CN108118077A - The technique that a kind of enzymatic hydrolysis salmon collagen prepares anti-oxidation peptide and antifreeze peptide - Google Patents
The technique that a kind of enzymatic hydrolysis salmon collagen prepares anti-oxidation peptide and antifreeze peptide Download PDFInfo
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- CN108118077A CN108118077A CN201611076917.5A CN201611076917A CN108118077A CN 108118077 A CN108118077 A CN 108118077A CN 201611076917 A CN201611076917 A CN 201611076917A CN 108118077 A CN108118077 A CN 108118077A
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- peptide
- antifreeze
- oxidation
- collagen
- oxidation peptide
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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
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- 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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
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- Peptides Or Proteins (AREA)
Abstract
The present invention designs the technique that a kind of collagen protein enzymolysis prepare anti-oxidation peptide and antifreeze peptide simultaneously, and method is to extract collagen by raw material of salmon skin, is utilizedVibrioThe extracellular protease crude enzyme liquid of the fermentation gained of sp.SQS2 3 is hydrolyzed into small peptide solution, by obtaining with protection myosin after 3000 Da super filter tube 4500 × g centrifugal ultrafiltrations 45min and slowing down its Ca2+The ATPase activity antifreeze peptide that decline acts in multigelation, after lower floor's ultrafiltration component being separated by 20 gel permeation chromatographies of Sephadex LH, obtain have remove DPPH free radicals, hydroxy radical and to DNA oxidative damages with the ability centainly inhibited, and oxygen radical absorbability detect in show concentration dependant antioxidation activity anti-oxidation peptide.LC MS technologies identify the antioxidant composition and contain 19 polypeptides.
Description
Technical field
The present invention relates to it is a kind of using microbial protease enzymolysis salmon collagen prepare simultaneously anti-oxidation peptide with it is freeze proof
The method of peptide, belongs to technical field of biotechnology.
Background technology
China ocean and freshwater vast area, aquatic resources enrich, and account for the 36% of World Water product population.With fishing
The development of industry, the development of Aquatic Product Process Industry are also promoted.According to statistics, there are about 20,000,000 tons of fishes for being rich in protein every year
Class byproduct, such as fish-bone, fish-skin, fin and fish scale, generate in process, they are most of directly buried abandon or
For making the low values product such as animal feed, fish meal, the serious wasting of resources is not only caused, also directly results in environmental pollution.
Therefore, the technique that high value added utilization is carried out to the discarded object that fish processing industry generates is developed, it is made to turn waste into wealth, is become
Alleviate the important means of resource and environment.
Collagen is widely present in the epidermis and bone tissue of animal, composition unit be procollagen, each procollagen
Molecule is made of three peptide chains, and peptide chain is α-helixstructure, and triple helix structure is formed in the form of parallel right-handed helix.Collagen egg
Most important feature is formation repeated arrangement with " Gly-X-Y " on amino acid composition in vain, i.e., every two amino acid residues
There is a glycine, X and Y is other amino acid residues.Traditionally the collagen of business application is mostly from Animal Skin
Bone, but because of diseases such as mad cattle disease, aftosas, people is made to generate doubt to livestock collagen products safety.Simultaneously because religion and
The factors such as custom, some areas use livestock collagen product with masses' refusal.Collagen content is very abundant in many aquatiles,
Also have the characteristics that some are different from mammal collagen, the content of hydroxyproline compares land in aquatic livestock collagen
Ground animal collagen is much lower, this so that the thermal stability of the triple-helix structure of aquatic livestock collagen is lower, at low temperature
It is more soluble in neutral salt solution or acid solution, it is easier to soluble collagen be made, provided for the extraction and application of collagen
Sharp condition.
In recent years, both at home and abroad studies have reported that successfully being prepared with different bioactivity using the protein in byproduct
Peptide quasi-molecule, become the research hotspot in food, medicine, skin care industry, including anti-oxidation peptide, blood pressure lowering peptide, antibacterial
Peptide, antifreeze peptide etc..The biologically active peptide species found from nature is limited, and extracted amount is few, and difficulty is big, it is difficult to realize industry
The production of change scale, and there are a large amount of different amino acid sequence informations in macro-molecular protein, albumen is made by biotechnology
Matter and polypeptide fracture, release the peptide fragment of different structure, this provides new grind to obtain potential biologically active peptide on a large scale
Study carefully thinking.
The low value albumen that collagen is enriched as a kind of quantity, derived from a wealth of sources, with reference to bio-enzyme engineering technology, exploitation one
Kind to the technique of the high-valued comprehensive utilization of collagen, collagen is hydrolyzed, obtain between protein and amino acids it
Between oligomeric active collagen peptide, have huge potential market value in medicine, skin care, health care, field of food.
The content of the invention
The present invention prepares the deficiency of active peptide technology for existing biological enzymolysis, provides a kind of outer egg of utilization extracellular microbial
The collagen that white enzyme enzymolysis is extracted from salmon skin, preparative separation goes out with different bioactivity simultaneously from different components
Collagen peptide process, make up to the purpose of comprehensive utilization of resources.
Step is as follows：
（One）The preparation of extracellular microbial exoproteinase：
1st, by extracellular protease bacteriaVibrioSp. SQS2-3 inoculations are in liquid seed culture medium, in 17 °C of levels
Shake culture 2-3 days in shaking table, rotating speed is 200 rpm, then by 2%（v/v）Inoculum concentration be inoculated in fermentation medium,
Shake culture 5 days in 17 °C of horizontal shakers, rotating speed are 180 rpm, and zymotic fluid is made；
2nd, zymotic fluid is poured into centrifuge tube progress low-temperature and high-speed centrifugation, centrifugal condition is 10000 × g, 4 °C, 30 min, so
After collect supernatant, pour into new centrifuge tube, 13000 × g, 4 °C centrifugation 15 min, repeat 2-3 times, until supernatant precipitate
It is less；Supernatant is added in the bag filter that molecule interception is 8000-14000 Da, is pressed from both sides and sealed with bag filter, Ran Houfang
It is 20 mM to put in concentration, and ice bath is dialysed 2 h in the Tris-HCl of pH 7.8, and it is saturating that the Tris-HCl then more renewed continues ice bath
2 h are analysed, finally impregnate bag filter with new Tris-HCl, and are placed in 4 °C of refrigerators 24 h that dialyse, obtain crude enzyme liquid, enzyme solution
It is dispensed into 1.5 ml Epperdof pipes, is placed in -20 °C of refrigerators and preserves for use.
（Two）The preparation of Isin glue collagen：
1st, the flesh of fish on fresh salmon skin is rejected, fish-skin is cut into and is about about 0.5 cm size strips of 4 cm wide, with 3 times of bodies
4 °C of product precooling ultrapure water fish-skin 2-3 times；
2nd, 20 g fish-skins are taken in 100 ml centrifuge tubes, 60 ml ultra-pure waters is added in, is then placed in 80 °C of water-baths and stands water
30 min are bathed, is filtered, filtrate is poured into 100 new ml centrifuge tubes, 12000 × g with four layers of emery cloth, 4 °C of centrifugations 15
Min takes supernatant to be added in the bag filter that molecule interception is 8000-14000 Da, is pressed from both sides and sealed with bag filter, be placed on 4 °
2 h of dialysis are stood in C ultra-pure waters, the ultra-pure water then more renewed continues to stand 16 h of dialysis；
3rd, after dialysing, liquid in bag filter is poured into the glass culture dish of a diameter of 12 cm, rested on straight in -20 °C of refrigerators
Freeze to liquid, be then placed in the vacuum freeze drier of precooling, set lyophilisation condition as -40 °C, 15 Pa, 24 h, into
Row freeze-drying, obtains collagen, is sealed with dry centrifuge tube in -20 °C of refrigerators for use.
（Three）The preparation and purification identification of biologically active peptide：
1st, according to enzyme-to-substrate ratio 1：10 （ml/mg）Collagen with crude enzyme liquid is mixed, is placed in 37 °C, 100 rpm
2 h are digested in constant temperature horizontal shaker；Enzymolysis liquid is placed in and heats 10 min in 95 °C of water-baths and inactivates enzyme, is cooled to room temperature
Afterwards, 10000 × g, 4 °C of centrifugation 5 min removal denaturation high molecular weight proteins, collects supernatant, is added to molecule interception as 3 kDa
Super filter tube in, 4500 × g centrifuges 45 min, collects super filter tube levels filtrate respectively, and upper strata filtrate has apparent anti-
Freeze effect；
2nd, sieve chromatography is carried out to lower floor's filtrate using Sephadex LH-20 gel chromatographic columns：Made with two volumes ultra-pure water
To flow the chromatographic column that balances each other（16 × 600 mm）, by bed volume 3-5% loadings, with ultrapure 3-4 times of volume of water elution, stream
Speed is 1 ml/min, and Detection wavelength is 220 nm, collects Peak Activity；
3rd, the Peak Activity component of collection is placed in -20 °C of refrigerators until liquid freezes, the vacuum for being then placed within precooling is cold
In lyophilizer, lyophilisation condition is set as -40 °C, and 15 Pa, 24 h are freeze-dried, and obtain the higher collagen antioxygen of purity
Change peptide.
Above-mentioned steps（One）Described in marine bacteriaVibrioSp. SQS2-3 is from Haikou City, Hainan Province Meilan District Qu Kou towns
Separation identification gained in the seawater of intertidal zone.
It is preferred according to the present invention, the step（1）In liquid seed culture medium component it is as follows, be parts by weight：Egg
White 0.5 part of peptone, 0.1 part of dusty yeast, Fe2(PO4)3 0.001 part of 100 parts of artificial seawater, pH 7.8.
It is preferred according to the present invention, the step（1）In fermentation medium component it is as follows, be parts by weight：Corn flour 1
Part, 0.5 part of wheat bran, 1 part of dregs of beans, Na2HPO4 0.05 part, KH2PO4 0.02 part, CaCl20.05 part, Na2CO3 0.05 part, people
50 parts of work seawater, pH 7.5-8.0.
The advantages of invention and good effect
The advantage of the invention is that the bacterium extracellular protease of untapped application is applied in protein digestion technique, and passing through
Single enzymolysis can obtain two kinds of peptide components with different bioactivity.It is embodied in：
1st, with the method for the invention prepare bacterium extracellular protease for this laboratory voluntarily ferment preparation, identification, belong to
Non-commercial enzyme can be used as innovative novel protease, reduce cost of the enzyme preparation in production technology；
2nd, with the recovery rate higher of the method for the invention collagen, and method is easier, and cost is lower, is suitble to fortune
For being prepared on a large scale proteolysis substrate；
3rd, only need progress ultrafiltration and gel permeation chromatography that can obtain respectively with the method for the invention isolating biologically active peptide
Obtain two kinds of bioactive compositions；
4th, the freeze proof peptide components being purified into the method for the invention preparation can effectively protect myosin in multigelation
In activity, meat product preservation, protein articles preserve on have development and application value；
5th, the anti-oxidation peptide being purified into the method for the invention preparation has stronger DPPH free radicals, hydroxy radical, mistake
Oxygen radical removing ability, while the ability that there is protection DNA to prevent oxidative damage.
Technical scheme is described further with reference to embodiment, but institute's protection domain of the present invention is without being limited thereto.
Marine bacteria described in embodimentVibrioSp. SQS2-3 is the tide from Haikou City, Hainan Province Meilan District Qu Kou towns
Between band seawater in separation identification gained.
Purchased from Sigma Co., USA, 1,10- Phen is purchased from Aladdin reagent for DPPH, AAPH, fluorescein sodium（Shanghai）
Co., Ltd, vitamin C are purchased from raw work bioengineering（Shanghai）Limited company, NaCl, MgSO4·7H20、KCl、
MgCl2·6H20、CaCl2·H20、Fe2(PO4)3Purchased from Sinopharm Chemical Reagent Co., Ltd., peptone, dusty yeast are purchased from
OXOID companies, the preparation raw material and fish-skin of fermentation medium, the myosin institute materials of extraction are commercially available.
Embodiment 1：
A kind of utilizationVibrioSp. SQS2-3 extracellular proteases enzymolysis salmon collagen prepares antifreeze peptide and anti-oxidation peptide
Method, step are as follows：
1st, by bacteriumVibrioSp. SQS2-3 inoculations shake training in liquid seed culture medium in 17 °C of horizontal shakers
It supports 2-3 days, rotating speed is 200 rpm, then by 2%（v/v）Inoculum concentration be inoculated in fermentation medium, in 17 °C of horizontal shakers
Middle shake culture 5 days, rotating speed are 180 rpm, and zymotic fluid is made；
Above-mentioned seed culture medium component is as follows, is parts by weight：0.5 part of peptone, 0.1 part of dusty yeast, Fe2(PO4)3 0.001
Part, 100 parts of artificial seawater, pH 7.8；
Above-mentioned fermentation medium component is as follows, is parts by weight：1 part of corn flour, 0.5 part of wheat bran, 1 part of dregs of beans, Na2HPO4 0.05
Part, KH2PO4 0.02 part, CaCl20.05 part, Na2CO3 0.05 part, 50 parts of artificial seawater, pH 7.5-8.0.
2nd, zymotic fluid is poured into and freezing ultracentrifugation is carried out in centrifuge tube, centrifugal condition be 10000 × g, 4 °C, 30
Then min collects supernatant, pour into new centrifuge tube, 12000 × g, and 4 °C of 15 min of centrifugation are repeated 2-3 times, until supernatant
It precipitates less；It collects supernatant and is added in the bag filter that molecule interception is 8000-14000 Da, pressed from both sides and sealed with bag filter,
Concentration is then placed within as 20 mM, 2 h of ice bath dialysis, the Tris-HCl then more renewed continue in the Tris-HCl of pH 7.8
Ice bath 2 h of dialysis, finally impregnate bag filter with new Tris-HCl, and are placed in 4 °C of refrigerators 24 h that dialyse, and obtain thick enzyme
Liquid；
3rd, the flesh of fish on fresh dace skin is rejected, fish-skin is cut into and is about about 0.5 cm size strips of 4 cm wide, with 3 times of volumes
4 °C of precooling ultrapure water fish-skins 2-3 times；
4th, 20 g degreasings fish-skins are taken in 100 ml centrifuge tubes, 60 ml ultra-pure waters is added in, is then placed within quiet in 80 °C of water-baths
30 min of water-bath is put, is filtered, filtrate is poured into 100 new ml centrifuge tubes, 12000 × g with four layers of emery cloth, 4 °C of centrifugations 15
Min takes supernatant to be added in the bag filter that molecule interception is 8000-14000 Da, is pressed from both sides and sealed with bag filter, be placed on 4 °
2 h of dialysis are stood in C ultra-pure waters, the ultra-pure water then more renewed continues to stand 16 h of dialysis, be freezed after the completion of dialysis,
And be placed in the vacuum freeze drier of precooling, lyophilisation condition is set as -40 °C, and 15 Pa, 24 h are freeze-dried, obtained
Obtain collagen；
5th, according to enzyme-to-substrate ratio 1：10（ml/mg）Collagen with crude enzyme liquid is mixed, is placed in 37 °C, 100 rpm
30,60,90,120,150 min of enzymolysis difference in constant temperature horizontal shaker；Enzymolysis liquid is placed in 95 °C of water-baths and heats 10 min
Inactivate enzyme, after being cooled to room temperature, 10000 × g, 4 °C of centrifugation 5 min removal denaturation high molecular weight proteins collect supernatant；
6th, the enzymolysis product solution of 50 μ l difference hydrolysis times is taken, adds in 100 μ l ninhydrins-sodium citrate solution, 90 °C add
20 min of heat take 50 μ l reaction solutions, add in 100 μ l, 50% normal propyl alcohols, OD values are detected under 570 nm after mixing, with different dense
The leucine solution of degree draws ninhydrin method as standard items and measures the standard curve of free amine group, and surveyed OD values are converted into
Free amine group concentration；
The results are shown in Figure 1.Salmon collagen existsVibrioSp. under the protease hydrolyzed effect of SQS2-3, with work
With time lengthening, the concentration of free amine group constantly rises, and illustrates that protein hydrolysis degree is higher and higher, and is reached after 120 min
To metastable degree.
7th, the 20 μ l of collagen enzymolysis product of different enzymolysis times are taken, are separately added into 95% ethanol solution of 100 μ l DPPH,
Then at room temperature sealing be protected from light 1 it is small when, 517 nm at detection absorbanceA i , sample is replaced with 20 μ l deionized waters
As blank control pipe, using 100 μ l, 95% ethyl alcohol DPPH solution is replaced to add in 20 μ l various concentrations filtrates as sample reference
Pipe, DPPH clearance rate calculation formula are as follows：
DPPH clearance rates=1- (A i-A j)/A 0 × 100%
Acquired results as shown in Fig. 2, the DPPH radical scavenging activities of product rise with the extension of enzymolysis time, and
Reach metastable activity after 120 min.
8th, the collagen enzymolysis reaction mixture of 120 min enzymolysis durations is taken, is added to the ultrafiltration that molecule interception is 3000 Da
Guan Zhong, 4500 × g centrifuge 45 min, collect super filter tube levels filtrate respectively；
9th, sieve chromatography is carried out to lower floor's filtrate using Sephadex LH-20 gel chromatographic columns：Made with two volumes ultra-pure water
To flow the chromatographic column that balances each other（16 × 600 mm）, by bed volume 3-5% loadings, with ultrapure 3-4 times of volume of water elution, stream
Speed is 1 ml/min, detects 220 nm absorbances, collects each group swarming, and carries out vacuum freeze drying, will be each with deionized water
Freeze-dried component is made into 200 μM.Liquid chromatography results are as shown in figure 3, lower floor's filtrate has 7 main polypeptide components；
10th, taking each 20 μ l of separation component respectively, 95% ethyl alcohol for then adding in 100 μ l DPPH is molten in 200 μ l centrifuge tubes
Liquid, then at room temperature sealing be protected from light 1 it is small when, 517 nm at detection absorbanceA i , sample is replaced with 20 μ l deionized waters
Product replace DPPH solution to add in 20 μ l various concentrations filtrates and join as sample as blank control pipe using 100 μ l, 95% ethyl alcohol
Than pipe, DPPH clearance rate calculation formula are as follows：
DPPH clearance rates=1- (A i-A j)/A 0 × 100%
The results are shown in Figure 4, and the DPPH free radical scavenging activities of component F5 are most strong, reach 73.29 ± 1.03%.
11st, 80 μ l are taken respectively in 200 μ l centrifuge tubes, add in 40 μ l, 2 mM FeSO4, the adjacent phenanthrene of 40 μ l, 2 mM
Sieve quinoline adds in 40 μ l, 0.03% H after abundant mixing2O2Start reaction, and in 37 °C of water-baths reaction 1 it is small when, in 536 nm
Go out to detect absorbance As, using deionized water sample is replaced to replace H as negative group with deionized water2O2As blank group, extinction
Degree is respectively An、Ab, Scavenging action to hydroxyl free radical calculation formula is as follows：
Scavenging action to hydroxyl free radical=(As – An) / (Ab– An) × 100%
The results are shown in Figure 5, and the Scavenging activity on hydroxyl free radical of component F5 is most strong, reaches 72.73 ± 3.34%.
12nd, take in 20 μ l active constituents F5 and 96 orifice plates, the fluorescein sodium of 150 μ l, 96 nM is added in, in 37 °C of water-baths
After preheating 5 min in pot, the AAPH solution of 30 μ l, 320 mM is added in, and 96 orifice plates are placed on the multifunctional enzyme of 37 °C of preheatings
It marks in instrument, persistently scans the fluorescence intensity of each sample, excitation wavelength is 485 nm, and detection launch wavelength is 538 nm, between the time
Every 1 min, continue 120 min.
Acquired results remove free radical as shown in fig. 6, component F5 has, the ability that protection fluorescein sodium is destroyed so that
Fluorescence decay is slowed down, and with the rise of constituents ratio, fluorescence decay is slower.
13rd, 4 μ l component F5 are taken, add in 8 μ l pET-22b Plasmid DNA, the FeSO of 3 μ l 2mM4, gently add after mixing
Enter the H of 4 μ l, 0.03 mM2O2Start reaction, be incubated 10 min in 37 °C of water-baths, then carried out with 1% agarose gel electrophoresis
Separation, deposition condition are 130 V, 30 min, then with 15 min of ethidium bromide staining, and DNA are detected in gel imaging system.
Component F5 is replaced as damage group using deionized water.
Acquired results with damage group as shown in fig. 7, compare, and the degree of injury of Plasmid DNA decreases after addition component F5.
14th, component F5 is analyzed and identified by LC-MS technologies, obtains the amino acid sequence of 19 polypeptides, sequence is such as
Shown in amino acid sequence table.
15th, fresh lean pork is taken to shred, is put into refiner, adds in the deionized water of two volumes, is turned with 12,000rpm
Speed homogenate 2min, takes out homogenate, with DEG C centrifugation 15min of 12,000 × g, 4, abandoning supernatant, the 1.2M of addition monoploid product
KCl solution is homogenized 2min with 12,000rpm rotating speeds, takes out homogenate, with DEG C centrifugation 15min of 12,000 × g, 4, supernatant is taken to obtain
Obtain myosin solution.With 1:1 liquor capacity ratio myosin solution and the upper strata ultrafiltrate of collagen enzymolysis product, lead to
The freeze thawing mode for crossing 15min and 37 DEG C of thawing 15min of -20 DEG C of freezings handles 3,6,9 Xun Huans respectively, takes molten after multigelation
25 μ l of liquid add in 15 μ l Tris-malatate (pH 7.0), 25 μ l 0.1M CaCl2, 172.5 μ l deionized waters, whirlpool shake
After swinging mixing, 12.5 μ l 20mM ATP are added in, abundant mixing reacts 10min at room temperature, and the TCA for adding in 125 μ l100g/L is whole
Only react.50 μ l reaction solutions are removed, add in 80 μ l, 2.5% ammonium molybdates, after shaking up, add in 20 μ l newly with 1% stannous chloride, constant volume
Absorbance A is detected at 720nm to 1ml, 25 DEG C of reaction 10minn(n=number of freezing and thawing).Myosin Ca2+- ATPase activity
It calculates as follows：
Ca2+- ATPase activity=An/A0× 100%
The results are shown in Figure 8, and myosin declines Ca with the increase of number of freezing and thawing2+- ATPase activity constantly declines, and adds in
After the upper strata ultrafiltrate of collagen enzymolysis product, Ca2+- ATPase activity, which declines, substantially to be slowed down.
In conclusion bacteriumVibrioSp. the extracellular protease of SQS2-3 secretions digests the salmon glue after 120 min
Original, can obtain the upper strata macromolecular component with Activity of Antifreeze by way of ultrafiltration and the lower floor with antioxidation activity is small
Molecular components.High activity polypeptide can be obtained after further being isolated and purified to small molecule component using the method for sieve chromatography
Component realizes the comprehensive utilization of collagen.
Description of the drawings
The relation of Fig. 1 collagen hydrolysates degree and enzymolysis time；
The DPPH Scavenging activities of the collagen enzymolysis product of Fig. 2 difference Degree of Enzymatic Hydrolysis；
Fig. 3 gel permeation chromatographies separate the chromatogram of 3 below kD components of enzymolysis product；
The DPPH Scavenging activities of Fig. 4 gel permeation chromatography each components；
The Hydroxyl radical-scavenging ability of Fig. 5 gel permeation chromatography each components；
The oxygen radical absorbability of Fig. 6 active constituents F5；
Protective effects of Fig. 7 active constituents F5 to the DNA of oxidation mediate injury；
The Activity of Antifreeze of 3 more than kD components of Fig. 8 collagens enzymolysis product.
SEQUENCE LISTING
<110>Central South University
<120>The technique that a kind of enzymatic hydrolysis salmon collagen prepares anti-oxidation peptide and antifreeze peptide simultaneously
<130> 1
<160> 19
<170> PatentIn version 3.5
<210> 1
<211> 10
<212> PRT
<213> Oncorhynchus mykiss
<400> 1
Pro Met Arg Gly Gly Gly Gly Tyr His Tyr
1 5 10
<210> 2
<211> 14
<212> PRT
<213> Salmo salar
<400> 2
Pro His Thr Gly Ile Phe Asn Val Pro Met Glu Gly His Tyr
1 5 10
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<213> Salmo salar
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Pro Gly Thr Ile Val Gly Ser Ala Ala Phe Asn
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<212> PRT
<213> Oncorhynchus mykiss
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Val Gly Lys Val Thr Asn Gly Gly Tyr His
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<211> 27
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<213> Oncorhynchus keta
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Gly Ile Thr Gly Pro Ala Gly Pro Arg Gly Pro Ala Gly Pro His Gly
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Pro Pro Gly Lys Asp Gly Arg Ala Gly Gly His
20 25
<210> 6
<211> 15
<212> PRT
<213> Oncorhynchus mykiss
<400> 6
Gly Glu Ser Gly Pro Ala Gly Pro Ala Gly Pro Asn Gly Pro Ala
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<213> Oncorhynchus mykiss
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Gly Ile Ala Gly Pro Ala Gly Pro Arg Gly Pro Ser Gly Pro Ala
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<213> Oncorhynchus mykiss
<400> 8
Ser Gly Pro Ala Gly Pro Ala Gly Pro Asn Gly Pro Ala
1 5 10
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<213> Oncorhynchus mykiss
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Ala Gly Ala Arg Ser Gly Ala Asp Val Asp Glu Gly Met Glu
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<213> Oncorhynchus keta
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Gly Leu Asn Gly Pro Ser Gly Pro Arg Gly Pro His Gly Ser
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Gly Pro Ala Gly Pro Arg Gly Pro Ala Gly Pro His Gly Pro Pro Gly
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Claims (12)
1. method that is a kind of while preparing anti-oxidation peptide and antifreeze peptide, it is characterised in that：It extracts glue by raw material of salmon skin
Collagen hydrolysate is obtained antioxygen by former albumen into small peptide using the extracellular protease obtained by microbial fermentation after separation
Change peptide and antifreeze peptide component.
2. method that is according to claim 1 a kind of while preparing anti-oxidation peptide and antifreeze peptide, it is characterised in that：Described
Collagen extracting method is using fresh 30 min of fleshing salmon skin of 80 °C of water bath processings, by dialysis with being freeze-dried
To collagen solids.
3. method that is according to claim 1 a kind of while preparing anti-oxidation peptide and antifreeze peptide, it is characterised in that：Described
Microorganism is the separation identification gained from the intertidal zone seawater in Haikou City, Hainan Province Meilan District Qu Kou townsVibrio sp. SQS2-
3。
4. method that is according to claim 1 a kind of while preparing anti-oxidation peptide and antifreeze peptide, it is characterised in that：Described
Extracellular microbial exoproteinase preparation method is by bacteriumVibrioSp. SQS2-3 inoculations are in liquid seed culture medium,
Shake culture 2-3 days in 17 °C of horizontal shakers, rotating speed is 200 rpm, then by 2%（v/v）Inoculum concentration be inoculated in fermented and cultured
In base, shake culture 5 days in 17 °C of horizontal shakers, rotating speed is 180 rpm, and zymotic fluid is made；Zymotic fluid is poured into centrifuge tube
In carry out freezing ultracentrifugation, centrifugal condition is 10000 × g, and 4 °C, then 30 min collect supernatant, pour into new centrifugation
Guan Zhong, 12000 × g, 4 °C of 15 min of centrifugation, repeat 2-3 times, until supernatant precipitation is less；It collects supernatant and is added to molecule
Interception is in the bag filter of 8000-14000 Da, is pressed from both sides and sealed with bag filter, is then placed within concentration as 20 mM, pH's 7.8
2 h of ice bath dialysis in Tris-HCl, the Tris-HCl then more renewed continue ice bath 2 h of dialysis, finally with new Tris-HCl
Bag filter is impregnated, and is placed in 4 °C of refrigerators 24 h that dialyse, obtains crude enzyme liquid.
5. method that is according to claim 4 a kind of while preparing anti-oxidation peptide and antifreeze peptide, it is characterised in that：Described
Seed culture medium component is as follows, is parts by weight：0.5 part of peptone, 0.1 part of dusty yeast, Fe2(PO4)3 It is 0.001 part, manually extra large
100 parts of water, pH 7.8.
6. method that is according to claim 4 a kind of while preparing anti-oxidation peptide and antifreeze peptide, it is characterised in that：Described
Fermentation medium component is as follows, is parts by weight：1 part of corn flour, 0.5 part of wheat bran, 1 part of dregs of beans, Na2HPO4 0.05 part,
KH2PO4 0.02 part, CaCl20.05 part, Na2CO3 0.05 part, 50 parts of artificial seawater, pH 7.5-8.0.
7. method that is according to claim 1 a kind of while preparing anti-oxidation peptide and antifreeze peptide, it is characterised in that：Described
Small peptide preparation method is according to enzyme-to-substrate ratio 1：10（ml/mg）Salmon collagen with crude enzyme liquid is mixed, is placed in
37 °C, 120 min are digested in 100 rpm constant temperature horizontal shakers；Enzymolysis liquid is placed in and heats 10 min in 95 °C of water-baths and makes enzyme
Inactivation, after being cooled to room temperature, 10000 × g, 4 °C of centrifugation 5 min removal denaturation high molecular weight proteins collect supernatant, obtain collagen
Small peptide.
8. method that is according to claim 1 a kind of while preparing anti-oxidation peptide and antifreeze peptide, it is characterised in that：Described
Anti-oxidation peptide and antifreeze peptide component separation method are as follows
A. the collagen obtained in claim 7 small peptide is taken to be added in the super filter tube that molecule interception is 3 kDa, 4500 × g
45 min are centrifuged, collect super filter tube levels filtrate respectively, upper strata is antifreeze peptide component；
B. above-mentioned lower floor's filtrate is taken, sieve chromatography is carried out to lower floor's filtrate using Sephadex LH-20 gel chromatographic columns：With
Two volumes ultra-pure water, which is used as, flows the chromatographic column that balances each other（16 × 600 mm）, by bed volume 3-5% loadings, use ultra-pure water
3-4 times of volume is eluted, flow velocity is 1 ml/min, detects 220 nm absorbances, collects Peak Activity, and carries out vacuum freeze drying,
Obtain anti-oxidation peptide component.
9. a kind of according to claim 1 or 8 while the method for preparing anti-oxidation peptide and antifreeze peptide, it is characterised in that：Institute
The anti-oxidation peptide stated with remove DPPH free radicals, hydroxy radical and to DNA oxidative damages with the ability centainly inhibited, and
And show dosage effect in the detection of oxygen radical absorbability.
10. a kind of according to claim 1 or 8 while the method for preparing anti-oxidation peptide and antifreeze peptide, it is characterised in that：Institute
The anti-oxidation peptide component LC-MS the results shows stated have 19 polypeptides.
11. 19 polypeptides according to claim 10, it is characterised in that：The polypeptid acid sequence such as specification ammonia
Shown in base acid sequence attachment.
12. a kind of according to claim 1 or 8 while the method for preparing anti-oxidation peptide and antifreeze peptide, it is characterised in that：Institute
The antifreeze peptide stated has protection myosin, slows down its Ca2+The effect of the decline of-ATPase activity in multigelation.
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| CN111323606A (en) * | 2020-03-16 | 2020-06-23 | 中南大学 | Method for detecting antioxidant effect of antioxidant by using oxidative damage of chromoprotein |
| CN112480207A (en) * | 2020-11-06 | 2021-03-12 | 浙江海洋大学 | Euphausia superba metal chelating peptide |
| CN113559238A (en) * | 2021-08-16 | 2021-10-29 | 北京戴域生物技术有限公司 | A pharmaceutical or cosmetic containing active peptide and having antiaging effect |
| CN113599492A (en) * | 2021-08-16 | 2021-11-05 | 北京戴域生物技术有限公司 | Anti-aging medicine or cosmetic and preparation method thereof |
| CN114634563A (en) * | 2022-02-16 | 2022-06-17 | 惠州华阳医疗器械有限公司 | Active collagen extracting solution, preparation method and system thereof, skin care product and preparation method thereof |
| CN114990095A (en) * | 2022-06-28 | 2022-09-02 | 天津天隆江大生物科技有限公司 | Complex enzyme preparation for producing antifreeze protein polypeptide and application thereof |
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| CN113559238B (en) * | 2021-08-16 | 2022-03-29 | 广州中科微晶生物科技有限责任公司 | A pharmaceutical or cosmetic containing active peptide and having antiaging effect |
| CN114634563A (en) * | 2022-02-16 | 2022-06-17 | 惠州华阳医疗器械有限公司 | Active collagen extracting solution, preparation method and system thereof, skin care product and preparation method thereof |
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