CN108624644A - A kind of squid active peptides - Google Patents
A kind of squid active peptides Download PDFInfo
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- CN108624644A CN108624644A CN201810401737.2A CN201810401737A CN108624644A CN 108624644 A CN108624644 A CN 108624644A CN 201810401737 A CN201810401737 A CN 201810401737A CN 108624644 A CN108624644 A CN 108624644A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
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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 a kind of squid active peptides, the amount ratio of the substance of branched-chain amino acid and aromatic amino acid is 28 32 in active peptides:1, average molecular weight is 1.1 3kDa.The preparation method of active peptides is：Squid albumen powder neutral proteinase and flavor protease are digested, ultrafiltration in enzymolysis process, addition in ultrafiltrate is obtained into clear filtrate with modified activated carbon decolorization, clear filtrate obtains condensing peptide liquid through nanofiltration, and condensing peptide liquid purifies to obtain squid active peptides through gel filtration chromatography and RP HPLC.It has the beneficial effect that：Squid active peptides color of the present invention is whiter, and no bitter peptides, sensory-acceptance is higher, has stronger antioxidant activity, purity and yield high；Preparation method, which has, puts into the advantage low, easy to operate, protein degradation effect is good, yield is high, is easy to industrial-scale production, has good application value and market potential.
Description
Technical field
The present invention relates to biotechnology, more particularly to a kind of squid active peptides.
Background technology
Squid, although traditionally they are referred to as fish, it is not fish in fact, but lives in the mollusk in ocean.
Squid belongs to Mollusca, Cephalopoda, and squid is generally called in squid section.Body colour is pale, and body cone, head is big, has filbert
Spot, front have 10 to touch foot, and Chang Chengqun cruises in deep about 20 meters of ocean.It is often active in shallow sea at the middle and upper levels, vertically moves
Range reaches over one hundred rice.Fine and tender taste, flavor is similar to abalone, but price is very low, is known as " abalones of the poor ".Westerner because
Squid epidermis is dark and variable, and squid is referred to as " devil fish "；Spaniard eats that squid is relatively more, and squid is processed into difference by they
The can of flavor, sleeve-fish sauce, squid loop etc.；American, which also begins to advocate in recent years, eats squid, they are processed into squid
The form of similar abalone is sold；Squid is popular in Japan, it has also become essential aquatic products, day in Japanese daily life
Squid is sized generally to refrigerated products, dried product, rare delicacies product, salt preserved product, heating bactericide product by I.In squid processing
The by-product of generation, such as skin, internal organ, eye and ink sac, all lose as waste, not only cause environmental pollution, can not
Improve the added value of squid processing.Squid whole body is all precious, and a large amount of collagen is contained in squid skin, can be used to extract collagen
Albumen；Contain the amino acid for promoting grass shrimp to take the photograph bait, crude fat in internal organ（The content of unsaturated fatty acid is very high）, protein（It can
For producing sleeve-fish sauce）；Prepared Chinese ink in ink sac has been shown to have antibacterial functions, the work(that also antitumor and enhancing is immunized
Energy.In order to make full use of the protein resource in squid, many researchers are explored generates active peptides using squid hydrolysis.Mesh
There are mainly two types of methods for crude protein in preceding hydrolysis squid：Chemical degradation method and enzyme hydrolysis method.Chemical degradation method is to utilize strong acid
Highly basic peptide bond achievees the purpose that protein hydrolysate, and experimental method is easy to operate, but reacts acutely not easy to control, often results in ammonia
The damage of base acid.Enzyme hydrolysis method reaction condition is mild, protein isolate can obtain low molecular polypeptide and amino acid well, so
To the favor of vast researcher.Existing zymolysis technique be all lay particular emphasis on control concentration of substrate, temperature, pH, enzyme dosage
Equal initial reactions condition, however as the progress of enzyme digestion reaction, concentration of substrate and pH are variations, and collagen polypeptide is caused to digest
Process there are problems that it is complicated, cumbersome, be difficult to control, therefore the prior art to the variation of these conditions in reaction process be difficult into
Row effectively control, while being also to take method to remove after enzyme digestion reaction to the control of bitter peptides, do not control during the reaction
The generation of bitter peptides, this constrains the intensive processing and application of squid to a certain extent.
Invention content
The purpose of the present invention is to provide a kind of color is whiter, no bitter peptides, sensory-acceptance is higher, has stronger anti-
Oxidation activity, purity and the high squid active peptides of yield.
The present invention in view of the above technology in the problem of mentioning, the technical solution taken is：
A kind of squid active peptides, the amount ratio of the substance of branched-chain amino acid and aromatic amino acid is 28-32 in active peptides:
1.The amount ratio of the substance of higher branched-chain amino acid and aromatic amino acid makes active peptides have stronger anti-oxidant work
Property, effectively free radical can be removed from body, make large biological molecule and biomembrane etc. from the damage of free radical, moreover it is possible to effectively
The activity for inhibiting more cruel oxidizing ferment, reduces the probability of browning food so that the active peptides can be widely used in cosmetics, health care
The fields such as food, pharmaceutical products and feed addictive.
Preferably, the average molecular weight of active peptides is 1.1-3kDa.
A kind of preparation method of squid active peptides, including enzymolysis, decoloration, nanofiltration, gel chromatography and RP-HPLC purifying,
Its specific steps are：
Enzymolysis step is：It is that deionized water is added into squid albumen powder by 2-5% by concentration of substrate, adjusts pH to 6.5-8.0, press
Enzyme concentration is that 6800-7000U/g and 3500-4200U/g is separately added into neutral proteinase and flavor protease, is then in temperature
2-4h, the ultrafiltration membrane ultrafiltration 10-20min that enzymolysis interval 30-50min is 3-5kDa with molecular cut off are digested at 50-55 DEG C, i.e.,
Ultrafiltrate is obtained, which uses neutral proteinase and flavor protease composite hydrolysis, can be carried out from different amino acid sites
Digestion to generate a large amount of active peptides, while leading to reinfocing effect between enzyme due to synergistic function, corresponding to produce
Raw polypeptide fragment function can further enhance, in the final enzymolysis efficiency for improving squid albumen, enzymolysis liquid the content of polypeptide and
The activity of active peptides；And small molecule product is removed with ultrafiltration during the reaction, it avoids and continues to be digested into as substrate
Small molecule product, and small molecule product is the source of bitter peptides, further reduces the generation of bitter peptides in this way, to reduce
The loss of product, and the concentration of product in enzymatic hydrolysis system can be reduced so that enzyme digestion reaction rate can continue to keep, and contract significantly
Short enzymolysis time, the yield of polypeptide is 38.42% in the step；
Decoloration：Ultrafiltrate is heated to 50-70 DEG C, adjusting pH is 5.5-6.5, and addition ethyl acetate is modified with oxygen sodium oxide molybdena
Modified activated carbon, additive amount are the 0.8-1.5% of squid protein by weight, and ultrasonic wave stirs 20-40min, and filtering obtains clear filtrate,
The absorption property of the step modified activated carbon is good, can be uniformly dispersed in ultrafiltrate, can under conditions of dosage is few, the time is short
Reach good decoloration deodorization effect, and active ingredient is not lost, can be so that active peptides to be whiter, sensory-acceptance is higher；
Nanofiltration：By NF membrane nanofiltration of the clear filtrate through 0.8-1kDa, the condensing peptide liquid of 0.8-1kDa or more is obtained, it is spare；
Gel filtration chromatography：Condensing peptide liquid is dissolved in the solution that distilled water is made into a concentration of 45-55mg/mL, 2-5 DEG C,
15-23min is centrifuged under 10000-15000r/min, removes insoluble impurities, chromatography is carried out 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, easy to operate to achieve the purpose that isolate and purify, and
It does not need anti-oxidation peptide to be combined with other substances, reduces the loss of anti-oxidation peptide in purification process, do not influence target components
Property is learned, the original activity of anti-oxidation peptide can be kept not to be damaged；
RP-HPLC purification steps are：By gel filtration chromatography zymolyte distilled water wiring solution-forming, carried out using high performance liquid chromatography
Separation, mobile phase are acetonitrile and aqueous tartaric acid solution, and the collection liquid for belonging to same peak is mixed, acetonitrile and trifluoroacetic acid are removed,
To get active peptides, above-mentioned chromatographic condition is for freeze-drying：A concentration of 80-100 μ g/mL, the sample size of solution are 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 analyze 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 active peptides, while sample size needed for the purification step is few, sample size can detach simultaneously using μ L as the order of magnitude
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, in decoloration ethyl acetate the lemon yellow containing 0.33-0.38wt% and 1.8-2.4wt% benzoic acid
Sodium.The addition of lemon yellow and sodium benzoate is conducive to accelerate the hydrolysis and hydrophily organic salt of ethyl acetate and oxygen sodium oxide molybdena
The movement of molecule so that distribution of the organic molecules of salt of hydrophily inside activated carbon also more extensively with uniformly, have more colors
Element and fishy smell substance are combined with hydrophilic surface groups in the form of oxygen key inside modified activated carbon；Activated carbon table can be improved simultaneously
The acid oxygen-containing functional group in face, and complexing can occur with pigment for these functional groups, increase the adsorbance to pigment, finally
Modified activated carbon processing can be so that active peptides be whiter, and sensory-acceptance is higher.
Preferably, decoloration is hydrophilic active carbon with modified activated carbon, preparation method is：It is 1 by solid-liquid ratio:8-
Activated carbon is put into the oxygen sodium hydroxide solution of a concentration of 0.8-1.2M by 12g/mL impregnates 45-50h, then quick with deionized water
Washing 2-3 times, then activated carbon is put into ethyl acetate, 45-50h is stirred at 75-85 DEG C, it is dry to get modified activated carbon.
By ethyl acetate and oxygen sodium oxide molybdena in activated carbon surface hydrolysis occurs for the step, then draws hydrophilic molecule sodium acetate
Enter to activated carbon surface, internal structure is mainly based on micropore so that modified activated carbon can be uniformly dispersed in ultrafiltrate, improve
Contact of the modified activated carbon with ultrafiltrate to quick adsorption pigment and fishy smell substance, while promoting space structure to disordering
" Turbostratic " conversion, expand the hole of activated carbon, improve aperture ratio, further increase the absorption property of activated carbon, make
It is whiter to obtain active peptides.
Preferably, RP-HPLC purification step mesotartaric acid concentration of aqueous solution is 0.08-0.12%, the acetonitrile and winestone
The volume ratio of aqueous acid is 20-70:1.
Preferably, the ratio of L-TARTARIC ACID and D- tartaric acid is 86-93 in RP-HPLC purification step mesotartaric acid:1.
Active peptides carry positive charge, can interact with remaining silanol group on silicagel column so that peak shape hangover is serious, matches containing special
Addition than L-TARTARIC ACID and the tartaric acid of D- tartaric acid can inhibit stationary phase residual silanol group effect so that active peptides exist
It can be stabilized in flow visualizing, obtain good peak shape, shorten analysis time, and it is good with other impurities to detach situation
It is good, reduce the waste of time and reagent；Due to tartaric acid can with metal ion formed complex compound, weaken metal ion to sun from
The suction-operated of sub- exchanger stationary phase, thus metal ion retention is made to reduce, improve the yield and purity of active peptides.
Compared with the prior art, the advantages of the present invention are as follows：1）Active peptides color of the present invention is whiter, no bitter peptides, sense
Official's acceptance is higher, and purity and yield are high, have stronger antioxidant activity, can be widely used in cosmetics, health food, doctor
The fields such as medicine product and feed addictive；2）The preparation method of the active peptides, which has, puts into low, easy to operate, protein degradation
The advantage that effect is good, yield is high, is easy to industrial-scale production, has good application value and market potential；3）In preparation
The absorption property of decoloration modified activated carbon is good, can be uniformly dispersed in enzymolysis liquid, can under conditions of dosage is few, the time is short
Reach good decoloration deodorization effect, and active ingredient is not lost, can be so that active peptides to be whiter, sensory-acceptance is higher；4）
The RP-HPLC purifying mobile phases make squid active peptides that can be stabilized in flow visualizing, obtain good peak
Shape shortens analysis time, and detaches with other impurities all right, so that metal ion retention is reduced, it is more to improve squid activity
The yield and purity of peptide.
Specific implementation mode
The present invention program is described further below by embodiment：
Embodiment 1：
A kind of squid active peptides, the amount ratio of the substance of branched-chain amino acid and aromatic amino acid is 30.53 in active peptides:
1, the average molecular weight of active peptides is 1156.5kDa.The amount ratio of the substance of higher branched-chain amino acid and aromatic amino acid
Value so that active peptides have stronger antioxidant activity, effectively free radical can be removed from body, make large biological molecule and
The damage from free radical such as biomembrane, moreover it is possible to which the activity for effectively inhibiting more cruel oxidizing ferment reduces the probability of browning food so that
The active peptides can be widely used in the fields such as cosmetics, health food, pharmaceutical products and feed addictive.
A kind of preparation method of squid active peptides, including enzymolysis, decoloration, nanofiltration, gel chromatography and RP-HPLC purifying,
Its specific steps are：
1）Enzymolysis step is：Deionized water is added into squid albumen powder for 5% by concentration of substrate, pH to 6.5 is adjusted, by enzyme
Amount is that 7000U/g and 3500U/g is separately added into neutral proteinase and flavor protease, and 2h is digested at being then 55 DEG C in temperature,
The ultrafiltration membrane ultrafiltration 20min that enzymolysis interval 50min is 3kDa with molecular cut off is to get ultrafiltrate, and the step is using neutral
Protease and flavor protease composite hydrolysis can carry out digestion from different amino acid sites, more to generate a large amount of activity
Peptide, while leading to reinfocing effect due to synergistic function between enzyme, the polypeptide fragment function of accordingly generating can be further
Enhance, the content of polypeptide and the activity of active peptides in the final enzymolysis efficiency for improving squid albumen, enzymolysis liquid；And it was reacting
Small molecule product is removed with ultrafiltration in journey, avoids and continues to be digested into small molecule product as substrate, and small molecule product is
The source of bitter peptides further reduces the generation of bitter peptides in this way, to reduce the loss of product, and can reduce enzymolysis
The concentration of product in system so that enzyme digestion reaction rate can continue to keep, and greatly shorten enzymolysis time, polypeptide in the step
Yield is 38.42%；
2）Decoloration：Ultrafiltrate is heated to 50 DEG C, it is 6.5 to adjust pH, the modification that addition ethyl acetate is modified with oxygen sodium oxide molybdena
Activated carbon, additive amount are the 0.8% of squid protein by weight, and ultrasonic wave stirs 40min, and filtering obtains clear filtrate, which is modified
The absorption property of activated carbon is good, can be uniformly dispersed in ultrafiltrate, can reach good under conditions of dosage is few, the time is short
Decoloration deodorization effect, and active ingredient is not lost, it can be so that active peptides be whiter, sensory-acceptance is higher；
3）Nanofiltration：By NF membrane nanofiltration of the clear filtrate through 0.8kDa, the condensing peptide liquid of 0.8kDa or more is obtained, it is spare；
4）Gel filtration chromatography：Condensing peptide liquid is dissolved in the solution that distilled water is made into a concentration of 55mg/mL, in 2 DEG C, 15000r/
15min is centrifuged under min, is removed insoluble impurities, is carried 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 chromatography zymolyte, 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, it is easy to operate to achieve the purpose that isolate and purify, and do not need anti-oxidation peptide
It is combined with other substances, reduces the loss of anti-oxidation peptide in purification process, do not influence target components chemical property, can keep anti-
The oxidation original activity of peptide is not damaged；
5）RP-HPLC purification steps are：By gel filtration chromatography zymolyte distilled water wiring solution-forming, using high performance liquid chromatography into
Row separation, mobile phase are acetonitrile and aqueous tartaric acid solution, and the collection liquid for belonging to same peak is mixed, acetonitrile and trifluoro second are removed
Acid is lyophilized to get active peptides, and above-mentioned chromatographic condition is：A concentration of 80 μ g/mL, the sample size of solution are 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, active peptides can be improved in energy fast separating and purifying target substance
Antioxidant activity, while sample size needed for the purification step is few, sample size using μ L as the order of magnitude, can detach simultaneously it is a variety of at
Point, can sample introduction repeatedly, and sample is not destroyed in separation process, is easily recycled, and the compositional purity of acquisition is higher.
The sodium benzoate of lemon yellow containing 0.38wt% and 1.8wt% in decoloration ethyl acetate.Lemon yellow and benzoic acid
The addition of sodium is conducive to accelerate ethyl acetate and the hydrolysis of oxygen sodium oxide molybdena and the movement of the organic molecules of salt of hydrophily so that parent
Distribution of the aqueous organic molecules of salt inside activated carbon also more extensively with uniformly, have more pigments and fishy smell substance in modification
It is combined in the form of oxygen key with hydrophilic surface groups inside activated carbon；The oxygen-containing function of acidity of activated carbon surface can be improved simultaneously
Group, and complexing can occur with pigment for these functional groups, increase the adsorbance to pigment, final modified activated carbon processing
Can be so that active peptides to be whiter, sensory-acceptance is higher.
Decoloration is hydrophilic active carbon with modified activated carbon, and preparation method is：It is 1 by solid-liquid ratio:12g/mL will be active
Charcoal, which is put into the oxygen sodium hydroxide solution of a concentration of 0.8M, impregnates 50h, then uses deionized water quick wash 2 times, then by activated carbon
It is put into ethyl acetate, 45h is stirred at 85 DEG C, it is dry to get modified activated carbon.The step is aoxidized by ethyl acetate and oxygen
In activated carbon surface hydrolysis occurs for sodium, and hydrophilic molecule sodium acetate is then introduced into activated carbon surface, internal structure master
It will be based on micropore so that modified activated carbon can be uniformly dispersed in ultrafiltrate, improve contact of the modified activated carbon with ultrafiltrate,
To quick adsorption pigment and fishy smell substance, while space structure being promoted to be converted to " Turbostratic " of disordering, expands activity
The hole of charcoal improves aperture ratio, further increases the absorption property of activated carbon so that active peptides are whiter.
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 RP-HPLC purification step mesotartaric acid:1.Active peptides carry
Positive charge can interact with remaining silanol group on silicagel column so that peak shape hangover is serious, containing special proportioning L-TARTARIC ACID with
The addition of the tartaric acid of D- tartaric acid can inhibit stationary phase residual silanol group effect so that active peptides are in flow visualizing
It can be stabilized, obtain good peak shape, shorten analysis time, and detach 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 metal ion retention is made to reduce, improve the yield and purity of active peptides.
Embodiment 2：
A kind of squid active peptides, the amount ratio of the substance of branched-chain amino acid and aromatic amino acid is 31.86 in active peptides:
1, the average molecular weight of active peptides is 2051.3kDa.
A kind of preparation method of squid active peptides, including enzymolysis, decoloration, nanofiltration, gel chromatography and RP-HPLC purifying,
Its specific steps are：
1）Enzymolysis step is：Deionized water is added into squid albumen powder for 3% by concentration of substrate, adjusts pH to 7.2 and presses enzyme concentration
It is separately added into neutral proteinase and flavor protease for 6950U/g and 4000U/g, 3h, enzyme are digested at being then 52 DEG C in temperature
The ultrafiltration membrane ultrafiltration 15min that solution interval 45min is 4kDa with molecular cut off is to get ultrafiltrate；
2）Decoloration：Ultrafiltrate is heated to 60 DEG C, it is 6.0 to adjust pH, the modification that addition ethyl acetate is modified with oxygen sodium oxide molybdena
Activated carbon, additive amount are the 1.2% of squid protein by weight, and ultrasonic wave stirs 30min, and filtering obtains clear filtrate；
3）Nanofiltration：By NF membrane nanofiltration of the clear filtrate through 0.9kDa, the condensing peptide liquid of 0.9kDa or more is obtained, it is spare；
4）Gel filtration chromatography：Condensing peptide liquid is dissolved in the solution that distilled water is made into a concentration of 50mg/mL, in 4 DEG C, 12000r/
20min is centrifuged under min, is removed insoluble impurities, is carried 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 chromatography zymolyte；
5）RP-HPLC purification steps are：By gel filtration chromatography zymolyte distilled water wiring solution-forming, using high performance liquid chromatography into
Row separation, mobile phase are acetonitrile and aqueous tartaric acid solution, and the collection liquid for belonging to same peak is mixed, acetonitrile and trifluoro second are removed
Acid is lyophilized to get active peptides, and above-mentioned chromatographic condition is：A concentration of 90 μ g/mL, the sample size of solution are 5 μ L, chromatographic column is
Agilent C18（250mm × 4.6mm, 5 μm）, column temperature be 30 DEG C, elution speed 1.0mL/min.
The sodium benzoate of lemon yellow containing 0.35wt% and 2.1wt% in decoloration ethyl acetate.
Decoloration is hydrophilic active carbon with modified activated carbon, and preparation method is：It is 1 by solid-liquid ratio:10g/mL will be active
Charcoal, which is put into the oxygen sodium hydroxide solution of a concentration of 1.0M, impregnates 48h, then uses deionized water quick wash 2 times, then by activated carbon
It is put into ethyl acetate, 48h is stirred at 80 DEG C, it is dry to get modified activated carbon.
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 45:1.
The ratio of L-TARTARIC ACID and D- tartaric acid is 90 in RP-HPLC purification step mesotartaric acid:1.
Embodiment 3：
A kind of squid active peptides, the amount ratio of the substance of branched-chain amino acid and aromatic amino acid is 28.56 in active peptides:
1, the average molecular weight of active peptides is 2987.0kDa.
A kind of preparation method of squid active peptides, including enzymolysis, decoloration, nanofiltration, gel chromatography and RP-HPLC purifying,
Its specific steps are：
1）Enzymolysis step is：Deionized water is added into squid albumen powder for 2% by concentration of substrate, pH to 8.0 is adjusted, by enzyme
Amount is that 6800U/g and 4200U/g is separately added into neutral proteinase and flavor protease, and 4h is digested at being then 50 DEG C in temperature,
The ultrafiltration membrane ultrafiltration 10min that enzymolysis interval 30min is 5kDa with molecular cut off is to get ultrafiltrate；
2）Decoloration：Ultrafiltrate is heated to 70 DEG C, it is 5.5 to adjust pH, the modification that addition ethyl acetate is modified with oxygen sodium oxide molybdena
Activated carbon, additive amount are the 1.5% of squid protein by weight, and ultrasonic wave stirs 20min, and filtering obtains clear filtrate；
3）Nanofiltration：By NF membrane nanofiltration of the clear filtrate through 1kDa, the condensing peptide liquid of 1kDa or more is obtained, it is spare；
4）Gel filtration chromatography：Condensing peptide liquid is dissolved in the solution that distilled water is made into a concentration of 45mg/mL, in 5 DEG C, 10000r/
23min is centrifuged under min, is removed insoluble impurities, is carried 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 chromatography zymolyte；
5）RP-HPLC purification steps are：By gel filtration chromatography zymolyte distilled water wiring solution-forming, using high performance liquid chromatography into
Row separation, mobile phase are acetonitrile and aqueous tartaric acid solution, and the collection liquid for belonging to same peak is mixed, acetonitrile and trifluoro second are removed
Acid is lyophilized to get active peptides, and above-mentioned chromatographic condition is：A concentration of 100 μ g/mL, the sample size of solution are 4 μ L, chromatographic column is
Agilent C18（250mm × 4.6mm, 5 μm）, column temperature be 35 DEG C, elution speed 0.8mL/min.
The sodium benzoate of lemon yellow containing 0.33wt% and 2.4wt% in decoloration ethyl acetate.
Decoloration is hydrophilic active carbon with modified activated carbon, and preparation method is：It is 1 by solid-liquid ratio:8g/mL is by activated carbon
It is put into the oxygen sodium hydroxide solution of a concentration of 1.2M and impregnates 45h, then use deionized water quick wash 3 times, then activated carbon is put
Enter in ethyl acetate, 50h is stirred at 75 DEG C, it is dry to get modified activated carbon.
RP-HPLC purification step mesotartaric acid concentration of aqueous solution is 0.08%, the volume of the acetonitrile and aqueous tartaric acid solution
Than being 70:1.
The ratio of L-TARTARIC ACID and D- tartaric acid is 86 in RP-HPLC purification step mesotartaric acid:1.
Routine operation in the operating procedure of the present invention is well known to those skilled in the art, herein without repeating.
Technical scheme 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 the spirit of the present invention,
Supplement or similar fashion replacement etc., should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of squid active peptides, it is characterised in that：The object of branched-chain amino acid and aromatic amino acid in the active peptides
The amount ratio of matter is 28-32:1.
2. a kind of squid active peptides according to claim 1, it is characterised in that：The average molecular weight of the active peptides
For 1.1-3kDa.
3. a kind of preparation method of squid active peptides, including enzymolysis, decoloration, nanofiltration, gel chromatography and RP-HPLC purifying,
It is characterized in that：The decolorization process is：The modified activated carbon that addition ethyl acetate in ultrafiltrate and oxygen sodium oxide molybdena are modified, surpasses
Sonic agitation, filtering, obtains clear filtrate.
4. a kind of preparation method of squid active peptides according to claim 3, it is characterised in that：The decolorization condition
For：Temperature is 50-70 DEG C, pH 5.5-6.5, and the additive amount of modified activated carbon is the 0.8-1.5% of squid albumen powder weight, when
Between be 20-40min.
5. a kind of preparation method of squid active peptides according to claim 3, it is characterised in that：In the ethyl acetate
The sodium benzoate of lemon yellow and 1.8-2.4wt% containing 0.33-0.38wt%.
6. a kind of preparation method of squid active peptides according to claim 3, it is characterised in that：The modified activated carbon
For hydrophilic active carbon, preparation method is：It is 1 by solid-liquid ratio:Activated carbon is put into a concentration of 0.8-1.2M's by 8-12g/mL
45-50h is impregnated in oxygen sodium hydroxide solution, washs, then activated carbon is put into ethyl acetate, 45-50h is stirred at 75-85 DEG C,
Drying is to get modified activated carbon.
7. a kind of preparation method of squid active peptides according to claim 3, it is characterised in that：The enzymolysis step
For：It is that deionized water is added into squid albumen powder by 2-5% by concentration of substrate, adjusts pH to 6.5-8.0, is 6800- by enzyme concentration
7000U/g and 3500-4200U/g is separately added into neutral proteinase and flavor protease, is digested at being then 50-55 DEG C in temperature
2-4h, ultrafiltration is to get ultrafiltrate in enzymolysis process.
8. a kind of preparation method of squid active peptides according to claim 3, it is characterised in that：The RP-HPLC is pure
Changing step is：It by gel filtration chromatography zymolyte distilled water wiring solution-forming, is detached using high performance liquid chromatography, mobile phase is
The collection liquid for belonging to same peak is mixed, removes acetonitrile and trifluoroacetic acid, is lyophilized to get squid by acetonitrile and aqueous tartaric acid solution
Active peptides.
9. a kind of preparation method of squid active peptides according to claim 8, it is characterised in that：The tartaric acid is water-soluble
The volume ratio of a concentration of 0.08-0.12% of liquid, the acetonitrile and aqueous tartaric acid solution is 20-70:1.
10. a kind of preparation method of squid active peptides according to claim 8, it is characterised in that：In the tartaric acid
The ratio of L-TARTARIC ACID and D- tartaric acid is 86-93:1.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109439716A (en) * | 2018-12-20 | 2019-03-08 | 湖南喜味佳生物科技有限公司 | A kind of preparation method of silver carp fish protein peptide |
| CN110684815A (en) * | 2019-08-15 | 2020-01-14 | 浙江海洋大学 | Preparation method of squid ink active peptide |
| CN117100674A (en) * | 2023-10-25 | 2023-11-24 | 广州安芮洁环保科技有限公司 | Black soldier fly extract and application thereof in preparation of antibacterial product |
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2018
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109439716A (en) * | 2018-12-20 | 2019-03-08 | 湖南喜味佳生物科技有限公司 | A kind of preparation method of silver carp fish protein peptide |
| CN109439716B (en) * | 2018-12-20 | 2021-06-29 | 湖南喜味佳生物科技有限公司 | Preparation method of silver carp protein peptide |
| CN110684815A (en) * | 2019-08-15 | 2020-01-14 | 浙江海洋大学 | Preparation method of squid ink active peptide |
| CN117100674A (en) * | 2023-10-25 | 2023-11-24 | 广州安芮洁环保科技有限公司 | Black soldier fly extract and application thereof in preparation of antibacterial product |
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Application publication date: 20181009 |