CN116463388A - Extraction method of hydrolyzed type II collagen peptide powder - Google Patents
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/10—Process efficiency
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Abstract
The invention provides an extraction method of hydrolyzed type II collagen peptide powder, which comprises the steps of pretreatment, degreasing treatment, enzymolysis, isoelectric precipitation and purification, wherein the method effectively improves the hydrolysis degree and the hydroxyproline extraction rate, and the isoelectric point of the prepared type II collagen is adjustable within the range of 4.4-6.2, so that the collagen has a triple helix structure, the prepared hydrolyzed type II collagen has higher protein and chondroitin sulfate (dry basis) contents and lower crude fat content, and provides a theoretical data basis for the next active function research of hydrolyzed type II collagen.
Description
Technical Field
The invention relates to the technical field of extraction of hydrolyzed collagen, in particular to an extraction method of hydrolyzed type II collagen peptide powder.
Background
Cartilage is mainly composed of chondrocytes and cell matrices, and has smooth, uniform and consistent surface. The cell matrix comprises water, proteoglycan and collagen fiber as main components. The collagen fibers in the cartilage are mainly type II collagen, are arranged in parallel in the matrix, and coarse and dense type II collagen fiber bundles are interwoven into a firm net frame, so that the integrity of the cartilage structure is maintained. Cartilage is unusual in that it contains a high proportion of glycine and proline residues. In particular 4-hydroxyproline and 5-hydroxylysine are very rare among other protein sources, which adds benefits to the intake of exogenous collagen.
The articular cartilage is mainly composed of collagen, proteoglycan, chondrocytes and the like, and the type II collagen is a main collagen type of cartilage, is a high-molecular long-chain fiber structure, so that the articular cartilage can form a collagen network in vivo and adsorbs various proteoglycan polymers. The network structure of collagen forms the framework of the articular cartilage, and other components such as proteoglycan and the like exist in the framework as a filler, so that the toughness and strength of the articular cartilage are improved. Collagen loss, loose network structure and even fracture, so that proteoglycan and other components are accelerated to be lost, articular cartilage is degenerated, and friction between bones is increased, thereby causing various joint diseases. With the increasing depth of collagen research, people find that increasing collagen intake can provide synthetic raw materials of articular cartilage, repair cartilage, promote regeneration of cartilage tissues, relieve joint pain, improve arthritis and other health-care functions, and has wider application prospects in the food industry.
The II collagen in the current market is formed by hydrolyzing fresh cartilage of animals, has large molecular weight, does not completely release nutrient components (II type collagen) in the cartilage, has poor water solubility, and is not easy to be absorbed by human bodies; meanwhile, the method is fundamentally different from the products, the hydrolysis type II collagen is formed by adopting fresh cartilage of animals after hydrolysis, the proportion of various active ingredients such as type II collagen, mucopolysaccharide, hyaluronic acid and the like in raw materials is kept purely natural and unchanged, but the method has poor hydrolysis effect, the small molecular weight proportion is small, the nutritional ingredients in the framework cannot be completely dissociated, and the human digestion and absorption rate is low, so that the utilization rate of the raw materials is reduced. In addition, the type II collagen prepared by the prior art is easy to destroy the triple helix structure collagen, most of the extracted collagen is a single peptide chain forming the collagen, or the triple helix structure collagen has low extraction rate, and the triple helix structure of the active collagen can not be reserved, and the extraction efficiency is improved.
Disclosure of Invention
In view of the above, the present invention provides an extraction method for hydrolyzed type II collagen peptide powder, which solves the above problems.
The technical scheme of the invention is realized as follows: an extraction method of hydrolyzed type II collagen peptide powder comprises the following steps: the method comprises the following steps:
s1, cartilage pretreatment: soaking cartilage periosteum in exudation agent at variable temperature, heating to 40-60deg.C for 20-40min, standing at normal temperature for 10-15min, heating again to 70-90deg.C for 16-35min, taking out, pulverizing to average particle diameter of 0.8-1.4mm to obtain cartilage powder, removing impurity protein, and removing telopeptide collagen;
s2, degreasing treatment: placing cartilage powder into glycidyl methacrylate or glycidyl acrylate, performing irradiation, degreasing and deodorization treatment in a cobalt 60-gamma ray irradiation field, and drying to obtain defatted cartilage powder with water content of 5-8%;
s3, enzymolysis: hydrolyzing the defatted cartilage powder with the aid of ultrasonic waves at a mass ratio of cartilage powder to complex enzyme of 5-9:1 at 28-44deg.C and pH of 7-9.0 for 3-6 hr under ultrasonic power of 400-600W, centrifuging to obtain supernatant to obtain clear collagen solution;
s4, isoelectric precipitation: regulating pH isoelectric point of the collagen clarified solution to 4.4-6.2, centrifuging, and recovering precipitate;
s5, purifying: adding water into the precipitate to adjust the solid matter concentration to 10-15%, adjusting pH to 6.3-7.5, ultrafiltering with 1-3kDa ultrafilter membrane at 3-6deg.C, nanofiltration of filtrate again, collecting the retentate, and spray drying to obtain hydrolyzed type II collagen peptide powder.
Further described, the exudation agent in the S1 is galactose glucuronic acid, rhamnose and acetic acid solution with the mass concentration of 10-30% in the mass ratio of 8-10:2-5:4-5.
Further illustratively, the mass to volume g/mL ratio of cartilage to exuding agent in S1 is 2-8:40.
Further, the temperature rising rate in the step S1 is 8-15 ℃/min.
Further, the cartilage in S1 is chicken breast cartilage, bovine tracheal cartilage, fish head bone cartilage, fish tail bone cartilage or fish ridge cartilage.
Further, the irradiation method in the step S2 is characterized in that the irradiation is carried out for three sections of time, each section of irradiation time is 30-45S, the interval is 15-20min, the irradiation quantity is 50-80kGy, the irradiation unevenness is less than 1.2, and the irradiation temperature is 18-25 ℃.
Further illustrated, the S3 composite decomposition enzyme is alkaline protease, trypsin and N-acetylmuramidase hydrolase with the mass ratio of 6-10:3-5:1-1.5, and the enzyme activities are all 5-9 multiplied by 10 5 cfu/g。
Further described, the inlet pressure of the S5 ultrafiltration is 0.1-0.3MPa, and the outlet pressure of the ultrafiltration is lower than the inlet pressure by 0.25-0.5kPa.
Further described, the S5 nanofiltration adopts an inorganic nanofiltration ceramic membrane with a molecular weight cutoff of 300-500Da, and amino acid and salt are removed by filtration.
Compared with the prior art, the invention has the beneficial effects that:
according to the method, the hydrolysis degree and the hydroxyproline extraction rate are effectively improved according to the steps of cartilage pretreatment, degreasing treatment, enzymolysis, isoelectric point precipitation and purification, and the specific exudation agent and the temperature change adjustment treatment are adopted, so that chondrocyte components in cartilage can be exuded, the raw materials are easy to be subjected to enzymolysis and extraction, and the enzymolysis product has small molecular weight and is concentrated; the glycidyl methacrylate or the glycidyl acrylate is added and irradiated, the irradiation rays can effectively kill microorganisms in degreasing, deodorization and degradation processes, and free radicals generated by molecular activation are generated after cartilage powder absorbs the irradiation rays in the irradiation process, and the free radicals act on macromolecular substances such as proteins to cause the breakage of molecular chains, so that the degradation of the macromolecular substances such as proteins is promoted, thereby forming micromolecular proteins, improving the degradation rate, improving the easy absorption degree of hydrolyzed II-type collagen, and promoting the oxidative removal of fat and fatty acid in the degreasing process, so that the yield of the collagen is improved;
the content of free amino acid in the enzymolysis product is low, the proportion of collagen with the molecular weight below 1000Da reaches 100%, the proportion of collagen peptide between 200 and 1000Da reaches more than 95%, the insoluble collagen is changed into water-soluble collagen by adopting a complex enzyme hydrolysis technology, so that the molecular weight of the product is reduced, the absorptivity of the product is effectively improved, the isoelectric point is changed, the isoelectric point of the prepared II-type collagen is adjustable within the range of 4.4 to 6.2, the collagen has a triple helix structure, no terminal peptide is contained, the collagen is in a single molecular state after the molecule is dissolved, the isoelectric point of the II-type collagen is focused, and the purification rate can be improved;
the protein and chondroitin sulfate (dry basis) contents in the hydrolyzed II-type collagen prepared by the invention are higher, and the crude fat content is lower, so that a theoretical data basis is provided for the next active function research on the hydrolyzed II-type collagen.
Detailed Description
In order to better understand the technical content of the present invention, the following provides specific examples to further illustrate the present invention.
The experimental methods used in the embodiment of the invention are conventional methods unless otherwise specified.
Materials, reagents, and the like used in the examples of the present invention are commercially available unless otherwise specified.
Example 1
An extraction method of hydrolyzed type II collagen peptide powder comprises the following steps:
s1, cartilage pretreatment: taking chicken breast cartilage to remove periosteum, placing the chicken breast cartilage into a exuding agent for variable temperature soaking, wherein the mass-to-volume g/mL ratio of cartilage to the exuding agent is 2:40, the exuding agent is galactose glucuronic acid, rhamnose and acetic acid solution with the mass concentration of 10% in a mass ratio of 8:2:4, heating to 40-60 ℃ for soaking for 20min, standing at normal temperature for 10-15min, heating to 70 ℃ for soaking for 16min again, heating to 8 ℃/min, taking out and crushing to obtain cartilage powder with the average particle size of 0.8 mm;
s2, degreasing treatment: placing cartilage powder into glycidyl methacrylate, performing irradiation, degreasing and deodorization treatment in a cobalt 60 gamma ray irradiation field, and drying to obtain defatted cartilage powder with water content of 5%, wherein the irradiation method comprises three sections of irradiation for 30s at intervals of 15min, the irradiation dose is 50kGy, the irradiation non-uniformity is less than 1.2, and the irradiation temperature is 18 ℃;
s3, enzymolysis: hydrolyzing the defatted cartilage powder with the aid of ultrasonic waves by using composite lytic enzyme, wherein the composite lytic enzyme comprises alkaline protease, trypsin and N-acetylmuramidase hydrolase with a mass ratio of 6:3:1, and the enzyme activities are all 5×10 5 cfu/g, wherein the mass ratio of cartilage powder to complex enzyme is 5:1, the enzymolysis temperature is 28 ℃, the enzymolysis is carried out for 3 hours under the condition of pH of 7.0, the ultrasonic power is 400W, and supernatant is centrifugally taken to obtain a collagen clear solution;
s4, isoelectric precipitation: regulating pH isoelectric point of the collagen clarified solution to 4.4, centrifuging, and recovering precipitate;
s5, purifying: adding water into the precipitate to adjust the solid matter concentration to 10%, adjusting the pH to 6.3, ultrafiltering at 3 ℃ by using a 1kDa ultrafiltration membrane, wherein the pressure of the liquid inlet of the ultrafiltration is 0.1MPa, the pressure of the liquid outlet of the ultrafiltration is lower than that of the liquid inlet by 0.25kPa, the filtrate is subjected to nanofiltration by adopting an inorganic nanofiltration ceramic membrane with the molecular weight cutoff of 300Da, and the trapped liquid is collected for spray drying to obtain the hydrolyzed type II collagen peptide powder.
Example 2
An extraction method of hydrolyzed type II collagen peptide powder comprises the following steps:
s1, cartilage pretreatment: taking chicken breast cartilage to remove periosteum, placing the chicken breast cartilage into a exuding agent for variable temperature soaking, wherein the mass-to-volume g/mL ratio of the cartilage to the exuding agent is 8:40, the exuding agent is galactose glucuronic acid, rhamnose and acetic acid solution with the mass concentration of 30% in a mass ratio of 10:5:5, heating to 60 ℃ for soaking 40min, standing at normal temperature for 15min, heating to 90 ℃ for soaking 35min again, heating to 15 ℃/min, taking out and crushing to obtain cartilage powder with the average particle size of 1.4 mm;
s2, degreasing treatment: placing cartilage powder into glycidyl acrylate, performing irradiation, degreasing and deodorization treatment in a cobalt 60 gamma ray irradiation field, and drying to obtain defatted cartilage powder with 8% of water content, wherein the irradiation method comprises three sections of irradiation time, each section of irradiation time is 45s, the interval is 20min, the irradiation dose is 80kGy, the irradiation non-uniformity is less than 1.2, and the irradiation temperature is 25 ℃;
s3, enzymolysis: hydrolyzing the defatted cartilage powder with the aid of ultrasonic waves by using composite lytic enzyme, wherein the composite lytic enzyme comprises alkaline protease, trypsin and N-acetylmuramidase hydrolase with a mass ratio of 10:5:1.5, and the enzyme activities are all 9×10 5 cfu/g, wherein the mass ratio of cartilage powder to complex enzyme is 9:1, the enzymolysis temperature is 44 ℃, the enzymolysis is carried out for 6 hours under the condition of pH value of 9.0, the ultrasonic power is 600W, and supernatant fluid is obtained by centrifugation, so that a collagen clear solution is obtained;
s4, isoelectric precipitation: regulating pH isoelectric point of the collagen clarified solution to 6.2, centrifuging, and recovering precipitate;
s5, purifying: adding water into the precipitate to adjust the solid matter concentration to 15%, adjusting the pH to 7.5, ultrafiltering at 6deg.C by using a 3kDa ultrafiltration membrane, wherein the pressure of the liquid inlet of the ultrafiltration is 0.3MPa, the pressure of the liquid outlet of the ultrafiltration is lower than that of the liquid inlet by 0.5kPa, nanofiltration is carried out on the filtrate by adopting an inorganic nanofiltration ceramic membrane with the molecular weight cutoff of 500Da, and the trapped liquid is collected for spray drying to obtain the hydrolyzed type II collagen peptide powder.
Example 3
An extraction method of hydrolyzed type II collagen peptide powder comprises the following steps:
s1, cartilage pretreatment: taking chicken breast cartilage to remove periosteum, placing the chicken breast cartilage into a exuding agent for variable temperature soaking, wherein the mass-to-volume g/mL ratio of the cartilage to the exuding agent is 5:40, the exuding agent is galactose glucuronic acid, rhamnose and acetic acid solution with the mass concentration of 20% in a mass ratio of 9:3:4, heating to 50 ℃ for 30min, standing at normal temperature for 12min, heating to 80 ℃ for 25min again, heating to 12 ℃/min, taking out and crushing to obtain cartilage powder with the average particle size of 1 mm;
s2, degreasing treatment: placing cartilage powder into glycidyl methacrylate, performing irradiation, degreasing and deodorization treatment in a cobalt 60 gamma ray irradiation field, and drying to obtain defatted cartilage powder with the moisture content of 7%, wherein the irradiation method comprises three sections of irradiation time, each section of irradiation time is 40s, the interval is 17min, the irradiation dose is 70kGy, the irradiation non-uniformity is less than 1.2, and the irradiation temperature is 22 ℃;
s3, enzymolysis: hydrolyzing the defatted cartilage powder with the aid of ultrasonic waves by using composite lytic enzyme, wherein the composite lytic enzyme comprises alkaline protease, trypsin and N-acetylmuramidase hydrolase with a mass ratio of 8:4:1.2, and the enzyme activities are all 7×10 5 cfu/g, wherein the mass ratio of the cartilage powder to the complex enzyme is 7:1, the enzymolysis temperature is 35 ℃, the enzymolysis is carried out for 5 hours under the condition of pH value of 8.0, the ultrasonic power is 500W, and supernatant fluid is obtained by centrifugation, so that a collagen clear solution is obtained;
s4, isoelectric precipitation: regulating pH isoelectric point of the collagen clarified solution to 5.0, centrifuging, and recovering precipitate;
s5, purifying: adding water into the precipitate to adjust the solid matter concentration to 12%, adjusting the pH to 6.0, ultrafiltering at 5 ℃ by using a 2kDa ultrafiltration membrane, wherein the pressure of the liquid inlet of the ultrafiltration is 0.2MPa, the pressure of the liquid outlet of the ultrafiltration is lower than that of the liquid inlet by 0.35kPa, the filtrate is subjected to nanofiltration by adopting an inorganic nanofiltration ceramic membrane with the molecular weight cutoff of 400Da, and the trapped liquid is collected for spray drying to obtain the hydrolyzed type II collagen peptide powder.
Example 4
The difference between the present example and example 3 is that the exudation agent is a solution of rhamnose with a mass-to-volume ratio g/mL of 3:4 and acetic acid with a mass concentration of 20%; in particular to an extraction method of hydrolyzed type II collagen peptide powder, which comprises the following steps:
s1, cartilage pretreatment: taking chicken breast cartilage to remove periosteum, placing the chicken breast cartilage into a exuding agent for variable temperature soaking, wherein the mass-to-volume g/mL ratio of cartilage to the exuding agent is 5:40, the exuding agent is rhamnose with the mass ratio of 3:4 and an acetic acid solution with the mass concentration of 20%, heating to 50 ℃ for soaking for 30min, standing at normal temperature for 12min, heating to 80 ℃ again for soaking for 25min, heating up to 12 ℃/min, taking out and crushing to obtain cartilage powder with the average particle diameter of 1 mm;
s2, degreasing treatment: placing cartilage powder into glycidyl acrylate, performing irradiation, degreasing and deodorization treatment in a cobalt 60 gamma ray irradiation field, and drying to obtain defatted cartilage powder with water content of 7%, wherein the irradiation method comprises three sections of irradiation time, each section of irradiation time is 40s, the interval is 17min, the irradiation dose is 70kGy, the irradiation non-uniformity is less than 1.2, and the irradiation temperature is 22 ℃;
s3, enzymolysis: hydrolyzing the defatted cartilage powder with the aid of ultrasonic waves by using composite lytic enzyme, wherein the composite lytic enzyme comprises alkaline protease, trypsin and N-acetylmuramidase hydrolase with a mass ratio of 8:4:1.2, and the enzyme activities are all 7×10 5 cfu/g, wherein the mass ratio of the cartilage powder to the complex enzyme is 7:1, the enzymolysis temperature is 35 ℃, the enzymolysis is carried out for 5 hours under the condition of pH value of 8.0, the ultrasonic power is 500W, and supernatant fluid is obtained by centrifugation, so that a collagen clear solution is obtained;
s4, isoelectric precipitation: regulating pH isoelectric point of the collagen clarified solution to 5.0, centrifuging, and recovering precipitate;
s5, purifying: adding water into the precipitate to adjust the solid matter concentration to 12%, adjusting the pH to 6.0, ultrafiltering at 5 ℃ by using a 2kDa ultrafiltration membrane, wherein the pressure of the liquid inlet of the ultrafiltration is 0.2MPa, the pressure of the liquid outlet of the ultrafiltration is lower than that of the liquid inlet by 0.35kPa, the filtrate is subjected to nanofiltration by adopting an inorganic nanofiltration ceramic membrane with the molecular weight cutoff of 400Da, and the trapped liquid is collected for spray drying to obtain the hydrolyzed type II collagen peptide powder.
Example 5
The difference between this embodiment and embodiment 3 is that the complex protease is alkaline protease, trypsin and pepsin with a mass ratio of 8:4:1.2, specifically an extraction method of hydrolyzed type II collagen peptide powder, comprising the following steps:
s1, cartilage pretreatment: taking chicken breast cartilage to remove periosteum, placing the chicken breast cartilage into a exuding agent for variable temperature soaking, wherein the mass-to-volume g/mL ratio of the cartilage to the exuding agent is 5:40, the exuding agent is galactose glucuronic acid, rhamnose and acetic acid solution with the mass concentration of 20% in a mass ratio of 9:3:4, heating to 50 ℃ for 30min, standing at normal temperature for 12min, heating to 80 ℃ for 25min again, heating to 12 ℃/min, taking out and crushing to obtain cartilage powder with the average particle size of 1 mm;
s2, degreasing treatment: placing cartilage powder into glycidyl acrylate, performing irradiation, degreasing and deodorization treatment in a cobalt 60 gamma ray irradiation field, and drying to obtain defatted cartilage powder with water content of 7%, wherein the irradiation method comprises three sections of irradiation time, each section of irradiation time is 40s, the interval is 17min, the irradiation dose is 70kGy, the irradiation non-uniformity is less than 1.2, and the irradiation temperature is 22 ℃;
s3, enzymolysis: hydrolyzing the defatted cartilage powder with the aid of ultrasonic waves by using composite lytic enzymes, wherein the composite lytic enzymes comprise alkaline protease, trypsin and pepsin with a mass ratio of 8:4:1.2, and the enzyme activities are all 7×10 5 cfu/g, wherein the mass ratio of the cartilage powder to the complex enzyme is 7:1, the enzymolysis temperature is 35 ℃, the enzymolysis is carried out for 5 hours under the condition of pH value of 8.0, the ultrasonic power is 500W, and supernatant fluid is obtained by centrifugation, so that a collagen clear solution is obtained;
s4, isoelectric precipitation: regulating pH isoelectric point of the collagen clarified solution to 5.0, centrifuging, and recovering precipitate;
s5, purifying: adding water into the precipitate to adjust the solid matter concentration to 12%, adjusting the pH to 6.0, ultrafiltering at 5 ℃ by using a 2kDa ultrafiltration membrane, wherein the pressure of the liquid inlet of the ultrafiltration is 0.2MPa, the pressure of the liquid outlet of the ultrafiltration is lower than that of the liquid inlet by 0.35kPa, the filtrate is subjected to nanofiltration by adopting an inorganic nanofiltration ceramic membrane with the molecular weight cutoff of 400Da, and the trapped liquid is collected for spray drying to obtain the hydrolyzed type II collagen peptide powder.
Comparative example 1
The difference between this comparative example and example 3 is that the exudation agent is not used for soaking in S1, specifically, an extraction method of hydrolyzed type II collagen peptide powder, comprising the following steps:
s1, cartilage pretreatment: removing periosteum from chicken breast cartilage, cleaning, and pulverizing to average particle diameter of 1mm to obtain cartilage powder;
s2, degreasing treatment: placing cartilage powder into glycidyl acrylate, performing irradiation, degreasing and deodorization treatment in a cobalt 60 gamma ray irradiation field, and drying to obtain defatted cartilage powder with water content of 7%, wherein the irradiation method comprises three sections of irradiation time, each section of irradiation time is 40s, the interval is 17min, the irradiation dose is 70kGy, the irradiation non-uniformity is less than 1.2, and the irradiation temperature is 22 ℃;
s3, enzymolysis: hydrolyzing defatted cartilage powder with compound lytic enzyme under assistance of ultrasonic wave, and complexingThe synthase and the catabolic enzyme are alkaline protease, trypsin and N-acetylmuramidase hydrolase with the mass ratio of 8:4:1.2, and the enzyme activities are all 7 multiplied by 10 5 cfu/g, wherein the mass ratio of the cartilage powder to the complex enzyme is 7:1, the enzymolysis temperature is 35 ℃, the enzymolysis is carried out for 5 hours under the condition of pH value of 8.0, the ultrasonic power is 500W, and supernatant fluid is obtained by centrifugation, so that a collagen clear solution is obtained;
s4, isoelectric precipitation: regulating pH isoelectric point of the collagen clarified solution to 5.0, centrifuging, and recovering precipitate;
s5, purifying: adding water into the precipitate to adjust the solid matter concentration to 12%, adjusting the pH to 6.0, ultrafiltering at 5 ℃ by using a 2kDa ultrafiltration membrane, wherein the pressure of the liquid inlet of the ultrafiltration is 0.2MPa, the pressure of the liquid outlet of the ultrafiltration is lower than that of the liquid inlet by 0.35kPa, the filtrate is subjected to nanofiltration by adopting an inorganic nanofiltration ceramic membrane with the molecular weight cutoff of 400Da, and the trapped liquid is collected for spray drying to obtain the hydrolyzed type II collagen peptide powder.
Comparative example 2
The difference between this comparative example and example 3 is that the temperature-variable soaking of S1 is replaced by isothermal soaking, and soaking is performed at 50 ℃ for 30min.
Comparative example 3
The difference between this comparative example and example 3 is that the degreasing treatment of S2 is carried out by immersing in 90 (wt)% ethanol solution for 3 hours, then placing in a shaking box and shaking at 25 ℃ and 200rpm for 3 hours, and the other steps are the same as in example 3.
Comparative example 4
The difference between this comparative example and example 3 is that the enzymolysis temperature of S3 is 50℃and the pH value is 8, and the enzymolysis is carried out for 4 hours.
Comparative example 5
The difference between this comparative example and example 3 is that isoelectric precipitation of S4 was not performed.
1. Hydrolysis type II collagen hydrolysis degree determination
The hydrolyzed proteins of examples 1 to 5 and comparative examples 1 to 3 were also mixed with an ninhydrin color former by ninhydrin colorimetric method to develop color, absorbance was measured at 570mm of an ultraviolet spectrophotometer, a hydrolysis degree standard curve was drawn, and the degree of hydrolysis of hydrolyzed type II collagen was measured according to the standard curve, and the degree of hydrolysis was calculated according to the following formula:
degree of hydrolysis of collagen type II (%) =millimoles of peptide bond cleaved per gram of protein after hydrolysis (mmol) -NH 2 Millimoles of peptide bond (mmol) per gram of raw protein ×100%
2. Determination of molecular weight
The molecular weight is measured by a high performance liquid chromatograph, and the chromatographic conditions are as follows:
chromatographic column: 300X 7.8mm;
mobile phase: acetonitrile, water and trifluoroacetic acid in the volume ratio of 40:60:0.05;
ultraviolet detection wavelength 220nm, flow rate 0.5mL/min, column temperature 30 ℃;
and calculating the relative molecular mass size and the distribution range of the sample according to the correction curve equation.
3. Collagen extraction yield
The hydroxyproline extraction rate was measured on the hydrolyzed type II collagen produced in examples 1 to 5 and comparative examples 1 to 4 of the present invention using the hydroxyproline extraction rate as an evaluation index.
The above measurement results are shown in the following table:
as can be seen from the table, compared with comparative examples 1 and 2, the specific exuding agent and the temperature change adjustment treatment are adopted, so that chondrocyte components in cartilage can be exuded, raw materials are easy to be subjected to enzymolysis and extraction, the molecular weight of enzymolysis products is small and concentrated, the free amino acid content of examples 1-3 is low, and the proportion of hydrolyzed collagen with the molecular weight less than 1000Da reaches 100%; compared with comparative example 3, the invention improves the traditional degreasing method, adds glycidyl methacrylate or glycidyl acrylate and irradiates, the irradiation rays can effectively kill microorganisms in degreasing, deodorization and degradation processes, and the cartilage powder absorbs the irradiation rays and then generates free radicals generated by molecular activation, and the free radicals act on high molecular substances such as proteins to cause the breakage of molecular chains, so as to promote the degradation of the high molecular substances such as proteins, thereby forming small molecular proteins, improving the degradation rate, improving the easy absorption degree of hydrolyzed type II collagen, promoting the oxidative removal of fat and fatty acid in the degreasing process, and improving the yield of collagen; compared with comparative example 4, the temperature of enzymolysis is raised to 50 ℃, and the too high temperature can hydrolyze the type II collagen into short peptide, oligopeptide or free amino acid in the enzymolysis process, which is unfavorable for enzymolysis; compared with comparative example 5, the isoelectric point of the prepared type II collagen is adjustable within the range of 4.4-6.2, so that the collagen has a triple helix structure, does not contain terminal peptide and is in a single molecular state after the molecule is dissolved, the isoelectric point of the type II collagen is focused, and the purification rate can be improved.
4. Basic component measurement
The obtained essential components of examples 1-3 were measured as follows:
(1) Moisture content: GB 5009.3-2016 direct drying method for determination of moisture in food safety national Standard food;
(2) Protein: GB 5009.5-2016 Kai's nitrogen determination method of determination of protein in food safety national Standard food;
(3) Ash content: GB 5009.4-2016 ashing method for measuring ash content in food safety national standard food;
(4) Crude fat: GB 5009.6-2016 determination of fat in food safety national Standard food by acid hydrolysis.
(5) Chondroitin sulfate: GB/T20365-2006 determination of chondroitin sulfate and glucosamine hydrochloride content by liquid chromatography.
The measurement results were as follows:
from the table, the protein and chondroitin sulfate (dry basis) contents in the hydrolyzed II-type collagen prepared by the invention are higher, the crude fat content is lower, and a theoretical data basis is provided for the next active function research on the hydrolyzed II-type collagen.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (9)
1. An extraction method of hydrolyzed type II collagen peptide powder is characterized in that: the method comprises the following steps:
s1, cartilage pretreatment: soaking cartilage periosteum in exudation agent at variable temperature, heating to 40-60deg.C for 20-40min, standing at normal temperature for 10-15min, heating to 70-90deg.C again for 16-35min, taking out, and pulverizing to average particle diameter of 0.8-1.4mm to obtain cartilage powder;
s2, degreasing treatment: placing cartilage powder into glycidyl methacrylate or glycidyl acrylate, performing irradiation, degreasing and deodorization treatment in a cobalt 60 gamma ray irradiation field, and drying to obtain defatted cartilage powder with water content of 5-8%;
s3, enzymolysis: hydrolyzing the defatted cartilage powder with the aid of ultrasonic waves at a mass ratio of cartilage powder to complex enzyme of 5-9:1 at 28-44deg.C and pH of 7-9.0 for 3-6 hr under ultrasonic power of 400-600W, centrifuging to obtain supernatant to obtain clear collagen solution;
s4, isoelectric precipitation: regulating pH isoelectric point of the collagen clarified solution to 4.4-6.2, centrifuging, and recovering precipitate;
s5, purifying: adding water into the precipitate to adjust the solid matter concentration to 10-15%, adjusting pH to 6.3-7.5, ultrafiltering with 1-3kDa ultrafilter membrane at 3-6deg.C, nanofiltration of filtrate again, collecting the retentate, and spray drying to obtain hydrolyzed type II collagen peptide powder.
2. The method for extracting hydrolyzed type II collagen peptide powder according to claim 1, wherein: the exudation agent in the S1 is galactose glucuronic acid, rhamnose and acetic acid solution with the mass concentration of 10-30% in the mass-volume ratio of g/mL of 8-10:2-5:4-5.
3. The method for extracting hydrolyzed type II collagen peptide powder according to claim 1, wherein: the mass volume g/mL ratio of the cartilage and the exudation agent in the S1 is 2-8:40.
4. The method for extracting hydrolyzed type II collagen peptide powder according to claim 1, wherein: the temperature rising rate in the step S1 is 8-15 ℃/min.
5. The method for extracting hydrolyzed type II collagen peptide powder according to claim 1, wherein: the cartilage in the S1 is chicken breast cartilage, ox trachea cartilage, fish head bone cartilage, fish tail bone cartilage or fish ridge cartilage.
6. The method for extracting hydrolyzed type II collagen peptide powder according to claim 1, wherein: the irradiation method in the step S2 is carried out in three sections of time, each section of irradiation time is 30-45S, the interval is 15-20min, the irradiation quantity is 50-80kGy, the irradiation unevenness is less than 1.2, and the irradiation temperature is 18-25 ℃.
7. The method for extracting hydrolyzed type II collagen peptide powder according to claim 1, wherein: the S3 complex decomposing enzyme is alkaline protease, trypsin and N-acetylmuramidase hydrolase with the mass ratio of 6-10:3-5:1-1.5, and the enzyme activities are 5-9 multiplied by 10 5 cfu/g。
8. The method for extracting hydrolyzed type II collagen peptide powder according to claim 1, wherein: the pressure of the liquid inlet of the S5 ultrafiltration is 0.1-0.3MPa, and the pressure of the liquid outlet of the ultrafiltration is lower than the pressure of the liquid inlet by 0.25-0.5kPa.
9. The method for extracting hydrolyzed type II collagen peptide powder according to claim 1, wherein: the S5 nanofiltration adopts an inorganic nanofiltration ceramic membrane with the molecular weight cut-off of 300-500 Da.
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