CN104131060B - Corbicula fluminea anti-oxidative peptide and preparation method thereof - Google Patents
Corbicula fluminea anti-oxidative peptide and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a corbicula fluminea anti-oxidative peptide and a preparation method thereof. The corbicula fluminea anti-oxidative peptide is prepared by deeply developing corbicula fluminea meat with a compound enzyme microwave synergistic controllable enzymolysis technology, is novel and has an anti-oxidative effect. The utilization rate of corbicula fluminea resources is improved, a new path is provided for deep processing of corbicula fluminea, the additional value of the corbicula fluminea is promoted, the income of peasants is increased, and meanwhile, better social and economic benefits can be gained on the aspect of promoting the development of the corbicula fluminea aquaculture industry.
Description
Technical field
The present invention relates to a kind of Corbicula fluminea anti-oxidation peptide and preparation method thereof, belongs to biological technical field.
Background technology
Corbicula fluminea, scientific name (corbicula fluminea), also known as Corbicula aurea Heude, golden Carnis Arca inflata, flat spiral shell etc., is bivalve,
It is one of important economic shellfish of China.Corbicula fluminea contains rich in protein（Crude protein content is up to more than 60% in dry sample）, sugar
The mineral that original, essential amino acids, taurine, multivitamin and calcium, phosphorus, ferrum, selenium etc. are acted on special physiological, also
Containing biological active substanceies such as bioactive peptide.
In recent years, the research that antioxidation active peptides are extracted from aquatic product protein also gradually spreads out, such as from squama fish, mackerel,
The peptide fragment with notable antioxidant activity is extracted in huge squid skin, white shrimp head and yellowfin tuna skelemin, this is fully demonstrate,proved
High-quality protein in open fire product has the potentiality for preparing anti-oxidation peptide.
Natural Antioxidant Peptides be after chemical antioxidant such as BHA, BHT, PG etc. and Natural Antioxidants such as
A kind of novel exogenous antioxidant after tea polyphenols, VC, carotenoid etc., it is to removing people's interior free yl, suppressing people
Asthenia has good effect, and safety non-toxic always.Not only molecular mass is little for anti-oxidation peptide, be easily absorbed by the body, and
Also soluble, low-viscosity, it is difficult to be formed the good functional characteristic such as gel, therefore anti-oxidation peptide just as a kind of emerging antioxidation
Agent and gradually praised highly by people.
At present, the utilization of Corbicula fluminea resource is limited to eat raw, makes the roughing aspect such as dry a species of small clam living in fresh water, salty a species of small clam living in fresh water and canned food, adds
Value is low.
China application CN 102160594A disclose a kind of Corbicula fluminea egg albumen powder and preparation method thereof, methods described include with
Lower step:Corbicula fluminea meat is cleaned;Rub into Corbicula fluminea meat paste;Endogenous enzymes and exogenous enzyme synergetic hydrolysis;Enzyme denaturing;Addition green tea powder and activity
Carbon decoloring deodorization;Membrane filtration;Concentrating under reduced pressure;Centrifugal spray drying.Corbicula fluminea egg albumen powder obtained by the method, suitably using in Corbicula fluminea
Source enzyme decomposes Corbicula fluminea soft tissue albumen, with reference to exogenous biological enzyme is added, reduces the bitterness of egg albumen powder, improves degree of hydrolysis, reduces
The consumption of exogenous enzyme, prepared Corbicula fluminea egg albumen powder, easily digestion, without stench bitterness.But there is enzymolysis in above-mentioned technical proposal
It is insufficient, the not high defect of the activity of gained Corbicula fluminea anti-oxidation peptide.
Therefore, the preparation method for being proposed to improve Corbicula fluminea anti-oxidation peptide is still needed.
The content of the invention
The first object of the present invention is to provide a kind of preparation method of Corbicula fluminea anti-oxidation peptide, by using compound enzyme microwave
Collaboration Controlled-enzymatic Hydrolysis technology carries out deep development to Corbicula fluminea meat, prepares Corbicula fluminea antioxidation new and with anti-oxidation efficacy
Peptide, can not only improve the utilization rate of Corbicula fluminea resource, and the deep processing for Corbicula fluminea provides a new approach, lift Corbicula fluminea and add
Value, promotes increasing peasant income, while will obtain more preferable Social and economic benef@in terms of the development of Corbicula fluminea aquaculture is promoted.
To achieve these goals, the present invention is adopted the following technical scheme that：
A kind of preparation method of Corbicula fluminea anti-oxidation peptide, it is characterised in that：Comprise the steps：
（1）Microwave cooperating compound enzyme coordinated enzymatic hydrolysis Corbicula fluminea meat, obtains zymolyte；
（2）Ultrafiltration removes high molecular weight protein in zymolyte；
（3）Filtered solution is passed sequentially through into glucose gel G-50 and G-25 to be isolated and purified, lyophilization is obtained final product.
Wherein, step（1）Specially：Fresh Corbicula fluminea meat or quick-freezing Corbicula fluminea meat are taken Jing after flowing water thaws and drains, by 1:2-1:6
(w/w) solid-liquid ratio adds water, and is homogenized, and adjusts pH to 6～8, adds 0.7% ~ 1.5% compound protease of Corbicula fluminea meat quality,
In 700 ~ 900W of microwave power, 20 ~ 35min is digested under the conditions of 55 ~ 60 DEG C, enzyme denaturing after the completion of enzymolysis, centrifugation takes supernatant, obtains
Zymolyte.
Preferred steps（1）Specially：Fresh Corbicula fluminea meat is taken, or quick-freezing Corbicula fluminea meat is Jing after flowing water thaws and drains, by 1:4(w/
W) solid-liquid ratio adds water, and is homogenized using the broken instrument of low-temperature ultrahigh-pressure continuous flow cell, adjusts pH to 6.8～7.2, adds river
1.0% ~ 1.2% compound protease of Carnis Corbicula fluminea quality, in microwave power 800W, digests 25 ~ 30min, enzymolysis under the conditions of 55 ~ 60 DEG C
After the completion of 15 ~ 20min of enzyme denaturing at 100 DEG C, be cooled to room temperature, 4000r/min is centrifuged 20 ~ 25min, takes supernatant, must digest
Thing.
Wherein, described compound protease is neutral protease and flavor protease in mass ratio 2 ~ 3:1, preferably 2:1
The mixture for arriving.Described neutral protease can adopt various neutral protease disclosed in prior art, including but do not limit to
In neutral protease（NOVO subpackages, CAS:9068-59-1 ACT:200000u/g EXP:Space biology section of 11/2014 Shanghai Austria
Skill company limited）.Flavor protease equally can be using various disclosed in prior art, including but not limited to, local flavor albumen
Enzyme（NOVO subpackages, ACT:15000u/g EXP:08/2014 Ao Yu bio tech ltd, Shanghai）, neutral protease belongs to
A kind of restriction endonuclease, with certain cleavage site；Compound protease etc., cleavage can be compounded out for some actual property applied
Point more wide spectrum, reaches the more preferable effect decomposed completely, and flavor protease, in the case of more finely positioning cleavage site,
Decomposite the aminoacid or peptide of some tool local flavors.Neutral protease and flavor protease in mass ratio 2 ~ 3:1 is digested, and is Jing
Different enzyme class screening tests are crossed, it is found that using both enzymes in the proportion Corbicula fluminea is carried out digesting the product for obtaining that its is total
Oxidation resistance is stronger, then both enzymes are carried out into single factor experiment by different proportion compounding, obtains neutral protease and local flavor
Protease in mass ratio 2:1 is digested, and the total antioxidant capacity of enzymatic hydrolysate is most strong.
Additionally, step of the present invention（1）It is preferred that being homogenized using the broken instrument of low-temperature ultrahigh-pressure continuous flow cell.
Preparation method of the present invention, step（2）Adopt ultrafilter membrane molecular weight cut off for 3KD cup type filter mistake
Filter.
Step（3）Specially：
By step（2）The filtered solution for obtaining by the glass chromatography column equipped with sephadex G -50, with distilled water to wash
De- liquid, flow velocity is 0.5 ~ 1mL/min, and by automatic fraction collector collection is in charge of, and one is collected per 5min and is managed, often pipe 3mL, point
The A280nm values of often pipe are not determined, with pipe number as abscissa, often the A280nm values of pipe are vertical coordinate mapping, obtain containing 5 peaks
The elution curve of value, merges the eluent of same detached peakses, 5 different components is obtained, by the most strong group of total antioxidant capacity
Divide and continue through the glass chromatography column equipped with sephadex G -25, with distilled water as eluent, flow velocity is 0.5 ~ 1mL/min,
Collection is in charge of by automatic fraction collector, one is collected per 5min and is managed, often pipe 3mL, determines respectively the A of often pipe280nmValue, to manage
Number is abscissa, often the A of pipe280nmIt is worth and is mapped for vertical coordinate, obtain the elution curve containing 3 peak values, merges same detached peakses
Eluent, obtain 3 different components.3 component Hydroxyl radical-scavenging abilities and DPPH radical scavenging activities are determined respectively
Power, collects the most strong eluant component lyophilization of Scavenging ability, as Corbicula fluminea anti-oxidation peptide.
More specifically such as：By step（2）The filtered solution for obtaining by equipped with sephadex G -50 glass chromatography column, with steam
Distilled water is eluent, and flow velocity is 0.5 ~ 1mL/min, and by automatic fraction collector collection is in charge of, and one is collected per 5min and is managed, often
Pipe 3mL, collects altogether 400 and manages, and the A of often pipe is determined respectively280nmValue, with pipe number as abscissa, the every A of pipe280nmIt is worth for vertical coordinate
Mapping, obtains the elution curve containing 5 peak values, merges the eluent of same detached peakses, obtains 5 different components, respectively
It is named as CF1、CF2、CF3、CF4And CF5, by the most strong CF of total antioxidant capacity2Continue through equipped with sephadex G -25
Glass chromatography column, with distilled water as eluent, flow velocity is 0.5 ~ 1mL/min, is in charge of collection by automatic fraction collector, often
5min collects one and manages, often pipe 3mL, 300 is collected altogether and is managed, and the A of often pipe is determined respectively280nmValue, with pipe number as abscissa, every pipe
A280nmIt is worth and is mapped for vertical coordinate, obtain the elution curve containing 3 peak values, merge the eluent of same detached peakses, obtains 3 not
Same component.3 component Hydroxyl radical-scavenging abilities and DPPH radical scavenging activities are determined respectively, are collected and are removed free radical energy
The most strong eluant component lyophilization of power, as Corbicula fluminea anti-oxidation peptide.
Wherein, described glass chromatography column is 2.0 × 100cm of Ф, and the volume of eluent is 4BV.
Wherein, step（3）Described lyophilization is：It is 5 ~ 10Pa in vacuum, temperature is -51 ~ -53 DEG C of bar
10 ~ 12h of vacuum lyophilization is carried out under part.
The second object of the present invention is the Corbicula fluminea anti-oxidation peptide for protecting said method to prepare.
Compared with prior art, the invention has the beneficial effects as follows:
（1）The Corbicula fluminea anti-oxidation peptide CF2a antioxidant activity of the present invention is high, and the reaction system hydroxyl radical free radical of 1mg/mL is clear
Except rate is up to 91.67%, DPPH free radical scavenging activities up to 67.15%.
（2）Corbicula fluminea anti-oxidation peptide CF2a prepared by the present invention, it is little with molecular weight, it is determined by sephadex G -75
Molecular weight is 1810Da, the characteristics of can be directly absorbed by the body, therefore can be widely applied to food, health product and cosmetics etc.
Field.
（3）Corbicula fluminea is homogenized using the broken instrument of low-temperature ultrahigh-pressure continuous flow cell, sample broke overall process all 4 DEG C ~
Carry out in 6 DEG C of low temperature water-baths, keep original species activity and performance；And pressure is up to 200MPa, Corbicula fluminea meat micronization is made, had
Beneficial to the contact of enzyme-to-substrate during enzymolysis, enzymolysis is promoted to be smoothed out.
（4）The preparation method of Corbicula fluminea anti-oxidation peptide provided by the present invention is digested using compound enzyme microwave cooperating, and microwave can
To strengthen the ability of compound protease enzymolysis protein, enzymolysis is promoted to be smoothed out, it is ensured that the peptide maximum journey with antioxidant activity
Degree is discharged.Enzymatic hydrolysis condition is simply controllable, and reaction condition is gentle, greatly shortens enzymolysis time, and anti-oxidation peptide yield is high；Institute
Obtain enzymolysis solution and be filtered to remove high molecular weight protein through the ultrafiltration cup of simple, intuitive, operate more convenient.Using sephadex G -50
Purification is carried out to polypeptide with G-25, the anti-oxidation peptide hydroxyl radical free radical clearance rate and DPPH free radical scavenging activities for obtaining is respectively enzyme
3.41 times and 2.65 times of the thick peptide of solution liquid.
（5）, with Corbicula fluminea as raw material, cheap, the anti-oxidation peptide for obtaining can substitute commercially available anti-oxidation peptide prepared by the present invention
Synthetic antioxidant, with this reduce production cost and save social resources, increase Corbicula fluminea added value, be the depth of Corbicula fluminea
Processing and utilization opens new way.
Specific embodiment
Embodiment 1
1st, quick-freezing Corbicula fluminea meat Jing flowing water thaw drain after, by 1:The solid-liquid ratio of 4 (w/w) adds water, and is connected using low-temperature ultrahigh-pressure
Afterflow cell crushing instrument is homogenized, and adjusts pH to 6.8, adds 1.0% compound protease of Corbicula fluminea meat quality（Neutral protease：
Flavor protease=2:1）, in microwave power 800W, 30min being digested under the conditions of 55 DEG C, enzymolysis is completed, enzyme denaturing 15 at 100 DEG C
Min, is cooled to room temperature, and 4000r/min centrifugation 20min take out supernatant.
2nd, the supernatant Jing ultrafilter membranes molecular weight cut off that step 1 is obtained is filtered for the cup type filter of 3KD, takes filtered solution.
3rd, the filtered solution for obtaining step 2 is by the glass chromatography column equipped with sephadex G -50, with distilled water to wash
De- liquid, flow velocity is 0.5mL/min, and by automatic fraction collector collection is in charge of, and one is collected per 5min and is managed, often pipe 3mL, is received altogether
Collection 400 is managed, and the A of often pipe is determined respectively280nmValue, with pipe number as abscissa, the every A of pipe280nmIt is worth and is mapped for vertical coordinate, is contained
Have an elution curve of 5 peak values, merge the eluent of same detached peakses, obtain 5 different components, be respectively designated as CF1,
CF2, CF3, CF4 and CF5, by the most strong CF2 of total antioxidant capacity the chromatography of the glass equipped with sephadex G -25 is continued through
Post, with distilled water as eluent, flow velocity is 0.5mL/min, and by automatic fraction collector collection is in charge of, and one is collected per 5min
Pipe, often pipe 3mL, collects altogether 300 and manages, and the A of often pipe is determined respectively280nmValue, with pipe number as abscissa, the every A of pipe280nmIt is vertical to be worth
Coordinate is mapped, and obtains the elution curve containing 3 peak values, merges the eluent of same detached peakses, obtains 3 different components point
CF2a, CF2b, CF2c are not named as it.3 component Hydroxyl radical-scavenging abilities and DPPH radical scavenging activities, group are determined respectively
CF2a, CF2b, CF2c scavenging hydroxyl ability of dividing is respectively 91.67%, 63.33%, 16.80%, removes the energy of DPPH free radicals
Power is respectively 67.15%, 36.35%, 15.61%.
4th, it is 5 Pa in vacuum to collect most strong eluant component CF2a of Scavenging ability, and temperature is -53 DEG C
Under the conditions of carry out vacuum lyophilization 12h and obtain powder, as Corbicula fluminea anti-oxidation peptide CF2a.
Corbicula fluminea anti-oxidation peptide CF2a manufactured in the present embodiment, it is little with molecular weight, it is determined by sephadex G -75
Molecular weight is 1810Da.Additionally, the Corbicula fluminea anti-oxidation peptide CF2a antioxidant activity is high, the reaction system hydroxyl radical free radical of 1mg/mL
Hydroxyl radical-scavenging ability of the clearance rate up to 91.67%, more than 1mg/mL BHT（84%）.In linoleic acid lipid peroxidation system
In oxidation resistance be 65%, higher than the non-oxidizability of BHT under similarity condition（60%）.
Embodiment 2
1st, quick-freezing Corbicula fluminea meat Jing flowing water thaw drain after, by 1:The solid-liquid ratio of 4 (w/w) adds water, and is connected using low-temperature ultrahigh-pressure
Afterflow cell crushing instrument is homogenized, and adjusts pH to 7.0, adds 1.1% compound protease of Corbicula fluminea meat quality（Neutral protease：
Flavor protease=2:1）, in microwave power 800W, 28min being digested under the conditions of 58 DEG C, enzymolysis is completed, enzyme denaturing 18min at 100 DEG C,
Room temperature is cooled to, 4000r/min centrifugation 22min take out supernatant.
2nd, the supernatant Jing ultrafilter membranes molecular weight cut off that step 1 is obtained is filtered for the cup type filter of 3KD, takes filtered solution.
3rd, the filtered solution for obtaining step 2 is by the glass chromatography column equipped with sephadex G -50, with distilled water to wash
De- liquid, flow velocity is 0.8mL/min, and by automatic fraction collector collection is in charge of, and one is collected per 5min and is managed, often pipe 3mL, is received altogether
Collection 400 is managed, and the A of often pipe is determined respectively280nmValue, with pipe number as abscissa, the every A of pipe280nmIt is worth and is mapped for vertical coordinate, is contained
Have an elution curve of 5 peak values, merge the eluent of same detached peakses, obtain 5 different components, be respectively designated as CF1,
CF2, CF3, CF4 and CF5, by the most strong CF2 of total antioxidant capacity the chromatography of the glass equipped with sephadex G -25 is continued through
Post, with distilled water as eluent, flow velocity is 0.8mL/min, and by automatic fraction collector collection is in charge of, and one is collected per 5min
Pipe, often pipe 3mL, collects altogether 300 and manages, and the A of often pipe is determined respectively280nmValue, with pipe number as abscissa, the every A of pipe280nmIt is vertical to be worth
Coordinate is mapped, and obtains the elution curve containing 3 peak values, merges the eluent of same detached peakses, obtains 3 different components；
It is respectively designated as CF2a, CF2b, CF2c.3 component Hydroxyl radical-scavenging abilities and DPPH radical scavenging activities are determined respectively,
Component CF2a, CF2b, CF2c scavenging hydroxyl abilities are respectively 87.56%, 58.45%, 17.82%, remove DPPH free radicals
Ability is respectively 64.78%, 32.75%, 16.71%.
4th, it is 8Pa in vacuum to collect most strong eluant component CF2a of Scavenging ability, and temperature is -52 DEG C of bar
Vacuum lyophilization 11h is carried out under part and obtains powder, as Corbicula fluminea anti-oxidation peptide CF2a.
Embodiment 3
1st, quick-freezing Corbicula fluminea meat Jing flowing water thaw drain after, by 1:The solid-liquid ratio of 4 (w/w) adds water, and is connected using low-temperature ultrahigh-pressure
Afterflow cell crushing instrument is homogenized, and adjusts pH to 7.2, adds 1.2% compound protease of Corbicula fluminea meat quality（Neutral protease：
Flavor protease=2:1）, in microwave power 800W, 25min being digested under the conditions of 60 DEG C, enzymolysis is completed, enzyme denaturing at 100 DEG C
20min, is cooled to room temperature, and 4000r/min centrifugation 25min take out supernatant.
2nd, the supernatant Jing ultrafilter membranes molecular weight cut off that step 1 is obtained is filtered for the cup type filter of 3KD, takes filtered solution.
3rd, the filtered solution for obtaining step 2 is by the glass chromatography column equipped with sephadex G -50, with distilled water to wash
De- liquid, flow velocity is 1mL/min, and by automatic fraction collector collection is in charge of, and one is collected per 5min and is managed, often pipe 3mL, is collected altogether
400 pipes, determine respectively the A of often pipe280nmValue, with pipe number as abscissa, the every A of pipe280nmIt is worth and is mapped for vertical coordinate, obtains containing 5
The elution curve of individual peak value, merges the eluent of same detached peakses, obtains 5 different components, be respectively designated as CF1, CF2,
CF3, CF4 and CF5, by the most strong CF2 of total antioxidant capacity the glass chromatography column equipped with sephadex G -25 is continued through, with
Distilled water is eluent, and flow velocity is 1mL/min, and by automatic fraction collector collection is in charge of, and one is collected per 5min and is managed, and is often managed
3mL, collects altogether 300 and manages, and the A of often pipe is determined respectively280nmValue, with pipe number as abscissa, the every A of pipe280nmIt is worth for vertical coordinate work
Figure, obtains the elution curve containing 3 peak values, merges the eluent of same detached peakses, obtains 3 different components；Order respectively
Entitled CF2a, CF2b, CF2c.3 component Hydroxyl radical-scavenging abilities and DPPH radical scavenging activities, component are determined respectively
CF2a, CF2b, CF2c scavenging hydroxyl ability is respectively 88.79%, 60.63%, 18.94%, removes the ability of DPPH free radicals
Respectively 66.54%, 34.78%, 17.81%.
4th, it is 10Pa in vacuum to collect most strong eluant component CF2a of Scavenging ability, and temperature is -51 DEG C
Under the conditions of carry out vacuum lyophilization 10h and obtain powder, as Corbicula fluminea anti-oxidation peptide CF2a.
Embodiment 4
Compared with Example 1, distinctive points are only that, in the present embodiment, take fresh Corbicula fluminea meat or quick-freezing Corbicula fluminea meat Jing flowing water
After defrosting is drained, by weight 1:2 solid-liquid ratio adds water, and is homogenized, and adjusts pH to 6, and add Corbicula fluminea meat quality 0.7% is answered
Hop protein enzyme, in microwave power 700W, under the conditions of 55 DEG C 20min is digested.Wherein, compound protease is neutral protease and local flavor
Protease in mass ratio 3:1 mixture for obtaining.
The preparation-obtained Corbicula fluminea anti-oxidation peptide of the present embodiment has molecular weight little, is easily absorbed by organisms；Antioxidation is lived
Property high, Hydroxyl radical-scavenging energy of the reaction system hydroxyl radical free radical clearance rate of 1mg/mL up to 87.55%, more than 1mg/mL BHT
Power（84%）.Oxidation resistance in linoleic acid lipid peroxidation system is 63.61%, higher than the antioxygen of BHT under similarity condition
The property changed（60%）.
Embodiment 5
Compared with Example 1, distinctive points are only that, in the present embodiment, take fresh Corbicula fluminea meat or quick-freezing Corbicula fluminea meat Jing flowing water
After defrosting is drained, by weight 1:6 solid-liquid ratio adds water, and is homogenized, and adjusts pH to 8, and add Corbicula fluminea meat quality 1.5% is answered
Hop protein enzyme, in microwave power 900W, digests 35min under the conditions of 60 DEG C, compound protease is neutral protease and local flavor albumen
Enzyme in mass ratio 2.5:1 mixture for obtaining.
The preparation-obtained Corbicula fluminea anti-oxidation peptide of the present embodiment has molecular weight little, is easily absorbed by organisms；Antioxidation is lived
Property high, Hydroxyl radical-scavenging energy of the reaction system hydroxyl radical free radical clearance rate of 1mg/mL up to 89.79%, more than 1mg/mL BHT
Power（84%）.Oxidation resistance in linoleic acid lipid peroxidation system is 64.76%, higher than the antioxygen of BHT under similarity condition
The property changed（60%）.
Comparative example 1
CN 102160594A disclosed embodiments 1, its preparation-obtained Corbicula fluminea egg albumen powder is a kind of mixed containing polypeptide
Compound, purity is low；Molecular weight is big, is difficult to be absorbed by organisms；And activity is low, its hydroxyl radical free radical clearance rate and DPPH free radicals are clear
Except rate is respectively 46.64% and 32.46%.
Embodiment in above-described embodiment can be further combined or replace, and embodiment is only to the present invention's
Preferred embodiment is described, and not the spirit and scope of the present invention are defined, without departing from design philosophy of the present invention
Under the premise of, the various changes and modifications that professional and technical personnel in the art make to technical scheme belong to this
Bright protection domain.
Claims (4)
1. a kind of preparation method of Corbicula fluminea anti-oxidation peptide, it is characterised in that：Comprise the steps：
（1）Microwave cooperating compound enzyme coordinated enzymatic hydrolysis Corbicula fluminea meat, obtains zymolyte；
（2）Ultrafiltration removes high molecular weight protein in zymolyte；
（3）Filtered solution is passed sequentially through into glucose gel G-50 and G-25 to be isolated and purified, lyophilization is obtained final product；
Step（1）Specially：Fresh Corbicula fluminea meat is taken, or quick-freezing Corbicula fluminea meat is Jing after flowing water thaws and drains, by weight 1:4 feed liquid
Than adding water, it is homogenized using the broken instrument of low-temperature ultrahigh-pressure continuous flow cell, is adjusted pH to 6.8～7.2, is added Corbicula fluminea meat quality
1.0% ~ 1.2% compound protease, in microwave power 800W, under the conditions of 55 ~ 60 DEG C digest 25 ~ 30min, 100 after the completion of enzymolysis
15 ~ 20min of enzyme denaturing at DEG C, is cooled to room temperature, and 4000r/min is centrifuged 20 ~ 25min, takes supernatant, obtains zymolyte；
Described compound protease is neutral protease and flavor protease in mass ratio 2 ~ 3:1 mixture for obtaining；
Step（2）Adopt ultrafilter membrane molecular weight cut off and filter for the cup type filter of 3KD；Step（3）Specially：
By step（2）The filtered solution for obtaining by equipped with sephadex G -50 glass chromatography column, with distilled water as eluent,
Flow velocity is 0.5 ~ 1mL/min, and by automatic fraction collector collection is in charge of, and one is collected per 5min and is managed, and often pipe 3mL, determines respectively
Every A280nm values of pipe, with pipe number as abscissa, often the A280nm values of pipe are vertical coordinate mapping, obtain washing containing 5 peak values
De- curve, merges the eluent of same detached peakses, obtains 5 different components, and the most strong component of total antioxidant capacity is continued
By the glass chromatography column equipped with sephadex G -25, with distilled water as eluent, flow velocity is 0.5 ~ 1mL/min, by certainly
Dynamic fraction collector is in charge of collection, one is collected per 5min and is managed, and often pipe 3mL, determines respectively the A of often pipe280nmValue, with pipe number as horizontal stroke
Coordinate, the often A of pipe280nmIt is worth and is mapped for vertical coordinate, obtain the elution curve containing 3 peak values, merges the eluting of same detached peakses
Liquid, obtains 3 different components；3 component Hydroxyl radical-scavenging abilities and DPPH radical scavenging activities are determined respectively, are collected
The most strong eluant component lyophilization of Scavenging ability, as Corbicula fluminea anti-oxidation peptide.
2. preparation method according to claim 1, it is characterised in that：Described glass chromatography column is 2.0 × 100cm of Ф,
The volume of eluent is 4BV.
3. preparation method according to claim 1 and 2, it is characterised in that：Step（3）Described lyophilization is：True
Reciprocal of duty cycle be 5 ~ 10Pa, temperature be -51 ~ -53 DEG C under conditions of carry out 10 ~ 12h of vacuum lyophilization.
4. the Corbicula fluminea anti-oxidation peptide prepared by claim 1-3 either method.
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| CN103265642A (en) * | 2013-05-13 | 2013-08-28 | 中国科学院海洋研究所 | Method for extracting active substance from sea cucumber processing waste |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103265642A (en) * | 2013-05-13 | 2013-08-28 | 中国科学院海洋研究所 | Method for extracting active substance from sea cucumber processing waste |
| CN103740792A (en) * | 2013-06-07 | 2014-04-23 | 浙江海洋学院 | Preparation method of Sinonovacula constricta polypeptide with antioxidation function and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 响应面法优化微波辅助酶解合浦珠母贝蛋白工艺;王晶;《食品科学》;20131028;第35卷(第10期);11-17 * |
| 洪泽湖野生河蚬抗氧化肽酶解工艺及清除自由基能力的研究;刘晶晶;《食品工业科技》;20130731;第34卷(第7期);第165-168页 * |
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