CN108396053A - A kind of industrialized producing technology of wood frog hide collagen oligopeptide - Google Patents
A kind of industrialized producing technology of wood frog hide collagen oligopeptide Download PDFInfo
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- CN108396053A CN108396053A CN201810262135.3A CN201810262135A CN108396053A CN 108396053 A CN108396053 A CN 108396053A CN 201810262135 A CN201810262135 A CN 201810262135A CN 108396053 A CN108396053 A CN 108396053A
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- 108010038807 Oligopeptides Proteins 0.000 title claims abstract description 51
- 102000015636 Oligopeptides Human genes 0.000 title claims abstract description 51
- 102000008186 Collagen Human genes 0.000 title claims abstract description 21
- 108010035532 Collagen Proteins 0.000 title claims abstract description 21
- 229920001436 collagen Polymers 0.000 title claims abstract description 21
- 241000191896 Rana sylvatica Species 0.000 title claims abstract description 18
- 238000005516 engineering process Methods 0.000 title claims abstract description 11
- 238000001914 filtration Methods 0.000 claims abstract description 32
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 31
- 238000004108 freeze drying Methods 0.000 claims abstract description 20
- 238000011049 filling Methods 0.000 claims abstract description 19
- 108090000790 Enzymes Proteins 0.000 claims abstract description 15
- 102000004190 Enzymes Human genes 0.000 claims abstract description 15
- 238000005238 degreasing Methods 0.000 claims abstract description 15
- 239000002994 raw material Substances 0.000 claims abstract description 14
- 230000000694 effects Effects 0.000 claims abstract description 10
- 239000012535 impurity Substances 0.000 claims abstract description 8
- 230000014759 maintenance of location Effects 0.000 claims abstract description 8
- 238000010612 desalination reaction Methods 0.000 claims abstract description 5
- 239000002002 slurry Substances 0.000 claims description 33
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 26
- 238000001728 nano-filtration Methods 0.000 claims description 25
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 22
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 20
- 239000012141 concentrate Substances 0.000 claims description 18
- 239000012530 fluid Substances 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 18
- 229940088598 enzyme Drugs 0.000 claims description 14
- 238000010438 heat treatment Methods 0.000 claims description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- 239000000047 product Substances 0.000 claims description 12
- 238000010792 warming Methods 0.000 claims description 12
- JGSARLDLIJGVTE-UHFFFAOYSA-N 3,3-dimethyl-7-oxo-6-[(2-phenylacetyl)amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid Chemical compound O=C1N2C(C(O)=O)C(C)(C)SC2C1NC(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-UHFFFAOYSA-N 0.000 claims description 9
- 238000000227 grinding Methods 0.000 claims description 9
- 239000000843 powder Substances 0.000 claims description 9
- 238000001223 reverse osmosis Methods 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 8
- 238000001976 enzyme digestion Methods 0.000 claims description 8
- 239000012634 fragment Substances 0.000 claims description 8
- 239000012465 retentate Substances 0.000 claims description 8
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- 238000002372 labelling Methods 0.000 claims description 7
- 150000003839 salts Chemical class 0.000 claims description 7
- 239000000706 filtrate Substances 0.000 claims description 6
- 239000012528 membrane Substances 0.000 claims description 6
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 108090000526 Papain Proteins 0.000 claims description 5
- 102000057297 Pepsin A Human genes 0.000 claims description 5
- 108090000284 Pepsin A Proteins 0.000 claims description 5
- 230000009849 deactivation Effects 0.000 claims description 5
- 239000012510 hollow fiber Substances 0.000 claims description 5
- 229940055729 papain Drugs 0.000 claims description 5
- 235000019834 papain Nutrition 0.000 claims description 5
- 229940111202 pepsin Drugs 0.000 claims description 5
- 238000010828 elution Methods 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 4
- 239000013527 degreasing agent Substances 0.000 claims description 3
- 239000004744 fabric Substances 0.000 claims description 3
- 210000004907 gland Anatomy 0.000 claims description 3
- 238000003801 milling Methods 0.000 claims description 3
- 238000005192 partition Methods 0.000 claims description 3
- 239000004033 plastic Substances 0.000 claims description 3
- 238000007711 solidification Methods 0.000 claims description 3
- 230000008023 solidification Effects 0.000 claims description 3
- 241001411320 Eriogonum inflatum Species 0.000 claims description 2
- 239000012065 filter cake Substances 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 15
- 230000008569 process Effects 0.000 abstract description 7
- 230000036541 health Effects 0.000 abstract description 6
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- 239000000490 cosmetic additive Substances 0.000 abstract description 2
- 238000013461 design Methods 0.000 abstract description 2
- 239000002778 food additive Substances 0.000 abstract 1
- 235000013373 food additive Nutrition 0.000 abstract 1
- 230000007062 hydrolysis Effects 0.000 abstract 1
- 238000006460 hydrolysis reaction Methods 0.000 abstract 1
- 238000011031 large-scale manufacturing process Methods 0.000 abstract 1
- 239000002417 nutraceutical Substances 0.000 abstract 1
- 235000021436 nutraceutical agent Nutrition 0.000 abstract 1
- 238000004519 manufacturing process Methods 0.000 description 9
- 230000006872 improvement Effects 0.000 description 7
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- 210000000988 bone and bone Anatomy 0.000 description 5
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 235000013372 meat Nutrition 0.000 description 3
- 229910001220 stainless steel Inorganic materials 0.000 description 3
- 239000010935 stainless steel Substances 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 239000002699 waste material Substances 0.000 description 3
- MVORZMQFXBLMHM-QWRGUYRKSA-N Gly-His-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CN=CN1 MVORZMQFXBLMHM-QWRGUYRKSA-N 0.000 description 2
- 206010054949 Metaplasia Diseases 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 244000019397 Pinus jeffreyi Species 0.000 description 2
- 235000013267 Pinus ponderosa Nutrition 0.000 description 2
- 235000006237 Pinus sabiniana Nutrition 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- 230000003796 beauty Effects 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 238000004061 bleaching Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000005452 food preservative Substances 0.000 description 2
- 235000019249 food preservative Nutrition 0.000 description 2
- 108010038983 glycyl-histidyl-lysine Proteins 0.000 description 2
- 238000007654 immersion Methods 0.000 description 2
- 230000009191 jumping Effects 0.000 description 2
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- 235000019419 proteases Nutrition 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 230000006641 stabilisation Effects 0.000 description 2
- 238000011105 stabilization Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108050004290 Cecropin Proteins 0.000 description 1
- 244000086443 Craterellus fallax Species 0.000 description 1
- 235000007926 Craterellus fallax Nutrition 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000000035 biogenic effect Effects 0.000 description 1
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- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
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- 230000007613 environmental effect Effects 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
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- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000011436 enzymatic extraction method Methods 0.000 description 1
- 230000005496 eutectics Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 239000011344 liquid material Substances 0.000 description 1
- 239000002932 luster Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 206010033675 panniculitis Diseases 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
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- 231100000719 pollutant Toxicity 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
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- 238000011160 research Methods 0.000 description 1
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- 230000000717 retained effect Effects 0.000 description 1
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- 238000000926 separation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
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- 210000004304 subcutaneous tissue Anatomy 0.000 description 1
- 238000005092 sublimation method Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
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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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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Toxicology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention provides a kind of industrialized producing technologies of wood frog hide collagen oligopeptide.Specific steps include：1, raw material, 2, impregnate softening, 3, rub, 4, degreasing, 5, defibrination, 6, decoloration, 7, saltout, 8, press filtration, 9, enzymolysis, 10, ultrafiltration oligopeptide, 11, press filtration retention concentration, desalination, removing impurities matter, 12, degerming, 13, it is filling, 14, freeze-drying, 15, pressure bottle cap and plug, label.The method of the present invention is simple for process, reasonable design, makes full use of the natural resources, it can be achieved that industrializing and large-scale production.The present invention uses food-grade albumen enzyme hydrolysis, safe, without side-effects；The present invention can be used as health products, function food additive or nutraceutical and directly take, while be also used as cosmetic additive agent safe handling.
Description
Technical field
The present invention relates to a kind of industrialized producing technologies of wood frog hide collagen oligopeptide, belong to functional health care product or health care food
Product and cosmetic additive agent field.
Background technology
Wood frog integrates beauty, medicinal, edible.Have the function of abundant medicinal and health and beauty, for a long time, people
It has all focused on the oviductus ranae of production high-quality, and has had ignored the utilization of tree frog skin by-product.Tree frog skin is main
Containing polypeptide, collagen and hyaluronic acid etc., it is referred to as " the huge storage library of biologically active peptide and biogenic amine, tree frog skin
Extracting its active peptide can be used for improving breathing and the excretory function of human skin.Be made cecropin can be used as food preservative and
Preservative.With being growing for the albumen oligopeptide market demand, developing has the active highly effective and safe environmental protection of biological health
Wood frog hide collagen oligopeptide production technology, significance is all had to scientific research and practical application.Tree frog skin accounts for wood frog gross weight
30% or so of amount contains the collagen compared with horn of plenty in tree frog skin.After wood frog processes oviductus ranae, a large amount of frog skin is made
It is dropped for waste, causes environmental pollution and the waste of living resources.
Invention content
The present invention is in the above context and provides a kind of industry of output capacity height, the high wood frog hide collagen oligopeptide of purity
Metaplasia production. art.
The present invention includes the following steps：1, raw material, 2, immersion softening, 3, rubbing, 4, degreasing, 5, defibrination, 6, decoloration, 7, salt
Analysis, 8, press filtration, 9, enzymolysis, 10, ultrafiltration oligopeptide, 11, press filtration retention concentration, desalination, removing impurities matter, 12, degerming, 13, it is filling,
14, freeze-drying, 15, pressure bottle cap and plug, labelling.It is digested using zymotechnic, collects the peptide fragment of suitable molecular weight, used
Ultrafiltration membrane technique prepares the wood frog hide collagen oligopeptide for having a variety of physiological active functions by concentration, vacuum freezedrying.
A kind of industrialized producing technology of wood frog hide collagen oligopeptide, steps are as follows：
1, raw material
Raw material selects fresh live body to take oil, removes the dry tree frog skin removed after body.
2, softening is impregnated
Dry tree frog skin is put into stainless steel rinse bath, pure water is injected, impregnates 48h-72 h, until the complete rehydration of tree frog skin,
As doughy state.
3, it rubs
It after pulling the soft tree frog skin of bull pine out water drenching, puts in meat grinder, is rubbed into the fragment that granularity is 0.1-0.2cm.
4, degreasing
By the degreaser soak degreasing 1-2d of the tree frog skin of rubbing 1.0-2.0% acetums（It）, finally use 30-50 DEG C of temperature
Water rinses 2 times, obtains the tree frog skin that degreasing is impregnated.
5, defibrination
Tree frog skin after degreasing is worn into slurries with bone mud barreling, particle is become and refines slurries in 20~50 μm of sizes.
6, it decolourizes
The activated carbon of the 3-5% of liquid weight is added in refining slurries so that activated carbon fully adsorbs, and obtains the fine grinding of decoloration
Slurries.
7, it saltouts
Fine grinding slurries obtained are adjusted into pH value 4.0~4.5, NaCl powder is added and saltouts, it is stirring while adding, make collagen egg
After white precipitation completely, standing 12~for 24 hours, obtain collagen mixed liquor.
8, press filtration
Mixed liquor is uniformly routed on sheet frame filter cloth, conveying mixed liquor starts 1~6h of press filtration, passes through the aperture regulation of outlet valve
Press filtration progress is controlled, filter cake is discarded, obtains transparent filtrate.
9, it digests
Slurries after press filtration are first added to the pepsin of slurry weight 0.1-2%, pH to pH 2~3 are adjusted, in 40-45 DEG C of temperature
Under the conditions of degree, after making enzyme digestion reaction 72h.The papain for adding slurry weight 0.5%~1% adjusts pH at 40-45 DEG C
To pH6-8, enzyme digestion reaction 5-10 hours is constantly stirred, is obtained containing oligopeptide mixed liquor.
10, ultrafiltration oligopeptide
Enzymolysis liquid is subjected to classification ultrafiltration through reverse osmosis ultrafiltration unit, the ultrafilter of molecular weight 75KDa is first passed through, using molecule
Measure the ultrafilter filtering of 3KDa.The peptide of suitable molecular weight segment enters collector through film, and the peptide of large fragment continues and enzyme effect
It digests again, then ultrafiltration obtains oligopeptide filtered fluid.Its temperature is warming up to 80~95 DEG C of progress high temperature enzyme deactivations after ultrafiltration.
11, press filtration retention concentration, desalination, removing impurities matter
It is concentrated in the nanofiltration device that the molecular weight that albumen oligopeptide filtrate is pumped into reverse osmosis nanofiltration unit is 500Da.Again will
Concentrate adds same volume water elution 3-5 times, obtains the nanofiltration retentate fluid for being concentrated into the minimum volume that water content only has 3-5%.
It will collect containing other substances and the nanofiltrations filtered fluid such as inorganic salts in raw material, carry out purified treatment, the nanofiltration retentate fluid of collection
For oligopeptide concentrate.
12, degerming
After concentrate is filtered using molecular weight 60-80KDa hollow fiber membrane ultrafiltration devices, enters back into 0.22 μm of micropore filter element and carried out
Filter, carries out degerming by way of retention.
13, filling
Oligopeptide concentrate after degerming is carried out filling with automatic filling machine, is fitted into and is cleaned in sterile 10ml cillin bottles, dosage
Control ensures the stabilization of net content within 5ml, per bottled amount 200mg, and pressurizes rubber stopper plug to state of partly jumping a queue, obtains
To canned product.
14, it is freeze-dried
（1）Pre-freeze cures
By canned concentrate cillin bottle, it is placed on the partition board in vacuum freeze drier body feed tank, pre-freeze solidification temperature -30
~-45 DEG C, and 5~8h is kept, so that moisture in concentrate is fully freezed, it is ensured that no liquid exists.
（2）Lyophilization：Freeze dryer starts vacuum system, and pressure in storehouse is evacuated to 30~50pa, takes slow heating, with
1 DEG C of heating rate per hour is slowly warming up to -20~-25 DEG C, keeps the temperature 6~8h, then with 2 DEG C per hour of heating rate, delay
It is slow to be warming up to -10 DEG C~-12 DEG C, 3~6h is kept the temperature, lyophilization is carried out.
（3）Parsing-desiccation：After lyophilization, then with 3 DEG C per hour of heating rate, 40~50 DEG C are slowly warming up to, pressure
Power is 10~20Pa.6~8h is kept the temperature, parsing-desiccation freeze-drying is completed, is taken out after the plastic lid that pressurizes.
15, bottle cap and plug, labelling are pressed
Aseptically mechanical gland bottle stopper after freeze-drying, makes it post label into labelling machine.Purity of protein height is made
Oligopeptide component content up to 99.5%, relative molecular mass less than 1000Da is averagely more than 85% tree frog skin oligopeptide finished product
Powder.
Compared with prior art, main advantages of the present invention are：
1, as a further improvement on the present invention, the present invention is to reduce the waste of wood frog resource, realizes the reasonable utilization of resource.
Dry tree frog skin softens by impregnating, and rubs, keeps raw material finer and smoother uniformly after the mill grinding of bone mud again, crushing effect is good, is follow-up
The oligomeric peptide content for improving extraction creates advantage.
2, in view of tree frog skin itself deposited a large amount of pigment, effective depigmentaton technical staff is not taken without processing or
Skill, products obtained therefrom are in Dark grey or grey black, influence presentation quality and application.As a further improvement on the present invention, of the invention
It is decolourized using activated carbon method, this method has good decolorizing effect, and activated carbon is cheap, can Reusability.By de-
Color overcomes the shortcomings of shade deviation existing for existing tree frog skin peptide product.
3, as a further improvement on the present invention, the present invention is saltoutd in the extraction of albumen oligopeptide using NaCl powder
Method first collagen is cemented out, and after saltouing carry out plate and frame filter press press filtration, then to press filtration obtain slurries carry out
Enzymolysis.Single enzyme and compound protease screening verification have been carried out to the enzyme used in wood frog collagen before enzymolysis, passed through verification
Selected the complex enzyme that pepsin and papain form to replace single enzyme, and after taking appropriate enzymolysis, ultrafiltration, again enzyme
The enzymatic extraction method repeatedly of solution destroys partial peptide key and forms the albumen oligopeptide that molecular weight is less than 1000Da, to not reaching
Extracting solution to small-molecular peptides realizes further enzymolysis, finally takes high temperature enzyme deactivation, and enzymolysis process design makes wood frog hide glue
Former protein breakdown averagely reaches 85% at the component content of the albumen oligopeptide less than 1000Da molecular weight.Thus improve wood frog
Hide collagen activity and utilization rate.
4, as a further improvement on the present invention, it is more than filtering method present invention employs reverse osmosis ultrafiltration unit, passes through
Macromolecular substances are retained repeatedly, and filtering, separation are achieved the purpose that through small-molecule substance.First pass through the super of molecular weight 75KDa
Filter filters, and is filtered using the ultrafilter of molecular weight 3KDa, and molecular cut off, will in 2000-100000Da protein peptides
1000Da small-molecular-weight segment oligopeptides below enter collector through film, and the peptide of large fragment continues and enzyme effect, then row is super
Filter.It ensure that the concentration output of oligomeric peptide product and making full use of for raw material.
5, as a further improvement on the present invention, the present invention uses molecular weight for the nanofiltration device of the reverse osmosis collecting and filtering apparatus of 500Da,
Oligopeptide is obtained by nanofiltration and retains concentrate, while making salt, impurity as nanofiltration filtered fluid is deviate from.It substantially increases
The purity of albumen oligopeptide so that albumen oligopeptide purity is up to 99.5%.Simultaneously by the nanofiltration containing salt, impurity of collection
Filtrate is pumped into biochemical treatment tank, carries out purified treatment, is discharged after up to standard so that production technology has reached environmentally protective standard.
6, as a further improvement on the present invention, the method that the present invention uses physics degerming, according to point of albumen oligopeptide
Son amount and bacteria molecule amount have selected molecular weight 60-80KDa hollow fiber membrane ultrafiltration devices to refilter, after filtering, into 0.22 micron
The small filter of degerming carries out degerming.The method of the degerming, which has, does not destroy active ingredient, does not increase other pollutions, efficient, degerming
Effect is good, effectively increases the shelf-life of the oligomeric peptide product of albumen.
7, as a further improvement on the present invention, the production of albumen oligopeptide of the present invention is in high-efficiency and continuous automated production
It is produced under system, entire technological process is mostly to be produced under sealing condition, such as filling using automatic filling machine progress, especially this hair
Bright freeze-drying takes slow heating technology, so that temperature in entire sublimation process is no more than eutectic point, ice crystal will not be caused to melt
Change and influence the quality of oligomeric Gly-His-Lys, ensure that the high activity of the oligomeric peptide product of albumen.Environment-friendly advantage of the present invention protrudes, and does not use
Toxic reagent, non-pollutant discharge reach clean manufacturing, environmentally protective, product safety health, with preferable industrialized production
Advanced and practicability.It is tree frog skin rationally using opening new approach, is wood frog hide collagen oligopeptide industrialization and scale
Metaplasia production provides effective process.
Specific implementation mode
Embodiment 1
A kind of industrialized producing technology of wood frog hide collagen oligopeptide, its step are as follows:
1, raw material
Raw material selects fresh live body to take oil, removes the Lin Wapi removed after body, removes subcutaneous tissue and adhesion object, cleans up,
Indoor Natural is dried or is dried, without putrid and deteriorated dry tree frog skin.
2, softening is impregnated
Dry tree frog skin is put into stainless steel rinse bath, pure water is injected, height of water level will be more than tree frog skin 10-15cm, stirring
Uniformly, 48h-72 h are impregnated, water are changed during immersion 2~4 times, until the complete rehydration of tree frog skin, becomes doughy state.
3, it rubs
It after pulling the soft tree frog skin of bull pine out water drenching, puts in meat grinder, is rubbed, it is 0.1-0.2cm to be rubbed into granularity
Fragment.
4, degreasing
By the tree frog skin of rubbing 1.0-2.0% acetum degreaser soak degreasing 1-2d, temperature is at 25-30 DEG C, per 2h-3h
It is sufficiently stirred 1 time, filters off supernatant liquid, it is primary to repeat degreasing as stated above, finally uses 30-50 DEG C of warm water rinse 2 times, obtains
Obtain the tree frog skin that degreasing is impregnated.
5, defibrination
Tree frog skin after degreasing is iterated through bone mud to grind 2-3 times, becomes particle in 20~50 μm of size grinding slurries.Water is added to arrive
500-1000L water level lines impregnate 1~2d, remove soaking water, and soaked tree frog skin is ground defibrination 1 time again in bone mud, is obtained
Finish milling slurry.
6, it decolourizes
Tree frog skin itself has special greyish black color and luster, is decolourized using regular activated carbon dust decolorising agent, in gained finish milling slurry
The activated carbon of the 3-5% of liquid weight is added in liquid, bleaching temperature is maintained at 50~60 DEG C, and bleaching time can be 30~
60min, decoloration will carry out under stiring so that activated carbon fully adsorbs, and obtains the fine grinding slurries of decoloration, and activated carbon will digest
It is removed when liquid is filtered by plate and frame filter press.
7, it saltouts
Fine grinding slurries obtained input 3000L is extracted into big tank, 100kg slurries add water 3000L, with 0.5mol/LHCl solution tune
PH value 4.0~4.5 is saved, is stirred evenly, NaCl powder is then added and saltouts, it is stirring while adding, keep 30r/min stirring slurries
Liquid, or so hour add 100kg sodium chloride.After collagen is precipitated completely, standing 12~for 24 hours, obtain collagen
Mixed serum.
8, press filtration
The sheet frame of plate and frame filter press is laid into No. 3927 filter clothes, 0.5% super-cell is added to the tank for filling enzymolysis liquid
In, it then stirs, is configured to the liquid material to be filtered of the enzymolysis liquid mixed liquor added with filter aid, it is later that mixed liquor is uniform
Be routed on sheet frame filter cloth, slowly open filter pressing pump inlet valve, outlet valve, start press filtration relay pump, pressure be 0.1~
1.0MPa, conveying mixed liquor start press filtration, control press filtration progress by the aperture regulation of outlet valve, filter is discarded after the completion of press filtration
Cake obtains transparent filtering slurries.
9, it digests
Slurries after press filtration are added in 2500L enzymatic vessels, add 7L water, 600L slurries about to add water 1400L according to every 3L slurries,
It takes compound protease to digest, the pepsin of slurry weight 0.1-2% is first added, dilute hydrochloric acid is added and adjusts pH to pH 2~3,
Under 40-45 DEG C of temperature condition, after making enzyme digestion reaction 72h.Then the papain of slurry weight 0.5%~1% is added,
At 40-45 DEG C, it is added under conditions of sodium hydroxide adjusting pH to pH6-8, continues enzyme digestion reaction 5-10 hours, in enzymolysis process not
It stirs disconnectedly, the mixed liquor containing a large amount of oligopeptides is obtained by enzymolysis.
10, ultrafiltration oligopeptide
Oligopeptide mixed liquor after enzymolysis is subjected to classification ultrafiltration through reverse osmosis ultrafiltration unit, first passes through the super of molecular weight 75KDa
Filter filters, and is filtered using the ultrafilter of molecular weight 3KDa.Ensure pressure 3.0-5.0MPa, flow velocity 1000- in ultra-filtration process
1200L/h adds water 800L ultrafiltration 2-3 times every time, and the peptide of suitable molecular weight segment enters collector through film after ultrafiltration, retention
The peptide of large fragment continues and enzyme effect digests again, then ultrafiltration obtains the peptide of suitable molecular weight segment.It is low that albumen is obtained repeatedly
Poly- peptide filtered fluid.Filtered fluid temperature is warming up to 80~95 DEG C of progress high temperature enzyme deactivations, 10~25min after ultrafiltration.
11, nanofiltration retention concentration, desalination, removing impurities matter
In the ultrafilter that the molecular weight that albumen oligopeptide filtrate is pumped into reverse osmosis nanofiltration unit is 500Da, holding pressure 2.5~
3.5MPa carries out reverse osmosis nanofiltration concentration under conditions of temperature is 15~45 DEG C, until nanofiltration retentate fluid is concentrated into water content only
There is the minimum volume of 3-5% or so, then concentrate is added into same volume water elution 3-5 times, same obtain is concentrated into water content only
There is the minimum volume nanofiltration retentate fluid of 3-5% or so.It is collected nanofiltration filtered fluid and nanofiltration retentate fluid respectively, collection is contained
The nanofiltrations filtered fluids such as other impurities substance and inorganic salts in raw material are pumped into biochemical treatment tank and carry out purified treatment, heel row up to standard
It puts, the nanofiltration retentate fluid of collection is tree frog skin oligopeptide concentrate, enters degerming process to it.
12, degerming
After being filtered using molecular weight 60-80KDa hollow fiber membrane ultrafiltration devices, enters back into 0.22 μm of micropore filter element and be filtered, pressing
Under the conditions of power 2.0-3.0 MPa, ultrafiltration feed velocity 1.2-1.8L/min, tree frog skin oligopeptide is concentrated by way of retention
Liquid carries out degerming, after degerming, is sealed into filling with stainless steel sealing bucket.
13, filling
Oligopeptide concentrate after degerming is carried out with automatic filling machine it is filling, using cleaning in sterile 10ml cillin bottles.West
Woods bottle first pass through ultraviolet light pass-through box enter ultrasonic bottle washing machine between wash bottle it is per minute wash 200 bottles of washed bottles and enter dryer exist
It is dried under 260 DEG C of 280 DEG C of preheating temperature, 300 DEG C of sterilising temp, heat preservation programs, completes 10,000 bottles of cillin bottle sterilizings per hour and dry
It is dry.Cillin bottle is filling into sterile beginning, and dosage controls within 5ml, ensures the stabilization of net content, per bottled amount 200mg,
Filling 200 bottles per minute, and after pressurized rubber plug to state of partly jumping a queue, canned product is entered in vacuum freeze drier
Freeze-drying.
14, it is freeze-dried
（1）Pre-freeze cures
By canned concentrate cillin bottle, it is placed on the partition board in vacuum freeze drier body feed tank, pre-freeze solidification temperature -30
~-45 DEG C, and 5~8h is kept, so that moisture in concentrate is fully freezed, it is ensured that no liquid exists.
（2）Lyophilization：Freeze dryer starts vacuum system, and pressure in storehouse is evacuated to 30~50pa, takes slow heating, with
1 DEG C of heating rate per hour is slowly warming up to -20~-25 DEG C, keeps the temperature 6~8h, then with 2 DEG C per hour of heating rate, delay
It is slow to be warming up to -10 DEG C~-12 DEG C, 3~6h is kept the temperature, lyophilization is carried out.
（3）Parsing-desiccation：After lyophilization, then with 3 DEG C per hour of heating rate, 40~50 DEG C are slowly warming up to, pressure
Power is 10~20Pa.6~8h is kept the temperature, parsing-desiccation freeze-drying is completed, is taken out after the plastic lid that pressurizes.
15, bottle cap and plug, labelling are pressed
Aseptically mechanical gland bottle cap and plug after freeze-drying, makes it post label into labelling machine.Purity of protein is made
Up to 99.5%, oligopeptide component content of the relative molecular mass less than 1000Da is averagely more than 85% tree frog skin oligopeptide finished product
Powder.
Embodiment 2
The dry tree frog skins of 100kg are weighed, 48h-72h is impregnated, until tree frog skin completes rehydration and becomes doughy state.It puts in meat grinder and twists
It is broken into the fragment that granularity is 0.1-0.2cm.With 1.0-2.0% acetum degreasing 1-2d, with 30-50 DEG C of warm water rinse 2 times.With
Bone mud barreling wears into particle and refines slurries in 20~50 μm of sizes.The decolorizing with activated carbon of the 3-5% of liquid weight is added.Adjust pH
Value 4.0~4.5 is added NaCl powder and saltouts, and with plate and frame filter press press filtration, will obtain the slurries after press filtration and slurries are first added
The pepsin of weight 0.1-2% adjusts pH to pH2~3, under 40-45 DEG C of temperature condition, after making enzyme digestion reaction 72h.Again plus
The papain for entering slurry weight 0.5%~1% adjusts pH to pH6-8 at 40-45 DEG C, and enzyme digestion reaction 5-10 hours obtains
Contain oligopeptide mixed liquor.Enzymolysis liquid is classified ultrafiltration, first passes through the ultrafilter of molecular weight 75KDa, using molecular weight 3KDa
Ultrafilter filtering.Its temperature is warming up to 80~95 DEG C of progress high temperature enzyme deactivations after ultrafiltration.It is again the nanofiltration of 500Da through molecular weight
It is concentrated in device.Elution 3-5 times, makes to be concentrated into the minimum volume that water content only has 3-5% or so.By impure and inorganic salts
Deng nanofiltration filtered fluid carry out purified treatment.Oligopeptide concentrate is filtered using molecular weight 60-80KDa hollow fiber membrane ultrafiltration devices
Afterwards, 0.22 μm of micropore filter element filtration sterilization is entered back into.Carried out with automatic filling machine it is filling, 10ml cillin bottle dosage control in 5ml
Within, per bottled amount 200mg.Vacuum freeze drier is freeze-dried.Purity of protein is made and is up to 99.5%, relative molecular mass
Oligopeptide component content less than 1000Da is averagely up to 85% tree frog skin oligopeptide finished powder.The dry tree frog skin raw materials of 100kg are flat
The oligomeric Gly-His-Lys of 4.662kg can be produced.
Claims (1)
1. a kind of industrialized producing technology of wood frog hide collagen oligopeptide, steps are as follows：
（1）Raw material
Raw material selects dry tree frog skin；
（2）Impregnate softening
48h-72 h are impregnated in water filling, until the complete rehydration of tree frog skin, becomes doughy state；
（3）It rubs
Rub into the fragment that granularity is 0.1-0.2cm；
（4）Degreasing
By the degreaser soak degreasing 1-2d of the tree frog skin of rubbing 1.0-2.0% acetums, finally 30-50 DEG C of warm water is used to float
It washes 2 times, obtains the tree frog skin that degreasing is impregnated；
（5）Defibrination
Particle is worn into 20~50 μm of grinding slurries；
（6）Decoloration
The activated carbon of the 3-5% of liquid weight is added in grinding slurry so that activated carbon fully adsorbs, and obtains the finish milling slurry of decoloration
Liquid；
（7）It saltouts
Grinding slurry obtained is adjusted into pH value 4.0~4.5, NaCl powder is added and saltouts, standing 12~for 24 hours, obtain collagen
Mixed liquor；
（8）Press filtration
Mixed liquor is uniformly routed on sheet frame filter cloth, conveying mixed liquor starts 1~6h of press filtration, discards filter cake, obtains transparent
Filtrate；
（9）Enzymolysis
Slurries after press filtration are first added to the pepsin of slurry weight 0.1-2%, adjust pH value to 2~3, in 40-45 DEG C of temperature
Under the conditions of degree, after making enzyme digestion reaction 72h, the papain of slurry weight 0.5%~1% is added, at 40-45 DEG C, adjusts pH
To 6-8, enzyme digestion reaction 5-10 hours is obtained containing oligopeptide mixed liquor value；
（10）Ultrafiltration oligopeptide
Enzymolysis liquid is subjected to classification ultrafiltration through reverse osmosis ultrafiltration unit, the ultrafilter of molecular weight 75KDa is first passed through, using molecule
The ultrafilter filtering of 3KDa is measured, the peptide of suitable molecular weight segment enters collector through film, and the peptide of large fragment continues and enzyme effect
It digests again, then ultrafiltration obtains oligopeptide filtered fluid, its temperature is warming up to 80~95 DEG C of progress high temperature enzyme deactivations after ultrafiltration；
（11）Press filtration retains concentration, desalination, removing impurities matter
It is concentrated in the nanofiltration device that the molecular weight that albumen oligopeptide filtrate is pumped into reverse osmosis nanofiltration unit is 500Da, then will
Concentrate adds same volume water elution 3-5 times, obtains the nanofiltration retentate fluid for being concentrated into the minimum volume that water content only has 3-5%,
It will collect containing other substances and the nanofiltrations filtered fluid such as inorganic salts in raw material, carry out purified treatment, the nanofiltration retentate fluid of collection
For oligopeptide concentrate；
（12）Degerming
After concentrate is filtered using molecular weight 60-80KDa hollow fiber membrane ultrafiltration devices, enters back into 0.22 μm of micropore filter element and carried out
Filter, carries out degerming by way of retention；
（13）It is filling
It is filling with cillin bottle；
（14）Freeze-drying
（a）Pre-freeze cures
By canned concentrate cillin bottle, it is placed on the partition board in vacuum freeze drier body feed tank, pre-freeze solidification temperature -30
~-45 DEG C, and 5~8h is kept, so that moisture in concentrate is fully freezed, it is ensured that no liquid exists；
（b）Lyophilization：Freeze dryer starts vacuum system, and pressure in storehouse is evacuated to 30~50pa, takes slow heating, with every small
When 1 DEG C of heating rate, be slowly warming up to -20~-25 DEG C, keep the temperature 6~8h, then with 2 DEG C per hour of heating rate, slowly rise
Temperature arrives -10 DEG C~-12 DEG C, keeps the temperature 3~6h, carries out lyophilization；
（c）Parsing-desiccation：After lyophilization, then with 3 DEG C per hour of heating rate, 40~50 DEG C are slowly warming up to, pressure is
10~20Pa；6~8h is kept the temperature, parsing-desiccation freeze-drying is completed, is taken out after the plastic lid that pressurizes；
（15）Press bottle cap and plug, labelling
Aseptically mechanical gland bottle stopper after freeze-drying, makes it post label into labelling machine；Purity of protein height is made
Oligopeptide component content up to 99.5%, relative molecular mass less than 1000Da is averagely more than 85% tree frog skin oligopeptide finished product
Powder.
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| WO2007044945A2 (en) * | 2005-10-13 | 2007-04-19 | Gu, Jennifer, L. | Mineral collagen chelates and methods of making and using same |
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