CN1207990C - Preparation method of sea cucumber mucopolysaccharide - Google Patents

Abstract

The present invention relates to food which is rich in sea cucumber mucopolysaccharide and a preparation method thereof. The present invention uses sea cucumbers as main raw materials, adopts alkaline hydrolysis, single exogenous enzyme enzymolysis, double exogenous enzyme enzymolysis or the combined enzymolysis of autoenzyme and exogenous enzyme, and then, separated mother liquid is dissolved by alcohol and purified so as to make mucopolysaccharide pure products. The mucopolysaccharide is added to wine to obtain sea cucumber wine; the mucopolysaccharide and the extracted solution of traditional Chinese medicine are mixed, and then, concentrating, granulating and baking are carried out so as to obtain granules; the mucopolysaccharide and the extracted solution of the traditional Chinese medicine are mixed, and then, clarifying, flavoring, filtering and sterilizing are carried out so as to obtain an oral liquid; the mucopolysaccharide and the extracted solution of the traditional Chinese medicine are mixed, and then, starch pressing slices are added so as to obtain tablets; the mucopolysaccharide and beta-Cyclodextrin are mixed and boiled to be dried in vacuum and packaged so as to obtain capsules. Small amount of mucopolysaccharide in the sea cucumbers is extracted, and the mucopolysaccharide is prepared into functional food with certain contents, so that the present invention can fully perform the functions of resisting cancer and improving immunity.

Description

Preparation method of sea cucumber mucopolysaccharide

Technical field The present invention relates to a kind of food, especially seafood food.

Background technology It is well known that sea cucumber mucopolysaccharide has the functions of enhancing immunity and anti-tumor. But most of the sea cucumber products sold in the market are whole ginseng products, though it contains sea cucumber mucopolysaccharide, its content is very little, it is difficult to bring into play its due effect.

SUMMARY OF THE INVENTION The present invention uses sea cucumbers as raw materials, adopts modern biotechnology and food high-tech to extract sea cucumber mucopolysaccharides, and makes them into a variety of functional health food.

1. Extraction of sea cucumber mucopolysaccharide

1. Raw materials and processing: Take dried sea cucumbers for foaming; or take salted sea cucumbers, cut them longitudinally along the notched side with a knife, remove the mouth and debris in the cavity, and wash them in clean water; or take fresh sea cucumbers , go directly to the viscera, tendons, and wash. Then add 0 to 5 times the volume of water to the above-mentioned sea, and use a tissue grinder to crush its wall.

2. Extraction method

①A combination of alkaline hydrolysis and single-enzyme hydrolysis

Add 0.2-0.8% solid K ₂ CO ₃ under the condition of 40-70°C, and hydrolyze for 1-5 hours under stirring. Then use 6 mol/L hydrochloric acid to adjust the pH to 7-9 (and keep the pH in this range during the enzymolysis process), add 0.3-0.5% trypsin (enzyme activity 5×10 ⁴ u/g) to maintain The temperature is 45-55°C, and the enzymolysis is carried out under stirring for 2-7 hours. Afterwards, it was rapidly cooled to below 30° C., and kept for 30 minutes to terminate the reaction. Adjust the pH to neutral with 6 mol/liter of hydrochloric acid, and separate at high speed (6000 rpm) for 15 minutes. The precipitate obtained is an insoluble hydrolyzate, which contains about 12% protein, which can be used as base material, and further Processed into powder, granule, tablet or processed into a series of products such as oral liquid. Slowly add 95% ethanol (30-80% by mass) to the mother liquor obtained by centrifugation, keep stirring at this time, and keep overnight at a temperature below 10°C. After centrifugation, the resulting supernatant can be distilled under reduced pressure. Not only can ethanol be recovered, but sea cucumber hydrolyzate can also be obtained, which can be used as a base material for further processing into various functional foods. % ethanol washing, acetone dehydration, drying at 40-70 DEG C or vacuum drying, the obtained product is the crude sea cucumber mucopolysaccharide.

②A combination of alkaline hydrolysis and double-enzyme hydrolysis

At 40-70°C, add 0.2-0.8% solid K ₂ CO ₃ , hydrolyze with stirring for 1-5 hours, then adjust the pH to 1-5 with 6 mol/L hydrochloric acid, add 0.3-0.5% pepsin (enzyme activity 7.8×10 ⁵ u/g), stirred and enzymolyzed in a water bath at 40-60°C for 3-10 hours while keeping the pH within the above-mentioned range. Then use 0.5 mol/L potassium hydroxide to adjust the pH to 7-9, add 0.3-0.5% trypsin (enzyme activity 5×10 ⁴ u/g) and stir in a water bath at 45-55°C for 2-7 hours while maintaining the pH within the above range. After enzymatic hydrolysis, quickly cool to below 30°C and keep for 30 minutes to terminate the reaction. Use 6 mol/L hydrochloric acid to adjust the pH value to neutral, and separate by high-speed centrifugation (6000 rpm) for 15 minutes. The precipitate is an insoluble hydrolyzate, and the mother liquor is collected. The remaining operations are the same as single-enzyme hydrolysis.

③Enzymatic hydrolysis with exogenous enzymes after autolysis

I Autolysis:

a. Put the sea cucumber slurry into a sterile enzymolysis tank, autolyze at pH 6-7.5, at 0-4°C for 24-48 hours;

or b. put the sea cucumber slurry into a sterile enzymolysis tank, and autolyze at pH 6-7.5 at room temperature for 6-8 hours;

Or c, after irradiating the sea cucumber pulp with ultraviolet light for 10-30 minutes, use a gradient of sodium chloride with a concentration of 0.06-0.08 mol/liter at pH 7.0-7.5 and a temperature of 40-65°C (5°C per minute) temperature is processed.

II Enzymatic hydrolysis with exogenous enzymes: adjust the pH of the self-solution obtained by one of the above three autolysis methods to 1-5 with 6 mol/L hydrochloric acid, add 0.3-0.5% pepsin, and enzymolyze 1 at 40-60°C ~2 hours. After enzymatic hydrolysis, quickly cool to below 30°C and keep for 30 minutes to terminate the reaction. Use 0.5 mol/L potassium hydroxide to adjust the pH value to neutral, high-speed centrifugation, the precipitate is an insoluble hydrolyzate, and the mother liquor is collected. The remaining operations are the same as single-enzyme hydrolysis.

④ Autolyses as exogenous enzymes combined with exogenous enzymes for enzymatic hydrolysis

I autolyzin extraction method: mash the fresh sea cucumber intestinal tissue, dissolve it in 2-15 times of 0.1M phosphate buffer (pH6-7.5), let stand and extract for 2-13 hours. Then centrifuge to remove the precipitate, slowly add solid ammonium sulfate to the supernatant to 10-30% saturation to dissolve it, salt out for 2-24 hours, refrigerate and centrifuge to remove the precipitate. Add solid ammonium sulfate to the supernatant to 80-95% saturation, salt out for 6-48 hours, then dialyze for 6-48 hours, centrifuge, take the precipitate, and dissolve the precipitate in 0.1M phosphate buffer (pH6-7.5 ), dialyze again for 6-48 hours, refrigerate and centrifuge to obtain a precipitate. The above process is all carried out at 10°C, and the precipitate is freeze-dried to obtain an autolyzed enzyme powder. The enzyme powder can also be purified with a G-25 gel column.

II Autolyzed Enzyme Hydrolysis: Add 0.3-3% of the above-mentioned autolyzed enzyme powder to sea cucumber pulp, enzymolyze at pH 6-8, temperature 15-30°C for 3-8 hours,

III Enzymatic hydrolysis with exogenous enzymes: adjust the pH to 1-5 with 6 mol/L hydrochloric acid, add 0.3-0.5% pepsin, enzymatically hydrolyze at 40-60°C for 1-2 hours, quickly cool to below 30°C after enzymatic hydrolysis, and keep for 30 minutes to terminate the reaction. Use 0.5 mol/L potassium hydroxide to adjust the pH value to neutral, high-speed centrifugation, the precipitate is insoluble hydrolyzate, collect the mother liquor, and the rest of the operation is the same as single-enzyme hydrolysis.

3. Purification: Dissolve the crude polysaccharide in 1:10-50 (g/ml) water, centrifuge to remove insoluble matter, add solid potassium acetate to the supernatant to make the concentration in the solution 0.5-4 mol/liter , Refrigerate overnight below 10°C. The precipitate was collected below 10°C. Dissolve it with half the volume of water before, adjust the pH to 9-11 with 0.5 mol/L potassium hydroxide, and add H ₂ O ₂ dropwise at 45-55°C to decolorize until the polysaccharide hydrolyzate turns light yellow as the end point. After cooling, remove the insoluble matter by centrifugation, cool the clear liquid to below 4°C, adjust the pH to 1.5-2.5 with 6 mol/L hydrochloric acid, remove the precipitate by centrifugation, and add solid potassium acetate to the clear liquid to make the concentration in the solution reach 0.5-4 mol/L, refrigerated overnight, collected the precipitate below 10°C, washed with ethanol, dehydrated with acetone, and dried to obtain pure mucopolysaccharide.

2. Using sea cucumber mucopolysaccharide as base material to prepare functional health food.

1. Preparation of sea cucumber wine: dissolve sea cucumber mucopolysaccharide in 4-60% wine to make the concentration 0.01-1000 mg/L.

2. Preparation of granules:

① Mixing: Mix the sea cucumber mucopolysaccharide base material with additives, the base material accounts for 0.05-20% of the mixture. These additives can be Chinese medicine extracts with health care effects, or dairy products, eggs, and vegetable proteins that increase nutritional value etc., can also be white granulated sugar, honey and citric acid etc. which improve the taste.

② Centrifugation and filtration: remove most of the residue and new insoluble matter produced by mixing. Before centrifugation, it should be processed by heat exchanger and cold exchanger, so that the precipitate produced by mixing can be formed as soon as possible. Before concentrating, a plate and frame filter is also used to remove suspended particles with a lower density.

③ Concentration: First use vacuum degree 86.25 ~ 98.64Kpa vacuum concentration, the temperature is lower than 50 ℃, vacuum concentration can speed up the concentration and maintain the color of the product. When the vacuum concentrate is both viscous and not too sticky to the container wall, use a jacketed pot to concentrate until its density is ≥ 1.0 g/ ^cm3 .

④Mixed granulation: The sucrose powder and soluble starch used need to be ground and sieved. Concentrate: soluble starch: sucrose powder = 1:1:2. Use edible alcohol to adjust the dry humidity, mix thoroughly in the mixer and granulate with a granulator after the dry humidity is moderate.

⑤Drying and packaging: After the selection of grains, dry at 60-80°C until the water content is lower than 4%, and the packaging materials should be moisture-proof materials.

3. Preparation of capsules

①Granulation: Microcapsule technology is used for granulation treatment, that is, mucopolysaccharide is added to the extract, and then 0.5-3% β-cyclodextrin is added, and boiled for 2-10 minutes, so that the residues in the hydrolyzate The fishy and astringent smell is wrapped by β-cyclodextrin, so that the unpleasant fishy and astringent smell is covered up, and the active ingredients in the hydrolyzate can be isolated from the external environment to prevent adverse quality changes, thereby effectively maintaining nutrients and other ingredients. Physiologically active substances.

②Drying: Vacuum freeze-drying can be used, that is, put the above-mentioned microencapsulated hydrolyzate into a freeze-dryer, control the temperature below -40°C, and vacuum degree of 133×10 ^-3 pa until the moisture content is lower than 4%. It can be concentrated by spray drying, first use vacuum concentration equipment to concentrate, control the vacuum degree at 86.25-98.64Kpa, and the temperature is 45-65°C. When concentrating, open the steam valve to slowly increase the temperature, and gradually increase the vacuum degree. When the solid content of the concentrated material reaches 4～ At 17%, spray drying was carried out. The temperature of the feed liquid for powder spraying is 45-50°C, the pressure of the feed pump is controlled at 19.6-49Kpa, the exhaust air temperature is 60-68°C, and the inlet air temperature is 150-180°C.

③ Cooling and packaging: Sieve off the lumps of the dried product, cool to room temperature in an environment with a relative humidity of 50-60%, and pack the cooled product into capsules.

Explanation: In the present invention, the soluble or insoluble protein hydrolyzate obtained by enzymatic hydrolysis can be prepared in the same way as the above method to prepare capsules.

4. Preparation of oral liquid

① Mixing: the sea cucumber mucopolysaccharide base material is mixed with the additive, the base material accounts for 0.05-20% of the mixture, and the description of the additive is the same as the preparation of the granule.

② Clarification: Add 0.1-0.5% pectin and 0-7% honey as flocculants to accelerate the precipitation of suspended particles and miscellaneous proteins in the solution. Honey can also be used as a flavoring agent. After adding, stir evenly, cool naturally, and let it stand to make the precipitation complete.

③ Filtration: Adopt centrifugation or pressure filtration to obtain clear liquid as mother liquor.

④ Seasoning: Dilute the mother liquor by 0-60 times, add 1-7% white granulated sugar (or 0.05-0.2% protein sugar, 6-12% sucrose), 0.02-0.4% citric acid, 0.01-0.05 % table salt, 0.1-0.2% vitamin C, 0.5-9% honey.

⑤ Clarification: Filtrate with diatomaceous earth under pressure, and then filter through two-stage microporous membranes with a pore size of 5 μm and 0.05 μm to completely remove fine suspended particles and bacteria in the nutrient solution.

⑥ Sterilization and packaging: Pasteurization (80°C, 30 minutes) and then filling.

Note: If the oral liquid is prepared from enzymatic soluble or insoluble protein hydrolyzate, although the same method as above can be used, it must be deodorized, that is, adding 0-0.2% citric acid and 0-0.2% For the mixed acid of malic acid, treat at 80-100°C for 10-30 minutes; or add 0.2-0.4% glucose and heat on a water bath at 90-100°C for 10-30 minutes.

5. Preparation of tablets

The sea cucumber mucopolysaccharide is mixed with other polysaccharides or Chinese herbal medicines (the preparation method is the same as that of granules), and 5-10% starch or starch filler is added before tableting, and the tablet is pressed.

The present invention has the following advantages:

1. Concentrate a small amount of sea cucumber mucopolysaccharides to make a functional food with a certain content, and then make it fully play the role of anti-cancer and enhancing immunity.

2. The method of combining sea cucumber autolysis with exogenous enzymes is adopted in the present invention, which not only reduces the cost, but also reduces the pollution of products introduced by exogenous enzymes.

Detailed ways

The preparation of example 1 sea cucumber wine

Take 10 kg of dried sea cucumbers for hair soaking, cut them longitudinally along the notched side with a knife, remove the ginseng mouth and debris in the cavity, and wash them in clean water. Then the body wall was crushed with a tissue masher, and 50 liters of water was added. At 40° C., 80 grams of solid potassium carbonate was added and hydrolyzed for 1 hour under stirring. Then adjust the pH to 7 with 6 mol/L hydrochloric acid, and keep the pH in this range during the enzymatic hydrolysis. Add 30 grams of trypsin (enzyme activity 5×10 ⁴ u/g) to keep the temperature at 45-47° C., and perform enzymolysis for 7 hours under agitation. Afterwards, rapidly cool to below 30°C and keep for 30 minutes to terminate the reaction. Adjust the pH to neutral with 6 mol/L hydrochloric acid. High-speed (6000 rpm) centrifugation to collect the mother liquor. Under continuous stirring, slowly add 8 kg of 95% ethanol, and keep overnight at low temperature (≤10°C). Afterwards, the precipitate was collected by centrifugation, washed with 90% ethanol in sequence, dehydrated with acetone, and then dried at a temperature of 40-70° C. to obtain sea cucumber crude polysaccharide. Dissolve the crude polysaccharide in 2 liters of water, centrifuge to remove insoluble matter, add 490 grams of potassium acetate to the supernatant, and refrigerate (below 10° C.) overnight. The precipitate was collected below 10°C and dissolved with 1 liter of water. Use 0.5 mol/L potassium hydroxide to adjust the pH to 9, keep 50°C and add hydrogen peroxide dropwise to decolorize until the polysaccharide hydrolyzate turns light yellow as the end point. After cooling, remove the insoluble matter by centrifugation, cool the clear liquid to about 0°C, adjust the pH to 1.5 with 6 mol/L hydrochloric acid, remove the precipitate by centrifugation, add 245 g/L potassium acetate to the clear liquid, and refrigerate overnight below 10°C. The precipitate was collected below 10°C, washed with ethanol, dehydrated with acetone, and dried to obtain pure sea cucumber mucopolysaccharide. Take 1 gram of the extracted pure mucopolysaccharide and dissolve it in 1 liter of 60-degree wine, and the product obtained is sea cucumber wine containing mucopolysaccharide.

The preparation of example 2 sea cucumber wine

Take 10 kg of dried sea cucumbers for hair soaking, cut them longitudinally along the notched side with a knife, remove the ginseng mouth and debris in the cavity, and wash them in clean water. Then the body wall was crushed with a tissue grinder, and 50 liters of water was added, and 20 grams of solid potassium carbonate was added at 40° C., and hydrolyzed for 5 hours under stirring. Then adjust the pH to 9 with 6 mol/L hydrochloric acid, and keep the pH in this range during the enzymatic hydrolysis. Add 50 grams of trypsin (enzyme activity 5×10 ⁴ u/g), keep the temperature at 53-55° C., and perform enzymatic hydrolysis for 2 hours while stirring. Afterwards, rapidly cool to below 30°C and keep for 30 minutes to terminate the reaction. Adjust the pH to neutral with 6 mol/L hydrochloric acid. High-speed (6000 rpm) centrifugation to collect the mother liquor. Under continuous stirring, slowly add 3 kg of 95% ethanol, and keep overnight at low temperature (≤10°C). Afterwards, the precipitate is collected by centrifugation, washed with 100% ethanol in sequence, dehydrated with acetone, and then dried under vacuum at 86.25-98.64Kpa to obtain sea cucumber crude polysaccharide. Dissolve the crude polysaccharide in 10 liters of water, centrifuge to remove insoluble matter, add 784 grams of potassium acetate to the supernatant, and refrigerate (below 10° C.) overnight. The precipitate was collected below 10°C and dissolved with 5 liters of water. Use 0.5 mol/L potassium hydroxide to adjust the pH to 11, keep 50°C and add hydrogen peroxide dropwise to decolorize until the polysaccharide hydrolyzate turns light yellow as the end point. After cooling, remove the insoluble matter by centrifugation, cool the clear liquid to about 0°C, adjust the pH to 2.5 with 6 mol/L hydrochloric acid, remove the precipitate by centrifugation, add 392 g/L potassium acetate to the clear liquid, and refrigerate overnight below 10°C. The precipitate was collected below 10°C, washed with ethanol, dehydrated with acetone, and dried to obtain pure sea cucumber mucopolysaccharide. 0.01 mg of the extracted mucopolysaccharide pure product is dissolved in 1 liter of 4-degree wine, and the obtained product is mucopolysaccharide-containing sea cucumber wine.

Preparation of example 3 sea cucumber mucopolysaccharide granule

Take 100 kilograms of salted sea cucumbers, cut them longitudinally along the notched side with a knife, remove the mouth and debris in the cavity, and wash them in clean water. Then its body wall was crushed with a tissue masher, and 500 liters of water was added, and 200 grams of solid potassium carbonate was added at 40° C., and hydrolyzed for 5 hours under stirring. Then adjust the pH to 1 with 6 mol/L hydrochloric acid, add 500 grams of pepsin (enzyme activity 7.8×10 ⁵ u/g) and stir in a water bath at 40°C for 10 hours for enzymolysis while keeping the pH within the above range. Then use 0.5 mol/L potassium hydroxide to adjust the pH to 7, add 500 grams of trypsin (enzyme activity 5×10 ⁵ u/g) and stir in a 45°C water bath for 2 hours, and keep the pH in the above range for further enzymolysis , after enzymatic hydrolysis, quickly cool to below 30°C and keep for 30 minutes to terminate the reaction. Adjust the pH to neutral with 6 mol/L hydrochloric acid, centrifuge at high speed (6000 rpm) for 15 minutes, and collect the mother liquor. Under constant stirring, slowly add 30 kilograms of ethanol, remaining operation is identical with example 1. Dissolve the crude polysaccharide in 60 liters of water, centrifuge to remove the insoluble matter, add 5880 grams of potassium acetate to the supernatant, and overnight below 10°C. The precipitate was collected below 10°C and dissolved with 30 liters of water. Use 6 mol/L potassium hydroxide to adjust the pH to 9, keep 50°C and add hydrogen peroxide dropwise to decolorize until the polysaccharide hydrolyzate turns light yellow as the end point, after cooling, centrifuge to remove insoluble matter, cool the clear liquid to about 0°C, and use 6 mol/L Adjust the pH to 1.5 with hydrochloric acid, centrifuge to remove the precipitate, add 2940 g of potassium acetate to the supernatant, and keep overnight below 10°C. The precipitate was collected below 10°C, washed with ethanol, dehydrated with acetone, and dried to obtain a pure polysaccharide.

Take another 4 kg of medlar, 4 kg of rhodiola and 40 kg of water to boil, keep the fire low for 1 hour, filter the filtrate, concentrate to 36 liters, add 95 liters of ethanol to the concentrated solution, let it stand for 10 hours, filter, and use Distill the ethanol to obtain the extract, take 12.5 grams of sea cucumber mucopolysaccharide obtained by the above method, mix with 25 kg of the extract obtained by the above method, centrifuge after being treated by the heat exchanger and the cold exchanger, and then use the plate frame The filter removes the suspended particles with low density, and then concentrates in vacuum with a vacuum degree of 86.25-96.64Kpa, and the temperature is lower than 50°C. When the vacuum concentrated liquid is both viscous and not too sticky to the container wall, use a jacketed pot to concentrate until its density is 1.0 g/ ^cm3 . Then mix the concentrate, soluble starch and sucrose powder in a ratio of 1:1:2, adjust the dry humidity with edible alcohol, mix thoroughly in the mixer and the dry humidity is moderate, and then granulate with a granulator. After the granulation is completed, dry it at 60-80°C until the water content is 3.8%, and finally pack it with aluminum foil.

Preparation of example 4 sea cucumber mucopolysaccharide granule

Take 100 kilograms of salted sea cucumbers, cut them longitudinally along the notched side with a knife, remove the mouth and debris in the cavity, and wash them in clean water. Then use a tissue grinder to break up its body wall, add 400 liters of water, and add 800 grams of solid potassium carbonate at 70° C., and hydrolyze for 1 hour under stirring. Then adjust the pH to 5 with 6 mol/L hydrochloric acid, add 300 g of pepsin (enzyme activity 7.8×10 ⁵ u/g) and stir in a water bath at 60°C for 3 hours for enzymolysis while keeping the pH within the above range. Then use 0.5 mol/L potassium hydroxide to adjust the pH to 9, add 300 grams of trypsin (enzyme activity 5×10 ⁵ u/g) and stir in a 55°C water bath for 7 hours, and keep the pH in the above range for further enzymolysis , after enzymatic hydrolysis, quickly cool to below 30°C and keep for 30 minutes to terminate the reaction. Adjust the pH to neutral with 6 mol/L hydrochloric acid, centrifuge at high speed (6000 rpm) for 15 minutes, and collect the mother liquor. Under constant stirring, slowly add 80 kilograms of ethanol, remaining operation is identical with example 2. The crude polysaccharide was dissolved in 60 liters of water, and the insoluble matter was removed by centrifugation. Add 5880 grams of potassium acetate to the supernatant, and keep it under 10°C overnight. Collect the precipitate below 10°C, then dissolve it with 30 liters of water, adjust the pH to 11 with 6 mol/L potassium hydroxide, keep 50°C and add hydrogen peroxide dropwise to decolorize until the polysaccharide hydrolyzate turns light yellow as the end point, after cooling, centrifuge to remove insoluble matter , the supernatant was cooled to about 0°C, and the pH value was adjusted to 2.5 with 6 mol/L hydrochloric acid, and the precipitate was removed by centrifugation, and 2940 g of potassium acetate was added to the supernatant, and the temperature was kept below 10°C overnight. The precipitate was collected below 10°C, washed with ethanol, dehydrated with acetone, and dried to obtain a pure polysaccharide.

Take another 4 kg of jujube, 4 kg of longan and 40 kg of water to boil, keep the fire low for 1 hour, filter the filtrate, concentrate to 20 liters, add 95 liters of ethanol to the concentrated solution, let it stand for 10 hours, filter, and distill the filtrate to 20 liters. Ethanol is distilled to obtain the extract, 3 kg of sea cucumber mucopolysaccharide obtained by the above method is mixed with 15 kg of the extract obtained by the above method, centrifuged after being treated by a heat exchanger and a cold exchanger, and then removed by a plate and frame filter Suspended particles with low density are then vacuum-concentrated with a vacuum degree of 86.25-96.64Kpa, and the temperature is lower than 50°C. When the vacuum concentrated liquid is both viscous and not too sticky to the container wall, use a jacketed pot to concentrate until its density is 1.0 g/ ^cm3 . Then mix the concentrate, soluble starch and sucrose powder in a ratio of 1:1:2, adjust the dry humidity with edible alcohol, mix thoroughly in the mixer and the dry humidity is moderate, and then granulate with a granulator. After the granulation is completed, dry it at 60-80°C until the water content is 4%, and finally pack it with aluminum foil.

Preparation of example 5 sea cucumber mucopolysaccharide oral liquid

Take 40 kg of fresh sea cucumbers, remove the viscera, tendons, and wash. Then use a tissue masher to crush its body wall, add 200 liters of water, and place it at a pH of 6 at room temperature for 6 hours to autolyze the sea cucumber, then adjust the pH to 1 with 6 mol/L hydrochloric acid, and add 120 grams of pepsin , enzymolysis at 40-45°C for 2 hours, after enzymolysis, quickly cool to below 30°C, keep for 30 minutes, stop the reaction, use 0.5 mol/L potassium hydroxide, adjust the pH value to neutral, and the remaining operations are the same as in Example 1 , to obtain pure mucopolysaccharides.

Take another 1.6 kg of rhodiola rosea, 1.6 kg of jujube and 16 kg of water to boil, keep the fire low for 1 hour, filter the filtrate, concentrate it to 8 liters, add 95 liters of ethanol to the concentrated solution and let it stand, distill the ethanol Get the extract, get 1.2 kilograms of sea cucumber mucopolysaccharides obtained by the above method, mix with 6 kilograms of extract obtained by the above method. Add 30 grams of pectin, stir evenly, cool naturally, and let stand for 2 hours to make the precipitation complete. Use centrifugation (4000 rpm, 15 minutes) to take the supernatant, dilute the solution 60 times, add 4.32 kg of sugar, 86.4 g of citric acid, 43.2 g of salt, 432 g of vitamin C, and 2.16 g of honey, mix well, and diatom The soil is pressure-filtered, and then filtered through two-stage microporous membranes with a pore size of 5 μm and 0.5 μm. Then pasteurize the filtrate at 80°C for 30 minutes, and then fill it.

Preparation of example 6 sea cucumber mucopolysaccharide oral liquid

Take 40 kg of fresh sea cucumbers, remove the viscera, tendons, and wash. Then use a tissue masher to crush its body wall, place it at a pH value of 6 at room temperature for 8 hours to autolyze the sea cucumber, then adjust the pH to 5 with 6 mol/L hydrochloric acid, add 150 grams of pepsin, and heat it at 55-60 ° C. Enzymolysis for 1 hour, after enzymolysis, quickly cool to below 30°C, keep for 30 minutes, stop the reaction, use 0.5 mol/L potassium hydroxide, adjust the pH value to neutral, and the remaining operations are the same as in Example 2 to obtain pure mucopolysaccharide .

Take another 1.6 kg of medlar, 1.6 kg of longan and 16 kg of water to boil, keep the fire low for 1 hour, filter the filtrate, concentrate it to 15 liters, add 95 liters of ethanol to the concentrated solution and let it stand, and distill the ethanol out to obtain For the extract, get 6 grams of sea cucumber mucopolysaccharide obtained by the above-mentioned method and mix it with 12 kilograms of the extract obtained by the above-mentioned method. Add 12 grams of pectin and 0.84 kilograms of honey, stir evenly, cool naturally, and let stand for 3 hours to make the precipitation complete. Use pressure filtration, take the filtrate, add 0.91 kg of sugar, 52 g of citric acid, 6.5 g of salt, 26 g of vitamin C, and 1.17 g of honey, mix well, filter with diatomaceous earth under pressure, and then pass through two stages with a pore size of 5 μm and 0.5 μm Microporous membrane filtration. Then pasteurize the filtrate at 80°C for 30 minutes, and then fill it.

The preparation of example 7 sea cucumber capsules

Take 40 kilograms of fresh sea cucumbers, remove internal organs, tendons, and wash. Then use a tissue masher to crush the body wall, add 80 liters of water, and beat well. The sea cucumber pulp was irradiated with ultraviolet rays for 10 minutes, and then autolyzed with a concentration of 0.06 mol/liter of sodium chloride at a pH of 7 and a temperature of 40 to 65°C (increased by 5°C every 3 minutes). . Afterwards, adjust the pH to 1 with 6 mol/L hydrochloric acid, add 160 g of pepsin for enzymatic hydrolysis for 2 hours, and after enzymatic hydrolysis, rapidly cool to below 30°C and keep for 30 minutes to terminate the reaction. The rest of the operations were the same as Example 1 to obtain the pure sea cucumber mucopolysaccharide.

Take another 80 kg of jujube, 80 kg of longan, add 800 liters of water, repeat the operation of preparing the extract in Example 3, and obtain 160 liters of extract, add 80 grams of mucopolysaccharide, then add 800 grams of β-cyclodextrin, and cook for 2 minutes , placed in a freeze dryer for drying, the temperature was controlled below -40°C, and the vacuum degree was 133×10 ^-3 pa, until the water content was 4%. Afterwards, lumps are removed by sieve, cooled to room temperature under an environment of 50% relative humidity, and the cooled product is packed into capsules.

The preparation of example 8 sea cucumber capsules

Take 40 kilograms of fresh sea cucumbers, remove internal organs, tendons, and wash. Then use a tissue masher to crush the body wall, add 120 liters of water, and beat well. The sea cucumber pulp was irradiated with ultraviolet rays for 30 minutes, and then autolyzed with a concentration of 0.08 mol/liter of sodium chloride at a pH of 7.5 and a temperature of 40 to 65°C (increased by 5°C every 3 minutes). . Then adjust the pH to 5 with 6 mol/L hydrochloric acid, add 180 grams of pepsin for enzymolysis for 1 hour, and after enzymolysis, quickly cool to below 30°C and keep for 30 minutes to terminate the reaction. The rest of the operations were the same as in Example 2 to obtain the pure sea cucumber mucopolysaccharide.

Take 80 kg of Lycium barbarum and 80 kg of rhodiola, add 800 liters of water, repeat the operation of preparing the extract in Example 3 to obtain 160 liters of extract, add 80 grams of mucopolysaccharide, and then add 4800 grams of β-cyclodextrin, cook For 10 minutes, control the vacuum at 86.25-98.64Kpa and the temperature at 45-65°C to concentrate. When the solid content in the concentrated material is 4%, start spray drying. The temperature of the powder spraying liquid is 45-50°C and the pressure of the material pump is 19.6 ～49.0Kpa, exhaust air temperature 60～68℃, inlet air temperature 150～180℃. Afterwards, lumps are removed by sieve, cooled to room temperature in an environment of 60% relative humidity, and the cooled product is packed into capsules.

Preparation of example 9 sea cucumber mucopolysaccharide tablet

Mash 200 kg of fresh sea cucumber intestinal tissue, dissolve it in 400 liters of 0.1M sodium phosphate buffer solution (pH6.0-7.5), let it stand for extraction for 13 hours, then centrifuge to remove the precipitate, and add solid ammonium sulfate to the supernatant To 80% saturation, salt out for 48 hours, then dialyze for 6 hours and centrifuge to get the precipitate, dissolve the precipitate in 0.1M phosphate buffer solution (pH6.0～7.5), dialyze again for 48 hours, and centrifuge to get the precipitate. It is carried out below 10°C, and the precipitate is freeze-dried to obtain autolysozyme powder.

Take another 50 kg of dried sea cucumber soaked in hair, cut it longitudinally along the side with the gap with a knife, remove the mouth and debris in the cavity, wash it in clean water, and then crush its body wall with a tissue grinder, and Add 250 liters of water and beat well. Add 150 grams of autolyzed enzyme extracted by the above method under stirring conditions, enzymolyze at pH 6 and temperature 15°C for 8 hours, then adjust the pH to 1 with 6 mol/L hydrochloric acid, add 150 grams of pepsin, and dissolve at 40 ~ Enzymolysis was carried out at 45°C for 2 hours, then rapidly cooled to below 30°C after enzymolysis, and kept for 30 minutes to terminate the reaction. The rest of the operations were the same as in Example 1 to obtain pure mucopolysaccharide. Take 100 grams of mucopolysaccharide, add it to the extract prepared by 400 grams of Rhodiola rosea, 200 grams of jujube, 200 grams of longan and 2000 ml of water, then add 5 grams of β-cyclodextrin, cook for 10 minutes, and then send Dry in a freeze dryer, control the temperature below -40°C, and vacuum at 133×10 ^-3 pa until the water content reaches 4%. Get 300 grams of the above-mentioned dry powder and add 30 grams of starch through a tablet press to obtain sea cucumber mucopolysaccharide tablets.

Preparation of example 10 sea cucumber mucopolysaccharide tablet

Mash 200 kg of fresh sea cucumber intestinal tissue, dissolve it in 3000 liters of 0.1M sodium phosphate buffer solution (pH6.0-7.5), let it stand for extraction for 2 hours, then centrifuge to remove the precipitate, and add solid ammonium sulfate to the supernatant To 95% saturation, salt out for 6 hours, then dialysis for 48 hours and centrifuge to get the precipitate, dissolve the precipitate in 0.1M phosphate buffer solution (pH6.0～7.5), dialyze again for 6 hours, and centrifuge to get the precipitate. It is carried out below 10°C, and the precipitate is freeze-dried to obtain autolysozyme powder.

Take another 50 kg of dried sea cucumber soaked in hair, cut it longitudinally along the side with the gap with a knife, remove the mouth and debris in the cavity, wash it in clean water, and then crush its body wall with a tissue grinder. Add 1500 grams of autolyzed enzyme extracted by the above method under stirring conditions, enzymolyze at pH 8 and 30°C for 3 hours, then adjust the pH to 5 with 6 mol/L hydrochloric acid, add 250 grams of pepsin, Enzymolysis was carried out at 60°C for 1 hour, then rapidly cooled to below 30°C after enzymolysis, and kept for 30 minutes to terminate the reaction. The rest of the operations are the same as in Example 2 to obtain pure mucopolysaccharide. Take 100 grams of mucopolysaccharide, add it to the extract prepared by 400 grams of Rhodiola rosea, 200 grams of jujube, 200 grams of longan and 2000 ml of water, then add 30 grams of β-cyclodextrin, cook for 2 minutes, and then send Dry in a freeze dryer, control the temperature below -40°C, and vacuum at 133×10 ^-3 pa until the water content reaches 4%. Get 300 grams of the above-mentioned dry powder and add 15 grams of starch through a tablet press to obtain sea cucumber mucopolysaccharide tablets.

Preparation of example 11 sea cucumber protein hydrolyzate drink

Take 40 kilograms of fresh sea cucumbers, remove internal organs, tendons, and wash. The body wall was then broken up with a tissue masher, and 200 liters of water was added. Autodissolve the sea cucumber slurry in a sterile enzymolysis tank for 24 hours at a pH of 6 and a temperature of 4°C, then adjust the pH to 5 with 6 mol/L hydrochloric acid, add 200 grams of pepsin, and enzyme at 40°C solution for 2 hours. After enzymatic hydrolysis, quickly cool to below 30°C and keep for 30 minutes to terminate the reaction. Use 0.5 mol/L potassium hydroxide to adjust the pH to neutral, centrifuge at high speed (6000 rpm), collect the mother liquor, slowly add 32 kg of 95% ethanol under constant stirring, and overnight at a low temperature below 10°C. Afterwards, the supernatant was obtained by centrifugation, and the ethanol was recovered by distillation under reduced pressure, and the remaining liquid was filtered, kept at 95-100° C. for 2 minutes, and sterilized for later use. Then insert Lactobacillus bulgaricus, the inoculation amount of bacteria seed liquid is 120 grams, and the fermentation temperature is 30°C. Simultaneously add 120 grams of skim milk powder and 120 grams of glucose at pH 4.0, when the acidity is 0.4%, the temperature is reduced to below 6° C., and the fermentation is stopped. Add 120 grams of white granulated sugar to the fermented liquid and mix evenly, and homogenize with a pressure of 20Mpa through a high-pressure homogenizer. The fermented liquid prepared by the above process is heated to 80° C., bottled while hot, sealed, and then cooled to room temperature after pasteurization (80° C., 30 minutes).

Preparation of example 12 sea cucumber mucopolysaccharide tablet

Take 40 kilograms of fresh sea cucumbers, remove internal organs, tendons, and wash. Then use a tissue masher to break its body wall, and the sea cucumber slurry is autolyzed in a sterile enzymolysis tank for 48 hours at a pH of 7.5 and a temperature of 0°C, and then adjusted to pH with 6 mol/L hydrochloric acid. 1. Add 120 grams of pepsin and hydrolyze at 60°C for 1 hour. After enzymatic hydrolysis, quickly cool to below 30°C and keep for 30 minutes to terminate the reaction. Adjust pH to neutral with 0.5 mol/L potassium hydroxide, centrifuge at high speed (6000 rpm), collect mother liquor, slowly add 12 kg of 95% ethanol under constant stirring, and overnight at low temperature below 10°C. Afterwards, the supernatant was obtained by centrifugation, and the ethanol was recovered by distillation under reduced pressure, and the remaining liquid was filtered, and sterilized by ultra-high temperature instantaneous heating method for future use. Then insert Lactobacillus bulgaricus, the inoculation amount of bacteria seed liquid is 40 grams, and the fermentation temperature is 50°C. Add 40 grams of skimmed milk powder and 20 grams of glucose simultaneously, and when the pH is 4.5 and the acidity is 0.2%, the temperature is reduced to below 6° C. to stop the fermentation. Add 40 grams of white granulated sugar to the fermented liquid and mix evenly, and homogenize with a pressure of 40Mpa through a high-pressure homogenizer. The fermented liquid prepared by the above process is heated to 80° C., bottled while hot, sealed, and then cooled to room temperature after pasteurization (80° C., 30 minutes).

Note: the percentages used in this application document are all percentages by weight.

Claims (2)

1, a kind of preparation method of sea cucumber mucopolysaccharide is characterized in that:

1. raw material and processing: get dried sea-cucumber and foam; Or get the salt marsh sea cucumber, and vertically cut open along a side jaggy with cutter, remove foreign material in ginseng mouth and the chamber, in clear water, clean; Or get fresh sea cucumber, directly remove internal organ, remove muscle, clean, add the water of 0～5 times of volume then to above-mentioned sea cucumber, with tissue mashing machine with its wall fragmentation,

2. extract sea cucumber mucopolysaccharide

A alkaline hydrolysis: be 40～70 ℃ in temperature and add 0.2～0.8% solid K ₂CO ₃, stir hydrolysis down 1～5 hour,

The b enzymolysis: transfer pH to 7～9 with 6 mol hydrochloric acid, adding quality hundred is that 0.3～0.5% trypsase constantly stirs, and maintains the temperature at 45～55 ℃, enzymolysis 2～7 hours, and speed is chilled to below 30 ℃ and kept 30 minutes afterwards,

C separates: transfer pH to neutral with 6 mol hydrochloric acid, 6000 rev/mins of high speed centrifugations separated 15 minutes, collected mother liquor,

D alcohol is molten: stir that slowly to add mass percent down be 30～80% 95% ethanol ,≤10 ℃ are spent the night,

E purifies: centrifugal collecting precipitation, and, in 40～70 ℃ of dryings or vacuum drying, get the sea cucumber mucopolysaccharide crude product after the acetone dehydration successively with the washing of 90～100% ethanol,

3. purifying: with the mucopolysaccharide dissolving crude product in 1: 10～50 grams per milliliter water, centrifugal, in clear liquid, add the solid potassium acetate, making its concentration in solution is 0.5～4 mol, refrigeration below 10 ℃ is spent the night, collecting precipitation below 10 ℃, and using last time again, the water of half volume dissolves it, transfer pH to 9～11 with 0.5 mol potassium hydroxide, keep 45～55 ℃ to drip H ₂O ₂The sugared at the most hydrolyzate that decolours is the light yellow terminal point that is, cooling is centrifugal, supernatant is cooled to below 4 ℃, transfer pH1.5～2.5 with 6 mol hydrochloric acid, centrifugal, in supernatant, add the solid potassium acetate, make its concentration in solution reach 0.5～4 mol, refrigeration below 10 ℃ is spent the night, collecting precipitation below 10 ℃, ethanol washing afterwards, the acetone dehydration, the dry pure product of mucopolysaccharide that get.

2, a kind of preparation method of sea cucumber mucopolysaccharide is characterized in that:

1. raw material and processing: get dried sea-cucumber and foam; Or get the salt marsh sea cucumber, and vertically cut open along a side jaggy with cutter, remove foreign material in ginseng mouth and the chamber, in clear water, clean; Or get fresh sea cucumber, directly remove internal organ, remove muscle, clean, add the water of 0～5 times of volume then to above-mentioned sea cucumber, with tissue mashing machine with its wall fragmentation,

2. extract sea cucumber mucopolysaccharide

A alkaline hydrolysis: be 40～70 ℃ in temperature and add 0.2～0.8% solid K ₂CO ₃, stir hydrolysis down 1～5 hour,

B enzymolysis: transfer pH1～5 with 6 mol hydrochloric acid, add 0.3～0.5% pepsin, 40～60 ℃ of stirred in water bath enzymolysis 3～10 hours, transfer pH to 7～9 with 0.5 mol potassium hydroxide again, add 0.3～0.5% trypsase, 45～55 ℃ of stirred in water bath enzymolysis 2～7 hours, speed was chilled to below 30 ℃ behind the enzymolysis, kept 30 minutes

C separates: transfer pH to neutral with 6 mol hydrochloric acid, 6000 rev/mins of high speed centrifugations separated 15 minutes, collected mother liquor,

D alcohol is molten: stir that slowly to add mass percent down be 30～80% 95% ethanol ,≤10 ℃ are spent the night,

E purifies: centrifugal collecting precipitation, and, in 40～70 ℃ of dryings or vacuum drying, get the sea cucumber mucopolysaccharide crude product after the acetone dehydration successively with the washing of 90～100% ethanol,

3. purifying: with the mucopolysaccharide dissolving crude product in 1: 10～50 grams per milliliter water, centrifugal, in clear liquid, add the solid potassium acetate, making its concentration in solution is 0.5～4 mol, refrigeration below 10 ℃ is spent the night, collecting precipitation below 10 ℃, and using last time again, the water of half volume dissolves it, transfer pH to 9～11 with 0.5 mol potassium hydroxide, keep 45～55 ℃ to drip H ₂O ₂The sugared at the most hydrolyzate that decolours is the light yellow terminal point that is, cooling is centrifugal, supernatant is cooled to below 4 ℃, transfer pH1.5～2.5 with 6 mol hydrochloric acid, centrifugal, in supernatant, add the solid potassium acetate, make its concentration in solution reach 0.5～4 mol, refrigeration below 10 ℃ is spent the night, collecting precipitation below 10 ℃, ethanol washing afterwards, the acetone dehydration, the dry pure product of mucopolysaccharide that get.