CN109234342A - The preparation method of turtle peptide - Google Patents
The preparation method of turtle peptide Download PDFInfo
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- CN109234342A CN109234342A CN201811093620.9A CN201811093620A CN109234342A CN 109234342 A CN109234342 A CN 109234342A CN 201811093620 A CN201811093620 A CN 201811093620A CN 109234342 A CN109234342 A CN 109234342A
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- shelled turtle
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 53
- 241000270666 Testudines Species 0.000 title claims abstract description 22
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- 241001482311 Trionychidae Species 0.000 claims abstract description 78
- 102000008186 Collagen Human genes 0.000 claims abstract description 54
- 108010035532 Collagen Proteins 0.000 claims abstract description 54
- 229920001436 collagen Polymers 0.000 claims abstract description 53
- 230000007071 enzymatic hydrolysis Effects 0.000 claims abstract description 34
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims abstract description 34
- 239000012528 membrane Substances 0.000 claims abstract description 33
- 238000009835 boiling Methods 0.000 claims abstract description 25
- 239000007788 liquid Substances 0.000 claims abstract description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 20
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 19
- 238000001728 nano-filtration Methods 0.000 claims abstract description 16
- RXUWDKBZZLIASQ-UHFFFAOYSA-N Puerarin Natural products OCC1OC(Oc2c(O)cc(O)c3C(=O)C(=COc23)c4ccc(O)cc4)C(O)C(O)C1O RXUWDKBZZLIASQ-UHFFFAOYSA-N 0.000 claims abstract description 15
- HKEAFJYKMMKDOR-VPRICQMDSA-N puerarin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1C1=C(O)C=CC(C2=O)=C1OC=C2C1=CC=C(O)C=C1 HKEAFJYKMMKDOR-VPRICQMDSA-N 0.000 claims abstract description 15
- 239000000047 product Substances 0.000 claims abstract description 13
- 239000007864 aqueous solution Substances 0.000 claims abstract description 11
- 238000001192 hot extrusion Methods 0.000 claims abstract description 11
- 239000006228 supernatant Substances 0.000 claims abstract description 11
- -1 tert-Butanol peroxide Chemical class 0.000 claims abstract description 11
- 239000004365 Protease Substances 0.000 claims abstract description 10
- UEDUENGHJMELGK-HYDKPPNVSA-N Stevioside Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@]12C(=C)C[C@@]3(C1)CC[C@@H]1[C@@](C)(CCC[C@]1([C@@H]3CC2)C)C(=O)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O UEDUENGHJMELGK-HYDKPPNVSA-N 0.000 claims abstract description 10
- 238000004108 freeze drying Methods 0.000 claims abstract description 10
- 229940013618 stevioside Drugs 0.000 claims abstract description 10
- OHHNJQXIOPOJSC-UHFFFAOYSA-N stevioside Natural products CC1(CCCC2(C)C3(C)CCC4(CC3(CCC12C)CC4=C)OC5OC(CO)C(O)C(O)C5OC6OC(CO)C(O)C(O)C6O)C(=O)OC7OC(CO)C(O)C(O)C7O OHHNJQXIOPOJSC-UHFFFAOYSA-N 0.000 claims abstract description 10
- 235000019202 steviosides Nutrition 0.000 claims abstract description 10
- 239000002994 raw material Substances 0.000 claims abstract description 9
- DKGAVHZHDRPRBM-UHFFFAOYSA-N tert-butyl alcohol Substances CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 claims abstract description 9
- 108090000526 Papain Proteins 0.000 claims abstract description 6
- 239000012153 distilled water Substances 0.000 claims abstract description 6
- 238000000605 extraction Methods 0.000 claims abstract description 6
- 239000012535 impurity Substances 0.000 claims abstract description 6
- 229940055729 papain Drugs 0.000 claims abstract description 6
- 235000019834 papain Nutrition 0.000 claims abstract description 6
- 229940088598 enzyme Drugs 0.000 claims description 19
- 102000004190 Enzymes Human genes 0.000 claims description 17
- 108090000790 Enzymes Proteins 0.000 claims description 17
- 230000007062 hydrolysis Effects 0.000 claims description 14
- 238000006460 hydrolysis reaction Methods 0.000 claims description 14
- JXOHGGNKMLTUBP-UHFFFAOYSA-N 3,4,5-trihydroxy-1-cyclohexene-1-carboxylic acid Chemical compound OC1CC(C(O)=O)=CC(O)C1O JXOHGGNKMLTUBP-UHFFFAOYSA-N 0.000 claims description 13
- 230000009849 deactivation Effects 0.000 claims description 10
- 235000013305 food Nutrition 0.000 claims description 7
- 239000000243 solution Substances 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 5
- 230000001376 precipitating effect Effects 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- 238000010411 cooking Methods 0.000 claims description 2
- 238000001125 extrusion Methods 0.000 claims description 2
- 230000014759 maintenance of location Effects 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims 1
- 150000002978 peroxides Chemical class 0.000 claims 1
- 238000010025 steaming Methods 0.000 claims 1
- 102000004169 proteins and genes Human genes 0.000 abstract description 11
- 108090000623 proteins and genes Proteins 0.000 abstract description 11
- 102000005158 Subtilisins Human genes 0.000 abstract description 8
- 108010056079 Subtilisins Proteins 0.000 abstract description 8
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 6
- 230000009286 beneficial effect Effects 0.000 abstract 1
- 238000000034 method Methods 0.000 description 15
- 238000010521 absorption reaction Methods 0.000 description 12
- 238000012545 processing Methods 0.000 description 9
- 150000001413 amino acids Chemical class 0.000 description 8
- 230000008569 process Effects 0.000 description 7
- 238000005259 measurement Methods 0.000 description 5
- 108091005804 Peptidases Proteins 0.000 description 4
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 4
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 4
- 238000000354 decomposition reaction Methods 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 238000001641 gel filtration chromatography Methods 0.000 description 4
- 239000003292 glue Substances 0.000 description 4
- 238000010438 heat treatment Methods 0.000 description 4
- 238000000703 high-speed centrifugation Methods 0.000 description 4
- 108020004707 nucleic acids Proteins 0.000 description 4
- 102000039446 nucleic acids Human genes 0.000 description 4
- 150000007523 nucleic acids Chemical class 0.000 description 4
- 229920001184 polypeptide Polymers 0.000 description 4
- 235000019419 proteases Nutrition 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000000796 flavoring agent Substances 0.000 description 3
- 235000019634 flavors Nutrition 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000003643 water by type Substances 0.000 description 3
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 102000015636 Oligopeptides Human genes 0.000 description 2
- 108010038807 Oligopeptides Proteins 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 241001313855 Bletilla Species 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 229920000832 Cutin Polymers 0.000 description 1
- 235000010575 Pueraria lobata Nutrition 0.000 description 1
- 241000219781 Pueraria montana var. lobata Species 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 150000001336 alkenes Chemical class 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000002929 anti-fatigue Effects 0.000 description 1
- 230000000767 anti-ulcer Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 238000013475 authorization Methods 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 235000019658 bitter taste Nutrition 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- SZIUXKBNJBUQDA-UHFFFAOYSA-N cyclohex-4-ene-1,2,3-triol Chemical compound OC1CC=CC(O)C1O SZIUXKBNJBUQDA-UHFFFAOYSA-N 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000019621 digestibility Nutrition 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000003925 fat Substances 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 235000013402 health food Nutrition 0.000 description 1
- 239000000413 hydrolysate Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000008935 nutritious Nutrition 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 235000019640 taste Nutrition 0.000 description 1
- 235000021404 traditional food Nutrition 0.000 description 1
- 230000005068 transpiration Effects 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 238000003809 water extraction Methods 0.000 description 1
- 230000003313 weakening effect Effects 0.000 description 1
Classifications
-
- 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]
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- 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)
- Microbiology (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses the preparation methods of turtle peptide, step are as follows: crush soft-shelled turtle raw material, it is added and contains 3, the aqueous solution of 4,5- trihydroxy -1- cyclohexene -1- formic acid and Puerarin is simultaneously put into twin-screw boiling heat extruder feed inlet together, and hot extrusion extrudes underflow object, add papain limited enzymatic hydrolysis, enzymolysis liquid is centrifuged, supernatant is collected, is freeze-dried to obtain soft-shelled turtle collagen dry product；It by soft-shelled turtle collagen and subtilopeptidase A, is dissolved in distilled water, sequentially adds tert-Butanol peroxide, stevioside, hydrolyze to obtain enzymolysis liquid；Enzymolysis liquid is obtained into soft-shelled turtle collagen peptide through decoloration, ultrafiltration membrane removal of impurities, nanofiltration membrane concentration, freeze-drying.Turtle peptide preparation method of the present invention is had the beneficial effect that without acid-base pretreatment, turtle proteins purity is high, soft-shelled turtle peptides extraction rate is high, extraction time is short.
Description
Technical field
The present invention relates to the deep process technology fields of soft-shelled turtle, more particularly, to the preparation method of turtle peptide.
Background technique
Soft-shelled turtle also known as soft-shelled turtle, the soft-shelled turtle enrich blood, strong bone, antifatigue, the effect of promoting longevity, are China's traditional foods, and
Develop the very good material of premium health food；The main nutrient composition of soft-shelled turtle is protein, fat, iron, calcium, animal glue, cutin
Bletilla multivitamin etc..Soft-shelled turtle contains very high protein, and about per kilogram has 165 grams of protein, and in food or drug
Protein to be generally digested liquid enzymatic hydrolysis and be oligopeptide or amino acid and be absorbed by organisms and utilize, therefore absorb slow, utilize
Rate is low, so due drug effect is also not achieved.
Collagen peptide is the hydrolysate of collagen, and general molecular weight is in 3000Dal or less.Collagen peptide has
Various physiological functions, the digestibility of collagen peptide almost up to 100%, can protect stomach lining and antiulcer, promote
It is metabolized into skin collagen, blood pressure is inhibited to rise, promote Ca2+Absorb and reduce serum cholesterol levels, anti-oxidant, anti-aging
Deng, therefore can be widely used in the industries such as medicine, health care product, cosmetics.
The method for extracting collagen peptide mainly has Hot water extraction, acid system, alkaline process, salt method and enzyme process etc., active peptide
Preparation method then have from native organism extract, by chemical method and recombinant DNA technology synthesis, extracorporeal hydrolysis protein
Deng.With the fast development of Enzymes Industry, enzymatic hydrolysis is more mild, safe, more single-minded than traditional acid system, alkaline process, not only
Degradation time is short, and product nutrition loss is less, and non-environmental-pollution.Currently, collagen protein enzymolysis mainly has single enzyme process and more
Enzyme process, multi-enzyme method are divided into mixed enzyme solution and stepwise discretization method again.The condition of enzymolysis and extraction active peptide is typically considered as being developed
Requirement of the product to molecular weight, the lesser product of molecular weight preferably use multi-enzyme method.
The prior art such as Authorization Notice No. is the Chinese invention patent of CN 102286589B, and it is oligomeric to disclose a kind of soft-shelled turtle
The extracting method of peptide, after being digested as raw material with alkali protease using soft-shelled turtle, then with flavor protease it is circumscribed and adjust flavor, enzymatic hydrolysis
Liquid carries out ultrafiltration, and alkali protease additional amount is the 0.2-5% of raw material soft-shelled turtle weight, and flavor protease additional amount is raw material soft-shelled turtle
The 0.1-2% of weight, retaining molecular weight are that the ultrafiltration membrane of 0.5-30 ten thousand dalton carries out ultrafiltration, and film concentrated spray is dry
To turtle oligopeptide；The invention is easy to operate, in good taste, at low cost, environment-friendly high-efficiency, is very suitable to industrialized production, still
The soft-shelled turtle collagen purity of protein obtained using traditional enzymatic isolation method is lower, so that hydrolysis is not thorough, thereby reduces turtle proteins
The yield of peptide, causes nutriment to waste.
Summary of the invention
The purpose of the present invention is to provide one kind to be not necessarily to acid-base pretreatment, turtle proteins are with high purity, soft-shelled turtle peptides extraction rate is high,
The preparation method of extraction time short turtle peptide.
The present invention in view of the above technology in the problem of mentioning, the technical solution taken are as follows:
The preparation method of turtle peptide is realized by following processing step:
Step 1: clean soft-shelled turtle raw material is crushed by pulverizer；
Step 2: together by the aqueous solution of smashed soft-shelled turtle, 3,4,5- trihydroxy -1- cyclohexene -1- formic acid and Puerarin
It puts into twin-screw boiling heat extruder feed inlet, is squeezed under 100-131 DEG C of boiling temperature, screw speed 50-80r/min
0.5-1h, hot extrusion extrude underflow object；
Step 3: squeezing in underflow object plus to account for the papain limited enzymatic hydrolysis of soft-shelled turtle weight 1-3%, control temperature
At 5-10 DEG C, bifrequency ultrasonic wave added enzymatic hydrolysis persistently stirs at low speed lower enzymatic hydrolysis 24-48 hours, boiling water bath enzyme deactivation, by enzymolysis liquid in
10000-13000r/min high speed centrifugation, takes supernatant, and precipitating repeats enzymatic hydrolysis operation 2-3 times, collects supernatant, be freeze-dried
Soft-shelled turtle collagen dry product；
Step 4: weighing the soft-shelled turtle collagen and 0.7-0.9 parts of subtilopeptidase As of 25-40 parts of freeze-dryings, be added to
It is completely dissolved in 100-150 parts of distilled water, sequentially adds tert-Butanol peroxide, stevioside, adjusting pH is 7.5-8.0, is placed in 45-55
In DEG C thermostat water bath, bifrequency ultrasonic wave added enzymatic hydrolysis, with boiling water bath enzyme deactivation 15-20 minutes after hydrolysis 1.5-2 hour, cooling
Enzymolysis liquid；The unique three strands of superhelixes of collagen, property is sufficiently stable, and general processing temperature and short time heating are all
It cannot make its decomposition, to cause its digestion and absorption more difficult, be not easy to be made full use of by human body, soft-shelled turtle collagen molecules are through water
The lesser polypeptide of relative molecular weight, small peptide, amino acid are primarily formed after solution, its absorption rate can be improved much after hydrolysis,
And the absorption of the other oroteins in food can be promoted；
Step 5: enzymolysis liquid is obtained into soft-shelled turtle collagen through decoloration, ultrafiltration membrane removal of impurities, nanofiltration membrane concentration, freeze-drying
Peptide.
Preferably, twin-screw cooking extrusion machine used in step 2 is food processing field universal machine.
Preferably, soft-shelled turtle content is the 85-90% of aqueous solution, 3,4,5- trihydroxy -1- cyclohexene -1- first in step 2
The content of acid is the 2-6% of aqueous solution, and the content of Puerarin is the 0.1-0.3% of aqueous solution；3,4,5- trihydroxy -1- hexamethylene
The special presence of alkene -1- formic acid and Puerarin can dissolve rapidly soft-shelled turtle cell under the action of helical screw agitator and hot boiling
Memebrane protein and fat, make membranolysis, isolate the nutrition such as the animal glue contained in soft-shelled turtle, nucleic acid, albumen, vitamin at
Point, improve hot extrusion efficiency；Simultaneously as its be rich in positive charge, ingredient that can be negatively charged with nucleic acid etc. combine, weakening
The combination of these substances and albumen finally may be used to improve the content and purity for squeezing out soft-shelled turtle collagen in underflow object
Improve the yield of soft-shelled turtle collagen peptide.
Preferably, the supersonic frequency of the enzymatic hydrolysis of bifrequency ultrasonic wave added used in step 3 and 4 be respectively 30-45kHz with
50-60kHz, the ultrasonic sound intensity are 0.4-0.5W/cm2；Bifrequency ultrasound can increase enzyme by reducing enzyme digestion reaction Ea and KM value
With the contact frequency and contact area of substrate, increase enzyme digestion reaction rate.
Preferably, the tert-Butanol peroxide being added in step 4, stevioside are respectively 0.003-0.007 parts, 0.001-
0.004 part；Micro tert-Butanol peroxide and stevioside generate synergistic effect, on the one hand can increase subtilopeptidase A and first
The active site that Isin glue collagen combines, improves the affinity of enzyme-to-substrate, accelerates the enzymatic hydrolysis rate of subtilopeptidase A, from
And reduce hydrolysis time, time cost and cost of labor are reduced, and transpiration removes when subsequent hydrolyzate boils, no
It can remain in collagen peptide product；On the other hand, subtilopeptidase A can be promoted the peptide chain of soft-shelled turtle collagen
It is disconnected from inside, the amino acid of active site of protein is exposed, and further the hydrophobic amino acid of peptide termini is cut off, is released
It releases, while increase small molecule amino acid dissociates degree, moreover it is possible to which the bitter taste for effectively reducing peptide improves the mouthfeel of product.
Preferably, ultrafiltration membrane used in step 5 is the ultrafiltration membrane that molecular cut off is 3000Dal, nanofiltration membrane is retention
Molecular weight is the nanofiltration membrane of 500Dal.
Compared with the prior art, the advantages of the present invention are as follows: (1) present invention using twin-screw boiling hot extrusion combine enzymatic hydrolysis
Soft-shelled turtle collagen is extracted, the special presence of 3,4,5- trihydroxy -1- cyclohexene -1- formic acid and Puerarin can speed up soft-shelled turtle
Membranolysis isolates the animal glue contained in soft-shelled turtle, nucleic acid, albumen, vitamins and other nutritious components, improves hot extrusion effect
Rate；Meanwhile, it is capable to the combination of these substances and albumen be weakened in conjunction with the ingredient negatively charged with nucleic acid etc., to improve
Squeeze out the content and purity of soft-shelled turtle collagen in underflow object；(2) in hydrolytic process, tert-Butanol peroxide and stevioside have association
Same-action, on the one hand can be improved the enzymatic hydrolysis rate of subtilopeptidase A, to reduce hydrolysis time, reduce the time at
On the other hand this and cost of labor can be improved the free degree of hydrophobic amino acid, improve the mouthfeel of product.
Specific embodiment
The present invention program is described further below by embodiment:
Embodiment 1:
The preparation method of turtle peptide is realized by following processing step:
(1) clean soft-shelled turtle raw material is crushed by pulverizer；
(2) aqueous solution of smashed soft-shelled turtle, 3,4,5- trihydroxy -1- cyclohexene -1- formic acid and Puerarin is thrown together
Enter in twin-screw boiling heat extruder feed inlet, squeezes 0.5h, hot extrusion under 100 DEG C of boiling temperature, screw speed 50r/min
Underflow object out；Wherein, by weight percentage, soft-shelled turtle content is 85%, 3,4,5- trihydroxy -1- cyclohexene -1- formic acid contents
It is 2%, puerarin content 0.1%；
(3) it squeezes in underflow object plus to account for the papain limited enzymatic hydrolysis of soft-shelled turtle weight 1%, controls temperature at 5 DEG C,
In supersonic frequency is 30kHz, the ultrasonic sound intensity is 0.4W/cm2Lower bifrequency ultrasonic wave added enzymatic hydrolysis, persistently stirs at low speed lower enzymatic hydrolysis 24
Hour, boiling water bath enzyme deactivation takes supernatant by enzymolysis liquid in 10000r/min high speed centrifugation, and precipitating repeats enzymatic hydrolysis operation 2 times, receives
Collect supernatant, is freeze-dried to obtain soft-shelled turtle collagen dry product；
(4) the soft-shelled turtle collagen and 0.7 part of subtilopeptidase A for weighing 25 parts of freeze-dryings, are added to 100 parts of distilled water
In be completely dissolved, sequentially add 0.003 part of tert-Butanol peroxide, 0.001 part of stevioside, adjusting pH is 7.5, is placed in 45 DEG C of thermostatted waters
In bath, in supersonic frequency is 50kHz, the ultrasonic sound intensity is 0.4W/cm2Lower bifrequency ultrasonic wave added enzymatic hydrolysis, after hydrolysis 1.5 hours
With boiling water bath enzyme deactivation 15 minutes, cooling enzymolysis liquid；The unique three strands of superhelixes of collagen, property is sufficiently stable, and one
As processing temperature and the short time heating cannot all make its decomposition, to cause its digestion and absorption more difficult, be not easy to be filled by human body
Divide and utilize, soft-shelled turtle collagen molecules primarily form the lesser polypeptide of relative molecular weight, small peptide, amino acid after hydrolysis, hydrolyze
Its absorption rate can be improved much afterwards, and can promote the absorption of the other oroteins in food；
(5) enzymolysis liquid of (4) step is obtained into soft-shelled turtle collagen egg through decoloration, ultrafiltration membrane removal of impurities, nanofiltration membrane concentration, freeze-drying
White peptide；Ultrafiltration membrane is the ultrafiltration membrane that molecular cut off is 3000Dal, and nanofiltration membrane is the nanofiltration membrane that molecular cut off is 500Dal.
Use the content of Coomassie Brilliant Blue measurement soft-shelled turtle collagen for 81.2%；Using high performance gel filtration chromatography
It is 95.3% that relative molecular mass, which is measured, less than the content of the soft-shelled turtle collagen peptide of 3000 dalton.
Embodiment 2:
The preparation method of turtle peptide is realized by following processing step:
(1) clean soft-shelled turtle raw material is crushed by pulverizer；
(2) aqueous solution of smashed soft-shelled turtle, 3,4,5- trihydroxy -1- cyclohexene -1- formic acid and Puerarin is thrown together
Enter in twin-screw boiling heat extruder feed inlet, squeezes 0.6h, hot extrusion under 123 DEG C of boiling temperature, screw speed 60r/min
Underflow object out；Wherein, by weight percentage, soft-shelled turtle content is 87%, 3,4,5- trihydroxy -1- cyclohexene -1- formic acid contents
It is 5%, puerarin content 0.2%；
(3) it squeezes in underflow object plus to account for the papain limited enzymatic hydrolysis of soft-shelled turtle weight 2%, controls temperature at 8 DEG C,
In supersonic frequency is 35kHz, the ultrasonic sound intensity is 0.4W/cm2Lower bifrequency ultrasonic wave added enzymatic hydrolysis, persistently stirs at low speed lower enzymatic hydrolysis 36
Hour, boiling water bath enzyme deactivation takes supernatant by enzymolysis liquid in 12000r/min high speed centrifugation, and precipitating repeats enzymatic hydrolysis operation 2 times, receives
Collect supernatant, is freeze-dried to obtain soft-shelled turtle collagen dry product；
(4) the soft-shelled turtle collagen and 0.8 part of subtilopeptidase A for weighing 30 parts of freeze-dryings, are added to 130 parts of distilled water
In be completely dissolved, sequentially add 0.005 part of tert-Butanol peroxide, 0.002 part of stevioside, adjusting pH is 7.8, is placed in 50 DEG C of thermostatted waters
In bath, in supersonic frequency is 55kHz, the ultrasonic sound intensity is 0.4W/cm2Lower bifrequency ultrasonic wave added enzymatic hydrolysis, after hydrolysis 1.8 hours
With boiling water bath enzyme deactivation 16 minutes, cooling enzymolysis liquid；The unique three strands of superhelixes of collagen, property is sufficiently stable, and one
As processing temperature and the short time heating cannot all make its decomposition, to cause its digestion and absorption more difficult, be not easy to be filled by human body
Divide and utilize, soft-shelled turtle collagen molecules primarily form the lesser polypeptide of relative molecular weight, small peptide, amino acid after hydrolysis, hydrolyze
Its absorption rate can be improved much afterwards, and can promote the absorption of the other oroteins in food；
(5) enzymolysis liquid of (4) step is obtained into soft-shelled turtle collagen egg through decoloration, ultrafiltration membrane removal of impurities, nanofiltration membrane concentration, freeze-drying
White peptide；Ultrafiltration membrane is the ultrafiltration membrane that molecular cut off is 3000Dal, and nanofiltration membrane is the nanofiltration membrane that molecular cut off is 500Dal.
Use the content of Coomassie Brilliant Blue measurement soft-shelled turtle collagen for 86.6%；Using high performance gel filtration chromatography
It is 96.8% that relative molecular mass, which is measured, less than the content of the soft-shelled turtle collagen peptide of 3000 dalton.
Embodiment 3:
The preparation method of turtle peptide is realized by following processing step:
(1) clean soft-shelled turtle raw material is crushed by pulverizer；
(2) aqueous solution of smashed soft-shelled turtle, 3,4,5- trihydroxy -1- cyclohexene -1- formic acid and Puerarin is thrown together
Enter in twin-screw boiling heat extruder feed inlet, 1h is squeezed under 131 DEG C of boiling temperature, screw speed 80r/min, hot extrusion extrudes
Underflow object；Wherein, by weight percentage, soft-shelled turtle content is 90%, and 3,4,5- trihydroxy -1- cyclohexene -1- formic acid contents are
6%, puerarin content 0.3%；
(3) it squeezes in underflow object plus to account for the papain limited enzymatic hydrolysis of soft-shelled turtle weight 3%, controls temperature 10
DEG C, in supersonic frequency is 45kHz, the ultrasonic sound intensity is 0.5W/cm2Lower bifrequency ultrasonic wave added enzymatic hydrolysis, persistently stirs at low speed lower enzyme
Solution 48 hours, boiling water bath enzyme deactivation take supernatant by enzymolysis liquid in 13000r/min high speed centrifugation, and precipitating repeats enzymatic hydrolysis operation 3
It is secondary, supernatant is collected, soft-shelled turtle collagen dry product is freeze-dried to obtain；
(4) the soft-shelled turtle collagen and 0.9 part of subtilopeptidase A for weighing 40 parts of freeze-dryings, are added to 150 parts of distilled water
In be completely dissolved, sequentially add 0.007 part of tert-Butanol peroxide, 0.004 part of stevioside, adjusting pH is 8.0, is placed in 55 DEG C of thermostatted waters
In bath, in supersonic frequency is 60kHz, the ultrasonic sound intensity is 0.5W/cm2Lower bifrequency ultrasonic wave added enzymatic hydrolysis, hydrolysis are used after 2 hours
Boiling water bath enzyme deactivation 20 minutes, cooling enzymolysis liquid；The unique three strands of superhelixes of collagen, property is sufficiently stable, generally
Processing temperature and the short time heating cannot all make its decomposition, to cause its digestion and absorption more difficult, be not easy abundant by human body
It utilizes, soft-shelled turtle collagen molecules primarily form the lesser polypeptide of relative molecular weight, small peptide, amino acid after hydrolysis, after hydrolysis
Its absorption rate can be improved very much, and can promote the absorption of the other oroteins in food；
(5) enzymolysis liquid of (4) step is obtained into soft-shelled turtle collagen egg through decoloration, ultrafiltration membrane removal of impurities, nanofiltration membrane concentration, freeze-drying
White peptide；Ultrafiltration membrane is the ultrafiltration membrane that molecular cut off is 3000Dal, and nanofiltration membrane is the nanofiltration membrane that molecular cut off is 500Dal.
Use the content of Coomassie Brilliant Blue measurement soft-shelled turtle collagen for 82.5%；Using high performance gel filtration chromatography
It is 96.0% that relative molecular mass, which is measured, less than the content of the soft-shelled turtle collagen peptide of 3000 dalton.
Comparative example 1:
Be added without 3,4,5- trihydroxy -1- cyclohexene -1- formic acid and Puerarin in step (2) aqueous solution, rest part and
Embodiment 2 is completely the same.
Use the content of Coomassie Brilliant Blue measurement soft-shelled turtle collagen for 70.9%；Using high performance gel filtration chromatography
It is 89.8% that relative molecular mass, which is measured, less than the content of the soft-shelled turtle collagen peptide of 3000 dalton.
By above measurement result it is found that the soft-shelled turtle collagen and relative molecular mass of embodiment 2 are less than 3000 dongles
The content of the soft-shelled turtle collagen peptide to pause is much higher than comparative example 1, shows 3,4,5- trihydroxy -1- cyclohexene -1- formic acid and pueraria lobata
Element has gain effect, can be improved the content and purity of soft-shelled turtle collagen, and then improves obtaining for soft-shelled turtle collagen peptide
Rate.
Routine operation in operating procedure of the invention is well known to those skilled in the art, herein without repeating.
Technical solution of the present invention is described in detail in embodiment described above, it should be understood that the above is only
For specific embodiments of the present invention, it is not intended to restrict the invention, all any modifications made in spirit of the invention,
Supplement or similar fashion substitution etc., should all be included in the protection scope of the present invention.
Claims (8)
1. the preparation method of turtle peptide, using twin-screw boiling hot extrusion combination enzymolysis and extraction soft-shelled turtle collagen, feature exists
In: in twin-screw boiling hot extrusion charging it is water-soluble comprising 3,4,5- trihydroxy -1- cyclohexene -1- formic acid and Puerarin
Liquid.
2. the preparation method of turtle peptide according to claim 1, it is characterised in that: sequentially include the following steps:
(1) soft-shelled turtle raw material is crushed；
(2) aqueous solution of smashed soft-shelled turtle, 3,4,5- trihydroxy -1- cyclohexene -1- formic acid and Puerarin is put into together double
In screw rod boiling heat extruder feed inlet, 0.5-1h is squeezed under 100-131 DEG C of boiling temperature, screw speed 50-80r/min,
Hot extrusion extrudes underflow object；
(3) it squeezes in underflow object plus to account for the papain limited enzymatic hydrolysis of soft-shelled turtle weight 1-3%, controls temperature in 5-10
DEG C, bifrequency ultrasonic wave added enzymatic hydrolysis, persistently stir at low speed lower enzymatic hydrolysis 24-48 hours, boiling water bath enzyme deactivation, by enzymolysis liquid at a high speed from
The heart, takes supernatant, and precipitating repeats enzymatic hydrolysis operation 2-3 times, collects supernatant, be freeze-dried to obtain soft-shelled turtle collagen dry product；
(4) the soft-shelled turtle collagen and 0.7-0.9 parts of subtilopeptidase As 25-40 parts being lyophilized, are added to 100-150 parts of steamings
It is completely dissolved in distilled water, sequentially adds tert-Butanol peroxide, stevioside, adjusting pH is 7.5-8.0, is placed in 45-55 DEG C of water bath with thermostatic control
In pot, bifrequency ultrasonic wave added enzymatic hydrolysis, with boiling water bath enzyme deactivation 15-20 minutes after hydrolysis 1.5-2 hour, cooling enzymolysis liquid；
(5) enzymolysis liquid is obtained into soft-shelled turtle collagen peptide through decoloration, ultrafiltration membrane removal of impurities, nanofiltration membrane concentration, freeze-drying.
3. the preparation method of turtle peptide according to claim 2, it is characterised in that: the twin-screw cooking extrusion machine is food
Product manufacture field universal machine.
4. the preparation method of turtle peptide according to claim 2, it is characterised in that: soft-shelled turtle content is water-soluble in (2)
The 85-90% of liquid, the content of 3,4,5- trihydroxy -1- cyclohexene -1- formic acid are the 2-6% of aqueous solution, and the content of Puerarin is water
The 0.1-0.3% of solution.
5. the preparation method of turtle peptide according to claim 2, it is characterised in that: bifrequency ultrasonic wave added in (3)
The supersonic frequency of enzymatic hydrolysis is 30-45kHz, and the ultrasonic sound intensity is 0.4-0.5W/cm2。
6. the preparation method of turtle peptide according to claim 2, it is characterised in that: bifrequency ultrasonic wave added in (4)
The supersonic frequency of enzymatic hydrolysis is 50-60kHz, and the ultrasonic sound intensity is 0.4-0.5W/cm2。
7. the preparation method of turtle peptide according to claim 2, it is characterised in that: the tertiary fourth of peroxide being added in (4)
Alcohol, stevioside are respectively 0.003-0.007 parts, 0.001-0.004 parts.
8. the preparation method of turtle peptide according to claim 2, it is characterised in that: ultrafiltration membrane is retention point in (5)
The ultrafiltration membrane that son amount is 3000Dal, nanofiltration membrane is the nanofiltration membrane that molecular cut off is 500Dal.
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|---|---|---|---|
| CN201811093620.9A CN109234342A (en) | 2018-09-19 | 2018-09-19 | The preparation method of turtle peptide |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114921515A (en) * | 2022-05-13 | 2022-08-19 | 纽斯葆广赛（广东）生物科技股份有限公司 | Preparation method of turtle oligopeptide |
| CN114990179A (en) * | 2022-04-11 | 2022-09-02 | 杭州萧山天福生物科技有限公司 | Preparation method and application of turtle egg active peptide freeze-dried powder |
-
2018
- 2018-09-19 CN CN201811093620.9A patent/CN109234342A/en not_active Withdrawn
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114990179A (en) * | 2022-04-11 | 2022-09-02 | 杭州萧山天福生物科技有限公司 | Preparation method and application of turtle egg active peptide freeze-dried powder |
| CN114990179B (en) * | 2022-04-11 | 2023-07-04 | 杭州萧山天福生物科技有限公司 | Preparation method and application of turtle egg active peptide freeze-dried powder |
| CN114921515A (en) * | 2022-05-13 | 2022-08-19 | 纽斯葆广赛（广东）生物科技股份有限公司 | Preparation method of turtle oligopeptide |
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Application publication date: 20190118 |