CN107164445A - Suppress fish-skin protein peptides of function and preparation method and application with DPP IV - Google Patents
Suppress fish-skin protein peptides of function and preparation method and application with DPP IV Download PDFInfo
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- CN107164445A CN107164445A CN201710429486.4A CN201710429486A CN107164445A CN 107164445 A CN107164445 A CN 107164445A CN 201710429486 A CN201710429486 A CN 201710429486A CN 107164445 A CN107164445 A CN 107164445A
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- fish
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- protein peptides
- skin protein
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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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- 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, cold insoluble globulin [CIG]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The present invention provides a kind of fish-skin protein peptides for suppressing function with DPP IV, and its amino acid sequence is respectively such as SEQ ID NO:Shown in 1 and 2.The present invention is directed to the fast development of current fish secondary industry, the quick increase of fish-skin protein resource quantity, according to the characteristic of fish-skin albumen, using fish-skin as raw material, by being pre-processed to fish-skin ultrasonic wave and HTHP etc., using multiple protein enzyme stepwise discretization technology, separated and high performance liquid chromatography separation technology by UF membrane, gel, sequence such as SEQ ID NO in gained fish-skin protein peptide powder:The content of fish-skin protein peptides shown in 1 and 2 accounts for more than the 50% of fish-skin protein peptide powder gross weight, establishes the preparation method of a set of simple efficient fish-skin protein peptides for suppressing function with DPP IV.
Description
Technical field
The present invention relates to fish DPP-IV peptide for inhibiting and its production and use, specifically, being related to one kind has
DPP-IV suppresses fish-skin protein peptides of function and preparation method and application.
Background technology
The function of blood sugar reduction factor refer to reduce blood sugar in diabetic patients concentration and improve the bioactivity of its symptom into
Point.More natural products class sugar deductive factor, mineral matter class sugar deductive factor, vitamins sugar deductive factor are studied at present, and it is acted on
Mechanism is different.Natural products class sugar deductive factor can be divided into flavonoids, active polysaccharide class, alkaloids, soap by chemical constitution
Glucoside, terpene, CLA, polypeptide etc..Since synthetic insulin, sulfonylurea drugs, biguanides, insulin increase
Quick dose, Glyco inhabiting agent etc. is various oral or medicine of injection is come out one after another, but chemicals often produces certain toxic side effect,
Natural hypoglycemic composition have action temperature and and it is lasting, property is stable, almost without toxic reaction, can also a variety of sugar-lowering components simultaneously
Deposit, integrate the advantages of working, enjoy patient and the favor with medical field, the main research side as the function of blood sugar reduction factor
To.
The content of the invention
It is an object of the invention to provide a kind of fish-skin protein peptides for suppressing function with DPP-IV and preparation method thereof.
It is a further object of the present invention to provide application of the fish-skin protein peptides in medicine, food and health products.
In order to realize the object of the invention, what the present invention was provided has the fish-skin protein peptides of DPP-IV suppression function, its amino
Acid sequence is respectively such as SEQ ID NO:Shown in 1 and 2.
Fish-skin protein peptide powder containing above-mentioned fish-skin protein peptides can be prepared as follows, and be comprised the following steps：
(1) freezing fish-skin is subjected to defrosting cleaning under the conditions of 8-12 DEG C, broken into fish-skin with beater after fish-skin cleaning
Slurry, adds the water of 1.5-2.5 times of fish-skin weight, and 30-60 minutes are incubated in 125-135 DEG C；
(2) 60-70 DEG C will be adjusted at a temperature of fish-skin protein milk, is carried out ultrasonically treated (ultrasonically treated to be intended to change fish-skin
The institutional framework of albumen)；Ultrasonically treated to be carried out in supersonic generator, the frequency of ultrasonic wave is 70-90kH, processing time
25-35 minutes；
(3) 115-125 DEG C will be adjusted in the temperature of fish-skin protein milk after ultrasound, 150-180 is cooked in 115-125 DEG C
Minute, it is then centrifuged for taking supernatant；
(4) compound protease is added into the supernatant of (3) according to fish-skin weight 0.10-0.20% ratio and carries out substep
Enzymolysis：The first step adds fish-skin weight 0.05-0.10% compound protease I into supernatant (by alkali protease and pancreas egg
White enzyme in mass ratio 1:1-2 is constituted, and wherein the enzyme activity of alkali protease is the U/g of 80-100 ten thousand, and the enzyme activity of trypsase is 50-60
Ten thousand U/g), digest 1.0-2.0h in 50-65 DEG C；Second step adds fish-skin weight 0.05-0.10%'s into above-mentioned enzymatic hydrolysis system
Compound protease II is (by bromelain and flavor protease in mass ratio 1:1 constitutes, and the enzyme activity of wherein bromelain is
The U/g of 20-30 ten thousand, the enzyme activity of flavor protease is the U/g of 40-60 ten thousand), digest 1.0-2.0h in 55-65 DEG C；Then at 95-98 DEG C
Lower insulation 10-20 minutes, is cooled to room temperature, is filtered with activated carbon, collects supernatant；
(5) hyperfiltration treatment is carried out to supernatant obtained by step (4), first with the ceramic membrane ultrafitration that aperture is 5000 dalton,
Molecular weight is less than 5000 albumen and peptide separation comes out, then is less than molecular weight for the filter filter membrane of 3000 dalton with aperture
The protein peptides of 3000 dalton are separated；
(6) protein peptides liquid of the molecular weight less than 3000 is again by the separation of Sephadex G-25 gels, and eluent is deionization
Water, eluting peak is detected under 280nm, collects the 2nd eluting peak, then divided with RP-HPLC RPLCs
From the peptide solution for taking collect for 15-17 minutes；
(7) peptide solution for obtaining step (6) is obtained containing fish-skin egg described in claim 1 by concentration, freeze-drying
The fish-skin protein peptide powder of white peptide.
In the present invention, RP-HPLC part is：0-5min, mobile phase is pure water；Mobile phase B：Containing 0.1%TFA (trifluoro second
Acid) acetonitrile, 5-10min Mobile phase Bs, from 0% to 35%, 10-15min, Mobile phase B is from 65% to 95%, 15-30min, stream
Dynamic phase B stops from 95% to 0%, 30min；Chromatographic column used is：Kromasil C18,5 μm, 4.6 × 250mm.
RP-HPLC chromatographic columns of the invention used are：Kromasil C18,5 μm, 4.6 × 250mm.
The main component of fish-skin protein peptide powder is measured by LC-MS/MS, sequence such as SEQ ID NO:Shown in 1 and 2
The contents of fish-skin protein peptides account for the 52-55% of fish-skin protein peptide powder gross weight.
Fish-skin of the present invention comes from silver carp, Tilapia mossambica.
The present invention also provides application of the fish-skin protein peptides in medicine, health food and food additives.
The present invention further provides contain such as SEQ ID NO:The medicine of fish-skin protein peptides, health food and food shown in 1 and 2
Product.
The present invention has advantages below：
(1) present invention carries out mashing processing to fish-skin, then carries out 125-135 DEG C of processing, in conjunction with specific frequency ultrasound
Wave technology handles fish-skin protein milk, then carries out 115-125 DEG C of high-temperature process, directly extracts soluble protein.
(2) present invention first extracts soluble protein, recycles protease hydrolyzed, total usage amount of protease is only
For the 0.10-0.20% of fish-skin weight.
(3) product safety of exploitation, the present invention is using numerous food level compound protease (alkali protease, tryptose
Enzyme, bromelain and flavor protease), in a mild condition, the fish-skin of specified molecular weight size is obtained by appropriateness enzymolysis
Protein peptides, without adjusting pH value, product is 100% fish-skin protein peptides.
(4) ratio that gained fish-skin protein peptides middle-molecular-weihydroxyethyl is less than 1500Da peptide is more than 90%.With preferable
DPP-IV suppresses function, its IC50Value is less than 0.55mg/mL.
(5) sequence such as SEQ ID NO in gained fish-skin protein peptide powder product:The content of fish-skin protein peptides shown in 1 and 2
Account for more than the 50% of fish-skin protein peptide powder gross weight.
(6) the fish-skin protein peptides that the present invention is provided can be widely used in functional food, special procure food as food additives
In.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment
In the conventional meanses that are well known to those skilled in the art of used technological means, raw materials used is commercial goods.
Alkali protease, trypsase, bromelain and the flavor protease used in following examples is purchased from respectively
Novozymes Company of Denmark (alkali protease, trypsase and flavor protease) and Pangbo Bioengineering Co Ltd, Nanning's (spinach
Trailing plants protease).
Embodiment 1 has the preparation method of the fish-skin protein peptides of DPP-IV suppression function
(1) from 100 grams of freezing silver carp skin, defrosting cleaning is carried out under the conditions of 12 DEG C, with meeting sanitary standard for drinking water
Clean water cleaning, fish-skin is broken into slurry with beater after fish-skin cleaning, the bar of the water of 1.5 times of addition fish-skin weight at 120 DEG C
60 minutes are incubated under part.
(2) temperature of fish-skin is adjusted to 70 DEG C, 35 is handled through ultrasonic wave (frequency is 60kH) in supersonic generator
Minute, change the institutional framework of fish-skin albumen.
(3) 120 DEG C and then by the temperature of fish-skin are adjusted to, is cooked 150 minutes under the conditions of 120 DEG C, by centrifuging and taking
Clear liquid.
(4) compound protease is added into the supernatant of (3) according to the ratio of fish-skin weight 0.15% and carries out stepwise discretization,
The first step added into supernatant fish-skin weight 0.10% compound protease (alkali protease and trypsase composition, they
Between mass ratio be 1：1, the wherein enzyme activity of alkali protease is 800,000 U/g, and the enzyme activity of trypsase is 600,000 U/g), 55
Enzyme digestion reaction 1.0h is carried out under conditions of DEG C；Then second step enzymolysis is carried out, fish-skin weight is added into above-mentioned enzymatic hydrolysis system
(bromelain and flavor protease composition, the mass ratio between them is 1 to 0.05% compound protease：1, wherein pineapple egg
The enzyme activity of white enzyme is 200,000 U/g, and the enzyme activity of flavor protease is 400,000 U/g), enzyme digestion reaction 1.0h is carried out under the conditions of 60 DEG C；
10 minutes are incubated at 95-98 DEG C, room temperature is cooled to, is filtered using activated carbon, supernatant is collected.
(5) supernatant obtained by step (4) is handled by two step hyperfiltration process, is 5000 dalton using aperture
Ceramic membrane ultrafitration, first by molecular weight be less than 5000 albumen and peptide separation come out, then with aperture for 3000 dalton filter
Film separates the protein peptides that molecular weight is less than 3000 dalton.
(6) it is the protein peptides liquid less than 3000 to take molecular weight, then by the separation of Sephadex G-25 gels, eluent is
Deionized water, eluting peak is detected under 280nm, collects the 2nd eluting peak, then use RP-HPLC RPLCs
Separated, the peptide solution for taking collect for 15-17 minutes.
(7) peptide solution for obtaining step (6) obtains fish-skin protein peptide powder by concentration, freeze-drying.Pass through LC-MS/
MS is measured to the main component of fish-skin protein peptides, and its amino acid sequence is respectively such as SEQ ID NO:Shown in 1 and 2, two kinds of fishes
Hide collagen peptide accounts for the 52.5% of fish-skin protein peptide powder gross weight.The molecular weight of wherein more than 90% fish-skin protein peptides is less than
1500Da.Embodiment 2 has the preparation method of the fish-skin protein peptides of anti-oxidation function
(1) from 500 grams of Java tilapia skin of freezing, defrosting cleaning is carried out under the conditions of 10 DEG C, fish-skin is taken, with meeting drinking water
The clean water cleaning of sanitary standard, breaks into slurry, the water for adding 2.0 times of fish-skin weight exists with beater after fish-skin cleaning by fish-skin
60 minutes are incubated under conditions of 125 DEG C.
(2) temperature of fish-skin is adjusted to 65 DEG C, 35 is handled through ultrasonic wave (frequency is 55kH) in supersonic generator
Minute, change the institutional framework of fish-skin albumen.
(3) 120 DEG C and then by the temperature of fish-skin are adjusted to, is cooked 180 minutes under the conditions of 120 DEG C, by centrifuging and taking
Clear liquid.
(4) compound protease is added into the supernatant of (3) according to the ratio of fish-skin weight 0.20% and carries out stepwise discretization,
The first step added into supernatant fish-skin weight 0.10% compound protease (alkali protease and trypsase composition, they
Between mass ratio be 1：1, the wherein enzyme activity of alkali protease is 900,000 U/g, and the enzyme activity of trypsase is 500,000 U/g), 55
Enzyme digestion reaction 0.5h is carried out under conditions of DEG C；Then second step enzymolysis is carried out, fish-skin weight is added into above-mentioned enzymatic hydrolysis system
(bromelain and flavor protease composition, the mass ratio between them is 1 to 0.10% compound protease：1, wherein pineapple egg
The enzyme activity of white enzyme is 300,000 U/g, and the enzyme activity of flavor protease is 500,000 U/g), enzyme digestion reaction 1.0h is carried out under the conditions of 65 DEG C；
10 minutes are incubated at 95 DEG C, room temperature is cooled to, is filtered using activated carbon, supernatant is collected.
(5) supernatant obtained by step (4) is handled by two step hyperfiltration process, is 5000 dalton using aperture
Ceramic membrane ultrafitration, first by molecular weight be less than 5000 albumen and peptide separation come out, then with aperture for 3000 dalton filter
Film separates the protein peptides that molecular weight is less than 3000 dalton.
(6) it is the protein peptides liquid less than 3000 to take molecular weight, then by the separation of Sephadex G-25 gels, eluent is
Deionized water, eluting peak is detected under 280nm, collects the 2nd eluting peak, then use RP-HPLC RPLCs
Separated, the peptide solution for taking collect for 15-17 minutes.
(7) peptide solution for obtaining step (6) obtains fish-skin protein peptide powder by concentration, freeze-drying.Pass through LC-MS/
MS is measured to the main component of fish-skin protein peptides, and its amino acid sequence is respectively such as SEQ ID NO:Shown in 1 and 2, two kinds of fishes
Hide collagen peptide accounts for the 53.2% of fish-skin protein peptide powder gross weight.The molecular weight of wherein more than 90% fish-skin protein peptides is less than
1500Da.The preparation method of fish-skin protein peptides of the embodiment 3 with anti-oxidation function
(1) from 1000 grams of Java tilapia skin of freezing, defrosting cleaning is carried out under the conditions of 8 DEG C, fish-skin is taken, with meeting drinking water
The clean water cleaning of sanitary standard, breaks into slurry, the water for adding 2.5 times of fish-skin weight exists with beater after fish-skin cleaning by fish-skin
30 minutes are incubated under conditions of 130 DEG C.
(2) temperature of fish-skin is adjusted to 60 DEG C, 35 is handled through ultrasonic wave (frequency is 50kH) in supersonic generator
Minute, change the institutional framework of fish-skin albumen.
(3) 125 DEG C and then by the temperature of fish-skin are adjusted to, is cooked 150 minutes under the conditions of 125 DEG C, by centrifuging and taking
Clear liquid.
(4) compound protease is added into the supernatant of (3) according to the ratio of fish-skin weight 0.20% and carries out stepwise discretization,
The first step added into supernatant fish-skin weight 0.10% compound protease (alkali protease and trypsase composition, they
Between mass ratio be 2：1, the wherein enzyme activity of alkali protease is 1,000,000 U/g, and the enzyme activity of trypsase is 550,000 U/g),
Enzyme digestion reaction 1.0h is carried out under conditions of 55 DEG C；Then second step enzymolysis is carried out, fish-skin weight is added into above-mentioned enzymatic hydrolysis system
(bromelain and flavor protease composition, the mass ratio between them is 2 to 0.10% compound protease：1, wherein pineapple egg
The enzyme activity of white enzyme is 250,000 U/g, and the enzyme activity of flavor protease is 600,000 U/g), enzyme digestion reaction 1.0h is carried out under the conditions of 65 DEG C.
10 minutes are incubated at 95 DEG C, room temperature is cooled to, is filtered using activated carbon, supernatant is collected；
(5) supernatant obtained by step (4) is handled by two step hyperfiltration process, is 5000 dalton using aperture
Ceramic membrane ultrafitration, first by molecular weight be less than 5000 albumen and peptide separation come out, then with aperture for 3000 dalton filter
Film separates the protein peptides that molecular weight is less than 3000 dalton.
(6) it is the protein peptides liquid less than 3000 to take molecular weight, then by the separation of Sephadex G-25 gels, eluent is
Deionized water, eluting peak is detected under 280nm, collects the 2nd eluting peak, then use RP-HPLC RPLCs
1 separation is carried out, the peptide solution for taking collect for 15-17 minutes.
(7) peptide solution for obtaining step (6) obtains fish-skin protein peptide powder by concentration, freeze-drying.Pass through LC-MS/
MS is measured to the main component of fish-skin protein peptides, and its amino acid sequence is respectively such as SEQ ID NO:Shown in 1 and 2, two kinds of fishes
Hide collagen peptide accounts for the 53.3% of fish-skin protein peptide powder gross weight.The molecular weight of wherein more than 90% fish-skin protein peptides is less than
1500Da。
The determination test of the fish-skin protein peptides DPP-IV rejection ability of the present invention of experimental example 1
Test specimen：Fish-skin DPP-IV rejection ability prepared by embodiment 1, embodiment 2, embodiment 3 is as follows
Carry out：
Sample is diluted to suitable concentration with 100mmol/L Tris-HCL (pH8.0) buffer solution, 25 μ L samples are drawn
Dilution is mixed with 25 μ L substrates (concentration is 1.6mmol/L), is added in 96 hole elisa Plates.It is incubated at 37 DEG C after 10min,
50 μ L DPP-IV enzyme liquids (enzyme activity is 8U/L) are added, it is accurate at 37 DEG C after mixing to be incubated 60min, 100 μ are added immediately
L1mol/L Acetic acid-sodium acetate (pH 4.0) buffer solution terminating reaction, in survey light absorption value A under 405nm, and according to following equation
Calculate the DPP-IV inhibiting rates of sample.
DPP-IV inhibiting rates %={ 1- (ASample-ASample blank is compareed)/(ANegative control-ANegative blank control)}×100
ASample：The light absorption value A for being example reaction liquid at 405nm；
ASample blank is compareed：The light absorption value A that DPP-IV enzyme liquids are compareed as sample blank is replaced using Tris-HCL buffer solutions；
ANegative control：Sample is replaced as the light absorption value of negative control using Tris-HCL buffer solutions；
ANegative blank control：DPP-IV enzyme liquids and sample are replaced as the light absorption value of negative blank control using Tris-HCL buffer solutions.
The IC that DPP-IV suppresses50Value is determined：
DPP-IV inhibiting rate of the determination sample under various concentrations, does recurrence bent with the logarithm value of peptide concentration and inhibiting rate
Line, obtains regression equation, thus calculates IC50, i.e., the concentration of peptide during 50% DPP-IV inhibition of enzyme activity.It the results are shown in Table 1.
The DPP-IV of the fish-skin protein peptides of the present invention of table 1 suppresses result of the test
The determination test of the fish-skin protein peptides antioxidation activity of the present invention of experimental example 2
Test specimen：Sample 1,2,3 is respectively embodiment 1, embodiment 2, the fish-skin protein peptides of the preparation of embodiment 3.
Carry out as follows：
(1) scavenging ability of DPPH free radical：20 μ g/mL fish-skin protein peptides 1.5mL is taken, 99.5% ethanol 1.5mL is added
With the ethanol solution 0.675mL containing 0.02%DPPH, vibration is mixed, at room temperature lucifuge water-bath 30min, is then examined under 517nm
Survey system light absorption value.Light absorption value is lower, and the ability that system removes DPPH free radicals is stronger.Blank control is by sample solution
1.5mL changes deionized water 1.5mL into.
DPPH radical scavenging activities %=((blank absorbency-sample light absorption value)/blank absorbency) × 100
(2) reducing power is determined：20 μ g/mL fish-skin protein peptides 1mL is taken, 0.2M phosphate buffers (pH 6.6) are added
2.5mL and 1% (mass percent) potassium ferricyanide solution 2.5mL, mix, then heat 20min in 50 DEG C of water-baths.Take out
Rapid cooling, adds 10% (mass percent) trichloroacetic acid (TCA) solution 2.5mL, is well mixed, then under 3000g from
Heart 10min.Supernatant 2.5mL is taken, deionized water 2.5mL and 1% (mass percent) liquor ferri trichloridi 0.5mL is added, fills
Divide and mix, 10min is reacted at room temperature, absorbance is determined with 700nm wavelength.Reducing power is light absorption value at available 700nm wavelength
Represent.
From table 2 it can be seen that fish-skin protein peptides of the present invention have certain oxidation resistance, under conditions of 20 μ g/mL,
Its scavenging ability of DPPH free radical reaches more than 87%, and reducing power reaches more than 0.87, is a kind of preferable anti-oxidation peptide.
The Oxidation Resistance Test Results of the fish-skin protein peptides of the present invention of table 2
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be modified or improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Suppress fish-skin protein peptides of function and preparation method and application with DPP-IV
<120>China Agricultural University
<130> KHP171112773.0
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 8
<212> PRT
<213>Fish
<400> 1
Ala Pro Arg Ala Val Phe Pro Ser
1 5
<210> 2
<211> 10
<212> PRT
<213>Fish
<400> 2
Leu Met Gln Ala Glu Ile Glu Glu Leu Arg
1 5 10
Claims (6)
1. suppress the fish-skin protein peptides of function with DPP-IV, it is characterised in that amino acid sequence is respectively such as SEQ ID NO:1 He
Shown in 2.
2. the preparation method of the fish-skin protein peptide powder containing fish-skin protein peptides described in claim 1, it is characterised in that including following
Step：
(1) freezing fish-skin is subjected to defrosting cleaning under the conditions of 8-12 DEG C, fish-skin is broken into slurry with beater after fish-skin cleaning, plus
Enter the water of 1.5-2.5 times of fish-skin weight, 30-60 minutes are incubated in 125-135 DEG C；
(2) 60-70 DEG C will be adjusted at a temperature of fish-skin protein milk, is carried out ultrasonically treated；It is ultrasonically treated in supersonic generator
Carry out, the frequency of ultrasonic wave is 70-90kH, processing time 25-35 minute；
(3) 115-125 DEG C will be adjusted in the temperature of fish-skin protein milk after ultrasound, 150-180 points is cooked in 115-125 DEG C
Clock, is then centrifuged for taking supernatant；
(4) compound protease is added into the supernatant of (3) according to fish-skin weight 0.10-0.20% ratio and carries out substep enzyme
Solution：The first step adds fish-skin weight 0.05-0.10% compound protease I into supernatant, and 1.0- is digested in 50-65 DEG C
2.0h；Second step adds fish-skin weight 0.05-0.10% compound protease II into above-mentioned enzymatic hydrolysis system, in 55-65 DEG C of enzyme
Solve 1.0-2.0h；Then 10-20 minutes are incubated at 95-98 DEG C, are cooled to room temperature, are filtered with activated carbon, collect supernatant；
(5) hyperfiltration treatment is carried out to supernatant obtained by step (4), first with the ceramic membrane ultrafitration that aperture is 5000 dalton, will divided
Albumen of the son amount less than 5000 and peptide separation come out, then with aperture for 3000 dalton filter filter membrane by molecular weight less than 3000
The protein peptides of dalton are separated；
(6) protein peptides liquid of the molecular weight less than 3000 is again by the separation of Sephadex G-25 gels, and eluent is deionized water,
Eluting peak is detected under 280nm, collects the 2nd eluting peak, then is separated with RP-HPLC RPLCs,
The peptide solution for taking collect for 15-17 minutes；
(7) peptide solution for obtaining step (6) is obtained containing fish-skin protein peptides described in claim 1 by concentration, freeze-drying
Fish-skin protein peptide powder；
Wherein, the compound protease I of step (4) is by alkali protease and trypsase in mass ratio 1:1-2 is constituted, its neutral and alkali
The enzyme activity of protease is the U/g of 80-100 ten thousand, and the enzyme activity of trypsase is the U/g of 50-60 ten thousand；Compound protease II is by bromelain
With flavor protease in mass ratio 1:1 composition, the wherein enzyme activity of bromelain are the U/g of 20-30 ten thousand, the enzyme activity of flavor protease
For the U/g of 40-60 ten thousand.
3. method according to claim 2, it is characterised in that the fish-skin comes from silver carp, Tilapia mossambica.
4. according to the method in claim 2 or 3, it is characterised in that step (6) RP-HPLC condition is：0-5min, stream
Dynamic is mutually pure water；Mobile phase B：Acetonitrile containing 0.1%TFA, 5-10min Mobile phase Bs, from 0% to 35%, 10-15min, flowing
Phase B stops from 65% to 95%, 15-30min, Mobile phase B from 95% to 0%, 30min；Chromatographic column used is：Kromasil
C18,5 μm, 4.6 × 250mm.
5. application of the fish-skin protein peptides in medicine, health food and food additives described in claim 1.
6. contain the medicine of fish-skin protein peptides, health food and food described in claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710429486.4A CN107164445B (en) | 2017-06-08 | 2017-06-08 | Fish skin protein peptide with DPP-IV inhibition function and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710429486.4A CN107164445B (en) | 2017-06-08 | 2017-06-08 | Fish skin protein peptide with DPP-IV inhibition function and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107164445A true CN107164445A (en) | 2017-09-15 |
| CN107164445B CN107164445B (en) | 2020-01-10 |
Family
ID=59824731
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108314705A (en) * | 2018-01-26 | 2018-07-24 | 海普诺凯营养品有限公司 | Inhibit sheep whey protein peptide, preparation method and its application of function with DPP-IV |
| CN108935912A (en) * | 2018-06-21 | 2018-12-07 | 中国农业大学 | A kind of fish protein peptide and preparation method thereof inhibited with DPP-IV with anti-fatigue effect |
| CN109182435A (en) * | 2018-10-23 | 2019-01-11 | 浙江海洋大学 | A kind of preparation method of biological source dipeptidyl peptidase-iv inhibitor |
| CN109776652A (en) * | 2019-01-30 | 2019-05-21 | 浙江省医学科学院 | Cod skin oligopeptides and its isolation and purification method and preparing the application in ɑ-glucosidase inhibitor and type II diabetes resisting drug |
| CN112931880A (en) * | 2021-02-24 | 2021-06-11 | 临沂华兴生物科技有限公司 | Glycosylated fish skin protein peptide for promoting growth of probiotics and reducing blood sugar and preparation method thereof |
| CN113151387A (en) * | 2021-04-16 | 2021-07-23 | 安徽国肽生物科技有限公司 | Cod skin collagen peptide with oxidation resistance and immunity enhancement functions and preparation method thereof |
| CN114711324A (en) * | 2021-02-04 | 2022-07-08 | 云南海王水产有限公司 | Fish scale collagen peptide with probiotic growth promoting and antioxidant effects and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108314705A (en) * | 2018-01-26 | 2018-07-24 | 海普诺凯营养品有限公司 | Inhibit sheep whey protein peptide, preparation method and its application of function with DPP-IV |
| CN108314705B (en) * | 2018-01-26 | 2020-07-14 | 海普诺凯营养品有限公司 | Sheep whey protein peptide with DPP-IV (dipeptidyl peptidase-IV) inhibition function, and preparation method and application thereof |
| CN108935912A (en) * | 2018-06-21 | 2018-12-07 | 中国农业大学 | A kind of fish protein peptide and preparation method thereof inhibited with DPP-IV with anti-fatigue effect |
| CN108935912B (en) * | 2018-06-21 | 2021-04-23 | 中国农业大学 | Fish meat protein peptide with DPP-IV inhibition and anti-fatigue functions and preparation method thereof |
| CN109182435A (en) * | 2018-10-23 | 2019-01-11 | 浙江海洋大学 | A kind of preparation method of biological source dipeptidyl peptidase-iv inhibitor |
| CN109182435B (en) * | 2018-10-23 | 2021-12-17 | 浙江海洋大学 | Preparation method of biological source dipeptidyl peptidase-IV inhibitor |
| CN109776652A (en) * | 2019-01-30 | 2019-05-21 | 浙江省医学科学院 | Cod skin oligopeptides and its isolation and purification method and preparing the application in ɑ-glucosidase inhibitor and type II diabetes resisting drug |
| CN109776652B (en) * | 2019-01-30 | 2020-08-18 | 浙江省医学科学院 | Codfish skin oligopeptide, separation and purification method thereof, and application of codfish skin oligopeptide in preparation of alpha-glucosidase inhibitor and anti-type II diabetes drug |
| CN114711324A (en) * | 2021-02-04 | 2022-07-08 | 云南海王水产有限公司 | Fish scale collagen peptide with probiotic growth promoting and antioxidant effects and preparation method thereof |
| CN112931880A (en) * | 2021-02-24 | 2021-06-11 | 临沂华兴生物科技有限公司 | Glycosylated fish skin protein peptide for promoting growth of probiotics and reducing blood sugar and preparation method thereof |
| CN113151387A (en) * | 2021-04-16 | 2021-07-23 | 安徽国肽生物科技有限公司 | Cod skin collagen peptide with oxidation resistance and immunity enhancement functions and preparation method thereof |
| CN113151387B (en) * | 2021-04-16 | 2022-08-16 | 安徽国肽生物科技有限公司 | Cod skin collagen peptide with oxidation resistance and immunity enhancement functions and preparation method thereof |
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