阅读说明：本技术 应用气浮法提取牛气管中硫酸软骨素钠和肽的工艺 (Process for extracting chondroitin sulfate sodium and peptide in bovine trachea by air floatation method ) 是由 谭岸 邹高远 朱小丰 谭金茂 于 2021-11-01 设计创作，主要内容包括：本发明公开了应用气浮法提取牛气管中硫酸软骨素钠和肽的工艺,属于生化制药技术领域,包括以下步骤：(1)牛气管蒸煮脱脂；(2)三步酶解；(3)气浮分层；(4)精滤；(5)过滤液的处理；(6)硫酸软骨素钠的精制；(7)肽的精制。本发明提供的工艺实现了直接用新鲜或干牛气管提取硫酸软骨素钠和肽,通过自絮凝气浮法使牛气管水解液快速分层,且过程可控,安全可行,省去了以往工艺中难度大、费用高的牛气管中软骨去杂工序,不另外添加絮凝剂,原料利用率高,减少了残留乳化油脂对后续过滤和树脂吸附的影响,且硫酸软骨素钠和肽的产率较高。(The invention discloses a process for extracting chondroitin sulfate sodium and peptide in a cattle trachea by applying an air floatation method, which belongs to the technical field of biochemical pharmacy and comprises the following steps: (1) cooking cattle trachea for degreasing; (2) carrying out three-step enzymolysis; (3) air floatation layering; (4) fine filtering; (5) treating the filtrate; (6) refining chondroitin sulfate sodium; (7) and (4) refining the peptide. The process provided by the invention realizes the direct extraction of sodium chondroitin sulfate and peptide by using a fresh or dry bovine trachea, quickly stratifies the bovine trachea hydrolysate by a self-flocculation air floatation method, is controllable, safe and feasible, omits the impurity removal process of the cartilage in the bovine trachea with high difficulty and high cost in the prior process, does not add a flocculating agent, has high utilization rate of raw materials, reduces the influence of residual emulsified oil on subsequent filtration and resin adsorption, and has higher yield of sodium chondroitin sulfate and peptide.)

1. The process for extracting the chondroitin sulfate sodium and the peptide in the bovine trachea by applying the air floatation method is characterized by comprising the following steps of: the method comprises the following steps:

(1) cooking cattle trachea for degreasing: cleaning and cutting the bovine trachea, adding water, stewing for 2-3h at the temperature of 105-;

(2) three steps of enzymolysis: carrying out three-step enzymolysis on bovine trachea pulp to obtain a bovine trachea hydrolysate;

(3) air floatation layering: pumping an air-floating agent solution into the cattle trachea hydrolysate to reach an isoelectric point under the conditions of sealing and stirring, heating to 80-90 ℃, preserving heat for 30min to inactivate hydrolase, then performing air-floating layering under reduced pressure to obtain layered cattle trachea hydrolysate, and discharging a lower layer clear solution;

(4) fine filtering: filtering the lower clear liquid by using 300-mesh diatomite as a filter aid and 400-mesh filter cloth to obtain filtrate;

(5) and (3) treating the filtrate: sequentially passing the filtrate through anion exchange resin and decolorizing resin to obtain permeate;

(6) refining of chondroitin sulfate sodium: eluting the adsorbed anion exchange resin with 10-15% by mass of saline solution to obtain an eluent; hydrolyzing the eluent again, filter-pressing, ultrafiltering, precipitating with ethanol, centrifuging, and drying to obtain pharmaceutical-grade sodium chondroitin sulfate;

(7) refining the bovine tracheal peptide: and (4) carrying out nanofiltration concentration, vacuum concentration and spray drying on the permeate to obtain the bovine trachea peptide.

2. The process of claim 1 for extracting chondroitin sulfate sodium and peptide in ox trachea by air floatation method, wherein: the cattle trachea in the step (1) is one of a fresh cattle trachea and a dry cattle trachea.

3. The process of claim 1 for extracting chondroitin sulfate sodium and peptide in ox trachea by air floatation method, wherein: the enzymes used in the three steps of enzymolysis in the step (2) are 2709 alkaline protease, papain and pepsin in sequence; 2709 the adding amount of alkaline protease is 0.2-0.3% of the fresh air pipe, the adding amount of papain is 0.2-0.3% of the fresh air pipe, the adding amount of pepsin is 0.1-0.2% of the fresh air pipe, and the adding amount of enzyme in the dry air pipe is 3 times of the fresh air pipe.

4. The process of claim 3 for extracting chondroitin sulfate sodium and peptide in ox trachea by air floatation method, wherein: the enzymolysis condition of the 2709 alkaline protease is as follows: the enzymolysis temperature is 45-55 deg.C, pH is 8-9, and the enzymolysis time is 2-4 h.

5. The process of claim 3 for extracting chondroitin sulfate sodium and peptide in ox trachea by air floatation method, wherein: the enzymolysis condition of the papain is as follows: the enzymolysis temperature is 60-65 deg.C, pH value is 5-7, and the enzymolysis time is 2-4 h.

6. The process of claim 3 for extracting chondroitin sulfate sodium and peptide in ox trachea by air floatation method, wherein: the enzymolysis condition of the pepsin is as follows: the enzymolysis temperature is 35-40 deg.C, pH value is 2-3, and enzymolysis time is 2-4 h.

7. The process of claim 1 for extracting chondroitin sulfate sodium and peptide in ox trachea by air floatation method, wherein: and (3) the air floating agent is sodium bicarbonate.

8. The process of claim 1 for extracting chondroitin sulfate sodium and peptide in ox trachea by air floatation method, wherein: the pH value of the isoelectric point in the step (3) is 4.0-5.0.

Technical Field

The invention belongs to the technical field of biochemical pharmacy, and particularly relates to a process for extracting chondroitin sulfate sodium and peptide in an ox trachea by applying an air floatation method.

Background

Sodium chondroitin sulfate (CS for short) is an acidic mucopolysaccharide, widely exists in ox bones, pig bones, chicken bones, shark bones and squid bones, belongs to one of glycosaminoglycans, and is a natural-source biological macromolecule. The sodium chondroitin sulfate consists of 50-70 disaccharide units, wherein the disaccharide units are formed by glucuronic acid and galactosamine through beta-1, 3 glycosidic bonds, and the disaccharide is connected and polymerized into macromolecules through the beta-1, 4 glycosidic bonds.

The sodium chondroitin sulfate has the physiological functions of reducing blood fat, resisting blood coagulation, resisting inflammation, resisting tumor and the like. The eye drops are mainly used for treating osteoarthritis, hyperlipidemia, angina, atherosclerosis and other diseases clinically, and can treat corneal injury, corneal ulcer or keratitis; sodium chondroitin sulfate is classified as a dietary supplement by the U.S. FDA on food and is used by australia as a nutraceutical to ameliorate or prevent joint disease.

Collagen is the most abundant animal protein in mammals, mainly exists in tissues such as bones, tendons, ligaments and skin, and is an important structural protein in connective tissues. The collagen consists of three peptide chains, and the three peptide chains form a composite helix in a special way, and can be hydrolyzed by protease after being denatured.

The peptide is protein hydrolysate, the collagen peptide indicated by the national food safety standard (GB 316452018) is the hydrolysate of animal tissue bone, tendon, skin, etc. rich in collagen, rich in hydroxyproline, and consists of more than 2 amino acids, the molecular weight is required to be less than 10000 and more than 90%, and the collagen peptide has good absorption performance and is a food raw material. Can be used in various fields such as food, health food, cosmetics, biological materials, microorganism culture medium, high-end feed, etc.

At present, chondroitin sulfate sodium is extracted from animal cartilage by an enzymolysis method in industrial production, and the method mainly comprises the steps of impurity removal, cleaning, material boiling, enzymolysis, deproteinization, filtration, centrifugation, ion exchange, ultrafiltration, alcohol precipitation, drying and the like. The bone oil is removed by floating on the feed liquid after the cartilage is cooked, and the oil removal rate is generally less than 85 percent. Separating sodium chondroitin sulfate dissolved in water from proteoglycan, hydrolyzing protein, separating peptide dissolved in water by anion resin adsorption or ultrafiltration membrane filtration, and respectively purifying and drying. Most of protein is hydrolyzed into peptide and amino acid by proteolytic enzyme, when unhydrolyzed protein is removed by isoelectric precipitation, part of larger emulsified oil particles adsorb the protein precipitated by isoelectric precipitation and bone dregs wrap part of aqueous solution to form cement-like substances, and sludge-bone dregs mixed precipitation is formed, the precipitated oil content is large, such as large bone dregs content, cement weight is heavier than water, sinking, such as extremely small bone dregs content, more oil content of cement, lighter than water, floating, such as moderate oil-slag ratio, and possibly forming suspended floccule or three to four layers. The daub layer can not be subjected to filter pressing, the daub layer is directly discharged after layering is finished, a large amount of micro emulsified oil and particles still exist in the hydrolysate clear solution layer, and more filter aids are needed for subsequent clear solution filtration, if unequal layering direct filtration is adopted, the filter cloth and the filter aids are more in use amount. The research on the method for increasing the layering rate and reducing the water content of the oil sludge layer and the using amount of the filter aid is a way for increasing the yield. Since reduction of fat and oil residues is a viable approach, removal of fat and muscle impurities containing much fat and oil as much as possible is required in the impurity removal step in production, and it is desired to increase the removal rate of fat and oil in cartilage. The residual fat in cartilage can be hydrolyzed by lipase, but the fat hydrolysis can only be carried out at an oil-water interface, and the residual fat in cartilage is easily wrapped and emulsified by protein after being dispersed in water due to the action of the protein as a surfactant, so the hydrolysis efficiency is not high.

The bovine trachea is composed of cartilage, muscle, connective tissue and mucous membranes. The cartilage is C-shaped cartilage ring, the gap is backward, the cartilage rings are connected by ligament, and the gap behind the rings is connected by smooth muscle and dense connective tissue. The dry trachea contains about 40-50% of protein, about 30-40% of fat and about 10-15% of polysaccharide. The cartilage, tendon and tendon with high collagen content in the bovine trachea are more, the tracheal cartilage after impurities are removed is more a high-quality collagen peptide production raw material, the peptide directly hydrolyzed by the bovine trachea mainly contains collagen peptide, and the detection index reaches the national standard of the collagen peptide.

The bovine tracheal cartilage has high chondroitin sulfate sodium content of 28-32% and oil content less than 10%, and is a high-quality chondroitin sulfate production raw material. Cartilage accounts for about 30% of the weight of the airway tube, but the cricoid cartilage is difficult to strip out. Therefore, the production of the sodium chondroitin sulfate from cattle generally selects cattle costal cartilage, nasal cartilage, scapular cartilage and the like which are easy to remove impurities.

The principle of the air flotation method, also called flotation, is to make the water generate a large amount of micro-bubbles to form a three-phase mixture of water, air and the removed substance, and promote the micro-bubbles to adhere to the removed micro-oil drops under the combined action of a plurality of forces such as interfacial tension, bubble rising buoyancy and hydrostatic pressure difference, and then the micro-bubbles float to the water surface because the density of the adhesive is less than that of the water, so that the oil particles in the water are separated and removed. In addition to the removal of oil in an emulsified state in sewage, the air-float process is widely used for removing impurities in sewage in a state of fine suspended particles having a density close to that of water. For example, the air-float process can be effectively used for the concentration of activated sludge; and removing suspended impurities in the sewage.

Therefore, the invention adopts a process for extracting chondroitin sulfate sodium and peptide in an ox gas tube by a self-flocculation air floatation method, and the air floatation condition is achieved by utilizing the principles of protein high-temperature denaturation precipitation, minimum solubility at isoelectric points, reduced emulsifying capacity after surface activity is reduced, continuous reduction of solubility of carbon dioxide in water at high temperature and reduced pressure and the like. In detail, the protein which is denatured at high temperature by proteolytic enzyme and is not hydrolyzed completely has the minimum solubility at the isoelectric point, and is separated out and bonded into small particles; meanwhile, the protein wrapped outside the emulsified oil is separated out, the emulsifying property is reduced, and the protein and the emulsified oil are mutually bonded into small particles; and raising the temperature and reducing the pressure to reduce the solubility of carbon dioxide to generate micro bubbles, and adhering the bubbles to the surfaces of the two small particles to quickly float upwards so as to quickly clarify and stratify the degreased hydrolysate of the bovine trachea. The process provided by the invention can be directly used for extracting fresh or dry bovine trachea, solves the problem that the process of the bovine trachea cartilage impurity removal procedure in the process of extracting chondroitin sulfate sodium and peptide in the bovine trachea is difficult to control, reduces the loss of the impurity removal process, and improves the yield of chondroitin sulfate and bovine trachea peptide.

Disclosure of Invention

The invention aims to provide a process for extracting chondroitin sulfate sodium and peptide in a cattle trachea by using an air floatation method, which improves the layering efficiency of a hydrolysate in the cattle trachea by using the air floatation method, thereby solving the influence of emulsified oil on subsequent filtration and resin adsorption, reducing the using amount of a filter aid and ensuring the stability and controllability of a production process.

The technical problems to be solved by the invention are as follows: the existing process for extracting sodium chondroitin sulfate and peptide from bovine tracheal cartilage has the problems of unstable extraction process and easy cartilage loss due to complex impurity separation process around the cartilage.

The invention can realize the purpose of directly extracting the chondroitin sulfate sodium and the peptide from the bovine trachea, and can be realized by the following technical scheme:

the process of extracting chondroitin sulfate sodium and peptide from ox trachea by air floating method includes the following steps:

(1) cooking cattle trachea for degreasing: cleaning a bovine trachea, cutting the bovine trachea into 2-10 cm sections, adding water, transferring the bovine trachea into an extraction tank, cooking the bovine trachea for 2-3h at the temperature of 105-plus-one 110 ℃, keeping the temperature and standing, slowly adding water at the temperature of 95 ℃, removing upper-layer grease from the top, and stirring the bovine trachea at the speed of 100-plus-one 150rpm for 3h to obtain bovine trachea slurry;

(2) three steps of enzymolysis: carrying out three-step enzymolysis on bovine trachea pulp to obtain a bovine trachea hydrolysate;

(3) air floatation layering: pumping an air-floating agent solution into the cattle trachea hydrolysate to reach an isoelectric point under the conditions of stirring and sealing, heating to 80-90 ℃, inactivating hydrolase for 30min, then performing air-floating layering under reduced pressure to obtain layered cattle trachea hydrolysate, and discharging a lower layer clear solution;

(4) fine filtering: filtering the lower clear liquid by using 300-mesh diatomite as a filter aid and 400-mesh filter cloth to obtain filtrate;

(5) and (3) treating the filtrate: sequentially passing the filtrate through anion exchange resin and decolorizing resin to obtain permeate;

(6) refining of chondroitin sulfate sodium: eluting the adsorbed anion exchange resin with 10-15% by mass of saline solution to obtain an eluent; hydrolyzing the eluent again, filter-pressing, ultrafiltering, precipitating with ethanol, centrifuging, and drying to obtain pharmaceutical-grade sodium chondroitin sulfate;

(7) refining the bovine tracheal peptide: and (4) carrying out nanofiltration, concentration and desalination, vacuum concentration and spray drying on the permeate to obtain the bovine tracheal peptide.

Further, the cattle trachea in the step (1) is a fresh cattle trachea or a dry cattle trachea, wherein 2-3 times of water is added into the fresh cattle trachea, and 6-7 times of water is added into the dry cattle trachea.

Further, the enzymes used in the three steps of enzymolysis in the step (2) are 2709 alkaline protease, papain and pepsin in sequence, the adding mass of the 2709 alkaline protease is 0.2-0.3% of the mass of the fresh trachea, the adding mass of the papain is 0.2-0.3% of the mass of the fresh trachea, the adding mass of the pepsin is 0.1-0.2% of the mass of the fresh trachea, and the adding amount of the enzyme in the dry trachea is 3 times of that in the fresh trachea.

Further, the enzymolysis conditions of the 2709 alkaline protease are as follows: the enzymolysis temperature is 45-55 deg.C, pH is 8-9, and the enzymolysis time is 2-4 h.

Further, the enzymolysis conditions of the papain are as follows: the enzymolysis temperature is 60-65 deg.C, pH is adjusted to 5-7, and the enzymolysis time is 2-4 h.

Further, the enzymolysis condition of the pepsin is as follows: the enzymolysis temperature is 35-40 deg.C, pH is adjusted to 2-3, and the enzymolysis time is 2-4 h.

Further, in the step (3), the air floating agent is one of sodium bicarbonate and sodium carbonate, preferably sodium bicarbonate.

Further, the air flotation layering in the step (3) is specifically operated in such a way that an air flotation agent solution is added to adjust the pH of the bovine trachea hydrolysate to the isoelectric point, the stirring is stopped after 30min, the temperature is raised to 80-90 ℃, and the mixture is kept stand for 1 h.

Further, the pH of the isoelectric point is 4.0 to 5.0.

Further, the anion exchange resin used in the step (5) is strong-base polyacrylic acid macroporous resin, and the decolorizing resin is weak-polarity polyacrylic acid macroporous resin.

Further, the step (6) is carried out again by using compound enzyme, preferably pancreatin; and (3) repeatedly adding water in the ultrafiltration for not less than 3 times, wherein the water diluted by repeatedly adding water is reverse osmosis water.

Further, the nanofiltration membrane used in the nanofiltration concentration process in the step (7) is a membrane with the molecular weight of 150 daltons.

The invention has the beneficial effects that:

according to the invention, sodium chondroitin sulfate and peptide can be directly extracted from bovine trachea, and on the basis of the technology of extracting sodium chondroitin sulfate and peptide from bovine cartilage by using an enzyme method in the prior art, through the improvement of a process route and process parameters, a hydrolysis method is controlled by three enzymes step by step to fully hydrolyze protein in bovine trachea, then an air floatation layering method is adopted, carbon dioxide generated by adding sodium bicarbonate serving as an air floatation agent is dissolved in water under an acidic condition, and when the temperature is raised to inactivate protease and the protease is kept still, tiny bubbles are generated by slowly reducing pressure and are uniformly adsorbed on the surfaces of suspended particles and drive the particles to float upwards, so that layering is realized, the layering process is fast and thorough, the interface of the floating oil sludge layer is clear, the clarity of the lower clear liquid is good, and the filtering is fast;

in addition, the air flotation method of carbon dioxide gas generated by adjusting isoelectric point with sodium bicarbonate has the following advantages: because the solubility of carbon dioxide in water is reduced along with the temperature and the pressure, the bubble generation speed and the generated bubble volume can be adjusted through the temperature and the pressure, so that the bubbles are controlled to be uniformly generated, the bubbles are promoted to be fully contacted with isoelectric flocculation protein and grease emulsified particles and adhered to the surfaces of the particles, the water absorption of the surfaces of the particles is reduced, the particles are promoted to float upwards quickly, the layering process is promoted, the interface of an oil sludge layer is clear, the water content of the oil sludge layer is reduced, the particles in a clear liquid layer are reduced, the influence of residual emulsified grease on subsequent filtration and resin adsorption is reduced, and the using amount of a filter aid is reduced;

in conclusion, the process for extracting chondroitin sulfate sodium and peptide in the cattle trachea by the self-flocculation air flotation method realizes the direct extraction of chondroitin sulfate sodium and peptide by using a fresh or dry cattle trachea, omits the procedure of removing impurities from cartilage, shortens the production time, improves the yield of the chondroitin sulfate sodium and the peptide, and improves the comprehensive benefit of the cattle trachea.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1: raw materials: fresh cattle in northeast of age less than 2 years

The process of extracting chondroitin sulfate sodium and peptide from ox trachea by air floating method includes the following steps:

(1) cooking cattle trachea for degreasing: cutting 6 tons of fresh cattle trachea into pieces (5 cm sections), adding 2 times of mass water, cooking for 2 hours at 105 ℃, preserving heat, standing for 6 hours, ejecting upper-layer grease by using 95 ℃ water, stirring for 3 hours at the speed of 120rpm, and stirring the cattle trachea solution into slurry to obtain cattle trachea slurry;

(2) three steps of enzymolysis: cooling bovine trachea slurry to 55 ℃, adjusting the pH value to 8.5, adding 2709 alkaline protease accounting for 0.3 percent of the mass of bovine trachea, and performing enzymolysis for 2 hours; heating to 65 ℃, adjusting the pH value to 6, adding papain with the mass of 0.2 percent of that of the bovine trachea, and carrying out enzymolysis for 2 hours; adjusting pH to 2, cooling to 37 deg.C, adding pepsin 0.1% of the quality of bovine trachea, and performing enzymolysis for 2 hr;

(3) air floatation layering: adding a saturated solution of sodium bicarbonate into 500 ml of the hydrolysate to reach an isoelectric point (pH value is 4-5) to obtain a small sample amount ratio; sealing the extraction tank, pumping a small-scale sodium bicarbonate saturated solution under stirring at 60rpm, adjusting the pH value to an isoelectric point, heating the temperature to 80 ℃, stirring for 30min, standing for 1h, slowly reducing the pressure, fully emptying, cooling, standing until the layering is basically stable to obtain a layered bovine trachea hydrolysate, and discharging a lower layer clear solution for filtration;

(4) fine filtering: adding a filter aid into the lower clear liquid, and performing precision filtration to obtain a filtrate; diatomite or active carbon is used as a filter cake of the filter aid, the thickness of the filter cake is 5mm, the filter aid is 300 meshes, and the filter cloth is 400 meshes;

(5) and (3) treating the filtrate: sequentially passing the filtrate through anion exchange resin (strong basic polyacrylic acid macroporous resin) and decolorizing resin (weak polar polyacrylic acid macroporous resin) to obtain permeate, controlling the flow rate of the filtrate to be 2 times of the volume per hour of the resin, wherein the resin dosage is 3 cubic meters respectively, and the filtrate is produced by Dow chemical company of America;

(6) refining of chondroitin sulfate sodium: eluting the anion exchange resin after adsorption with 10% of saline solution (the dosage of the saline solution is 1 time of the volume of the resin) by mass fraction to obtain eluent; hydrolyzing the eluate again (pancreatin 0.5%), filter-pressing, ultrafiltering (3000 dalton membrane), precipitating with ethanol, dehydrating, centrifuging, and drying to obtain pharmaceutical-grade sodium chondroitin sulfate; using diatomite as a filter aid, performing ultrafiltration concentration and desalination on the hydrolysate filter pressing filtrate to 2 cubic meters by adding reverse osmosis water in an amount which is 1 time that the filtrate is subjected to filter pressing again, repeating the process for 4 times, performing alcohol precipitation on the cut-off solution (adding edible alcohol until the concentration of the alcohol is 70 percent, stirring the mixture for 30 minutes, standing the mixture for 30 minutes, taking precipitate), performing secondary dehydration (adding edible alcohol into the precipitate and stirring the precipitate until the concentration is 85 percent once and 90 percent once), and then performing centrifugal dealcoholization; vacuum drying at 60 deg.C to water content below 10% to obtain the final product;

(7) refining the bovine tracheal peptide: desalting and concentrating the decolorized resin absorption permeate through nanofiltration, wherein the membrane cut-off molecular weight is 150 daltons, carrying out double-effect vacuum concentration (below 60 ℃) on the concentrated cut-off solution, and carrying out spray drying on the concentrated solution (the specific weight of the concentrated solution is 1.15, the air inlet temperature is 175 ℃, and the air outlet temperature is 85 ℃) to obtain the bovine tracheal peptide.

Example 2: the cattle are dry, Xinjiang in the production area and less than 3 years old.

The process of extracting chondroitin sulfate sodium and peptide from ox trachea by air floating method includes the following steps:

(1) cooking cattle trachea for degreasing: chopping 2 tons of bovine trachea (2 cm sections), adding 6 times of mass water, boiling for 3 hours at 108 ℃, preserving heat, standing for 6 hours, ejecting upper-layer grease by using 95 ℃ water, stirring for 3 hours at the speed of 120rpm, and stirring the solution of the bovine trachea into slurry to obtain bovine trachea slurry;

(2) three steps of enzymolysis: cooling bovine trachea slurry to 55 ℃, adjusting the pH value to 8.5, adding 2709 alkaline protease accounting for 0.9 percent of the mass of bovine trachea, and performing enzymolysis for 2 hours; heating to 65 ℃, adjusting the pH value to 6, adding papain with the mass of 0.6 percent of that of the bovine trachea, and carrying out enzymolysis for 2 hours; adjusting pH to 2.0, cooling to 37 deg.C, adding pepsin 0.3% of the quality of the bovine trachea, and performing enzymolysis for 2 hr;

(3) air floatation layering: adding a saturated solution of sodium bicarbonate into 500 ml of the hydrolysate to reach an isoelectric point (pH value is 4-5) to obtain a small sample amount ratio; sealing the extraction tank, pumping a small-scale sodium bicarbonate saturated solution under stirring at 60rpm, adjusting the pH value to an isoelectric point, heating the temperature to 85 ℃, stirring for 30min, standing for 1h, slowly reducing the pressure, fully emptying, cooling, standing until the layering is basically stable to obtain a layered bovine trachea hydrolysate, and discharging a lower layer clear solution;

(4) fine filtering: adding a filter aid into the lower clear liquid, and performing precision filtration to obtain a filtrate; diatomite or active carbon is used as a filter cake of the filter aid, the thickness of the filter cake is 5mm, the filter aid is 300 meshes, and the filter cloth is 400 meshes;

(5) and (3) treating the filtrate: sequentially passing the filtrate through anion exchange resin (strong basic polyacrylic acid macroporous resin) and decolorizing resin (weak polar polyacrylic acid macroporous resin) to obtain permeate, controlling the flow rate of the filtrate to be 2 times of the volume per hour of the resin, wherein the resin dosage is 3 cubic meters respectively, and the filtrate is produced by Dow chemical company of America;

(6) refining of chondroitin sulfate sodium: eluting the adsorbed anion exchange resin with 15% by mass of saline (the amount of the saline is 1 time of the volume of the resin) to obtain an eluate; hydrolyzing the eluate again (pancreatin 0.5%), filter-pressing, ultrafiltering (3000 dalton membrane), precipitating with ethanol, dehydrating, centrifuging, and drying to obtain pharmaceutical-grade sodium chondroitin sulfate; using diatomite as a filter aid, performing ultrafiltration concentration and desalination on the hydrolysate filter pressing filtrate to 2 cubic meters by adding reverse osmosis water in an amount which is 1 time that the filtrate is subjected to filter pressing again, repeating the process for 3 times, performing alcohol precipitation on the cut-off solution (adding edible alcohol until the concentration of the alcohol reaches 70 percent, stirring the mixture for 30 minutes, standing the mixture for 30 minutes, taking precipitate), performing secondary dehydration (adding edible alcohol into the precipitate and stirring the precipitate until the concentration reaches 85 percent once and 90 percent once), and then performing centrifugal dealcoholization; vacuum drying at 60 deg.C to water content below 10% to obtain the final product;

(7) refining the bovine tracheal peptide: desalting and concentrating the decolorized resin absorption permeate through nanofiltration, wherein the membrane cut-off molecular weight is 150 daltons, carrying out double-effect vacuum concentration (below 60 ℃) on the concentrated cut-off solution, and carrying out spray drying on the concentrated solution (the specific weight of the concentrated solution is 1.20, the air inlet temperature is 175 ℃, and the air outlet temperature is 85 ℃) to obtain the bovine tracheal peptide.

Example 3: fresh bovine trachea, Shandong of origin, age of cattle 1-2 years, other same as example one.

Example 4: air pipe of dry cattle, birth area inner Mongolia, cattle age 1-2 years, other same as example two.

Example 5: the cattle are dry, Xinjiang in the production area and less than 3 years old.

The process of extracting chondroitin sulfate sodium and peptide from ox trachea by air floating method includes the following steps:

(1) cooking cattle trachea for degreasing: cutting 6kg of bovine trachea into pieces (2 cm sections), adding 6 times of mass of water, boiling for 3 hours at 108 ℃, preserving heat, standing for 6 hours, ejecting upper-layer grease by using 95 ℃ water, stirring for 3 hours at the speed of 120rpm, and stirring the solution of the bovine trachea into slurry to obtain bovine trachea slurry;

the rest is the same as in example 2.

Comparative example 1: the cattle are dry, Xinjiang in the production area and less than 3 years old.

The same procedure was followed as in example 5, except that the pH was adjusted with sodium hydroxide in (3).

Yield and performance testing

The yields of sodium chondroitin sulfate and bovine tracheal peptide obtained in examples 1-5 and comparative example 1 are shown in Table 1.

The properties of the sodium chondroitin sulfate obtained in examples 1 to 5 and comparative example 1 were measured, and the data are shown in Table 2.

The bovine tracheal peptides obtained in examples 1 to 5 and comparative example 1 were tested according to the GB31645-2018 standard, and the data are shown in Table 3.

TABLE 1 chondroitin sulfate sodium and bovine tracheal peptide yields Table

TABLE 2 summary of sodium chondroitin sulfate assay data

TABLE 3 summary of bovine tracheal peptide assay data

As can be seen from the data in Table 1, the yields of chondroitin sulfate sodium and bovine tracheal peptide in examples 1-5 were higher than that in comparative example 1, and from the data in tables 2 and 3, the masses of chondroitin sulfate sodium and bovine tracheal peptide obtained in examples 1-5 were superior to those obtained in comparative example 1, and the floc volume in comparative example 1 was 14.5cm³In example 5, the volume of flocs was 1.8cm³The layering standing time is shortened from 120min to 30min, and the data show that the process provided by the invention realizes the direct extraction of the sodium chondroitin sulfate and the peptide by using a fresh or dry cattle trachea, shortens the production time, improves the yield of the sodium chondroitin sulfate and the peptide, and improves the comprehensive benefit of the cattle trachea.

In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

The foregoing is illustrative and explanatory only and is not intended to be exhaustive or to limit the invention to the precise embodiments described, and various modifications, additions, and substitutions may be made by those skilled in the art without departing from the scope of the invention or exceeding the scope of the claims.