CN106916870A - A kind of preparation method of the cartilage extracts of the collagen types of II containing non denatured - Google Patents
A kind of preparation method of the cartilage extracts of the collagen types of II containing non denatured Download PDFInfo
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- 102000008186 Collagen Human genes 0.000 title claims abstract description 49
- 108010035532 Collagen Proteins 0.000 title claims abstract description 49
- 229920001436 collagen Polymers 0.000 title claims abstract description 49
- 239000000284 extract Substances 0.000 title claims abstract description 39
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- 238000000034 method Methods 0.000 claims abstract description 28
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 28
- 230000001954 sterilising effect Effects 0.000 claims abstract description 26
- 238000001035 drying Methods 0.000 claims abstract description 25
- 238000005238 degreasing Methods 0.000 claims abstract description 22
- 239000007788 liquid Substances 0.000 claims abstract description 22
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims abstract description 18
- 235000007119 Ananas comosus Nutrition 0.000 claims abstract description 16
- 244000189799 Asimina triloba Species 0.000 claims abstract description 14
- 235000006264 Asimina triloba Nutrition 0.000 claims abstract description 14
- 235000009467 Carica papaya Nutrition 0.000 claims abstract description 14
- 102000004190 Enzymes Human genes 0.000 claims abstract description 14
- 108090000790 Enzymes Proteins 0.000 claims abstract description 14
- 229940088598 enzyme Drugs 0.000 claims abstract description 14
- 239000002002 slurry Substances 0.000 claims abstract description 10
- 238000001914 filtration Methods 0.000 claims abstract description 8
- 230000007062 hydrolysis Effects 0.000 claims abstract description 7
- 238000006460 hydrolysis reaction Methods 0.000 claims abstract description 7
- 238000007670 refining Methods 0.000 claims abstract description 6
- 102000005741 Metalloproteases Human genes 0.000 claims abstract description 4
- 108010006035 Metalloproteases Proteins 0.000 claims abstract description 4
- 102000057297 Pepsin A Human genes 0.000 claims abstract description 4
- 108090000284 Pepsin A Proteins 0.000 claims abstract description 4
- 108091005804 Peptidases Proteins 0.000 claims abstract description 4
- 239000004365 Protease Substances 0.000 claims abstract description 4
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims abstract description 4
- 239000003513 alkali Substances 0.000 claims abstract description 4
- 229940111202 pepsin Drugs 0.000 claims abstract description 4
- 102000005158 Subtilisins Human genes 0.000 claims abstract description 3
- 108010056079 Subtilisins Proteins 0.000 claims abstract description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 25
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 18
- 239000012530 fluid Substances 0.000 claims description 16
- 241000234671 Ananas Species 0.000 claims description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 239000000463 material Substances 0.000 claims description 10
- 238000005237 degreasing agent Methods 0.000 claims description 8
- 239000013527 degreasing agent Substances 0.000 claims description 8
- 238000000227 grinding Methods 0.000 claims description 8
- 239000013618 particulate matter Substances 0.000 claims description 8
- 238000004108 freeze drying Methods 0.000 claims description 7
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 230000000249 desinfective effect Effects 0.000 claims description 4
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 claims description 4
- 229940033663 thimerosal Drugs 0.000 claims description 4
- 238000004140 cleaning Methods 0.000 claims description 3
- 238000000643 oven drying Methods 0.000 claims description 3
- 101000905241 Mus musculus Heart- and neural crest derivatives-expressed protein 1 Proteins 0.000 claims description 2
- 239000005708 Sodium hypochlorite Substances 0.000 claims description 2
- 238000007598 dipping method Methods 0.000 claims description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Substances [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 2
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 2
- 230000009967 tasteless effect Effects 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims 1
- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 abstract description 8
- 229920001287 Chondroitin sulfate Polymers 0.000 abstract description 8
- 229940059329 chondroitin sulfate Drugs 0.000 abstract description 8
- 238000011084 recovery Methods 0.000 abstract description 3
- 244000099147 Ananas comosus Species 0.000 abstract 1
- 238000011031 large-scale manufacturing process Methods 0.000 abstract 1
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- 102000002734 Collagen Type VI Human genes 0.000 description 2
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- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 230000009182 swimming Effects 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
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- 230000005784 autoimmunity Effects 0.000 description 1
- 239000002585 base Substances 0.000 description 1
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- 108090000623 proteins and genes Proteins 0.000 description 1
- 201000003068 rheumatic fever Diseases 0.000 description 1
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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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- 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
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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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 provides a kind of preparation method of the cartilage extracts of the collagen types of II containing non denatured, it is characterised in that the method is comprised the following steps successively：Degreasing, sterilization, homogenate, enzymolysis, filtering, drying；Wherein, in the enzymolysis step：The pH of the slurry obtained in refining step is adjusted to 2.5~8.5, add 0.001% the 2% of cartilage weight enzyme, the pawpaw and/or pineapple low temperature for adding the 1/20~1/500 of cartilage weight filter the clear liquid of acquisition after squeezing the juice, digest 12~48h, and hydrolysis temperature is 25 50 DEG C；The enzyme is preferably one or more in pepsin, subtilopeptidase A, alkali protease, metalloproteinases.Cartilage extracts purity that the preparation method that the present invention is provided is obtained is high, recovery rate is high, easy large-scale production and rich in non denatured typeⅡ Collagen and chondroitin sulfate.
Description
Technical field
The present invention relates to a kind of preparation method of cartilage extracts, more particularly to a kind of typeⅡ Collagen containing non denatured
Cartilage extracts preparation method.
Background technology
Collagen is the important part of the tissues such as skin, bone and joint, is metazoan extracellular
The main component of matrix.27 kinds of different types of collagens are issued from mammal at present, wherein identical by three
α chains composition typeⅡ Collagen be primarily present in cartilaginous tissue and vitreum in, therefore typeⅡ Collagen regarded
It is a kind of structural functionality element of cartilage.
Current market is generally that, by the collagen after hydrolysis, its main component is polypeptide, molecule in sale collagen
Amount is distributed in the range of 200-20000Da.Although these polypeptides also have certain holding moisture of skin, enhancing bone density and
Strengthen immunity etc. is acted on, but has no the effect for the rheumatic arthritis that prevention and treatment cause by the change of autoimmunity function,
Also without the effect of prevention and treatment DA.
Non denatured typeⅡ Collagen is the albumen with complete triple-helix structure, when human body takes in the Collagen Type VI of non denatured II
After albumen, the immune tolerance of vivo immunization cell especially lymphocyte can improve, and be due to taking the photograph the reason for immune tolerance
Contain antigenic determinant in the typeⅡ Collagen for entering.Antigenic determinant is a special structural units of antigen, can be resisted
Body recognizes and binds that, when antibody is tied to large biological molecule, antibody can not recognize whole organism or material, but can recognize
Mark is simultaneously bound.Active antigens decision base is bundled in aggregate nodules and is stored in small intestine as a tissue, goes stimulation to exempt from
Epidemic disease cell, information transmission molecule such as cell factor and participate in Proliferation, Differentiation chemotactic factor (CF) and suppress cell secretion disinthibiting inflammation.
Also there is the extraction process of some non denatured typeⅡ Collagens both at home and abroad at present, but or its raw material do not removed
It is miscellaneous to wait pretreatment or do not processed foreign protein that recovery rate is low or preservation condition is restricted.
The content of the invention
One of the object of the invention is at least to overcome the one kind in drawbacks described above, there is provided a kind of new containing the Collagen Type VI of non denatured II
The preparation method of the cartilage extracts of albumen.
Therefore, the invention provides a kind of preparation method of the cartilage extracts of the collagen types of II containing non denatured, it is special
Levy and be, the method is comprised the following steps successively：Degreasing, sterilization, homogenate, enzymolysis, filtering, drying；Wherein, in the enzymolysis step
In rapid：The pH of the slurry obtained in refining step is adjusted to 2.5~8.5, adds the 0.001%-2%'s of cartilage weight
Enzyme, the pawpaw and/or pineapple low temperature for adding the 1/20~1/500 of cartilage weight filters the clear liquid of acquisition after squeezing the juice, and enzymolysis 12~
48h, hydrolysis temperature is 25-50 DEG C.
The present invention provide preparation method obtain cartilage extracts purity it is high, recovery rate is high, easy to maintain, easy scale metaplasia
Produce, and rich in non denatured typeⅡ Collagen and chondroitin sulfate.
Brief description of the drawings book
Fig. 1 is the transmission electron microscope picture of the non denatured II collagen types that embodiment 1 is obtained.
Fig. 2 represents the result of the western traces of the II collagen types that embodiment 1 and 2 is obtained, wherein left side swimming lane is
The II collagen types that embodiment 2 is obtained, right lanes are the II collagen types that embodiment 1 is obtained.
Specific embodiment
The invention provides a kind of preparation method of the cartilage extracts of the collagen types of II containing non denatured, its feature exists
In the method is comprised the following steps successively：Degreasing, sterilization, homogenate, enzymolysis, filtering, drying；Wherein, in the enzymolysis step
In：The pH of the slurry obtained in refining step is adjusted to 2.5~8.5, adds the 0.001%-2%'s of cartilage weight
Enzyme, the pawpaw and/or pineapple low temperature for adding the 1/20~1/500 of cartilage weight filters the clear liquid of acquisition after squeezing the juice, and enzymolysis 12~
48h, hydrolysis temperature is 25-50 DEG C.
Present inventor has been surprisingly found that, digested under above-mentioned enzymatic hydrolysis condition, is obtained in that the non-of higher concentration
Denatured II-type collagen.
In enzymolysis step, the enzyme can use any enzyme used by this area, but in one kind preferred embodiment
In, the enzyme is preferably one or more in pepsin, subtilopeptidase A, alkali protease, metalloproteinases；Most
Preferably pepsin.
In a preferred embodiment, the consumption of institute's enzyme is the 0.5-2% of cartilage weight, and pawpaw and/or pineapple are low
Temperature filters the clear liquid of acquisition consumption after squeezing the juice is the 1/20-1/50 of cartilage weight, and enzymolysis time is 12~20h, hydrolysis temperature
25~40 DEG C.
In another preferred embodiment, obtain mixed is filtered after the clear liquid of addition is pawpaw and pineapple low temperature is squeezed the juice
Clear liquid is closed, wherein, the weight ratio of pawpaw and pineapple is 1-10：1, preferably 3-6：1.Under the optimum condition, the cartilage of acquisition
Non denatured typeⅡ Collagen concentration is higher in extract.
In a preferred embodiment, crushed after the drying step, wherein,
Degreasing：Cartilage fourth shape thing 0.2h~6h is soaked with certain density degreasing agent, is then cleaned to cleaning with water
The pH of water to 5.5~8.5；
Sterilization：With the cartilage fourth shape thing 1~3 time after finite concentration medicining liquid dipping degreasing, then cleaned to cartilage with water
Fourth shape thing is tasteless；
Homogenate：Cartilage fourth shape thing after sterilization is carried out into homogenized, the granularity of cartilage is 50~300 mesh after homogenate；
Filtering：Filtered using 5~100 eye mesh screens, large particulate matter in removal solution collects filtered fluid；
Dry：The filtered fluid that will be collected by filtration carries out low temperature drying, and drying temperature is not higher than 30 DEG C, and cartilage is obtained after drying
Extract；
Crush：Dried cartilage extracts carry out ultramicro grinding below 4 DEG C, the mesh number control of crushed material 100 mesh-
500 mesh.
In another preferred embodiment, the degreasing agent is NaOH solution, KOH solution, Na2CO3Solution, NaCl
One or more in solution, KCl solution.
In another preferred embodiment, the concentration of the degreasing agent is 0.01~3 weight %, the degreasing agent with
The weight ratio of cartilage is 1~10:1
In another preferred embodiment, the thimerosal used in sterilisation step is sodium hypochlorite and/or dioxygen
Water, in 100ppm~2000ppm, disinfecting time is controlled in 0.2h~12h for the concentration control of thimerosal in disinfecting process.
In another preferred embodiment, refining step is carried out below 37 DEG C.
In another preferred embodiment, low temperature drying method is roller drying method, low temperature fourdrinier wire oven drying method, freezing
Seasoning.
In another preferred embodiment, when the step of having precooling, precooling temperature is not higher than -10 DEG C, after drying
The water content control of cartilage extracts is below 6%.
In another preferred embodiment, the preparation method is pre-processed before degreasing, purer to obtain
Net collagen.The pretreatment can be carried out according to prior art, and the preferably pretreatment includes：Deposited on removal cartilage raw material
Fat, manadesma, the meat of residual or the non-cartilaginous material such as red bone, be then cut into the fourth shape thing of 2mm~20mm.
In another preferred embodiment, the raw material cartilage for using can be chicken cartilage, duck cartilage, ox cartilage or its
Mixture.
Embodiment 1
(1) pre-process：Chick sternal cartilage 3.5kg is chosen, chicken, manadesma and red bone after defrosting on removing cartilage, Ran Houli
The fourth shape of 3*3mm is cut into dicer.
(2) degreasing：With the Na of 0.01% concentration eventually2CO3With the mixed defatting agent solution 7kg of 2%NaCl, normal temperature degreasing 4h,
Cleaned to pH6.5 with purified water after degreasing draining.
(3) sterilize：Plus concentration 400ppm liquor natrii hypochloritises start sterilization, sterilize 2 times, each 12h, sterilization is used after terminating
Purified water is cleaned to without sterilization also smell and detected without chlorine residue, and pH is controlled in 6.5-7.0.
(4) it is homogenized：The cartilage fourth shape thing of step (3) treatment is added into 6kg purified waters, cartilage is worn into 100- with colloid mill
200 mesh.
(5) digest：The slurries pH that step (4) is obtained is adjusted to 2.5,70 grams of pepsins are added, adds 60 low temperature to squeeze
Filtered after juice acquisition fresh pawpaw clear liquid and 10g low temperature squeeze the juice after filter the fresh pineapple clear liquid of acquisition, in temperature, 25 DEG C are entered
Row enzymolysis 20h.
(6) filter：Filtered using 5 eye mesh screens, large particulate matter in removal solution collects filtered fluid.
(7) dry：The filtered fluid that step (6) is collected is carried out into vacuum freeze drying, precooling temperature is not higher than -40 DEG C, done
Dry process finishing temperature control obtains cartilage extracts at 30 DEG C after freezing.
(8) crush：Cartilage extracts after freeze-drying carry out ultramicro grinding, the mesh number control of crushed material below 4 DEG C
In 200 mesh, dried frozen aquatic products is obtained.
Use the method for GB5009.5 to detect that collagen content is 55.2%, chondroitin sulfate is detected using GB/T20365
Cellulose content is 7.9%, uses ELISA method to detect (kit supplier is Shanghai Jiang Lai bio tech ltd) non denatured
TypeⅡ Collagen content is 11.3%.The form of non denatured typeⅡ Collagen is detected using transmission electron microscope, as a result such as Fig. 1 institutes
Show.II collagen types are detected using western traces, as a result as shown in Fig. 2 right lanes.
Embodiment 2
(1) pre-process：Fresh duck chest cartilage 20kg is chosen, duck, manadesma and red bone after defrosting on removing cartilage, then
The fourth shape of about 2mm*2mm is cut into using dicer
(2) degreasing：With the KCl solution 200kg of final concentration of 0.01% NaOH and 0.9%, normal temperature degreasing 6h, Ran Houyong
Purified water cleans pH6.0.
(3) sterilize：Plus concentration 500ppm liquor natrii hypochloritises (hydrogen peroxide containing 100ppm) start sterilization, sterilize 3 times, often
Secondary 6h, sterilization is cleaned to without sterilization also smell with purified water after terminating and detected without chlorine residue, and pH is controlled in 6.5-7.0.
(4) it is homogenized：To the purified water that 40kg is added in the cartilage fourth shape thing that step (3) is processed, cartilage is ground with colloid mill
Into about 100-200 mesh.
(5) digest：The slurries pH that step (4) is obtained is adjusted to 8.5, plus 0.2 gram of alkali protease, 1kg low temperature is added
The fresh pineapple clear liquid of acquisition is filtered after squeezing the juice, in temperature 50 C, enzymolysis 12h is carried out.
(6) filter：Filtered using 100 eye mesh screens, large particulate matter in removal solution collects filtered fluid.
(7) dry：Dried by low temperature fourdrinier wire oven drying method after the filtered fluid cooling shaping that step (6) is collected, precooling temperature
- 10 DEG C of degree, drying process finishing temperature is controlled at 30 DEG C, and cartilage extracts are obtained after drying.
(8) crush：Cartilage extracts after freeze-drying carry out ultramicro grinding, the mesh number control of crushed material below 4 DEG C
In 180 mesh or so, the dried frozen aquatic products of certain dry weight is obtained.
Use the method for GB5009.5 to detect that collagen content is 45.6%, chondroitin sulfate is detected using GB/T20365
Cellulose content is 4.8%, uses ELISA method to detect (kit supplier is Shanghai Jiang Lai bio tech ltd) non denatured
TypeⅡ Collagen content is 3.9%.II collagen types are detected using western traces, as a result such as Fig. 2 left sides swimming lane institute
Show.
Embodiment 3
(1) pre-process：Chick sternal cartilage 20kg is chosen, then chicken, manadesma and red bone after defrosting on removing cartilage utilize
Dicer is cut into the fourth shape of 3*3mm.
(2) degreasing：With the solution 100kg of whole 0.2%NaOH and 0.6%NaCl, normal temperature degreasing 0.2h, purified water is then used
Cleaning pH6.0.
(3) sterilize：Plus concentration 2000ppm liquor natrii hypochloritises start sterilization, sterilize 1 time, each 0.2h, after sterilization terminates
Cleaned with purified water to without sterilization also smell and detected without chlorine residue, pH is controlled in 6.5-7.0.
(4) it is homogenized：To the purified water that 50kg is added in the cartilage fourth shape thing that step (3) is processed, cartilage is ground with colloid mill
Into 100-200 mesh.
(5) digest：The slurries pH that step (4) is obtained is adjusted to 7.0, plus 20g subtilopeptidase As, adds 200g low
Temperature filtered after squeezing the juice acquisition fresh pawpaw clear liquid and 20g low temperature squeeze the juice after filter the fresh pineapple clear liquid of acquisition, in temperature 40
DEG C carry out enzymolysis 24h.
(6) filter：Filtered using 50 eye mesh screens, large particulate matter in removal solution collects filtered fluid.
(7) dry：The filtered fluid that step (6) is collected is carried out into vacuum freeze drying, precooling temperature is not higher than -30 DEG C, done
Dry process finishing temperature control obtains cartilage extracts at 20 DEG C after freezing.
(8) crush：Cartilage extracts after freeze-drying carry out ultramicro grinding, the mesh number control of crushed material below 4 DEG C
In 200 mesh, dried frozen aquatic products is obtained.
Use the method for GB5009.5 to detect that collagen content is 53.6%, chondroitin sulfate is detected using GB/T20365
Cellulose content is 9.2%, uses ELISA method to detect (kit supplier is Shanghai Jiang Lai bio tech ltd) non denatured
TypeⅡ Collagen content is 10.3%.
Embodiment 4
(1) pre-process：Chick sternal cartilage 70kg is chosen, then chicken, manadesma and red bone after defrosting on removing cartilage utilize
Dicer is cut into the fourth shape of 3*3mm.
(2) degreasing：With the solution 140kg of whole 0.15%KOH and 0.3%KCl, normal temperature degreasing 2h is then clear with purified water
Wash pH6.0.
(3) sterilize：Plus concentration 100ppm liquor natrii hypochloritises (hydrogen peroxide containing 1000ppm) starts sterilization, sterilize 2 times, often
Secondary 0.5h, sterilization is cleaned to without sterilization also smell with purified water after terminating and detected without chlorine residue, and pH is controlled in 6.5-7.0.
(4) it is homogenized：To the purified water that 140kg is added in the cartilage fourth shape thing that step (3) is processed, cartilage is ground with colloid mill
Into 100-200 mesh.
(5) digest：The slurries pH that step (4) is obtained is adjusted to 7.5, plus 21g metalloproteinases, adds 700g low temperature to squeeze
The fresh pineapple clear liquid that acquisition is filtered after juice carries out enzyme 16h for 40 DEG C in temperature.
(6) filter：Filtered using 50 eye mesh screens, large particulate matter in removal solution collects filtered fluid.
(7) dry：The filtered fluid that step (6) receives disaggregation is carried out into vacuum freeze drying, precooling temperature is not higher than -30 DEG C,
Drying process finishing temperature is controlled at 20 DEG C, and cartilage extracts are obtained after freezing.
(8) crush：Cartilage extracts after freeze-drying carry out ultramicro grinding, the mesh number control of crushed material below 4 DEG C
In 200 mesh, dried frozen aquatic products is obtained.
Use the method for GB5009.5 to detect that collagen content is 48.3%, chondroitin sulfate is detected using GB/T20365
Cellulose content is 6.8%, uses ELISA method to detect (kit supplier is Shanghai Jiang Lai bio tech ltd) non denatured
TypeⅡ Collagen content is 8.3%.
Embodiment 5
1) pre-process：Chick sternal cartilage 30kg is chosen, then chicken, manadesma and red bone after defrosting on removing cartilage utilize
Dicer is cut into the fourth shape of 5*5mm.
(2) degreasing：With the solution 90kg of whole 0.05%NaOH and 0.1%KCl, normal temperature degreasing 1h is then clear with purified water
Wash pH6.0.
(3) sterilize：Plus concentration 300ppm liquor natrii hypochloritises 60kg starts sterilization, sterilize 2 times, each 8h, sterilization terminates
Cleaned with purified water afterwards to without sterilization also smell and detected without chlorine residue, pH is controlled in 6.5-7.0.
(4) it is homogenized：To the purified water that 70kg is added in the cartilage fourth shape thing that step (3) is processed, cartilage is ground with colloid mill
Into 100-200 mesh.
(5) digest：The slurries pH that step (4) is obtained is adjusted to 3.0, plus 150g pepsins, adds 135g low temperature to squeeze
Filtered after juice acquisition fresh pawpaw clear liquid and 45g low temperature squeeze the juice after filter the fresh pineapple clear liquid of acquisition, in temperature, 25 DEG C are entered
Row enzyme 16h.
(6) filter：Filtered using 50 eye mesh screens, large particulate matter in removal solution collects filtered fluid.
(7) dry：The filtered fluid that step (6) is collected is carried out into vacuum freeze drying, precooling temperature is not higher than -30 DEG C, done
Dry process finishing temperature control obtains cartilage extracts at 25 DEG C after freezing.
(8) crush：Cartilage extracts after freeze-drying carry out ultramicro grinding, the mesh number control of crushed material below 4 DEG C
In 200 mesh, dried frozen aquatic products is obtained.
Use the method for GB5009.5 to detect that collagen content is 57.3%, chondroitin sulfate is detected using GB/T20365
Cellulose content is 12.3%, uses ELISA method to detect (kit supplier is Shanghai Jiang Lai bio tech ltd) non-change
Property typeⅡ Collagen content be 12.6%.
Embodiment 6
1) pre-process：Ox cartilage 5kg is chosen, beef, manadesma and red bone on cartilage are removed after defrosting, then using dicing
Machine is cut into the fourth shape of 5*5mm.
(2) degreasing：With the solution 20kg of whole 0.3%NaOH and 1%NaCl, normal temperature degreasing 3h, then cleaned with purified water
pH6.0。
(3) sterilize：Plus concentration 500ppm liquor natrii hypochloritises 10kg starts sterilization, sterilize 3 times, each 1.5h, sterilization knot
Shu Houyong purified waters are cleaned to without sterilization also smell and detected without chlorine residue, and pH is controlled in 6.5-7.0.
(4) it is homogenized：To the purified water that 10kg is added in the cartilage fourth shape thing that step (3) is processed, cartilage is ground with colloid mill
Into 100-200 mesh.
(5) digest：The slurries pH that step (4) is obtained is adjusted to 3.0, plus 75g pepsins, adds 25g low temperature to squeeze the juice
Filter afterwards acquisition fresh pawpaw clear liquid and 25g low temperature squeeze the juice after filter the fresh pineapple clear liquid of acquisition, in temperature, 25 DEG C are carried out
Enzyme 20h.
(6) filter：Filtered using 50 eye mesh screens, large particulate matter in removal solution collects filtered fluid.
(7) dry：The filtered fluid that step (6) receives disaggregation is carried out into vacuum freeze drying, precooling temperature is not higher than -30 DEG C,
Drying process finishing temperature is controlled at 30 DEG C, and cartilage extracts are obtained after freezing.
(8) crush：Cartilage extracts after freeze-drying carry out ultramicro grinding, the mesh number control of crushed material below 4 DEG C
In 200 mesh, dried frozen aquatic products is obtained.
Use the method for GB5009.5 to detect that collagen content is 54.8%, chondroitin sulfate is detected using GB/T20365
Cellulose content is 10.1%, uses ELISA method to detect (kit supplier is Shanghai Jiang Lai bio tech ltd) non-change
Property typeⅡ Collagen content be 10.3%.
Claims (10)
1. a kind of preparation method of the cartilage extracts of the collagen types of II containing non denatured, it is characterised in that the method is wrapped successively
Include following steps：Degreasing, sterilization, homogenate, enzymolysis, filtering, drying；Wherein, in the enzymolysis step：Will be in refining step
The pH of the slurry of acquisition is adjusted to 2.5~8.5, adds the enzyme of the 0.001%-2% of cartilage weight, adds the 1/ of cartilage weight
20~1/500 pawpaw and/or pineapple low temperature filter the clear liquid of acquisition after squeezing the juice, digest 12~48h, and hydrolysis temperature is 25-50
℃；The enzyme is preferably one or more in pepsin, subtilopeptidase A, alkali protease, metalloproteinases.
2. the preparation method of the cartilage extracts of a kind of collagen types of II containing non denatured according to claim 1, it is special
Levy and be, the consumption of the enzyme is the 0.5-2% of cartilage weight, and pawpaw and/or pineapple low temperature filter the clear liquid of acquisition after squeezing the juice
Consumption for cartilage weight 1/20-1/50, enzymolysis time be 12~20h, 25~40 DEG C of hydrolysis temperature.
3. the preparation method of the cartilage extracts of a kind of collagen types of II containing non denatured according to claim 1, it is special
Levy and be, the mixing clear liquid of acquisition is filtered after the clear liquid of addition is pawpaw and pineapple low temperature is squeezed the juice, wherein, the weight of pawpaw and pineapple
Amount is than being 1-10：1, preferably 3-6：1.
4. the preparation of the cartilage extracts of a kind of collagen types of II containing non denatured according to claim 1-3 any one
Method, it is characterised in that crushed after the drying step, wherein,
Degreasing：Cartilage fourth shape thing 0.2h~6h is soaked with certain density degreasing agent, the water for then being cleaned to cleaning with water
PH to 5.5~8.5；
Sterilization：With the cartilage fourth shape thing 1~3 time after finite concentration medicining liquid dipping degreasing, then cleaned with water to cartilage fourth shape
Thing is tasteless；
Homogenate：Cartilage fourth shape thing after sterilization is carried out into homogenized, the granularity of cartilage is 50~300 mesh after homogenate；
Filtering：Filtered using 5~100 eye mesh screens, large particulate matter in removal solution collects filtered fluid；
Dry：The filtered fluid that will be collected by filtration carries out low temperature drying, and drying temperature is not higher than 30 DEG C, and obtaining cartilage after drying extracts
Thing；
Crush：Dried cartilage extracts carry out ultramicro grinding below 4 DEG C, and the mesh number of crushed material is controlled in 100-500 mesh.
5. the preparation method of the cartilage extracts of a kind of collagen types of II containing non denatured according to claim 4, it is special
Levy and be, the degreasing agent is certain density NaOH solution, KOH solution, Na2CO3In solution, NaCl solution, KCl solution
One or more.
6. the preparation method of the cartilage extracts of a kind of collagen types of II containing non denatured according to claim 5, it is special
Levy and be, the concentration of the degreasing agent is 0.01~3 weight %, and the degreasing agent is 1~10 with the weight ratio of cartilage:1.
7. the preparation method of the cartilage extracts of a kind of collagen types of II containing non denatured according to claim 4, it is special
Levy and be, the thimerosal used in sterilisation step is sodium hypochlorite and/or hydrogen peroxide, the concentration control of thimerosal in disinfecting process
System is controlled in 0.2h~12h in 100ppm~2000ppm, disinfecting time.
8. the preparation method of the cartilage extracts of a kind of collagen types of II containing non denatured according to claim 4, it is special
Levy and be, refining step is carried out below 37 DEG C.
9. the preparation method of the cartilage extracts of a kind of collagen types of II containing non denatured according to claim 4, it is special
Levy and be, low temperature drying method is roller drying method, low temperature fourdrinier wire oven drying method, freeze-drying.
10. the preparation method of the cartilage extracts of a kind of collagen types of II containing non denatured according to claim 9, it is special
Levy and be, when the step of having precooling, precooling temperature is not higher than -10 DEG C, and the water content control of cartilage extracts is 6% after drying
Below.
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| CN107625788A (en) * | 2017-10-10 | 2018-01-26 | 北京市营养源研究所 | A kind of chick sternal cartilage and preparation method and application |
| CN108522949A (en) * | 2018-01-04 | 2018-09-14 | 嘉兴纽迪康生物科技有限公司 | A kind of active biological product and its processing method |
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| CN109384842A (en) * | 2018-12-25 | 2019-02-26 | 美泰科技（青岛）股份有限公司 | A kind of preparation method of non denatured II collagen of industrialization |
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| WO2022226731A1 (en) * | 2021-04-26 | 2022-11-03 | 北京盛美诺生物技术有限公司 | Pretreatment method for quantitative detection of undenatured type ii collagen in collagen product or cartilage, and application |
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