adds 10g water); Itself and the aqueous solution raw product of low molecule sea cucumber fucoidin are decoloured to yellow or colourless with ydrogen peroxide 50 respectively, and pH value of solution is 10～11 during decolouring, 60 ℃ of temperature; The whole mass concentration of the ydrogen peroxide 50 that adds is 4%, after the decolouring pH value of solution is adjusted to 2, removes acidic protein; Centrifugal, get supernatant.
(11) DHF is refining: adding calcium chloride to whole mass concentration in the HF solution after will decolour is 6%, adds ethanol then and carries out alcohol precipitation, and adding the whole mass concentration of alcoholic acid is 70%, centrifugal after, pure hypostasis is dehydrated, promptly get sea cucumber and hang down the molecule fucoidin.
(12) DHG is refining: adding whole mass concentration in the HG solution after will decolouring is the Potassium ethanoate of 4M, is positioned under 0～10 ℃ of low temperature, to there being deposition to separate out; It is centrifugal under low temperature to take out the back; After centrifugal, centrifugal sediment is dehydrated, promptly obtain the low molecule ammonia polyose of candy of sea cucumber.(13) the low molecular weight sea cucumber ammonia polyose of candy after making with extra care is a white powder, is prone to the moisture absorption, is prone to be dissolved in the water, and the aqueous solution is water white.Viscosity-average molecular weight is 22000.
Low molecular weight sea cucumber Fucose after refining is a white powder, is prone to the moisture absorption, is prone to be dissolved in the water, and the aqueous solution is water white.Viscosity-average molecular weight is 42000.
Embodiment 7
(1) raw material is handled: get fresh and alive sea cucumbers, after gilling, the sea cucumber body wall is cleaned up, place encloses container.
(2) gel: said vesse in 120～130 ℃, is heated 10min.
(3) vacuum lyophilization: the sea cucumber after the gel is carried out lyophilize, and moisture is 10%.
(4) coarse reduction and (5) micronizing are all with embodiment 1.
(6) nano-pulverization: the sea cucumber super-fine powder that will pass through comminution by gas stream carries out nanometer with the high-energy ball milling kibbler to be pulverized, and the pulverizing time is 20h.
(7) enzymolysis: after proportionally 1: 10 ratio adds with nanometer sea cucumber powder and water (weight ratio), stir, add the mass ratio 1g of neutrality property proteolytic enzyme according to nanometer sea cucumber powder and neutrality property proteolytic enzyme: 0.8mg; PH6.7～7, hydrolysis temperature are 50 ℃, carry out enzymolysis 1h after; Add Sumizyme MP again, add the mass ratio 1g of Sumizyme MP according to nanometer sea cucumber powder and Sumizyme MP: 1mg, pH 7～8; 65 ℃ of temperature are carried out enzymolysis 1h, again temperature are adjusted into 45 ℃; Add trypsinase, add the mass ratio 1g of trypsinase according to nanometer sea cucumber powder and pancreatin: 10mg, pH 8～9; Carry out enzymolysis 1h, after enzyme reaction finishes enzyme digestion reaction liquid is heated 10min in 100 ℃.Enzymolysis product is carried out spinning, get the enzymolysis supernatant.
(8) alcohol precipitation with separate: according to the volume of enzymolysis supernatant, will wherein add sodium-chlor and reach 8% to whole mass concentration, and add ethanolic soln and carry out alcohol precipitation, the alcohol concn in the final enzymolysis solution is 70%, and low-temperature centrifugation obtains the total Crude polysaccharides of degraded sea cucumber.
(9) thick DHG, thick DHF separation of polysaccharides: the low molecule Crude polysaccharides of the sea cucumber that makes is added water dissolve; The ratio of amount of water is that 1 gram Crude polysaccharides adds 10ml water, and the volume according to Crude polysaccharides solution adds the Potassium ethanoate that whole mass concentration is 5M then, is positioned under 0～10 ℃ of low temperature; To being arranged, deposition separates out; It is centrifugal under low temperature to take out the back, and being precipitated as after centrifugal promptly obtains low molecule sea cucumber ammonia polyose of candy raw product, and supernatant is the aqueous solution raw product of low molecule sea cucumber fucoidin.
(10) decolouring: will add water by the low molecule sea cucumber ammonia polyose of candy raw product that (9) obtain and dissolve (ratio of amount of water is that 1 gram Crude polysaccharides adds 10g water); Itself and the aqueous solution raw product of low molecule sea cucumber fucoidin are decoloured to yellow or colourless with ydrogen peroxide 50 respectively, and pH value of solution is 9～10 during decolouring, 50 ℃ of temperature; The whole mass concentration of the ydrogen peroxide 50 that adds is 5%, after the decolouring pH value of solution is adjusted to 2, removes acidic protein; Centrifugal, get supernatant.
(11) DHF is refining: adding calcium acetate to whole mass concentration in the HF solution after will decolour is 8%, adds ethanol then and carries out alcohol precipitation, and adding the whole mass concentration of alcoholic acid is 80%, centrifugal after, pure hypostasis is dehydrated, promptly get sea cucumber and hang down the molecule fucoidin
(12) DHG is refining: adding whole mass concentration in the HG solution after will decolouring is the Potassium ethanoate of 5M, is positioned under 0～10 ℃ of low temperature, to there being deposition to separate out; It is centrifugal under low temperature to take out the back; After centrifugal, centrifugal sediment is dehydrated, promptly obtain the low molecule ammonia polyose of candy of sea cucumber.
(13) the low molecular weight sea cucumber ammonia polyose of candy after making with extra care is a white powder, is prone to the moisture absorption, is prone to be dissolved in the water, and the aqueous solution is water white.Viscosity-average molecular weight is 30000.
Low molecular weight sea cucumber Fucose after refining is a buff powder, is prone to the moisture absorption, is prone to be dissolved in the water, and the aqueous solution is light yellow.Viscosity-average molecular weight is 51000.
Embodiment 8:
(1) raw material is handled: get fresh and alive sea cucumbers, after gilling, the sea cucumber body wall is cleaned up, place encloses container.
(2) gel: said vesse in 105～110 ℃, is heated 40min.
(3) vacuum lyophilization: the sea cucumber after the gel is carried out lyophilize, and moisture is 9%.
(4) coarse reduction, (5) micronizing and (6) nano-pulverization are all with embodiment 7.
(7) enzymolysis: after proportionally 1: 9 ratio adds with nanometer sea cucumber powder and water (weight ratio), stir, add Sumizyme MP; Add the mass ratio 1g of Sumizyme MP according to nanometer sea cucumber powder and Sumizyme MP: 10mg, pH 7～8,65 ℃ of temperature; Carry out enzymolysis 1h, again temperature is adjusted into 45 ℃, add trypsinase; Add the mass ratio 1g of trypsinase according to nanometer sea cucumber powder and pancreatin: 100mg; PH 8～9, carry out enzymolysis 1h, after enzyme reaction finishes enzyme digestion reaction liquid heated 10min in 100 ℃.Enzymolysis product is carried out spinning, get the enzymolysis supernatant.
(8) alcohol precipitation with separate: according to the volume of enzymolysis supernatant; To wherein add calcium acetate to whole mass concentration and reach 2.5%; The salt that adds can be sodium-chlor, calcium acetate etc., adds ethanolic soln and carries out alcohol precipitation, and the ethanol mass concentration in the final enzymolysis solution is 60%; Low temperature (0～10 ℃) is centrifugal, obtains the total Crude polysaccharides of degraded sea cucumber.
(9) the total Crude polysaccharides of sea cucumber is refining: the low total Crude polysaccharides of molecule of the sea cucumber that makes is added water dissolve, the ratio of amount of water is that 1 gram Crude polysaccharides adds 20ml water, decolours to yellow or colourless with ydrogen peroxide 50; PH value of solution is 10 during decolouring, 50 ℃ of temperature, and the whole mass concentration of the ydrogen peroxide 50 of adding is 1%; After the decolouring pH value of solution is adjusted to 2; Remove acidic protein, centrifugal, get supernatant.
(10) sea cucumber total polysaccharides separation and purification: adding calcium acetate to whole mass concentration in the total Crude polysaccharides aqueous solution of the sea cucumber after will decolouring is 2.5%; Add ethanol then and carry out alcohol precipitation, adding the whole mass concentration of alcoholic acid is 70%, centrifugal after; Pure hypostasis is dehydrated, promptly get the sea cucumber total polysaccharides.
(11) the sea cucumber total polysaccharides is a white powder after making with extra care, and is prone to the moisture absorption, is prone to be dissolved in the water, and the aqueous solution is water white.Viscosity-average molecular weight is 46000.
Embodiment 9:
(1) raw material is handled: get fresh and alive sea cucumbers, after gilling, sea cucumber body wall and internal organ are cleaned up respectively, place encloses container.
(2) gel: said vesse in 100～105 ℃, is heated 120min.
(3) vacuum lyophilization: the sea cucumber after the gel is carried out lyophilize, and moisture is 3%.
(4) coarse reduction, (5) micronizing and (6) nano-pulverization are all with embodiment 7.
(7) enzymolysis: after proportionally 1: 6 ratio adds with nanometer sea cucumber powder and water (weight ratio), stir, add Sumizyme MP; Add the mass ratio 1g of Sumizyme MP according to nanometer sea cucumber powder and Sumizyme MP: 5mg, pH 7～8,65 ℃ of temperature; Carry out enzymolysis 1h, again temperature is adjusted into 45 ℃, add trypsinase; Add the mass ratio 1g of trypsinase according to nanometer sea cucumber powder and pancreatin: 1000mg; PH 8～9, carry out enzymolysis 1h, after enzyme reaction finishes enzyme digestion reaction liquid heated 10min in 100 ℃.Enzymolysis product is carried out spinning, get the enzymolysis supernatant.
(8) alcohol precipitation with separate: according to the volume of enzymolysis supernatant; To wherein add sodium chloride to whole mass concentration and reach 4%, and add ethanolic soln and carry out alcohol precipitation, the ethanol mass concentration in the final enzymolysis solution is 50%; Low temperature (0 ℃～10 ℃) is centrifugal, obtains the total Crude polysaccharides of degraded sea cucumber.
(9) the total Crude polysaccharides of sea cucumber is refining: the low total Crude polysaccharides of molecule of the sea cucumber that makes is added water dissolve, the ratio of amount of water is that 1 gram Crude polysaccharides adds 20ml water, decolours to yellow or colourless with ydrogen peroxide 50; PH value of solution is 11 during decolouring, 50 ℃ of temperature, and the whole mass concentration of the ydrogen peroxide 50 of adding is 3%; After the decolouring pH value of solution is adjusted to 2; Remove acidic protein, centrifugal, get supernatant.
(10) sea cucumber total polysaccharides separation and purification: adding sodium chloride to whole mass concentration in the total Crude polysaccharides aqueous solution of the sea cucumber after will decolouring is 2.5%; Add ethanol then and carry out alcohol precipitation, adding the whole mass concentration of alcoholic acid is 60%, centrifugal after; Pure hypostasis is dehydrated, promptly get the sea cucumber total polysaccharides.
(11) the sea cucumber total polysaccharides is a white powder after making with extra care, and is prone to the moisture absorption, is prone to be dissolved in the water, and the aqueous solution is water white.Viscosity-average molecular weight is 52000.
Claims (5)
1. the preparation method of a low molecular weight sea cucumber polysaccharide is characterized in that process step is:
(1) raw material is handled: sea cucumber is cut off clean, rubbed, place encloses container;
(2) heating gel: 70～130 ℃, heating 1min～20h;
(3) lyophilize: the sea cucumber after the gel is carried out lyophilize, and moisture is less than 10%;
(4) coarse reduction: the sea cucumber after the lyophilize carry out coarse reduction to 10～300 orders;
(5) micronizing: the Stichopus japonicus powder of coarse reduction carry out micronizing to 100～3000 orders with micronizer mill;
(6) nano-pulverization: the sea cucumber super-fine powder that will pass through comminution by gas stream carries out nanometer with the high-energy ball milling kibbler to be pulverized, the pulverizing time be 4～20h to median size be 100～200nm;
(7) enzymolysis and separation: nanometer Stichopus japonicus powder and water are dissolved in water with 1: 3～10 mass ratioes; In the nanometer Stichopus japonicus powder: proteolytic enzyme is that the ratio of 1g: 0.1～1010mg adds proteolytic enzyme and under corresponding pH value, carrying out enzymolysis in 40～70 ℃; Per step enzyme digestion reaction carries out 1～5h; The enzyme that goes out of then heating, 0～10 ℃ of high speed centrifugation is got supernatant;
Said enzymolysis carries out enzymolysis for using single enzyme, two enzyme or three enzymes: proteolytic enzyme is selected bromeline, Sumizyme MP, neutral protease or trypsinase for use when using single enzyme enzymolysis; Select Sumizyme MP and trypsinase for use when using two enzyme enzymolysis, during with two enzymes or three enzyme enzymolysis, adopt the substep enzyme solution to carry out enzymolysis; Its enzymolysis is Sumizyme MP-trypsinase, Sumizyme MP-Sumizyme MP in proper order during two enzyme enzymolysis; Proteolytic enzyme is Sumizyme MP, neutral protease and trypsinase when using three enzyme enzymolysis; Its enzymolysis is neutral protease-Sumizyme MP-trypsinase in proper order;
Wherein the required condition of protease hydrolyzed is respectively: bromeline, and pH6～7, hydrolysis temperature is 40 ℃; Sumizyme MP, pH7～8, hydrolysis temperature is 65 ℃; Neutral protease, pH6.7～7, hydrolysis temperature is 50 ℃; Trypsinase, pH8～9, hydrolysis temperature is 45 ℃;
(8) alcohol precipitation with separate: according to the quality of enzymolysis supernatant; To wherein add salt to whole mass concentration and reach 1～8%; The salt that adds is sodium-chlor or calcium acetate, adds ethanolic soln and carries out alcohol precipitation, and the ethanol mass concentration in the final enzymolysis solution is 20～70%; 0～10 ℃ centrifugal, obtains the total Crude polysaccharides of degraded sea cucumber;
(9) decolouring: the low molecule Crude polysaccharides of the sea cucumber that makes is added water dissolve, Crude polysaccharides with the quality ratio is: 1: 5～20, decolour to yellow or colourless with ydrogen peroxide 50; PH value of solution is 8～12 during decolouring; 40～60 ℃ of temperature, the whole mass concentration of the ydrogen peroxide 50 of adding is 0.1～5.0%, after the decolouring pH value of solution is adjusted to 2; The centrifuging and taking supernatant is to remove acidic protein;
(10) purifying: adding salt to whole mass concentration in the total Crude polysaccharides aqueous solution of the sea cucumber after will decolouring is 1～8%; The salt that adds is selected from sodium-chlor or calcium acetate; Add ethanol then and carry out alcohol precipitation, adding the whole mass concentration of alcoholic acid is 40～80%, centrifugal after; Pure hypostasis is dehydrated, and sub-sea cucumber total polysaccharides promptly makes low score;
(11) low molecule sea cucumber fucoidin hangs down separating of molecule ammonia polyose of candy and refining with sea cucumber:
(i) separate: the low molecule sea cucumber total polysaccharides that will make adds water by 1: 5～20 mass ratio and dissolves; Quality according to low molecule sea cucumber total polysaccharides solution adds Potassium ethanoate or the Repone K dissolving that whole mass concentration is 1～5M then; 0～10 ℃ of low temperature settle is separated out; Centrifugal, be precipitated as low molecule sea cucumber ammonia polyose of candy raw product; Supernatant is the aqueous solution raw product of low molecule sea cucumber fucoidin;
(ii) decolouring: the aqueous solution raw product that will be dissolved in water into solution and low molecule sea cucumber fucoidin from the low molecule sea cucumber ammonia polyose of candy raw product that step (i) separation obtains all decolours respectively with ydrogen peroxide 50; PH value of solution is 8～12 during decolouring; 40～60 ℃ of temperature; The whole mass concentration of the ydrogen peroxide 50 that adds is 0.1～5.0%, after the decolouring pH value of solution is adjusted to 2, gets supernatant;
Said low molecule sea cucumber ammonia polyose of candy raw product is dissolved in water into solution, and the mass ratio of raw sugar and water is 1: 5～20;
(iii) the low molecule fucoidin of sea cucumber is refining: adding salt to whole mass concentration in the aqueous solution solution of crude product of the low molecule sea cucumber fucoidin after will decolouring is 1～8%; The salt that adds is sodium-chlor or calcium acetate; Add ethanol then and carry out alcohol precipitation; Adding the whole mass concentration of alcoholic acid is 40～80%, centrifugal after, hypostasis through dehydrate the low molecule fucoidin of sea cucumber;
(iv) the low molecule ammonia polyose of candy of sea cucumber is refining: adding whole mass concentration in the low molecule ammonia polyose of candy solution after will decolouring is the Potassium ethanoate of 1～5M; Separate out in 0～10 ℃ of low temperature settle; Centrifugal under the low temperature, throw out obtains the low molecule ammonia polyose of candy of sea cucumber through dehydrating.
2. according to the preparation method of the said a kind of low molecular weight sea cucumber polysaccharide of claim 1, it is characterized in that in process step (2) the heating gel it being that sea cucumber is heated 1h down in 100～105 ℃ in encloses container.
3. according to the preparation method of the said a kind of low molecular weight sea cucumber polysaccharide of claim 1, it is characterized in that in process step (3) lyophilize it being that the sea cucumber after the gel is carried out lyophilize to moisture less than 3%.
4. according to the preparation method of the said a kind of low molecular weight sea cucumber polysaccharide of claim 1, it is characterized in that in process step (6) nano-pulverization it being the sea cucumber super-fine powder through comminution by gas stream to be carried out nanometer with the high-energy ball milling kibbler pulverize 10～12h.
5. the preparation method of a low molecular weight sea cucumber polysaccharide is characterized in that process step is:
(1) raw material is handled: sea cucumber is cut off clean, rubbed, place encloses container;
(2) heating gel: 70～130 ℃, heating 1min～20h;
(3) lyophilize: the sea cucumber after the gel is carried out lyophilize, and moisture is less than 10%;
(4) coarse reduction: the sea cucumber after the lyophilize carry out coarse reduction to 10～300 orders;
(5) micronizing: the Stichopus japonicus powder of coarse reduction carry out micronizing to 100～3000 orders with micronizer mill;
(6) nano-pulverization: the sea cucumber super-fine powder that will pass through comminution by gas stream carries out nanometer with the high-energy ball milling kibbler to be pulverized, the pulverizing time be 4～20h to median size be 100～200nm;
(7) enzymolysis and separation: the mass ratio of nanometer Stichopus japonicus powder and water is to add the water mixing at 1: 7, and the ratio of nanometer sea cucumber powder and enzyme is that the ratio of 1g: 0.1～10mg adds Sumizyme MP Acalase, and pH carries out enzymolysis 0.1～5h under 7～8,40～70 ℃; With pH regulator to 8～10; The mass ratio of nanometer sea cucumber powder and enzyme is 1 gram: 10～1000mg adds trypsinase; At 40～70 ℃ of enzymolysis 0.1～5h; After enzyme reaction finishes with enzyme digestion reaction liquid in 90～100 ℃ of heating 1～20min enzyme that goes out, enzymolysis product is carried out spinning, get the enzymolysis supernatant;
(8) alcohol precipitation with separate: according to the quality of enzymolysis supernatant; To wherein add salt to whole mass concentration and reach 1～8%; The salt that adds is sodium-chlor or calcium acetate, adds ethanolic soln and carries out alcohol precipitation, and the ethanol mass concentration in the final enzymolysis solution is 20～70%; 0～10 ℃ centrifugal, obtains the total Crude polysaccharides of degraded sea cucumber;
(9) decolouring: the low molecule Crude polysaccharides of the sea cucumber that makes is added water dissolve, Crude polysaccharides with the quality ratio is: 1: 5～20, decolour to yellow or colourless with ydrogen peroxide 50; PH value of solution is 8～12 during decolouring; 40～60 ℃ of temperature, the whole mass concentration of the ydrogen peroxide 50 of adding is 0.1～5.0%, after the decolouring pH value of solution is adjusted to 2; The centrifuging and taking supernatant is to remove acidic protein;
(10) purifying: adding salt to whole mass concentration in the total Crude polysaccharides aqueous solution of the sea cucumber after will decolouring is 1～8%; The salt that adds is sodium-chlor, calcium acetate; Add ethanol then and carry out alcohol precipitation, adding the whole mass concentration of alcoholic acid is 40～80%, centrifugal after; Pure hypostasis is dehydrated, and sub-sea cucumber total polysaccharides promptly makes low score;
(11) low molecule sea cucumber fucoidin hangs down separating of molecule ammonia polyose of candy and refining with sea cucumber:
(i) separate: the low molecule sea cucumber total polysaccharides that will make adds water by 1: 5～20 mass ratio and dissolves; Quality according to low molecule sea cucumber total polysaccharides solution adds Potassium ethanoate or the Repone K dissolving that whole mass concentration is 1～5M then; 0～10 ℃ of low temperature settle is separated out; Centrifugal, be precipitated as low molecule sea cucumber ammonia polyose of candy raw product; Supernatant is the aqueous solution raw product of low molecule sea cucumber fucoidin;
(ii) decolouring: the aqueous solution raw product that will be dissolved in water into solution and low molecule sea cucumber fucoidin from the low molecule sea cucumber ammonia polyose of candy raw product that step (i) separation obtains all decolours respectively with ydrogen peroxide 50; PH value of solution is 8～12 during decolouring; 40～60 ℃ of temperature; The whole mass concentration of the ydrogen peroxide 50 that adds is 0.1～5.0%, after the decolouring pH value of solution is adjusted to 2, gets supernatant;
Said low molecule sea cucumber ammonia polyose of candy raw product is dissolved in water into solution, and the mass ratio of raw sugar and water is 1: 5～20;
(iii) the low molecule fucoidin of sea cucumber is refining: adding salt to whole mass concentration in the aqueous solution solution of crude product of the low molecule sea cucumber fucoidin after will decolouring is 1～8%; The salt that adds is sodium-chlor or calcium acetate; Add ethanol then and carry out alcohol precipitation; Adding the whole mass concentration of alcoholic acid is 40～80%, centrifugal after, hypostasis through dehydrate the low molecule fucoidin of sea cucumber;
(iv) the low molecule ammonia polyose of candy of sea cucumber is refining: adding whole mass concentration in the low molecule ammonia polyose of candy solution after will decolouring is the Potassium ethanoate of 1～5M; Separate out in 0～10 ℃ of low temperature settle; Centrifugal under the low temperature, throw out obtains the low molecule ammonia polyose of candy of sea cucumber through dehydrating.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN102002110B (en) * | 2010-11-24 | 2012-09-05 | 刘全胜 | Preparation method of polysaccharides from sea cucumber |
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| CN114560958B (en) * | 2022-04-21 | 2023-05-12 | 温州大学 | Sea cucumber polysaccharide extraction method and application of sea cucumber polysaccharide |
| CN116531549A (en) * | 2023-03-02 | 2023-08-04 | 大连深蓝肽科技研发有限公司 | Low molecular weight sea cucumber polysaccharide lung targeting microsphere, preparation method and anticoagulation application thereof |
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