CN113234181A - Preparation method of chondroitin sulfate - Google Patents
Preparation method of chondroitin sulfate Download PDFInfo
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- CN113234181A CN113234181A CN202110487356.2A CN202110487356A CN113234181A CN 113234181 A CN113234181 A CN 113234181A CN 202110487356 A CN202110487356 A CN 202110487356A CN 113234181 A CN113234181 A CN 113234181A
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0069—Chondroitin-4-sulfate, i.e. chondroitin sulfate A; Dermatan sulfate, i.e. chondroitin sulfate B or beta-heparin; Chondroitin-6-sulfate, i.e. chondroitin sulfate C; Derivatives thereof
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Abstract
The invention discloses a preparation method of chondroitin sulfate, belonging to the technical field of biology. The application takes the cartilage and/or trachea of livestock and poultry as raw materials, and combines the modern biological enzymolysis technology and separation and extraction technology to prepare the high-purity chondroitin sulfate and the protein peptide. The process is as follows: placing the mixture of cartilage and/or trachea in a reaction tank for cooking, adding sodium hydroxide to adjust the pH value to 8.0-9.5, adding trypsin and lipase for primary enzymolysis, and primarily removing impurities; then adding compound protease to carry out secondary enzymolysis, and specifically hydrolyzing the protein. Filtering the enzymolysis solution, and performing ultrafiltration, concentration and drying on the supernatant to obtain the high-purity chondroitin sulfate. And (4) performing nanofiltration desalination, concentration and drying on the ultrafiltration permeate to obtain the collagen oligopeptide. The continuous two-stage enzymolysis and membrane separation technology is utilized to realize the efficient separation and preparation of chondroitin sulfate and protein peptide, the process is simple and easy to operate, the production period is short, the product purity is high, and the method is suitable for factory production and application.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for extracting chondroitin sulfate from animal cartilage and trachea.
Background
Chondroitin Sulfate (CS) is an acidic mucopolysaccharide whose basic units are D-glucuronic acid and N-acetyl-D-galactosamine sulfate. Chondroitin sulphate generally contains 50-70 disaccharide basic structures and has a molecular weight of about 10-50 kDa. Chondroitin sulfate has different physiological activities, and can be widely applied to industries such as medical treatment, medicines, foods, cosmetics and the like. In clinic, chondroitin sulfate can be used for treating neuralgia, arthritis, ulcer healing and other diseases due to good anti-inflammatory property, and can be used as a dietary supplement in the food industry.
At present, a great deal of research is carried out on methods for extracting chondroitin sulfate from animal cartilage such as fish, pigs, cattle, sheep and the like at home and abroad, but the research on preparing chondroitin sulfate by taking a mixture of cartilage and trachea as a raw material is less. The poultry such as chicken, duck and the like has short growth cycle and large breeding amount, and the chondroitin sulfate content of the slaughter processing by-product cartilage is high. The pig trachea is a byproduct after slaughter and also contains rich chondroitin sulfate, but is often treated as waste after slaughter and cannot be fully utilized.
Chondroitin sulfate exists in cartilage in the form of proteoglycan, and cartilage membrane and cartilage matrix contain a large amount of type I collagen and type II collagen, respectively. Therefore, the impurity mainly removed during the extraction of chondroitin sulfate is protein, and chondroitin sulfate is released from glycoprotein by breaking the glycosidic bond between the sugar and the peptide. In the prior art, alkali extraction, enzymolysis, ultrasonic assistance and the like are commonly adopted to extract chondroitin sulfate, and the alkali extraction mainly breaks the covalent bond between a polysaccharide chain and protein to break the sugar-peptide bond of proteoglycan so as to release the chondroitin sulfate; the concentration of alkali has great influence on the yield and the purity of the chondroitin sulfate, the concentration of alkali liquor is small, the production period for preparing the chondroitin sulfate is long, and the prepared sample solution is turbid. The reason is that collagen, mucin and the like in cartilage can not be completely hydrolyzed under the mild condition of dilute alkali, and the collagen, mucin and the like are soluble in neutral salt and weak acid solution and are not easy to be removed in the solution, and meanwhile, small molecular impurities such as polypeptide and the like in the product are easy to precipitate along with chondroitin sulfate, so that the yield and the purity of the product are influenced. If the concentration of the alkali liquor is too high, the chondroitin sulfate is easily degraded into oligosaccharide with smaller molecular weight and has dark color. Therefore, enzyme is added for synergic extraction, and the enzyme has the function of hydrolyzing the specificity of protein by utilizing protease to hydrolyze collagen, mucin and the like into amino acid; when protease is added for degradation, the reaction condition is mild, so that the chondroitin sulfate is not degraded, and the activity of the chondroitin sulfate is ensured; meanwhile, the protein in the solution and on the chondroitin sulfate long chain can be further hydrolyzed to generate soluble micromolecule peptide, amino acid and other substances. In the production of chondroitin sulfate, proteins, one of the most important impurities in chondroitin sulfate, must be removed as far as possible, since they are mainly present in the form of proteoglycans. Generally, the nitrogen content of the product is difficult to meet the requirement through one-time enzymolysis, the hydrolysis can be enhanced by adopting the compound enzyme, and the enzyme with wide peptide bonds is selected as the enzyme, so that the better enzymolysis effect is favorably obtained. Generally, a product obtained after enzymolysis is discharged as wastewater in a processing process, so that the environment is polluted and precious resources are wasted, and therefore, the small molecular peptides obtained after enzymolysis need to be fully recycled and utilized at a high value, and the comprehensive utilization value of raw materials is improved.
Animal cartilage or trachea have complex chemical components, and contain various macromolecular substances such as fat, glycogen and the like, which interfere the separation and purification of chondroitin sulfate, so that impurities in raw materials are degraded by specific enzymes such as collagenase, lipase and the like, and the purity of the chondroitin sulfate is improved.
Chinese patent CN111253503A discloses a method for extracting chondroitin sulfate from ray by ultrasonic-assisted alkali salt-enzymolysis, wherein the ultrasonic time is 180 min, the alkali liquor concentration is 2.3%, the enzymolysis is carried out for 12 h, and the optimal extraction rate can reach 43.32%. Although the extraction rate is high, the method is difficult to be applied to large-scale production. Patent CN110922503A discloses an extraction method of pig and cattle nasal bone
The process of the chondroitin sulfate comprises the steps of crushing cartilage, adding alkali liquor to adjust the pH value to 10-14, soaking at 15-20 ℃ for 25-35 h, performing solid-liquid separation, adding a complex enzyme of alkaline protease, neutral protease, lipase and pancreatin into an extracting solution to react for 5-10 h, and performing separation and purification to obtain the chondroitin sulfate with the purity of 93.5% -93.7%. The process adopts a complex enzymolysis method, but the pretreatment of the raw materials takes longer time, and the temperature is kept between 15 and 20 ℃ when the raw materials are required to be soaked, so that the energy consumption is large. The time of later enzymolysis separation is long, and the enzymolysis efficiency is not high. In patent CN109280095A, poultry connective tissue is boiled in water, dried and crushed, added with alkali liquor with the volume 6 times of that of 2-6% for leaching to obtain alkali extract, the pH value is adjusted to be neutral, then compound enzymolysis (alkaline protease, flavourzyme and lipase) is carried out, after enzyme deactivation, lipase is added for enzymolysis to obtain enzymolysis liquid, chondroitin sulfate is obtained through activated carbon adsorption and protein removal, the yield reaches more than 12%, and the product purity reaches 95.5%. The production process combines alkali extraction and secondary enzymolysis of complex enzyme, the obtained product has high purity, but a large amount of alkali liquor needs to be added in the alkali extraction process, acid needs to be added in the enzymolysis process to adjust the pH value, so that the salt content in the extracting solution is high, the protein hydrolysate is not effectively utilized, the difficulty of wastewater treatment is increased, and the activated carbon adopted by adsorption can only be used as solid waste to be treated, so that the resource waste and the environmental pollution are caused. Chinese patent CN102533915B discloses a method for preparing chondroitin sulfate and collagen polypeptide by enzymolysis of animal cartilage, which adopts anion exchange resin to recover collagen polypeptide, and the molecular weight of the obtained collagen polypeptide is centralized below 4000 Da, thereby realizing high-value utilization of cartilage. However, the ion exchange resin needs to consume a large amount of salt eluent in industrial production, and needs to be regenerated after being used, so that the operation is complicated. Chinese patent CN111533826A proposes a method for simultaneously extracting chondroitin sulfate and collagen peptide from pig bone, wherein the raw material is extracted twice at 115-120 ℃ and 130-135 ℃ respectively, and the enzymolysis is carried out for 60 min each time. Carrying out primary enzymolysis for 2 hours by adopting alkaline protease and trypsin, adsorbing the enzymolysis liquid for 3-5 hours by using resin, and washing to remove salt to obtain a chondroitin sulfate solution; adding keratinase, bromelain and neutral protease, performing secondary enzymolysis for 2 hr, and filtering with ceramic membrane (1000 Da) to obtain ossein peptide. The yield of the obtained chondroitin sulfate is 17.8-18.5%, the purity is more than 99.8%, the yield of the collagen peptide is 23.3-26.1%, and the molecular weight is 150-600 Da. The method can obtain the chondroitin sulfate with higher purity, but the extraction temperature is higher, the extraction time is longer, the chondroitin sulfate structure is easy to damage in the extraction process, and degradation or deepening of the color of the product can be caused. Although the resin adsorption has strong specificity, the elution and regeneration process needs to consume a large amount of resources, the operability of factory production is not strong, the energy consumption is large, and the cost is high. The production process needs to be carried out for multiple times of filtration and separation, the time consumption is long, and the production continuity is not high.
The analysis of the prior art has the following problems and defects:
(1) the existing method for extracting chondroitin sulfate has low enzymolysis specificity and low enzymolysis efficiency.
(2) A large amount of acid-base reagents are introduced into the existing method for extracting chondroitin sulfate, so that the subsequent desalting burden is increased, the quality of the chondroitin sulfate is influenced, the economic benefit of enterprise production is influenced, and the environmental pollution is caused.
(3) The existing method for extracting chondroitin sulfate has the disadvantages of complex process, long production period, unsuitability for continuous production in factories and low operability.
Disclosure of Invention
The invention aims to fully utilize the wastes in slaughtering and processing, adopts a two-stage enzymolysis method to fully hydrolyze protein: in the first-stage enzymolysis, the interference of impurities such as fat and the like is reduced while the sugar-peptide bond is hydrolyzed by adding pancreatin and lipase complex enzyme, and the preliminary separation is carried out; and then continuing to fully hydrolyze the protein by using the compound protease subjected to secondary enzymolysis to obtain high-quality chondroitin sulfate and collagen oligopeptide, wherein the obtained collagen oligopeptide is intensively distributed below 1000 Da, and the oligopeptide with the molecular weight range has good stability and high bioactivity. The process of the invention aims at overcoming the defects of incomplete enzymolysis, long enzymolysis time, difficult control of process parameters and high alkali liquor concentration in the prior art, which influence the purity of chondroitin sulfate and collagen peptide, and combining the ultrafiltration and nanofiltration separation technology to purify the product, thereby reducing the environmental pollution and the production cost, improving the comprehensive utilization value of the livestock processing waste, realizing the green, environment-friendly and economic preparation, and being suitable for industrial large-scale production.
The purpose of the invention can be realized by the following technical scheme:
a method for producing chondroitin sulfate in animal cartilage and/or trachea, comprising the steps of:
(1) cleaning: putting cartilage/trachea according to the mass ratio of 1:1-1:5, and cleaning.
(2) And (3) cooking: soaking cartilage and trachea in water in a cooking pot, heating to 80-100 deg.C, decocting in water for 1-3 hr to peel off muscle tissue on cartilage surface.
(3) Cooling: cooling the cooked material liquid to 45-55 ℃.
(4) First-stage enzymolysis: and (4) adding a small amount of sodium hydroxide into the feed liquid obtained in the step (3) to adjust the feed liquid to be alkalescent, adding the complex enzyme into the reaction tank according to the proportion of 1.0-4.0% of the mass of the raw materials, keeping stirring, and reacting for 2.0-5.0 h at the reaction temperature of 45-55 ℃.
(5) Secondary enzymolysis: after the reaction in the step (4) is finished, adding the complex enzyme according to the proportion of 0.20-0.40% of the mass of the raw materials, keeping stirring, and carrying out enzymolysis reaction for 1.5-4.0 h. After the second-stage enzymolysis reaction is finished, the temperature is raised to 70-85 ℃, and the mixture is settled and inactivated for 0.5-1.5 h.
(6) Filtration and ultrafiltration: and (5) filtering the enzymolysis liquid obtained in the step (5), collecting filtrate, washing filter residues for 1-2 times, combining the filtrates, filtering through an ultrafiltration membrane, and respectively collecting concentrated solution and permeate after ultrafiltration.
(7) Alcohol precipitation: and (4) adding 95% ethanol with the volume of 3-5 times that of the concentrated solution obtained in the step (6), settling for 0.5-1.0 h, centrifuging, collecting precipitate, drying to obtain a chondroitin sulfate finished product, wherein the ethanol can be recycled.
(8) And (3) recovering a byproduct of collagen peptide: and (4) desalting the permeate obtained in the step (6) through nanofiltration, concentrating and drying to obtain a collagen peptide by-product.
And (4) the pH value of the weak base is 8.00-9.50, the primary hydrolysis complex enzyme is a compound of pancreatin and lipase, and the pancreatin is porcine pancreas.
And (5) the secondary hydrolysis complex enzyme is a mixture of collagenase and one or more of neutral protease, papain and flavourzyme. Preferably a mixture of neutral protease and collagenase with the mass ratio of 2:1-5: 3.
The ultrafiltration membrane in the step (6) preferably has the molecular weight cutoff of 3000-5000 Da and the operation pressure of 4-8 bar.
The drying mode of the steps (7) and (8) is one of spray drying, forced air drying, vacuum drying and freeze drying, preferably vacuum drying and spray drying, and the vacuum drying temperature is preferably 55-70 ℃.
The molecular weight cut-off of the nanofiltration membrane in the step (8) is preferably 100-500 Da, and the operating pressure is preferably 8-12 bar.
Compared with the prior art, the invention has the following advantages:
(1) the invention uses the mixture of cartilage and trachea of livestock and poultry as the extraction raw material, increases the source of chondroitin sulfate and improves the added value of processing waste.
(2) The pH value of the feed liquid is adjusted to be alkalescent by primary enzymolysis, the enzymolysis time is prolonged, the pH condition is favorable for enzymolysis, the breakage of sugar peptide bonds under the alkalescent condition is also favorable without destroying chondroitin sulfate, the hydrolysis effect is improved, and impurities are better removed.
(3) The primary enzymolysis compound enzyme is a compound of pancreatin and lipase, the pancreatin is a compound of various enzymes (pancreatic amylase, pancreatic lipase, trypsin and the like), and the lipase is added to enhance the hydrolysis effect on glycogen, fat and protein in the raw materials, improve the enzymolysis efficiency, carry out primary separation and reduce the interference of subsequent separation. And the pig pancreas is adopted to replace industrial trypsin for enzymolysis, so that the method is simple and easy to obtain, the mixing of chemical impurities is reduced, and the high-value utilization of resources is realized.
(4) The pH and temperature conditions after the first-stage enzymolysis reaction are suitable for the second-stage enzymolysis reaction, the temperature of the reaction tank does not need to be repeatedly adjusted or acid and alkali are added to adjust the pH of the feed liquid, the structural damage of the chondroitin sulfate in the extraction process is avoided, the biological activity of the chondroitin sulfate is greatly reserved, and the operation steps are simplified. The addition of acid and alkali is reduced, so that the quality of chondroitin sulfate and by-product polypeptide is improved, and the production cost and the sewage treatment cost are reduced.
(5) According to the invention, according to the characteristic of complex chemical components of the raw materials, one or more of collagenase, neutral protease, papain and pepsin are selected for compounding, and the enzymes used have wide action peptide bonds, thereby being beneficial to obtaining better enzymolysis effect.
(6) In the extraction process, the continuous two-stage enzymolysis method is adopted, repeated filtering separation and temperature and pH adjustment are not needed in the production process, the reaction time is shortened, the extraction process is simplified, the continuous production is facilitated, and the method is suitable for industrial large-scale production.
(7) In the extraction process, the specific complex enzyme is selected for step-by-step two-stage enzymolysis according to the material characteristics, so that the enzymolysis efficiency is improved, the membrane pollution in the subsequent separation process is effectively reduced, the separation efficiency is improved, and the separation cost is saved.
(8) The invention adopts ultrafiltration technology to separate and purify the chondroitin sulfate feed liquid, is favorable for separating small molecular impurities and salt in the feed liquid, and effectively improves the purity of the chondroitin sulfate.
(9) The method recycles the permeated liquid after ultrafiltration through a nanofiltration process to obtain the collagen oligopeptide with low salt, high purity, white color and no peculiar smell, the molecular weight of the obtained oligopeptide is intensively distributed at 1000 Da, and the product is stable, high in bioactivity and high in availability. The nanofiltration equipment is simple and easy to operate, effectively recovers byproducts, and realizes high-value utilization of resources.
Drawings
FIG. 1 is a high performance liquid chromatography assay of chondroitin sulfate extract provided in the examples of the present invention.
FIG. 2 is a molecular weight distribution diagram of a protein peptide provided in an embodiment of the present invention.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.
Example 1:
500 g of cartilage and 500 g of trachea are weighed and washed 2 times with drinking water. Adding into a cooking pot, adding distilled water, soaking, heating to 100 deg.C, and cooking for 2 hr. Cooling to 45 deg.C, adding a small amount of sodium hydroxide to adjust pH to 8.50.
Adding 35 g of pig pancreas and 5 g of lipase into the cooking liquid, and slowly stirring with a stirring paddle, wherein the temperature is kept at 45-50 ℃, and reacting for 4 hours.
To the above feed solution, a mixed enzyme containing neutral protease and collagenase (the mass ratio of both enzymes in the mixed enzyme is 2: 1) was added in an amount of 0.20% by mass of the raw materials, and the reaction was carried out at 45 ℃ for 2.5 hours. (the pH is stable after the first-stage enzymolysis is finished, the second-stage enzymolysis reaction is suitable, and the pH is adjusted without adding acid and alkali), after the enzymolysis is finished, the feed liquid is heated to 80 ℃, the feed liquid is filtered after standing for 1.0 h, the filtrate is collected, the filter residue is washed by pure water with the volume of 1-2 times, and the filtrate and the washing liquid are combined.
And (3) carrying out ultrafiltration membrane filtration on the feed liquid, wherein the ultrafiltration process conditions are as follows: the operating pressure was 4 bar, the molecular weight cut-off was 5000 Da and the temperature was room temperature (25 ℃). Filtering with ultrafiltration membrane, and collecting membrane permeate and concentrated solution.
Adding 95% ethanol by mass into the ultrafiltration concentrated solution to make the ethanol concentration in the feed liquid reach 75%, standing for precipitation for 1.0 h, centrifuging, collecting the precipitate, and vacuum drying at 55 deg.C for 4.0 h to obtain chondroitin sulfate finished product. And (5) the ethanol enters a recovery tank for recycling.
Concentrating the ultrafiltration permeate through a nanofiltration membrane, wherein the nanofiltration process conditions are as follows: the pressure was 8 bar, the molecular weight cut-off was 100 Da and the temperature was room temperature (25 ℃). Concentrating the nanofiltration concentrated solution by a double-effect evaporation concentrator, and then carrying out spray drying to obtain a byproduct collagen peptide.
The yield of chondroitin sulfate was calculated according to the following formula, and the content of chondroitin sulfate was measured according to chromatography (FIG. 2).
Yield = (chondroitin sulfate product mass/weighed raw material mass (wet weight)). 100%
The detection shows that the yield of the chondroitin sulfate product is 28.8%. The chondroitin sulfate content was 99.3%. The collagen peptide yield was 10.5% and the ash content was 1.4%.
Compared with the traditional process, the process adopted in the embodiment is simple, and the reaction temperature and the pH do not need to be repeatedly adjusted. The continuous two-stage enzymolysis method can effectively improve the enzymolysis efficiency, reduce the interference of impurities such as fat and the like, has strong specificity, is beneficial to fully hydrolyzing protein, and improves the yield and the purity of chondroitin sulfate. The first-stage enzymolysis uses pig pancreas as a substitute of a chemical enzyme preparation, so that resources are reasonably utilized, and the production cost is reduced.
Example 2:
250 g of cartilage and 750 g of trachea are weighed and washed 2 times with drinking water. Adding into a cooking pot, adding distilled water, soaking, heating to 100 deg.C, and cooking for 2 hr. Cooling to 50 deg.C, adding a small amount of sodium hydroxide to adjust pH to 9.00.
30 g of pig pancreas and 10 g of lipase are added into the cooking feed liquid, and a stirring paddle is used for stirring at a low speed, the temperature is kept at 50 ℃, and the reaction is carried out for 2.0 h.
To the above-mentioned feed solution, a mixed enzyme containing collagenase and flavourzyme (the mass ratio of both enzymes in the mixed enzyme is 5: 3) was added in an amount of 0.40% by mass of the raw materials, and the reaction was carried out at 50 ℃ for 2.0 hours. (the pH is stable after the first-stage enzymolysis is finished, the second-stage enzymolysis reaction is suitable, and the pH is adjusted without adding acid and alkali additionally) after the enzymolysis is finished, the feed liquid is heated to 80 ℃, the feed liquid is filtered after standing for 1.0 h, the filtrate is collected, the filter residue is washed by pure water with the volume of 1-2 times, and the filtrate and the washing liquid are combined.
And (3) carrying out ultrafiltration membrane filtration on the feed liquid, wherein the ultrafiltration process conditions are as follows: the operating pressure was 8 bar, the molecular weight cut-off was 3000 Da and the temperature was room temperature (25 ℃). Filtering with ultrafiltration membrane, and collecting membrane permeate and concentrated solution.
Adding 95% ethanol by mass into the ultrafiltration concentrated solution to make the ethanol concentration in the feed liquid reach 75%, standing for precipitation for 1.0 h, centrifuging, collecting the precipitate, and vacuum drying at 65 deg.C for 4 h to obtain chondroitin sulfate finished product. And (5) the ethanol enters a recovery tank for recycling.
Concentrating the ultrafiltration permeate through a nanofiltration membrane, wherein the nanofiltration process conditions are as follows: the pressure was 12 bar, the molecular weight cut-off was 100 Da and the temperature was room temperature (25 ℃). Concentrating the nanofiltration concentrated solution by a double-effect evaporation concentrator, and then carrying out spray drying to obtain a byproduct collagen peptide.
The yield of chondroitin sulfate was calculated according to the following formula, and the content of chondroitin sulfate was measured according to chromatography.
Yield = (chondroitin sulfate product mass/weighed raw material mass (wet weight)). 100%
The detection shows that the yield of the chondroitin sulfate product is 29.7%. The chondroitin sulfate content was 99.4%. The collagen peptide yield was 8.5% and the ash content was 1.4%.
Compared with the traditional process, the process adopted in the embodiment is simple, the reaction temperature and the pH do not need to be repeatedly adjusted, the interference of impurities in the raw materials can be effectively reduced by secondary enzymolysis, the full hydrolysis of protein is facilitated, and the yield and the purity of the chondroitin sulfate are improved. The pig pancreas is taken as a substitute of a chemical enzyme preparation for the first-stage enzymolysis, so that resources are reasonably utilized, and the production cost is reduced.
Comparative example
This example uses a single enzymatic hydrolysis to prepare chondroitin sulfate. The specific implementation method comprises the following steps: 500 g of cartilage and 500 g of trachea are weighed and washed 2 times with drinking water. Adding into a cooking pot, adding distilled water, soaking, heating to 100 deg.C, and decocting in water for 4 hr. Cooling to 45-50 deg.C, adding sodium hydroxide to adjust pH to 8.00-9.00, and maintaining the temperature. Adding 30 g of pig pancreas according to 3.0 percent of the mass of the raw materials, and slowly stirring with a stirring paddle, keeping the temperature at 45-50 ℃, and reacting for 1.5 h. Then adding sodium hydroxide to adjust the pH value of the feed liquid to 10.0-11.0, and adding 3 g of alkaline protease according to the proportion of 0.30 percent of the raw material. The temperature is maintained at 45-50 ℃, the pH value of the feed liquid is maintained at 10.0-11.0, and the reaction lasts for 2.0 h. After enzymolysis, heating the feed liquid to 80 ℃, standing for 1.0 h, filtering, collecting filtrate, washing filter residue with 1-2 times of pure water, and combining the filtrate and water washing liquid. And (3) carrying out ultrafiltration membrane filtration on the feed liquid, wherein the ultrafiltration process conditions are as follows: the operating pressure was 5 bar, the molecular weight cut-off was 5000 Da and the temperature was room temperature (25 ℃). Filtering with ultrafiltration membrane, and collecting membrane permeate and concentrated solution. Adding 95% ethanol by mass into the ultrafiltration concentrated solution to make the ethanol concentration in the feed liquid reach 75%, standing for precipitation for 1.0 h, centrifuging, collecting the precipitate, and vacuum drying at 55 deg.C for 4 h to obtain chondroitin sulfate finished product. And (5) the ethanol enters a recovery tank for recycling. Concentrating the ultrafiltration permeate through a nanofiltration membrane, wherein the nanofiltration process conditions are as follows: the pressure was 10 bar, the molecular weight cut-off was 100 Da and the temperature was room temperature (25 ℃). Concentrating the nanofiltration concentrated solution by a double-effect evaporation concentrator, and then carrying out spray drying to obtain a byproduct collagen peptide.
The chondroitin sulfate product yield obtained in the example is 18.2% by detection. The chondroitin sulfate content is 85.6%. The collagen peptide yield was 5.5% and the ash content was 6.9%.
The continuous two-stage enzymolysis method disclosed by the patent is proved to be high in chondroitin sulfate extraction rate and purity, capable of obtaining low-ash high-quality collagen peptide by-products and high in operability, and beneficial to industrial large-scale production.
Claims (6)
1. A preparation method of chondroitin sulfate is characterized by comprising the following steps: the method comprises the steps of taking a mixture of animal cartilage and/or trachea as a production raw material, carrying out continuous two-stage composite enzymolysis, carrying out ultrafiltration separation, carrying out alcohol precipitation and centrifugal drying on the obtained ultrafiltration concentrated solution to obtain chondroitin sulfate, and treating the filtrate obtained by ultrafiltration by a nanofiltration membrane to prepare a collagen oligopeptide product.
2. The method for preparing chondroitin sulfate of claim 1, wherein the chondroitin sulfate production raw material is a mixture of chicken, duck, pig or cattle cartilage and trachea.
3. The method for preparing chondroitin sulfate according to claim 1, comprising the steps of:
(1) cooking and cooling: soaking cartilage and trachea in water in a cooking pot, heating to 80-100 deg.C, decocting for 1-3 hr to peel off muscle tissue on cartilage surface, and cooling the decoction to 45-55 deg.C;
(2) first-stage enzymolysis: adding sodium hydroxide to adjust the pH value of the feed liquid to be alkalescent, adding primary complex enzyme for enzymolysis, and primarily removing impurities;
(3) secondary enzymolysis: adding compound protease into the enzymolysis liquid in the step (2) for continuous secondary enzymolysis, specifically hydrolyzing protein, breaking sugar-peptide bonds, and further purifying;
(4) filtering, ultrafiltration and nanofiltration: and (4) filtering the enzymolysis liquid obtained in the step (3), performing ultrafiltration, concentration, alcohol precipitation and drying on the filtrate to obtain chondroitin sulfate, and performing nanofiltration, desalination, concentration and drying on the ultrafiltration permeate to obtain the collagen oligopeptide.
4. The method for preparing chondroitin sulfate as claimed in claim 3, wherein the weak base in step (2) is that the pH value of the feed liquid is 8.0-9.5, the primary complex enzyme is pancreatin and lipase, the addition amount of sodium hydroxide is 0.1% -1.0% of the mass of the raw materials, the addition amount of pancreatin and lipase is 1.0% -4.0% of the mass of the raw materials, the reaction temperature is 45-55 ℃, and the reaction time is 2.0-5.0 h.
5. The primary enzymolysis according to claim 3, wherein the trypsin preparation is replaced by porcine pancreas, and the amount of the added trypsin preparation is 1.0-4.0% of the mass of the raw materials.
6. The continuous secondary enzymolysis according to claim 3, characterized in that the complex enzyme in step (3) is a mixture of collagenase and one or more of neutral protease, papain, flavourzyme and pepsin in a mass ratio of 1:2:1-1:5:3, the reaction temperature is 45-55 ℃, and the addition amount is 0.20-0.50% of the mass of the raw materials.
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| CN112321750A (en) * | 2020-12-09 | 2021-02-05 | 美泰科技（青岛）股份有限公司 | Method for controlling content of chondroitin sulfate by adjusting conductivity of feed liquid |
| CN113637720A (en) * | 2021-09-10 | 2021-11-12 | 山东广昊生物制品有限公司 | Preparation method of desalted cartilage oligopeptide powder |
| CN113896811A (en) * | 2021-11-01 | 2022-01-07 | 湖南伍星生物科技有限公司 | Process for extracting chondroitin sulfate sodium and peptide in bovine trachea by air floatation method |
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| CN113637720A (en) * | 2021-09-10 | 2021-11-12 | 山东广昊生物制品有限公司 | Preparation method of desalted cartilage oligopeptide powder |
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Denomination of invention: A preparation method of chondroitin sulfate Effective date of registration: 20230529 Granted publication date: 20221227 Pledgee: Postal Savings Bank of China Limited Yinan County Sub-branch Pledgor: Shandong Guanghao Biological Products Co.,Ltd. Registration number: Y2023980041820 |