CN109295145A - A kind of preparation method of Ultra-low molecular weight yak collagen peptide - Google Patents
A kind of preparation method of Ultra-low molecular weight yak collagen peptide Download PDFInfo
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- CN109295145A CN109295145A CN201811285514.0A CN201811285514A CN109295145A CN 109295145 A CN109295145 A CN 109295145A CN 201811285514 A CN201811285514 A CN 201811285514A CN 109295145 A CN109295145 A CN 109295145A
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- 102000008186 Collagen Human genes 0.000 title claims abstract description 61
- 108010035532 Collagen Proteins 0.000 title claims abstract description 61
- 229920001436 collagen Polymers 0.000 title claims abstract description 60
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 58
- 238000002360 preparation method Methods 0.000 title claims abstract description 23
- 238000000034 method Methods 0.000 claims abstract description 25
- 230000007071 enzymatic hydrolysis Effects 0.000 claims abstract description 16
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims abstract description 16
- 238000007654 immersion Methods 0.000 claims abstract description 8
- 239000000047 product Substances 0.000 claims abstract description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 36
- 239000004365 Protease Substances 0.000 claims description 23
- 102000004190 Enzymes Human genes 0.000 claims description 17
- 108090000790 Enzymes Proteins 0.000 claims description 17
- 229940088598 enzyme Drugs 0.000 claims description 17
- 239000000706 filtrate Substances 0.000 claims description 12
- 108091005804 Peptidases Proteins 0.000 claims description 10
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 10
- 230000008569 process Effects 0.000 claims description 10
- 235000019419 proteases Nutrition 0.000 claims description 10
- 108010004032 Bromelains Proteins 0.000 claims description 8
- 235000019835 bromelain Nutrition 0.000 claims description 8
- 230000009849 deactivation Effects 0.000 claims description 8
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 6
- 239000004519 grease Substances 0.000 claims description 6
- 235000019834 papain Nutrition 0.000 claims description 6
- 108090000526 Papain Proteins 0.000 claims description 5
- 229940055729 papain Drugs 0.000 claims description 5
- 238000012545 processing Methods 0.000 claims description 5
- 238000007598 dipping method Methods 0.000 claims description 3
- 239000000284 extract Substances 0.000 claims description 3
- 244000189799 Asimina triloba Species 0.000 claims description 2
- 235000006264 Asimina triloba Nutrition 0.000 claims description 2
- 235000009467 Carica papaya Nutrition 0.000 claims description 2
- 239000003814 drug Substances 0.000 claims description 2
- 229940079593 drug Drugs 0.000 claims description 2
- 235000013305 food Nutrition 0.000 claims description 2
- 230000036541 health Effects 0.000 claims description 2
- 238000010438 heat treatment Methods 0.000 claims description 2
- 238000002791 soaking Methods 0.000 claims description 2
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 claims 1
- 102000002322 Egg Proteins Human genes 0.000 claims 1
- 108010000912 Egg Proteins Proteins 0.000 claims 1
- 235000014103 egg white Nutrition 0.000 claims 1
- 210000000969 egg white Anatomy 0.000 claims 1
- 210000002540 macrophage Anatomy 0.000 abstract description 14
- 108010031099 Mannose Receptor Proteins 0.000 abstract description 5
- 238000001914 filtration Methods 0.000 abstract description 4
- 108010010803 Gelatin Proteins 0.000 abstract description 2
- 229920000159 gelatin Polymers 0.000 abstract description 2
- 239000008273 gelatin Substances 0.000 abstract description 2
- 235000019322 gelatine Nutrition 0.000 abstract description 2
- 235000011852 gelatine desserts Nutrition 0.000 abstract description 2
- 230000036039 immunity Effects 0.000 abstract description 2
- 235000015097 nutrients Nutrition 0.000 abstract description 2
- 238000001694 spray drying Methods 0.000 abstract description 2
- 239000000413 hydrolysate Substances 0.000 abstract 1
- 230000004936 stimulating effect Effects 0.000 abstract 1
- 241001416153 Bos grunniens Species 0.000 description 42
- 102000004196 processed proteins & peptides Human genes 0.000 description 11
- 238000000605 extraction Methods 0.000 description 10
- 230000000052 comparative effect Effects 0.000 description 9
- 239000007788 liquid Substances 0.000 description 9
- 238000000926 separation method Methods 0.000 description 8
- 229920001184 polypeptide Polymers 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 230000035617 depilation Effects 0.000 description 6
- 238000007792 addition Methods 0.000 description 5
- 238000004587 chromatography analysis Methods 0.000 description 5
- 239000012535 impurity Substances 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 238000001035 drying Methods 0.000 description 4
- 238000011049 filling Methods 0.000 description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 210000000952 spleen Anatomy 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 210000001541 thymus gland Anatomy 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 239000004568 cement Substances 0.000 description 2
- 238000005352 clarification Methods 0.000 description 2
- 239000006071 cream Substances 0.000 description 2
- 238000005202 decontamination Methods 0.000 description 2
- 230000003588 decontaminative effect Effects 0.000 description 2
- 238000005238 degreasing Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000005057 refrigeration Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- 108090000145 Bacillolysin Proteins 0.000 description 1
- 241001247317 Bos mutus Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 235000008733 Citrus aurantifolia Nutrition 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 102100025354 Macrophage mannose receptor 1 Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 1
- 235000011941 Tilia x europaea Nutrition 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000004332 deodorization Methods 0.000 description 1
- 230000002951 depilatory effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 230000009246 food effect Effects 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 239000002655 kraft paper Substances 0.000 description 1
- 239000004571 lime Substances 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 238000000643 oven drying Methods 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 239000002893 slag Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- 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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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
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- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Gastroenterology & Hepatology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Toxicology (AREA)
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- Biophysics (AREA)
- Medicinal Chemistry (AREA)
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Cosmetics (AREA)
Abstract
The invention discloses a kind of preparation methods of Ultra-low molecular weight yak collagen peptide, belong to nutrient and healthcare products technical field.The preparation method of Ultra-low molecular weight collagen peptide of the invention is the Yak-skin Gelatin hydrolysate for obtaining yak skin by techniques such as diluted alkaline immersion, high temperature and pressure slurrying, filtering, enzymatic hydrolysis, concentration and spray drying, and the content of Ultra-low molecular weight collagen peptide (< 300D) is greater than 75%.Ultra-low molecular weight yak collagen peptide of the invention can be in conjunction with the mannose receptor on macrophage, and stimulating expression of macrophage increment promotes immunity.
Description
Technical field
The present invention relates to a kind of preparation methods of Ultra-low molecular weight yak collagen peptide, belong to nutrient and healthcare products technical field.
Background technique
Yak is the peculiar ox kind of Qinghai-Tibet Platean, studies have shown that Yak-skin Gelatin original has content more, and bioactivity is strong etc.
Plurality of advantages.Especially Ultra-low molecular weight peptide has been found that blood circulation can be entered directly through intestinal wall without decomposing, thus
By the direct absorbed intact of human body, therefore the effect of food is favourable.Collagen or collagen peptide are produced using yak skin, especially production is super
Low molecular weight collagen peptide is, it can be achieved that yak skin higher value application.
Traditional collagen peptide preparation method technical process is de- for depilation-degreasing-boiling-rubbing-degradation-separation-
Salt-concentration-spray drying, process flow is complicated, and depilation degreasing also frequently with various solvents, is polluted larger.In addition to this, western
Regional height above sea level is hidden, air pressure is low, causes water boiling point to be substantially reduced, therefore conventional collagen peptide preparation method is not appropriate for Tibetan area
The requirement of environmental protection and development of resources.Therefore, suitable highlands is developed, can make full use of resource, the pollution of generation
Less technique has great importance.
In current yak collagen extraction technique, the extraction including conventional kraft collagen peptide has more research and report
Road.Typically, if the process flow in a kind of preparation method (application number CN 106148466A) of yak collagen is to incite somebody to action
Yak tissue crushes, enzymatic hydrolysis, then isolates and purifies collagen peptide from the tissue fluid that enzymatic hydrolysis obtains using chromatography, although can obtain
To collagen, but chromatography industrially produces high higher cost in enormous quantities；A kind of grading extraction of bovine collagen protein peptides
Process flow in technique (application number CN107446981A) is that ultrasonic wave carries out primary extract and is separated by solid-liquid separation, obtain filtered fluid,
Filter residue primary enzymolysis, obtain the enzyme deactivation of primary enzymolysis liquid, separation insoluble matter, removal of impurities, obtain primary purification liquid concentration sterilization,
It is dry, obtain a protein peptide powder；Secondary enzymolysis obtains secondary enzymatic solution enzyme deactivation, separation insoluble matter, removal of impurities, obtains secondary
The sterilization of refined liquid concentration, drying, obtain secondary protein peptide powder, although technique anacidity alkali pollution used and discharge, surpass
Sound wave extraction method is industrially realized relatively difficult.As in conclusion totally existing in existing collagen peptide extraction process
In addition to this problems such as extraction process is cumbersome and/or quantity of wastewater effluent is big is directed to highlands in existing patent
The preparation process that distinctive low pressure, environmental issue are developed.
Summary of the invention
To solve the above problems, the present invention one kind is provided can be in highlands scale processing, simple process, pollution
Small Ultra-low molecular weight yak collagen peptide production method.Creative use high temperature and pressure slurrying of the present invention realizes that step depilation is de-
Rouge, it is intended to reduce the pollution of extraction process, improve the bioactivity of product.It is pre-processed specifically, using diluted alkaline, makes yak
The structure of ox-hide becomes loose, then is aided with high temperature and pressure, keeps its degradable at rubber cement, and the hair being attached on skin at this time
Naturally fall off, at this time only need to by filtering can be by hair removing.Then rubber cement is refrigerated, lubricant component can be cold at low temperature
Form block, only needs simply filtering that can remove grease at this time.
The first purpose of the invention is to provide a kind of preparation method of Ultra-low molecular weight yak collagen peptide, including it is as follows
Step:
(1) it pre-processes: by the decontamination of yak skin and cleaning up, handled with dipping by lye；By the yak skin after immersion treatment
It cleans up；
(2) high temperature slurrying: by step (1) treated yak skin in 115~125 DEG C of 0.08~0.1mP, temperature conditions
1~5h of lower processing, after treatment filter defeathering, then chilling treatment while hot, and filtering removes grease after chilling treatment, collect filter
Liquid；
(3) enzyme process extract: the filtrate for taking step (2) to obtain, be added protease digested, centrifuge separation supernatant with it is residual
Supernatant is heated enzyme deactivation by slag, obtains Ultra-low molecular weight yak collagen peptide.
In one embodiment of the invention, the addition protease hydrolyzed is that bromelain and pawpaw is added
Protease carries out complex enzyme hydrolysis.
In one embodiment of the invention, the addition protease hydrolyzed specifically first be added filtrate quality 5~
15% 30~40 DEG C of 4~8h of enzymatic hydrolysis of bromelain；Add 30~40 DEG C of papain of filtrate quality 5~15%
Digest 4~8h.
In one embodiment of the invention, the lye is one of sodium hydroxide or potassium hydroxide or two
Kind.
In one embodiment of the invention, the concentration of the lye is 0.1~1% (w/v).
In one embodiment of the invention, the yak skin is derived from the peculiar ox kind yak wild yad in Tibet,
For the wild yak resources of characteristic of Tibetan areas.
In one embodiment of the invention, in step (1), the diluted alkaline immersion treatment be using 0.1~
1~3h of soaking with sodium hydroxide of 1% (w/v).
In one embodiment of the invention, in step (2), the chilling treatment is refrigerated at 0~10 DEG C
10~20h.
In one embodiment of the invention, in step (3), the heating enzyme deactivation is that supernatant is heated to 80
~90 DEG C make enzyme-deactivating.
In one embodiment of the invention, the preparation method further includes the steps that refining collagen peptide, tool
Body are as follows: the Ultra-low molecular weight yak collagen peptide that step (3) obtains is concentrated, is refined and is dried.
In one embodiment of the invention, the concentration is centrifugal concentrating, ultrafiltration concentration, appointing in concentrated by rotary evaporation
It anticipates one kind.
In one embodiment of the invention, the purification is using appointing in active carbon, diatomite, ion exchange resin
A kind of pair of collagen peptide of anticipating carries out deodorization decoloring.
In one embodiment of the invention, the drying is freeze-drying, is spray-dried, appointing in oven drying
It anticipates one kind.
A second object of the present invention is to provide a kind of Ultra-low molecular weight yak collagen peptides that the method is prepared.
In one embodiment of the invention, Ultra-low molecular weight yak collagen peptide (< 300D) content is greater than
75%.
Third object of the present invention is to provide the Ultra-low molecular weight yak collagen peptides in food, drug, health care product
In application.
The beneficial effects of the present invention are:
1, the step depilation degreasing method that the present invention uses, process is simple, high-efficient, and the low pressure on plateau can be overcome to ask
It is conventional caused by topic to cook the inadequate problem of temperature, and preparation flow is simple compared with chemical method depilation, no high concentration soda acid
It uses, technique greenization meets the requirement of Tibet region green production and discharge.
2, products obtained therefrom low molecule collagen peptides extraction rate of the present invention is higher than 75%, and molecular weight is low, and bioactivity is good.
Detailed description of the invention
Fig. 1 is the GPC testing result of 2 yak collagen peptide of the embodiment of the present invention；
Fig. 2 is the GPC testing result of 1 yak collagen peptide of comparative example；
Fig. 3 is the GPC testing result of 2 yak collagen peptide of comparative example；
Fig. 4 is the GPC testing result of 3 yak collagen peptide of comparative example.
Specific embodiment
The present invention will be further explained below with reference to the attached drawings and specific examples, so that those skilled in the art can
It to better understand the invention and can be practiced, but illustrated embodiment is not as a limitation of the invention.
Embodiment 1: the hair removal effect of yak skin compares
Using the hair removal effect of simultaneously comparative chemistry depilation and this patent epilation, the method is as follows: (1) referring to patent
Chemical depilation in 201710089525.0, proportionally 100 parts of water add 12 parts of vulcanized sodium and 30 parts of white lime mixed preparings de-
Hair cream, then by yak skin tiling on the ground, surface is downward, and hair side is upward, configured depilatory cream film on hair side,
Hair is manually pushed away after stacking 5 hours, is rinsed well；(2) it is cleaned up the decontamination of yak skin and repeatedly in this patent, with 0.1~
The NaOH of 1% (w/v) impregnates 1h；It is cleaned up repeatedly after immersion treatment.Yak skin is in 0.1MP high temperature and pressure item after handling
1h is handled under part, after treatment filters defeathering while hot.
The different epilation comparison results of table 1
Embodiment 2: the preparation of yak collagen peptide
After taking yak skin to clean up repeatedly, 2h is impregnated with the NaOH of 0.5% (w/v), immersion treatment is cleaned dry repeatedly
Only, clean yak skin is handled into 3h under 0.08mP high pressure, 115 DEG C of high temperature, after treatment filters while hot, 4 DEG C of refrigerations
Overnight, filtered through gauze remove grease, collect filtrate, according to every 25g organize be added 2g bromelain amount, 35 DEG C of enzymatic hydrolysis 6h, so
2g papain is added by every 25g tissue afterwards, 35 DEG C of enzymatic hydrolysis 6h, after enzymatic hydrolysis, being heated to 80 DEG C inactivates enzyme, is centrifuged
Separation is concentrated, dry.And collagen peptide molecular weight obtained is detected.Testing result is shown in Fig. 1 and table 2.The result shows that
Collagen peptide content of the molecular weight less than 300d accounts for 75%.
2 yak collagen peptide GPC testing result of table
Embodiment 3:
After taking yak skin to clean up repeatedly, 3h is impregnated with the NaOH of 0.1% (w/v), immersion treatment is cleaned dry repeatedly
Only, clean yak skin is handled into 5h under 0.09mP high pressure, 120 DEG C of high temperature, after treatment filters while hot, 4 DEG C of refrigerations
Overnight, filtered through gauze remove grease, collect filtrate, according to every 10g organize be added 1g bromelain amount, 30 DEG C of enzymatic hydrolysis 8h, so
1g papain is added by every 10g tissue afterwards, 30 DEG C of enzymatic hydrolysis 8h, after enzymatic hydrolysis, being heated to 80 DEG C inactivates enzyme, is centrifuged
Separation is concentrated, dry.Obtain Ultra-low molecular weight yak collagen peptide.
Embodiment 4:
After taking yak skin to clean up repeatedly, 1h is impregnated with the NaOH of 1% (w/v), immersion treatment cleans up repeatedly,
Clean yak skin is handled into 1h under 0.1mP high pressure, 125 DEG C of high temperature, after treatment filters while hot, 4 DEG C of refrigerated overnights,
Filtered through gauze removes grease, collects filtrate, and the amount that 1 g bromelain is added is organized according to every 10g, then 40 DEG C of enzymatic hydrolysis 4h are pressed
Every 10g tissue is added 1g papain, 40 DEG C of enzymatic hydrolysis 4h, and after enzymatic hydrolysis, being heated to 90 DEG C inactivates enzyme, is centrifugated,
Concentration, it is dry.Obtain Ultra-low molecular weight yak collagen peptide.
Comparative example 1:
According to the method for publication CN106148466A, i.e., by yak ox head skin crush, be added 1% pepsin with
1% trypsin digestion, hydrolysis temperature are -4 DEG C, are centrifuged after enzymatic hydrolysis, and revolving speed is 11000 rpm, are centrifuged 10min, obtain
Collagen liquid, then collagen liquid is chromatographed, chromatography pressure is 0.08MP, and chromatography temperature is -4 DEG C, is freezed after chromatography dry
It is dry, using GPC detection molecules amount, as a result see Fig. 2.
Comparative example 2:
According to the method for publication CN107446981A, clean to raw material oil removing, under the conditions of pH7-8,100 DEG C
500W ultrasonic wave extraction 8h, is separated by solid-liquid separation, and filtrate cools to 65 DEG C of 0.1% Bacillus subtilis neutral protease hydrolyzeds of addition
Active carbon removal of impurities is added in 4h, 80 DEG C of processing 30min enzyme deactivations, and clarification is again heated to 65 DEG C of 0.1% papains of addition, enzyme
4h is solved, is centrifugated, concentration is drying to obtain yak collagen peptide, using GPC detection molecules amount, as a result sees Fig. 3.
Comparative example 3:
Substantially according to the method for publication CN107446981A, protease is replaced with to protease of the invention: to original
Expect oil removing removal of impurities, the 500W ultrasonic wave extraction 8h under the conditions of pH7-8,100 DEG C is separated by solid-liquid separation, and filtrate cools to 40 DEG C of additions
8% bromelain digests 4h, and 80 DEG C of processing 30min enzyme deactivations are added active carbon removal of impurities, clarification, then 8% wood is added at 35 DEG C
Melon protease digests 4h, is centrifugated, and concentration is drying to obtain yak collagen peptide, using GPC detection molecules amount, as a result sees figure
4。
3 distinct methods collagen peptide molecular weight of table compares
Embodiment 5: activation of the ultra-low molecular collagen polypeptide to macrophage mannose receptor
Collagen polypeptide macrophage made from embodiment 2 is co-cultured, following experimental group is arranged: (1) prepared by embodiment 2
Obtained ultra-low molecular collagen polypeptide+macrophage is incubated for altogether；(2) commercially available common molecular collagen polypeptide+macrophage is incubated altogether
It educates；(3) macromolecular collagen peptide+macrophage is incubated for altogether；(4) after mannose+macrophage preincubate 30min again with ultralow point
Sub- collagen polypeptide is incubated for altogether；(5) macrophage control group is incubated for counts afterwards to cell for 24 hours altogether, different real as the result is shown
Test a group cell quantity are as follows: 1 > 2 > 3 ≈, 4 ≈ 5.Comparative experiments group 1,2,3, shows that collagen peptide is smaller, to the growth promotion of macrophage
Effect is better；Furthermore comparative experiments group 1 and 4, research have shown that Macrophage Surface has mannose receptor, when being located in advance with mannose
Manage macrophage after, mannose can preferentially occupy the mannose receptor of Macrophage Surface, make its no longer with collagen polypeptide knot
It closes, therefore causes 4 cell quantity of experimental group less than experimental group 1.Experimental data shows that ultra-low molecular collagen polypeptide can be with macrophage
Cell surface mannose receptor combines, the proliferation of activating macrophage.
Embodiment 6:
Using inbred mouse, 18-22g, half male and half female, every group 10.Control group and experimental group are set, using stomach-filling
Mode gives animal subject, and daily timing stomach-filling is primary.Control group stomach-filling distilled water, experimental group stomach-filling embodiment 2 are prepared
Ultra-low molecular weight collagen peptide solution.Mouse is put to death after 30 days, takes thymus gland and spleen, is weighed, thymus index is calculated and refers to spleen
Number, discovery experimental mice thymus gland refer to that index and spleen index is higher than control group, illustrate that Ultra-low molecular weight collagen peptide can be improved immune device
Official's index has activation to immunity of organism.
Embodiment described above is only to absolutely prove preferred embodiment that is of the invention and being lifted, protection of the invention
Range is without being limited thereto.Those skilled in the art's made equivalent substitute or transformation on the basis of the present invention, in this hair
Within bright protection scope.Protection scope of the present invention is subject to claims.
Claims (10)
1. a kind of preparation method of Ultra-low molecular weight yak collagen peptide, which comprises the steps of:
(1) it pre-processes: yak skin is handled with dipping by lye；Yak skin after immersion treatment is cleaned up；
(2) high temperature slurrying: by step (1), treated that yak skin is handled under the conditions of 0.08~0.1mP, 115~125 DEG C of temperature
1~5h, after treatment filter defeathering, and chilling treatment is filtered to remove grease after chilling treatment, collect filtrate；
(3) enzyme process extracts: the filtrate for taking step (2) to obtain, and protease is added and is digested, and is then centrifuged for removing residue, will be upper
Reset and add hot enzyme deactivation, obtains Ultra-low molecular weight yak collagen peptide.
2. preparation method according to claim 1, which is characterized in that the protease is bromelain and pawpaw egg
White enzyme.
3. preparation method according to claim 2, which is characterized in that the addition protease hydrolyzed is specifically first added
30~40 DEG C of 4~8h of enzymatic hydrolysis of bromelain of filtrate quality 5~15%；Add the Papain of filtrate quality 5~15%
30~40 DEG C of 4~8h of enzymatic hydrolysis of enzyme.
4. preparation method according to claim 1, which is characterized in that the lye is sodium hydroxide, in potassium hydroxide
One or two mixing.
5. preparation method according to claim 1, which is characterized in that in step (1), the dipping by lye processing is
Using 1~3h of soaking with sodium hydroxide of 0.1~1% (w/v).
6. preparation method according to claim 1, which is characterized in that in step (2), the chilling treatment is 0
10~20h is refrigerated at~10 DEG C.
7. preparation method according to claim 1, which is characterized in that in step (3), the heating enzyme deactivation is will be upper
Being heated to 80~90 DEG C makes enzyme-deactivating clearly.
8. preparation method according to claim 1, which is characterized in that the preparation method further includes carrying out essence to collagen peptide
The step of processed, specifically: the Ultra-low molecular weight yak collagen peptide that step (3) obtains is concentrated, is refined and is dried.
9. a kind of Ultra-low molecular weight yak collagen peptide that preparation method according to any one of claims 1 to 8 is prepared.
10. application of the Ultra-low molecular weight yak collagen peptide as claimed in claim 9 in food, drug, health care product.
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