CN108823268A - The preparation method of fish collagen antioxidant peptide - Google Patents
The preparation method of fish collagen antioxidant peptide Download PDFInfo
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- CN108823268A CN108823268A CN201810408295.4A CN201810408295A CN108823268A CN 108823268 A CN108823268 A CN 108823268A CN 201810408295 A CN201810408295 A CN 201810408295A CN 108823268 A CN108823268 A CN 108823268A
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- 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]
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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
Abstract
The invention discloses the preparation methods of fish collagen antioxidant peptide, the specific steps are that：Fish-skin is pre-processed through oxygen sodium hydroxide solution and aqueous isopropanol, acid extracts to obtain collagen protein powder, it is digested again through clostridiopetidase A and flavor protease, ultrafiltration membrane ultrafiltration is used in enzymolysis process, ultrafiltrate is through nanofiltration membrane nanofiltration, obtain condensing peptide liquid, gel filtration chromatography zymolyte is obtained through gel filtration chromatography, then distilled water wiring solution-forming is used, is separated using high performance liquid chromatography, mobile phase is acetonitrile and aqueous tartaric acid solution, the collection liquid for belonging to same peak is mixed, acetonitrile and tartaric acid are removed, is lyophilized to get fish collagen antioxidant peptide.It has the beneficial effect that：Preparation method of the present invention has that albumen enzyme dosage is few, enzymolysis time is short, and protein degradation effect is good, anti-oxidation peptide yield and purity is high, the higher advantage of sensory-acceptance, can reduce the generation of bitter peptides and the loss of product.
Description
Technical field
The present invention relates to field of biotechnology, the in particular to preparation method of fish collagen antioxidant peptide.
Background technique
Vast territory and abundant resources in China, and waters is vast, aquatic resources very abundant, in recent years, with the improvement of living standards, people
The diversity and practicability of aquatic resources demand are gradually increased, to promote the rapid development of China's fish processing industry.But
It is that can generate a large amount of fish pomace, such as fish-skin, fish scale, internal organ, fish head etc. while fish processing industry high speed development,
These leftover bits and pieces account for about the 40% ~ 50% of raw material fish.Relevant information shows nutrient substance rich in fish pomace,
The a large amount of wasting of resources is not only caused in this way, if dealt with improperly it is also possible to causing serious environmental pollution.Therefore, how
The great attention of domestic and international researcher can efficiently have been caused using the nutrient substance in fish pomace.In fish-skin
The content of collagen reaches as high as 80% of total protein or more, and fat content is extremely low, in order to make full use of in fish-skin
Protein resource, many researchers, which are explored, generates collagen using fish-skin hydrolysis.Crude protein master in hydrolytic fish skin at present
It will be there are two types of method：Chemical degradation method and enzyme hydrolysis method.Chemical degradation method is to reach protein hydrolysate using strong acid and strong base peptide bond
Purpose, experimental method is easy to operate, but reacts acutely not easy to control, often results in the damage of amino acid.Enzyme hydrolysis method is anti-
Mild condition is answered, protein isolate low molecular polypeptide and amino acid can be obtained well, so obtaining the blueness of vast researcher
It looks at.
Existing zymolysis technique is all to lay particular emphasis on the initial reactions conditions such as control concentration of substrate, temperature, the dosage of pH, enzyme,
However as the progress of enzyme digestion reaction, concentration of substrate and pH are variations, cause collagen polypeptide enzymolysis process to exist complicated, numerous
It is trivial, be difficult to the problem of controlling, therefore the prior art is difficult to control effectively to the variation of these conditions in reaction process, simultaneously
Control to bitter peptides is also to take method to remove after enzyme digestion reaction, without controlling the generation of bitter peptides during the reaction.
Summary of the invention
That the purpose of the present invention is to provide a kind of albumen enzyme dosages is few, enzymolysis time is short, and protein degradation effect is good, antioxygen
Change peptide yield and purity is high, sensory-acceptance are higher, reduces the fish collagen antioxidant peptide of the generation of bitter peptides and the loss of product
Preparation method.
The present invention in view of the above technology in the problem of mentioning, the technical solution taken is：
The preparation method of fish collagen antioxidant peptide, including pretreatment of raw material, collagen protein powder preparation, enzymatic hydrolysis, nanofiltration, gel column layer
Analysis and RP-HPLC purifying, the specific steps are that：
Pretreatment of raw material：Tissue mashing after fish-skin is shredded is 1 by solid-liquid ratio:It is 0.08- that concentration, which is added, in fish-skin by 4-6g/mL
It in the oxygen sodium hydroxide solution of 0.13M, impregnates and constantly stirs, rinsed with circulating water to neutrality after 1.5-2.5h, then press solid-liquid ratio
It is 1:The aqueous isopropanol that concentration is 2-5% is added in 1.5-2.5g/mL, is cleaned repeatedly with distilled water to neutrality after impregnating 10-15h
Filtered through gauze afterwards sufficiently drains to get pretreatment fish-skin, and fish collagen antioxidant peptide is before preparation, because wherein containing many miscellaneous eggs
Bletilla fat, so to carry out pre-treatment first to remove these foreign proteins, fat etc., can reduce substance of this kind to collagen antioxygen
Change the influence of peptide；
Collagen protein powder preparation：It is 1 by solid-liquid ratio:It is 0.2-0.5M acetic acid solution that concentration is added into pretreatment fish-skin by 8-11,
Revolving speed is stirring and leaching 5-7h under 130-170r/min, and centrifuging and taking supernatant precipitates and continues quadratic acid extraction, centrifuged supernatant
Vacuum distillation is freeze-dried up to collagen protein powder, which makes collagen remain its unique superhelix, property
Stablize, the recovery rate of collagen is up to 23.46%, prepares for next enzymatic hydrolysis；
Enzymatic hydrolysis：It is that deionized water is added into collagen protein powder by 8-10% by concentration of substrate, pH to 7.4-8.6 is adjusted, by enzyme concentration
It is separately added into clostridiopetidase A and flavor protease for 2.4-3.5% and 1.6-2.0%, digests 10- at being then 30-40 DEG C in temperature
15h, enzymatic hydrolysis interval 1-2h is with the ultrafiltration membrane ultrafiltration 10-20min that molecular cut off is 8-12kDa to get ultrafiltrate, the step
It is digested using clostridiopetidase A and flavor protease, clostridiopetidase A can be opened exclusively under the premise of not destroying other protein
The distinctive superhelix of collagen, and flavor protease can carry out digestion from different amino acid sites, to generate big
The antioxidation polypeptide of amount, while leading to reinfocing effect due to synergistic function between enzyme, the polypeptide fragment accordingly generated
Function can further enhance, the content of polypeptide and antioxidation polypeptide in the final enzymolysis efficiency for improving fish-skin albumen, enzymolysis liquid
Activity；And small molecule product is removed with ultrafiltration during the reaction, it avoids and continues to be digested into small molecule product as substrate,
And small molecule product is the source of collagen bitter peptides, further reduces the generation of bitter peptides in this way, to reduce production
The loss of object, and can reduce the concentration of product in enzymatic hydrolysis system, so that enzyme digestion reaction rate can continue to keep, greatly shorten
Enzymolysis time；
Nanofiltration：Nanofiltration membrane nanofiltration by ultrafiltrate through 1-1.2kDa obtains the condensing peptide liquid of 1-1.2kDa or more, spare；
Gel filtration chromatography：By condensing peptide liquid be dissolved in distilled water be made into concentration be 45-55mg/mL solution, 2-5 DEG C,
It is centrifuged 15-23min under 10000-15000r/min, removes insoluble impurities, carries out chromatography in supernatant loading to pillar,
It is eluted with distilled water, elution fraction is collected according to the absorbance curve under 280nm, wherein there is highest radicals scavenging
Active peak is gel filtration chromatography zymolyte, and this method is different according to movement speed of the enzymolysis liquid in chromatographic column, macromolecular
Component is first eluted, and is eluted after the component of small molecule, thus achieve the purpose that isolate and purify, it is easy to operate, and
Anti-oxidation peptide is not needed in conjunction with other substances, reduces the loss of anti-oxidation peptide in purification process, does not influence target components
Property is learned, the original activity of anti-oxidation peptide is able to maintain and is not damaged；
RP-HPLC purifying：By gel filtration chromatography zymolyte distilled water wiring solution-forming, separated using high performance liquid chromatography,
Mobile phase is acetonitrile and aqueous tartaric acid solution, and the collection liquid for belonging to same peak is mixed, acetonitrile and tartaric acid are removed, is lyophilized, i.e.,
Fish collagen antioxidant peptide is obtained, above-mentioned chromatographic condition is：The concentration of solution is 80-100 μ g/mL, sample volume is 4-6 μ L, chromatographic column
For Agilent C18（250mm × 4.6mm, 5 μm）, column temperature be 25-35 DEG C, elution speed 0.8-1.2mL/min, this method tool
Have that analysis speed is fast, advantage of high resolution, high sensitivity, good separating effect, can fast separating and purifying target substance, can be improved
The antioxidant activity of fish collagen antioxidant peptide, while sample size needed for the purification step is few, sample volume, can be same using μ L as the order of magnitude
When separate Multiple components, can sample introduction repeatedly, and sample is not destroyed in separation process, is easily recycled, and the compositional purity of acquisition is higher；
Preferably, RP-HPLC purification step mesotartaric acid concentration of aqueous solution is 0.08-0.12%, the acetonitrile and winestone sour water
The volume ratio of solution is 20-70:1.
Further preferably, the ratio of L-TARTARIC ACID and D- tartaric acid is 86-93 in tartaric acid:1.Fish collagen antioxidant peptide band
There is positive charge, can interact with silanol group remaining on silicagel column, so that peak shape hangover is serious, contains special proportion L-TARTARIC ACID
Addition with the tartaric acid of D- tartaric acid is able to suppress stationary phase residual silanol group effect, so that fish collagen antioxidant peptide is flowing
It can be stabilized in phase system, obtain good peak shape, shorten analysis time, and separate with other impurities all right, subtracted
The waste of time and reagent is lacked；Since tartaric acid can form complex compound with metal ion, weakens metal ion and cation is handed over
The suction-operated of agent stationary phase is changed, thus reduces metal ion retention, improves the yield and purity of fish collagen antioxidant peptide.
Preferably, deionized water contains 0.2-0.4M potassium chloride and 3-5mM sodium carboxymethylcellulose in enzymolysis step.Chlorine
Changing potassium and sodium carboxymethylcellulose may make in enzymatic hydrolysis system metal ion and acid ion by the interaction with protein,
So that clostridiopetidase A and flavor protease are able to maintain its structure and are not destroyed, to play enzymolysis, and be conducive to protease
And the combination of substrate and catalytic hydrolysis reaction improve enzymatic hydrolysis rate, and the presence of sodium carboxymethylcellulose is avoided that protein
It is intermolecular close, dispersing uniformity of the protein in enzymatic hydrolysis system is improved, the touch opportunity of protease and substrate is improved, into
And the content of anti-oxidation peptide and the activity of anti-oxidation peptide in raising enzymatic hydrolysis rate enzymolysis efficiency, enzymolysis liquid.
Compared with the prior art, the advantages of the present invention are as follows：1）Preparation method of the present invention have put into it is low, easy to operate,
Protein degradation effect is good, anti-oxidation peptide yield is high, the higher advantage of sensory-acceptance, is easy to industrial-scale production, has
Good application value and market potential；2）The preparation method is digested using clostridiopetidase A and flavor protease, fish-skin albumen
Enzymolysis efficiency, the content of antioxidation polypeptide and activity are higher in enzymolysis liquid；3）The preparation method is in enzymolysis process with super
Small molecule product is filtered out, the generation of bitter peptides and the loss of product can be reduced, and can reduce product in enzymatic hydrolysis system
Concentration greatly shorten enzymolysis time so that enzyme digestion reaction rate can continue to keep；4）Mobile phase is used in RP-HPLC purifying
So that fish collagen antioxidant peptide can be stabilized in flow visualizing, good peak shape is obtained, shortens analysis time, and with
Other impurities separation is all right, reduces metal ion retention, improves the yield and purity of fish collagen antioxidant peptide.
Specific embodiment
The present invention program is described further below by embodiment：
Embodiment 1：
The preparation method of fish collagen antioxidant peptide, including pretreatment of raw material, collagen protein powder preparation, enzymatic hydrolysis, nanofiltration, gel column layer
Analysis and RP-HPLC purifying, the specific steps are that：
1）Pretreatment of raw material：Tissue mashing after fish-skin is shredded is 1 by solid-liquid ratio:It is 0.13M that concentration, which is added, in fish-skin by 4g/mL
Oxygen sodium hydroxide solution in, impregnate and constantly stir, rinsed with circulating water to neutrality after 1.5h, then by solid-liquid ratio be 1:2.5g/
The aqueous isopropanol that concentration is 2% is added in mL, and filtered through gauze after being cleaned repeatedly to neutrality after immersion 15h with distilled water sufficiently drips
Dry to get pretreatment fish-skin, fish collagen antioxidant peptide is before preparation, because wherein containing many foreign proteins and fat, so will be first
Pre-treatment is carried out to remove these foreign proteins, fat etc., influence of the substance of this kind to fish collagen antioxidant peptide can be reduced；
2）Collagen protein powder preparation：It is 1 by solid-liquid ratio:8 are added concentration into pretreatment fish-skin as 0.5M acetic acid solution, and revolving speed is
Stirring and leaching 7h under 130r/min, centrifuging and taking supernatant, precipitating continue quadratic acid extraction, and centrifuged supernatant vacuum distillation is cold
Jelly is drying to obtain collagen protein powder, which makes collagen remain its unique superhelix, and property is stablized, collagen egg
White recovery rate is up to 23.46%, prepares for next enzymatic hydrolysis；
3）Enzymatic hydrolysis：Deionized water is added into collagen protein powder for 8% by concentration of substrate, adjusts pH to 8.6, is by enzyme concentration
2.4% and 2.0% is separately added into clostridiopetidase A and flavor protease, and 15h is digested at being then 30 DEG C in temperature, and enzymatic hydrolysis interval 1h, which is used, to be cut
Stay the ultrafiltration membrane ultrafiltration 10min that molecular weight is 12kDa to get ultrafiltrate, the step using clostridiopetidase A and flavor protease into
Row enzymatic hydrolysis, clostridiopetidase A can exclusively open the distinctive superhelix of collagen under the premise of not destroying other protein,
And flavor protease can carry out digestion from different amino acid sites, to generate a large amount of antioxidation polypeptide, while between enzyme
Lead to reinfocing effect due to synergistic function, the polypeptide fragment function of accordingly generating can further enhance, final to improve
The content of polypeptide and the activity of antioxidation polypeptide in the enzymolysis efficiency of fish-skin albumen, enzymolysis liquid；And ultrafiltration is used during the reaction
Small molecule product is removed, avoids and continues to be digested into small molecule product as substrate, and small molecule product is collagen hardship
The source of gustin further reduces the generation of bitter peptides in this way, to reduce the loss of product, and can reduce enzymatic hydrolysis body
The concentration of product greatly shortens enzymolysis time so that enzyme digestion reaction rate can continue to keep in system；
4）Nanofiltration：Nanofiltration membrane nanofiltration by ultrafiltrate through 1.2kDa obtains the condensing peptide liquid of 1.2kDa or more, spare；
5）Gel filtration chromatography：Condensing peptide liquid is dissolved in distilled water and is made into the solution that concentration is 45mg/mL, in 5 DEG C, 10000r/
It is centrifuged 23min under min, removes insoluble impurities, carries out chromatography in supernatant loading to pillar, washed with distilled water
It is de-, elution fraction is collected according to the absorbance curve under 280nm, wherein the peak with highest free radical scavenging activity is gel
Column chromatographs zymolyte, and this method is different according to movement speed of the enzymolysis liquid in chromatographic column, and the component of macromolecular is first eluted down
Come, is eluted after the component of small molecule, thus achieve the purpose that isolate and purify, it is easy to operate, and do not need anti-oxidation peptide
In conjunction with other substances, reduce the loss of anti-oxidation peptide in purification process, do not influence target components chemical property, is able to maintain anti-
The oxidation original activity of peptide is not damaged；
6）RP-HPLC purifying：By gel filtration chromatography zymolyte distilled water wiring solution-forming, divided using high performance liquid chromatography
From, mobile phase is acetonitrile and aqueous tartaric acid solution, the collection liquid for belonging to same peak mixed, acetonitrile and tartaric acid are removed, is lyophilized,
Up to fish collagen antioxidant peptide, above-mentioned chromatographic condition is：The concentration of solution is 80 μ g/mL, sample volume is 6 μ L, chromatographic column is
Agilent C18（250mm × 4.6mm, 5 μm）, column temperature be 25 DEG C, elution speed 1.2mL/min, this method have analysis speed
Fast, high resolution, high sensitivity, good separating effect advantage is spent, it is anti-that collagen can be improved in energy fast separating and purifying target substance
The antioxidant activity of peptide is aoxidized, while sample size needed for the purification step is few, sample volume can separate more simultaneously using μ L as the order of magnitude
Kind of ingredient, can sample introduction repeatedly, and sample is not destroyed in separation process, is easily recycled, and the compositional purity of acquisition is higher；
RP-HPLC purification step mesotartaric acid concentration of aqueous solution is 0.08%, and the volume ratio of the acetonitrile and aqueous tartaric acid solution is
70:1。
The ratio of L-TARTARIC ACID and D- tartaric acid is 86 in above-mentioned tartaric acid:1.Fish collagen antioxidant peptide has positive charge,
It can interact with silanol group remaining on silicagel column, so that peak shape hangover is serious, contain special proportion L-TARTARIC ACID and D- winestone
The addition of the tartaric acid of acid is able to suppress stationary phase residual silanol group effect, so that fish collagen antioxidant peptide is in flow visualizing
It can be stabilized, obtain good peak shape, shorten analysis time, and separate with other impurities all right, reduce the time
With the waste of reagent；Since tartaric acid can form complex compound with metal ion, weakens metal ion and cation-exchanger is fixed
The suction-operated of phase, thus reduce metal ion retention, improve the yield and purity of fish collagen antioxidant peptide.
Deionized water contains 0.4M potassium chloride and 3mM sodium carboxymethylcellulose in enzymolysis step.Potassium chloride and carboxymethyl are fine
Tieing up plain sodium may make that metal ion and acid ion are by the interaction with protein in enzymatic hydrolysis system, so that clostridiopetidase A and wind
Taste protease is able to maintain its structure and is not destroyed, to play enzymolysis, and is conducive to the combination of albumen enzyme-to-substrate simultaneously
Catalytic hydrolysis reaction improves enzymatic hydrolysis rate, and the presence of sodium carboxymethylcellulose is avoided that approaching between protein molecule, mentions
Dispersing uniformity of the high protein in enzymatic hydrolysis system improves the touch opportunity of protease and substrate, and then improves enzymatic hydrolysis rate
The activity of the content of anti-oxidation peptide and anti-oxidation peptide in enzymolysis efficiency, enzymolysis liquid.
Embodiment 2：
The preparation method of fish collagen antioxidant peptide, including pretreatment of raw material, collagen protein powder preparation, enzymatic hydrolysis, nanofiltration, gel column layer
Analysis and RP-HPLC purifying, the specific steps are that：
1）Feed pretreatment step is：Tissue mashing after fish-skin is shredded is 1 by solid-liquid ratio:Concentration is added in fish-skin by 5g/mL
It in the oxygen sodium hydroxide solution of 0.1M, impregnates and constantly stirs, rinsed with circulating water to neutrality after 2.0h, then pressing solid-liquid ratio is 1:
The aqueous isopropanol that concentration is 4% is added in 2.0g/mL, and filtered through gauze after being cleaned repeatedly to neutrality after immersion 12h with distilled water is filled
Divide and drains to get pretreatment fish-skin；
2）Collagen protein powder preparation step is：It is 1 by solid-liquid ratio:10 is molten for 0.3M acetic acid to addition concentration in fish-skin is pre-processed
Liquid, revolving speed are stirring and leaching 6h under 150r/min, and centrifuging and taking supernatant precipitates and continues quadratic acid extraction, and centrifuged supernatant subtracts
Pressure distillation, is freeze-dried up to collagen protein powder；
3）Enzymatic hydrolysis：Deionized water is added into collagen protein powder for 8% by concentration of substrate, adjusts pH to 8.0, is by enzyme concentration
3.0% and 1.8% is separately added into clostridiopetidase A and flavor protease, and 12h is digested at being then 35 DEG C in temperature, and enzymatic hydrolysis interval 1.5h is used
Molecular cut off is the ultrafiltration membrane ultrafiltration 15min of 10kDa to get ultrafiltrate；
4）Nanofiltration：Nanofiltration membrane nanofiltration by ultrafiltrate through 1.1kDa obtains the condensing peptide liquid of 1.1kDa or more, spare；
5）Gel filtration chromatography：Condensing peptide liquid is dissolved in distilled water and is made into the solution that concentration is 50mg/mL, in 4 DEG C, 12000r/
It is centrifuged 20min under min, removes insoluble impurities, carries out chromatography in supernatant loading to pillar, washed with distilled water
It is de-, elution fraction is collected according to the absorbance curve under 280nm, wherein the peak with highest free radical scavenging activity is gel
Column chromatographs zymolyte；
6）RP-HPLC purifying：By gel filtration chromatography zymolyte distilled water wiring solution-forming, divided using high performance liquid chromatography
From, mobile phase is acetonitrile and aqueous tartaric acid solution, the collection liquid for belonging to same peak mixed, acetonitrile and tartaric acid are removed, is lyophilized,
Up to fish collagen antioxidant peptide, above-mentioned chromatographic condition is：The concentration of solution is 90 μ g/mL, sample volume is 5 μ L, chromatographic column is
Agilent C18（250mm × 4.6mm, 5 μm）, column temperature be 30 DEG C, elution speed 1.0mL/min.
RP-HPLC purification step mesotartaric acid concentration of aqueous solution is 0.1%, the volume of the acetonitrile and aqueous tartaric acid solution
Than being 40:1.
The ratio of L-TARTARIC ACID and D- tartaric acid is 90 in above-mentioned tartaric acid:1.
Deionized water contains 0.3M potassium chloride and 4mM sodium carboxymethylcellulose in enzymolysis step.
Embodiment 3：
The preparation method of fish collagen antioxidant peptide, including pretreatment of raw material, collagen protein powder preparation, enzymatic hydrolysis, nanofiltration, gel column layer
Analysis and RP-HPLC purifying, the specific steps are that：
1）Pretreatment of raw material：Tissue mashing after fish-skin is shredded is 1 by solid-liquid ratio:It is 0.08M that concentration, which is added, in fish-skin by 6g/mL
Oxygen sodium hydroxide solution in, impregnate and constantly stir, rinsed with circulating water to neutrality after 2.5h, then by solid-liquid ratio be 1:1.5g/
The aqueous isopropanol that concentration is 5% is added in mL, and filtered through gauze after being cleaned repeatedly to neutrality after immersion 10h with distilled water sufficiently drips
It does to get pretreatment fish-skin；
2）Collagen protein powder preparation：It is 1 by solid-liquid ratio:11 to pretreatment fish-skin in be added concentration be 0.2M acetic acid solution, revolving speed
For stirring and leaching 5h under 170r/min, centrifuging and taking supernatant, precipitating continues quadratic acid extraction, and centrifuged supernatant is evaporated under reduced pressure,
It is freeze-dried up to collagen protein powder；
3）Enzymatic hydrolysis：Deionized water is added into collagen protein powder for 10% by concentration of substrate, adjusts pH to 7.4, is by enzyme concentration
3.5% and 1.6% is separately added into clostridiopetidase A and flavor protease, and 10h is digested at being then 40 DEG C in temperature, and enzymatic hydrolysis interval 2h, which is used, to be cut
Stay the ultrafiltration membrane ultrafiltration 20min that molecular weight is 8kDa to get ultrafiltrate；
4）Nanofiltration：Nanofiltration membrane nanofiltration by ultrafiltrate through 1kDa obtains the condensing peptide liquid of 1kDa or more, spare；
5）Gel filtration chromatography：Condensing peptide liquid is dissolved in distilled water and is made into the solution that concentration is 55mg/mL, in 2 DEG C, 15000r/
It is centrifuged 15min under min, removes insoluble impurities, carries out chromatography in supernatant loading to pillar, washed with distilled water
It is de-, elution fraction is collected according to the absorbance curve under 280nm, wherein the peak with highest free radical scavenging activity is gel
Column chromatographs zymolyte；
6）RP-HPLC purifying：By gel filtration chromatography zymolyte distilled water wiring solution-forming, divided using high performance liquid chromatography
From, mobile phase is acetonitrile and aqueous tartaric acid solution, the collection liquid for belonging to same peak mixed, acetonitrile and tartaric acid are removed, is lyophilized,
Up to fish collagen antioxidant peptide, above-mentioned chromatographic condition is：The concentration of solution is 100 μ g/mL, sample volume is 4 μ L, chromatographic column is
Agilent C18（250mm × 4.6mm, 5 μm）, column temperature be 35 DEG C, elution speed 0.8mL/min.
RP-HPLC purification step mesotartaric acid concentration of aqueous solution is 0.12%, the volume of the acetonitrile and aqueous tartaric acid solution
Than being 20:1.
The ratio of L-TARTARIC ACID and D- tartaric acid is 93 in above-mentioned tartaric acid:1.
Deionized water contains 0.2M potassium chloride and 5mM sodium carboxymethylcellulose in enzymolysis step.
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 (10)
1. the preparation method of fish collagen antioxidant peptide, including pretreatment of raw material, collagen protein powder preparation, enzymatic hydrolysis, nanofiltration, gel column
Chromatography and RP-HPLC purifying, it is characterised in that：The RP-HPLC purification step is：By gel filtration chromatography zymolyte distilled water
Wiring solution-forming is separated using high performance liquid chromatography, and mobile phase is acetonitrile and aqueous tartaric acid solution, will belong to the receipts at same peak
Liquid collecting mixing, removes acetonitrile and tartaric acid, is lyophilized to get fish collagen antioxidant peptide.
2. the preparation method of fish collagen antioxidant peptide according to claim 1, it is characterised in that：The aqueous tartaric acid solution
Concentration is 0.08-0.12%, and the volume ratio of the acetonitrile and aqueous tartaric acid solution is 20-70:1.
3. the preparation method of fish collagen antioxidant peptide according to claim 1, it is characterised in that：L- wine in the tartaric acid
The ratio of stone acid and D- tartaric acid is 86-93:1.
4. the preparation method of fish collagen antioxidant peptide according to claim 1, it is characterised in that：The chromatographic condition is：
The concentration of solution is 80-100 μ g/mL, sample volume is 4-6 μ L, chromatographic column is Agilent C18（250mm × 4.6mm, 5 μm）, column
Temperature is 25-35 DEG C, elution speed 0.8-1.2mL/min.
5. the preparation method of fish collagen antioxidant peptide according to claim 1, it is characterised in that：The pretreatment of raw material step
Suddenly it is：Tissue mashing after fish-skin is shredded is 1 by solid-liquid ratio:The oxygen oxygen that concentration is 0.08-0.13M is added in fish-skin by 4-6g/mL
Change in sodium solution, impregnate and constantly stir, rinsed with circulating water to neutrality after 1.5-2.5h, then pressing solid-liquid ratio is 1:1.5-
The aqueous isopropanol that concentration is 2-5% is added in 2.5g/mL, filters, drips after being cleaned repeatedly with distilled water to neutrality after immersion 10-15h
It does to get pretreatment fish-skin.
6. the preparation method of fish collagen antioxidant peptide according to claim 1, it is characterised in that：The collagen protein powder system
It is for step：It is 1 by solid-liquid ratio:It is 0.2-0.5M acetic acid solution, stirring and leaching 5- that concentration is added into pretreatment fish-skin by 8-11
7h, centrifuging and taking supernatant, precipitating continue quadratic acid extraction, and centrifuged supernatant vacuum distillation is freeze-dried up to collagen
Powder.
7. the preparation method of fish collagen antioxidant peptide according to claim 1, it is characterised in that：The enzymolysis step is：
It is that deionized water is added into collagen protein powder by 8-10% by concentration of substrate, adjusts pH to 7.4-8.6, is 2.4- by enzyme concentration
3.5% and 1.6-2.0% is separately added into clostridiopetidase A and flavor protease, digests 10-15h at being then 30-40 DEG C in temperature, digests
The ultrafiltration membrane ultrafiltration 10-20min that interval 1-2h is 8-12kDa with molecular cut off is to get ultrafiltrate.
8. the preparation method of fish collagen antioxidant peptide according to claim 7, it is characterised in that：The deionized water contains
0.2-0.4M potassium chloride and 3-5mM sodium carboxymethylcellulose.
9. the preparation method of fish collagen antioxidant peptide according to claim 1, it is characterised in that：The nano-filtration step is：
Nanofiltration membrane nanofiltration by ultrafiltrate through 1-1.2kDa obtains the condensing peptide liquid of 1-1.2kDa or more, spare.
10. the preparation method of fish collagen antioxidant peptide according to claim 1, it is characterised in that：The gel filtration chromatography
Step is：Condensing peptide liquid is dissolved in distilled water and is made into the solution that concentration is 45-55mg/mL, centrifuged supernatant loading to pillar
Interior carry out chromatography, is eluted with distilled water, collects elution fraction according to the absorbance curve under 280nm, has highest
The peak of free radical scavenging activity is gel filtration chromatography zymolyte.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109627323A (en) * | 2018-12-27 | 2019-04-16 | 江苏隆力奇生物科技股份有限公司 | Snakeskin collagen peptide and its preparation and application |
| CN113080466A (en) * | 2021-04-09 | 2021-07-09 | 南昌大学 | Preparation of Fe3O4Method for preparing magnetic biological composite material of collagen mixed peptide-soybean isoflavone covalent compound |
| CN113603768A (en) * | 2021-07-14 | 2021-11-05 | 烟台德胜海洋生物科技有限公司 | Preparation method of fish-derived collagen |
| CN114106151A (en) * | 2021-12-10 | 2022-03-01 | 中基惠诚科技有限公司 | Preparation method of antioxidant active collagen peptide |
| CN115669792A (en) * | 2021-07-30 | 2023-02-03 | 中国海洋大学 | Method for preparing debitterized low-molecular-weight mucin by using sturgeon tendon |
-
2018
- 2018-05-02 CN CN201810408295.4A patent/CN108823268A/en not_active Withdrawn
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109627323A (en) * | 2018-12-27 | 2019-04-16 | 江苏隆力奇生物科技股份有限公司 | Snakeskin collagen peptide and its preparation and application |
| CN113080466A (en) * | 2021-04-09 | 2021-07-09 | 南昌大学 | Preparation of Fe3O4Method for preparing magnetic biological composite material of collagen mixed peptide-soybean isoflavone covalent compound |
| CN113603768A (en) * | 2021-07-14 | 2021-11-05 | 烟台德胜海洋生物科技有限公司 | Preparation method of fish-derived collagen |
| CN113603768B (en) * | 2021-07-14 | 2024-01-23 | 烟台德胜海洋生物科技有限公司 | Preparation method of fish-source collagen |
| CN115669792A (en) * | 2021-07-30 | 2023-02-03 | 中国海洋大学 | Method for preparing debitterized low-molecular-weight mucin by using sturgeon tendon |
| CN114106151A (en) * | 2021-12-10 | 2022-03-01 | 中基惠诚科技有限公司 | Preparation method of antioxidant active collagen peptide |
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