CN101979652A - Mactra quadrangularis protein polypeptide with antioxidant activity and preparation method and application thereof - Google Patents
Mactra quadrangularis protein polypeptide with antioxidant activity and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses mactra quadrangularis protein polypeptide with antioxidant activity and a preparation method and application thereof. The mactra quadrangularis protein polypeptide is prepared by the following steps of: washing mactra quadrangularis clam, grinding and homogenizing; adding trypsin in an amount which is 0.05 to 0.5 percent of the weight of the homogenized solution; performing enzymolysis at the pH of between 7.5 and 8.5 and at the temperature of between 45 and 55 DEG C for 30 to 90 minutes; deactivating the enzyme in water bath at the temperature of between 90 and 100 DEG C for 10 minutes; centrifuging; ultra-filtering the supernatant; intercepting the ultra-filtrate with molecular weight of between 10 and 50kDa; concentrating under reduced pressure; and performing spray-drying. Experiment results show that the mactra quadrangularis protein polypeptide provided by the invention has high antioxidant activity and small molecular weight and can be easily absorbed by human bodies after being taken; and through a great amount of experimental screening, the provided preparation method of the mactra quadrangularis protein polypeptide has rational technological parameter design and high operability, can fully utilize the rich resources of the mactra quadrangularis and has great economic effect and social effect.
Description
Technical field
The present invention relates to Mactra veneriformis, what be specifically related to that the Mactra veneriformis proteolysis obtains has polypeptide of anti-oxidant activity and its production and application.
Background technology
Mactra veneriformis is square horse jade-like stone clam, white clam or Mactra quadrangularis again, is commonly called as white a species of small clam living in fresh water, mud a species of small clam living in fresh water, cloth dove head, belongs to Mollusca, lamellibranchiata, Eulamellibranchia, clam section.Shell slightly is quadrangle, and two shells equate that side expands, and the shell face is smooth to be white in color.The abdominal foot prosperity, no byssus moves in the silt of shallow sea, is the living shellfish mollusk in a kind of common sea, is distributed widely in coastal marine site, China north and south.The many beach of jiangsu coast, Mactra veneriformis are the advantage shellfish, have realized propagating artificially on a large scale, and output is up to 80000 tons/year.Mactra veneriformis is a kind of important edible economic shellfish, and the tender deliciousness of its software meat is nutritious, inner protein, fat, carbohydrate, calcium, iron, phosphorus and multivitamin.Mactra veneriformis is not only edible, also has medicinal, health care value.Traditional Chinese medical theory thinks that the clam flavor is salty, cold in nature, nontoxic, returns stomach, liver, urinary bladder channel, and the multiple function that cures mainly is arranged.Mainly concentrate on food-processing, aquaculture, kind about the research of Mactra veneriformis and the aspect such as define.Both at home and abroad see with the software to be raw material processing can, seasonings, fodder additives etc. more, and be that medicine, the healthcare products research and development of the Mactra veneriformis of target are less with the pharmacodynamic feature at the Application and Development of Mactra veneriformis.The research of Mactra veneriformis active substance focuses mostly at small molecules chemical ingredients (as taurine and EPA), and is to its bioactive macromolecule material, less such as protein and STUDY ON POLYSACHAROSE report.
Many reactive oxygen species be can form in the normal aerobic metabolism process in the organism, ultra-oxygen anion free radical, hydroxyl radical free radical, fat oxyradical, hydrogen peroxide etc. comprised; In addition, organism also can pass through exogenous intrusion such as infection, ionizing radiation, atmospheric pollution and produce active oxygen.The activity in vivo oxyradical has certain function, as immunity and signal conductive process.But too much active oxygen radical just has destruction, have the free radical of unpaired electron on can the trigger cell film unsaturated fatty acid ester matter peroxidation and combine with ferment on the film, the destroy integrity that causes cytolemma changes structure and permeability; In addition, free radical can cause protein denaturation with intracellular proteins react equally, and then makes that normal function can't carry out in the cell, and free radical also can attack dna molecular, causes transgenation.Big quantity research proves, the genesis mechanism of aging and aging relevant disorders such as cancers, cardiovascular, autoimmune disorder and sacroiliitis etc. produces too much with interior free yl or removing free radical ability drop has confidential relation.Therefore make full use of the Mactra veneriformis resource, will utilize enzymolysis process that it is processed as the Mactra veneriformis protein of main component, it be significant to make the protein polypeptide that has the anti-oxidant function activity and be easy to absorption of human body then.
Summary of the invention:
Goal of the invention: technical problem to be solved by this invention is to overcome the deficiencies in the prior art, provide a kind of anti-oxidant function good, the Mactra veneriformis protein polypeptide that human body easily absorbs, another object of the present invention provide preparation method and its application of this Mactra veneriformis protein polypeptide.
Technical scheme: in order to realize above purpose, the technical solution used in the present invention is:
A kind of Mactra veneriformis protein polypeptide with anti-oxidant activity, it prepares by the following method: at first get the Mactra veneriformis software and clean and to blend homogenate, the trypsinase that adds suitable homogenate weight 0.05%～0.5%, at pH7.5 to 8.5, enzyme digestion reaction is 30 to 90 minutes under 45～55 ℃ of conditions of temperature, then 90～100 ℃ of water-bath inactivators 10 minutes, centrifugal, get supernatant liquor and carry out ultrafiltration, molecular weight cut-off is the ultrafiltrated of 10～50kDa, concentrating under reduced pressure, spraying drying get the Mactra veneriformis protein polypeptide.
Preparation method with Mactra veneriformis protein polypeptide of anti-oxidant activity provided by the invention specifically may further comprise the steps:
At first getting the Mactra veneriformis software cleans, blend homogenate, get homogenate, add the trypsinase of suitable homogenate weight 0.05%～0.5%, at pH7.5 to 8.5, enzyme digestion reaction is 30 to 90 minutes under 45～55 ℃ of conditions of temperature, at go out enzyme 10 minutes of 90～100 ℃ of water-baths, centrifugal then, get supernatant liquor and carry out ultrafiltration, molecular weight cut-off is the ultrafiltrated of 10～50kDa, and the ultrafiltrated concentrating under reduced pressure promptly gets the Mactra veneriformis protein polypeptide.
As preferred version, above-described preparation method with Mactra veneriformis protein polypeptide of anti-oxidant activity, described tryptic add-on are 0.25%～0.5% of Mactra veneriformis homogenate weight.
As other preferred version, above-described preparation method with Mactra veneriformis protein polypeptide of anti-oxidant activity, wherein the pH value of enzyme digestion reaction is 8.5.
As other preferred version, above-described preparation method with Mactra veneriformis protein polypeptide of anti-oxidant activity, wherein the temperature of enzyme digestion reaction is 45 degree, the time of enzyme digestion reaction is 70～90 minutes.
As preferred version, described preparation method with Mactra veneriformis protein polypeptide of anti-oxidant activity, wherein the centrifugal gained supernatant liquor of enzymolysis solution carries out ultrafiltration, holds back and obtains the ultrafiltrated that molecular weight is 10～30kDa, promptly gets the Mactra veneriformis protein polypeptide.
For fully studying the preparation method of Mactra veneriformis protein polypeptide, the following experiment screening of the present invention's process:
One, the screening experiment of proteolytic ferment
1.1 experiment material
Mactra veneriformis: be collected in Nantong Mao Jiagang (ocean, Jiangsu Province and aquatic products institute provide).
1.2 main agents
Enzyme for screening comprises: stomach en-, papoid, trypsinase, available from Chemical Reagent Co., Ltd., Sinopharm Group; Neutral protease: Nanjing Ao Duofuni bio tech ltd; Pierce BCA protein quantification test kit: U.S. Thermo company; Hexichol is for bitter taste hydrazides (DPPH): Alfa company;
1.3 key instrument and equipment
BP 211D electronic balance: German Sartorious company; Rotavapor R-210 Rotary Evaporators: German BUCHI company; Spectra Max 190 microplate reader: U.S. AD company; Hollow-fibre membrane (molecular weight cut-off 10,20,50kDa): membrane filtration technique company limited is liked to give birth in Tianjin; JJ-2 type tissue is smashed refiner to pieces: the glorious instrument in Jintan City, Jiangsu Province manufacturing company; Avanti J-25 high speed freezing centrifuge: German Backman company; The electrodeless constant speed stirrer of DW-1 reinforcement: Nanjing Cole's plant and instrument company limited.
2. method
2.1 Mactra veneriformis enzymolysis solution preparation
The Mactra veneriformis software is cleaned and is blended homogenate, the water that adds 3 times of weight is made into suspension, adding enzyme activity unit more respectively is 4 kinds of lytic enzymes (trypsinase, papoid, neutral protease and stomach en-) of 500IU, enzyme concentration is 0.2% of a homogenate weight, mark separately top condition (trypsinase: 50 ℃, pH=8.0; Papoid: 55 ℃, pH=5.5; Neutral protease: 50 ℃, pH=7.0; Stomach en-: 37 ℃, carry out 2 hours enzyme digestion reactions under pH=2.0).Enzymolysis finishes the back 90 ℃ of water-bath inactivators 10 minutes, and centrifugal 15 minutes of 4000rpm gets supernatant liquor and carries out ultrafiltration (molecular weight cut-off 50kDa), and the ultrafiltrated concentrate under reduced pressure at low temperature is to former homogenate volume, four kinds of different Mactra veneriformis enzymolysis solutions.
2.2 the mensuration of reducing power
Adopt the prussian blue reaction method to measure the reducing power of different Mactra veneriformis enzymolysis solutions.Add given the test agent, solvent control 2.5ml in the colorimetric cylinder respectively, other add phosphate buffered saline buffer (0.2mol/L, pH=7) and each 2.5mL of the 10mg/ml Tripotassium iron hexacyanide, behind the mixing in 50 ℃ of water-baths 20 minutes, add 100mg/ml trichoroacetic acid(TCA) 2.5ml then, 3000rpm is centrifugal 10 minutes behind the mixing.Accurately pipette supernatant liquor 2.5ml, add distilled water 2.0ml and 1mg/ml iron trichloride 0.5ml, measure absorbance in the 700nm place behind the mixing, the big more reducing power that shows of absorbance (OD value) is strong more.
2.3 hydroxy radical qiao removing ability is measured
Adopt different enzymolysis solutions that the experiment in vitro of the hydroxy radical qiao clearance rate of Fenton system generation is measured.Get the FeSO of 0.2ml
4-EDTA mixed solution (10.0mmol/L) adds 2-deoxyribosyl solution (10.0mmol/L) and the 0.6ml Mactra veneriformis enzymolysis solution of people 0.5ml in test tube, (pH=7.4 0.1mol/L) is settled to 1.8mL, adds people 0.2mLH again with phosphoric acid buffer
2O
2(10.0mol/L), mixing is placed in 37 ℃ of waters bath with thermostatic control and reacts 1h.Add 2.8% (w/w) trichoroacetic acid(TCA) (TCA) solution 1.0mL then, under 4000rpm centrifugal 20 minutes, get supernatant liquor 2.0ml in another test tube, add 1.0% (w/w) thiobarbituricacid (TBA) solution 1.0ml, the rearmounted boiling water bath reaction of mixing 15 minutes, absorbance is surveyed in 5 times of cooling back dilutions at 532nm wavelength place.Enzymolysis solution is represented with clearance rate (SA/%) the removing effect of hydroxy radical qiao, is calculated as follows:
Clearance rate SA (%)=[(Ac-As)/(Ac-A0)] * 100%
In the formula: Ac is not for adding the absorbancy of scavenging agent, and As is the absorbancy behind the adding Mactra veneriformis enzymolysis solution, A
0Absorbancy for reagent blank.
2.4DPPH radical scavenging activity is measured
Getting Mactra veneriformis enzymolysis solution 1ml joins respectively in the clean 10ml tool plug test tube, add 0.6mmol/L DPPH methanol solution 0.5ml again, replenish volume to 5ml with methyl alcohol then, light absorption value A was measured in room temperature lucifuge reaction back in the 517nm place in 30 minutes, blank group replaces DPPH solution with the equal-volume methanol solution, control group replaces sample solution with equal-volume distilled water, and with equal-volume distilled water and the blank zeroing of methyl alcohol mixed liquor.Be calculated as follows:
Clearance rate SA (%)=[1-(Ai-Aj)/A
0] * 100%
In the formula: A
0Be control group absorbancy, A
iBe sample sets absorbancy, A
jBe blank group absorbancy.
2.5 ultra-oxygen anion free radical removing ability is measured
Adopt pyrogallol autoxidation method to measure.Measure 50mmol/L, pH 8.20Tris-HCl damping fluid 4.7ml, add 0.1ml Mactra veneriformis enzymolysis solution, placed 25 ℃ of water bath heat preservations 20 minutes, and added pyrogallol solution (preparing) 0.2ml of the 3mmol/L of 25 ℃ of pre-temperature then, pour in the cuvette after shaking up rapidly with 10mmol/LHCl, under 319nm, measured light absorption value once every 1 minute, coreaction 9 minutes is blank zeroing with 10mmol/L HCl solution, and control group replaces sample with the equal-volume deionized water.Make the absorbancy regression equation of change curve in time, its slope is the autoxidation speed V of pyrogallol, is calculated as follows sample to O
-2Clearance rate.
Clearance rate SA (%)=[1-V
1/ V
0] * 100%
In the formula: V
1Be the absorbancy of sample sets rate over time, V
0Be blank group group absorbancy rate over time.
3. result and discussion
3.1 the screening of enzyme: concrete experimental result is as shown in table 1:
The resistance of oxidation of table 14 kind of protease hydrolyzed liquid (concentration 5mg/ml,
N=3)
Show by table 1 experimental result, the present invention screening 4 in the proteolytic ferment, the Mactra veneriformis enzymolysis solution of trypsin hydrolyzing all is better than papoid, neutral protease and stomach en-to the removing ability of DPPH free radical, hydroxy radical qiao and ultra-oxygen anion free radical.And show that from experimental result the pepsic enzymolysis solution reducing power of Mactra veneriformis is the strongest, and the removing ability of DPPH free radical, hydroxy radical qiao and ultra-oxygen anion free radical is better than papoid and neutral protease.So the preferred trypsinase of proteolytic enzyme or the stomach en-of hydrolysis Mactra veneriformis, as preferred plan, the preferred trypsinase of lytic enzyme.
Because the hydroxy radical qiao clearance rate is the important indicator of reflection antioxygenation, so the present invention carries out the optimization and the investigation of trypsin digestion technology to remove the hydroxy radical qiao ability as investigating index.
Two, the experiment of single factor of trypsin digestion technology
1, temperature experiment of single factor
The Mactra veneriformis software is cleaned and is blended homogenate, the water that adds 3 times of weight is made into suspension, add the trypsinase that enzyme activity unit is 500IU more respectively, enzyme concentration is 0.5% of a homogenate weight, at pH 8.0, under 120 minutes enzyme digestion reaction time, investigate temperature factor to product polypeptide anti-oxidant activity influence experiment, investigate 40 ℃ respectively, 45 ℃, 50 ℃, 55 ℃, the Mactra veneriformis hydrolyzed peptide that 60 ℃ and 65 ℃ of enzymolysis obtain is to the clearance rate activity of hydroxy radical qiao, see shown in Figure 1, by the result as can be known, hydroxy radical qiao clearance rate peaking in the time of temperature 45-50 ℃, this is that the active polypeptide yield is the highest, so anti-oxidant activity is the strongest because enzyme is alive the strongest under this temperature; When temperature more than 50 ℃ the time, temperature begins the enzyme restraining effect of having lived, making degradation of substrates is that the speed of purpose product descends.Therefore, the tryptic hydrolysis temperature of optimum Mactra veneriformis albumen is 45～50 ℃.
2, time experiment of single factor
The Mactra veneriformis software is cleaned and is blended homogenate, the water that adds 3 times of weight is made into suspension, add the trypsinase that enzyme activity unit is 500IU more respectively, enzyme concentration is 0.5% of a homogenate weight, at pH 8.0, under 50 ℃ of the temperature, investigate of the influence experiment of enzyme digestion reaction time to product polypeptide anti-oxidant activity, investigate Mactra veneriformis protein polypeptide that 20 minutes, 40 minutes, 60 minutes, 80 minutes, 100 minutes, 120 minutes enzymolysis obtain clearance rate activity respectively, see shown in Figure 2 hydroxy radical qiao.Experimental result shows, prolongation along with enzymolysis time, the hydroxy radical qiao clearance rate of enzymolysis solution increases gradually, and reaction times hydroxy radical qiao clearance rate in the time of 80 minutes is in peak value, but the time is when surpassing 100 minutes, prolongation along with the reaction times, clearance rate descends on the contrary, and this is because in time prolongation, under the condition of hydrolysing component, active polypeptide is further become the little peptide of non-activity by enzymolysis, so the enzymolysis solution overall activity reduces.Therefore, selecting enzymolysis time is to be advisable in 70～90 minutes.
3, pH experiment of single factor
The Mactra veneriformis software is cleaned and is blended homogenate, the water that adds 3 times of weight is made into suspension, add the trypsinase that enzyme activity unit is 500IU more respectively, enzyme concentration is 0.5% of a homogenate weight, 50 ℃ of following enzymolysis of temperature 80 minutes, investigate the influence experiment of enzyme digestion reaction pH, investigate the pH value respectively and be respectively Mactra veneriformis protein polypeptide that 6.5,7.0,7.5,8.0,8.5,9.0 enzymolysis obtain clearance rate activity, see shown in Figure 3 hydroxy radical qiao to product polypeptide anti-oxidant activity.Experimental result by Fig. 3 shows, 7.5～8.5 of alkaline pH scopes, the hydroxy radical qiao clearance rate is more excellent, when the pH value is 8.5, hydroxy radical qiao clearance rate maximum, and pH successively decreases greater than 8.5 o'clock hydroxy radical qiao clearance rates, illustrates at pH about 7.5～8.5 it is that enzyme acts on the optimum range that substrate produces bioactive peptide.Therefore, optimum Mactra veneriformis proteolysis pH value is 7.5～8.5.
4, enzyme concentration experiment of single factor
The Mactra veneriformis software is cleaned and is blended homogenate, the water that adds 3 times of weight is made into suspension, add the trypsinase that a certain amount of enzyme activity unit is 500IU more respectively, pH=8.5,50 ℃ of following enzymolysis of temperature 80 minutes, investigate the influence experiment of enzyme concentration to product polypeptide anti-oxidant activity, investigate enzyme-substrate and be respectively Mactra veneriformis protein polypeptide that 0.05%, 0.1%, 0.25%, 0.5%, 0.75% o'clock enzymolysis obtains clearance rate activity, see shown in Figure 4 hydroxy radical qiao than (enzyme-substrate is than the per-cent that accounts for the homogenate gross weight by enzyme concentration).Experimental result shows that the hydroxy radical qiao clearance rate of homogenate filtration component only is not 41.23% when enzyme-added, and can be up to reaching 79.7% than the hydroxy radical qiao clearance rate that is 0.05% o'clock enzymolysis product at enzyme-substrate; Along with the increase of enzyme-substrate ratio, the hydroxy radical qiao clearance rate of product increases gradually, increases to 0.25% when above at the enzyme-substrate ratio, and the hydroxy radical qiao clearance rate of product reaches maximum; Yet, increasing enzyme-substrate than surpassing at 0.25% o'clock, the hydroxy radical qiao clearance rate of product presents downtrending.Its former because: along with its substrate of increase of enzyme concn reaches capacity gradually, when the enzyme concn supersaturation occurring, oversaturated enzyme just may act on has active polypeptide, makes its degraded generate the little peptide of non-activity, and the content of reduction bioactive peptide.Therefore, to account for Mactra veneriformis albumen homogenate weight percent be 0.25%～0.5% to be advisable to the trypsinase add-on.
Three, Mactra veneriformis trypsin digestion technology orthogonal experiment
The present invention chooses enzymolysis pH value, temperature, enzyme concentration and four factors of enzymolysis time, and each factor is selected 3 levels near single factor The selection result numerical value, select L9 (3 for use
4) orthogonal test, trypsin hydrolyzing Mactra veneriformis technology is further optimized.Experimental result is shown in table 2 and table 3.
Table 2L9 (3
4) the range analysis table of orthogonal experiments
Table 3 trypsin digestion thing is to the Hydroxyl Radical Scavenging analysis of variance table
Definite coefficient=0.980 of equation (adjust and determine coefficient=0.922)
By variance analysis in R value and the table 3 in the table 2 as can be known: the secondary factors that influences the enzymolysis solution Hydroxyl Radical Scavenging is: pH value＞temperature＞hydrolysis time＞enzyme concentration, further test of significance shows that free radical scavenging reaches conspicuous level (P＜0.05) to pH value (factor C) to product.Therefore show according to Orthogonal experiment results that the enzymatic hydrolysis condition that Mactra veneriformis albumen trypsin digestion thing is removed ability the best to hydroxy radical qiao is that pH is 8.5,45 ℃ of temperature, 90 minutes time, the enzyme-substrate ratio is 0.5%.Show that trypsin digestion Mactra veneriformis provided by the invention prepares the reasonableness and the science of antioxidation polypeptide technology.
Show that through experimental study Mactra veneriformis protein polypeptide provided by the invention has good anti-oxidant activity, therefore Mactra veneriformis protein polypeptide of the present invention can be applicable to prepare the antioxidant and anti-aging medicine.
Mactra veneriformis protein polypeptide provided by the invention can become drug forms such as granule, capsule, tablet dose, pill with the Mactra veneriformis protein polypeptide with pharmaceutically acceptable preparing carriers.
When Mactra veneriformis protein polypeptide provided by the invention is made tablet,, add magnesium stearate lubricant when needing, mix Mactra veneriformis protein polypeptide and lactose or W-Gum, whole grain, compressing tablet is made tablet then.
When Mactra veneriformis protein polypeptide provided by the invention is made capsule Mactra veneriformis protein polypeptide and carrier lactose or W-Gum are mixed whole grain, the encapsulated then capsule of making.
When Mactra veneriformis protein polypeptide provided by the invention is made granule, Mactra veneriformis protein polypeptide and thinner lactose or W-Gum, cyclodextrin are mixed, sieve, whole grain, drying is made granule.
Mactra veneriformis protein polypeptide with anti-oxidant activity of the present invention also can be in the application in preparation antioxidant and anti-aging healthcare products or the foodstuff additive.
Beneficial effect: the Mactra veneriformis protein polypeptide with anti-oxidant activity provided by the invention has the following advantages:
Mactra veneriformis protein polypeptide with anti-oxidant activity provided by the invention, experimental result show to have good anti-oxidant activity, and the protein polypeptide molecular weight that obtains behind the enzymolysis is little, are easier to after human body is taken absorb; Preparation method with anti-oxidant activity Mactra veneriformis protein polypeptide provided by the invention, through a large amount of experiment screenings, each parameter designing of gained technology is reasonable, can make full use of the Mactra veneriformis affluent resources, Mactra veneriformis hmw protein enzymolysis had both been had the strong anti-oxidation activity, be easy to the protein polypeptide of absorption of human body again; Mactra veneriformis protein polypeptide provided by the invention can be used to be prepared into antioxidant and anti-aging medicine or antioxidant and anti-aging healthcare products or foodstuff additive, has wide range of applications, and has good economy and social effect.
Description of drawings
Fig. 1 is the correlated results figure of differing temps and free radical scavenging activity in the temperature experiment of single factor.
Fig. 2 is the correlated results figure of different time and free radical scavenging activity in the time experiment of single factor.
Fig. 3 is the correlated results figure of different pH values and free radical scavenging activity in the pH experiment of single factor.
Fig. 4 is the correlated results figure of different enzyme concentrations and free radical scavenging activity in the enzyme concentration experiment of single factor.
Embodiment:
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand that the described concrete material proportion of embodiment, processing condition and result thereof only are used to illustrate the present invention, and should also can not limit the present invention described in detail in claims.
1 one kinds of Mactra veneriformis protein polypeptides of embodiment with anti-oxidant activity, it is to prepare by the following method:
Concrete steps are: the Mactra veneriformis software is cleaned, blend homogenate, get homogenate, add the trypsinase that accounts for Mactra veneriformis homogenate weight 0.25% then, at pH 8.5, enzymolysis is 80 minutes under 45 ℃ of conditions of temperature, and enzymolysis finishes the back at go out enzyme 10 minutes of 90 ℃ of water-baths, centrifugal 15 minutes of 4000rpm, get supernatant liquor and carry out ultrafiltration, molecular weight cut-off is the ultrafiltrated of 10-30kDa, the ultrafiltrated concentrating under reduced pressure, and spraying drying gets the Mactra veneriformis protein polypeptide.
2 one kinds of Mactra veneriformis protein polypeptides of embodiment with anti-oxidant activity, it is to prepare by the following method:
Concrete steps are: the Mactra veneriformis software is cleaned, blend homogenate, get homogenate, add the trypsinase that accounts for Mactra veneriformis homogenate weight 0.5% then, at pH 8.0, enzymolysis is 90 minutes under 55 ℃ of conditions of temperature, and enzymolysis finishes the back at go out enzyme 10 minutes of 100 ℃ of water-baths, centrifugal 15 minutes of 4000rpm, get supernatant liquor and carry out ultrafiltration, molecular weight cut-off is the ultrafiltrated of 10-50kDa, the ultrafiltrated concentrating under reduced pressure, and spraying drying gets the Mactra veneriformis protein polypeptide.
3 one kinds of Mactra veneriformis protein polypeptides of embodiment with anti-oxidant activity, it is to prepare by the following method:
Concrete steps are: the Mactra veneriformis software is cleaned, blend homogenate, get homogenate, add the trypsinase that accounts for Mactra veneriformis homogenate weight 0.1% then, at pH 7.5, enzymolysis is 70 minutes under 55 ℃ of conditions of temperature, and enzymolysis finishes the back at go out enzyme 10 minutes of 90 ℃ of water-baths, centrifugal 15 minutes of 4000rpm, get supernatant liquor and carry out ultrafiltration, molecular weight cut-off is the ultrafiltrated of 30-50kDa, the ultrafiltrated concentrating under reduced pressure, and spraying drying gets the Mactra veneriformis protein polypeptide.
4 one kinds of Mactra veneriformis protein polypeptides of embodiment with anti-oxidant activity, it is to prepare by the following method:
Concrete steps are: the Mactra veneriformis software is cleaned, blend homogenate, get homogenate, add the trypsinase that accounts for Mactra veneriformis homogenate weight 0.5% then, at pH 8.5, enzymolysis is 80 minutes under 45 ℃ of conditions of temperature, and enzymolysis finishes the back at go out enzyme 10 minutes of 100 ℃ of water-baths, centrifugal 15 minutes of 4000rpm, get supernatant liquor and carry out ultrafiltration, molecular weight cut-off is the ultrafiltrated of 20-50kDa, the ultrafiltrated concentrating under reduced pressure, and spraying drying gets the Mactra veneriformis protein polypeptide.
Embodiment 5 anti-oxidant experiments
1. material and equipment
Implement 4 kinds of Mactra veneriformis protein polypeptides that 1-4 prepares; Pierce BCA protein quantification test kit: U.S. Thermo company; Hexichol is for bitter taste hydrazides (DPPH): Alfa company.BP 211D electronic balance: German Sartorious company; Spectra Max190 microplate reader: U.S. AD company; Constant temperature perspective tank, Shanghai laboratory apparatus head factory; The UV-1700 spectrophotometer, Japanese Shimadzu company.
2. method
2.1 test solution preparation
Get the foregoing description 1-4 preparation 4 in the Mactra veneriformis protein polypeptide, precision weighing, adding distil water are mixed with the test solution of 100 μ g/ml.
2.2 the mensuration of reducing power
Adopt the prussian blue reaction method to measure the reducing power of different Mactra veneriformis enzymolysis solutions.Add given the test agent, solvent control 2.5ml in the colorimetric cylinder respectively, other add phosphate buffered saline buffer (0.2mol/L, pH=7) and each 2.5mL of the 10mg/ml Tripotassium iron hexacyanide, behind the mixing in 50 ℃ of water-baths 20 minutes, add 100mg/ml trichoroacetic acid(TCA) 2.5ml then, 3000rpm is centrifugal 10 minutes behind the mixing.Accurately pipette supernatant liquor 2.5ml, add distilled water 2.0ml and 1mg/ml iron trichloride 0.5ml, measure absorbance in the 700nm place behind the mixing, the big more reducing power that shows of absorbance (OD value) is strong more.
2.3 hydroxy radical qiao removing ability is measured
Adopt different enzymolysis solutions that the experiment in vitro of the hydroxy radical qiao clearance rate of Fenton system generation is measured.Get the FeSO of 0.2ml
4-EDTA mixed solution (10.0mmol/L) adds 2-deoxyribosyl solution (10.0mmol/L) and the 0.6ml need testing solution of people 0.5ml in test tube, (pH=7.4 0.1mol/L) is settled to 1.8mL, adds people 0.2mLH again with phosphoric acid buffer
2O
2(10.0mol/L), mixing is placed in 37 ℃ of waters bath with thermostatic control and reacts 1h.Add 2.8% (w/w) trichoroacetic acid(TCA) (TCA) solution 1.0mL then, under 4000rpm centrifugal 20 minutes, get supernatant liquor 2.0ml in another test tube, add 1.0% (w/w) thiobarbituricacid (TBA) solution 1.0ml, the rearmounted boiling water bath reaction of mixing 15 minutes, absorbance is surveyed in 5 times of cooling back dilutions at 532nm wavelength place.Enzymolysis solution is represented with clearance rate (SA/%) the removing effect of hydroxy radical qiao, is calculated as follows:
Clearance rate SA (%)=[(Ac-As)/(Ac-A
0)] * 100%
In the formula: Ac is not for adding the absorbancy of scavenging agent, and As is the absorbancy behind the adding trial-product, A
0Be the barren absorbancy.
2.4DPPH radical scavenging activity is measured
Getting need testing solution 1ml joins respectively in the clean 10ml tool plug test tube, every Guan Zhongzai adds 0.6mmol/L DPPH methanol solution 0.5ml, replenish volume to 5ml with methyl alcohol then, light absorption value A was measured in room temperature lucifuge reaction back in the 517nm place in 30 minutes, blank group replaces DPPH solution with the equal-volume methanol solution, control group replaces sample solution with equal-volume distilled water, and with equal-volume distilled water and the blank zeroing of methyl alcohol mixed liquor.Be calculated as follows:
Clearance rate SA (%)=[1-(Ai-Aj)/A
0] * 100%
In the formula: A
0Be control group absorbancy, A
iBe sample sets absorbancy, A
jBe blank group absorbancy.
2.5 ultra-oxygen anion free radical removing ability is measured
Adopt pyrogallol autoxidation method to measure.Measure the Tris-HCl damping fluid 4.7ml of 50mmol/L, pH=8.20, the need testing solution that adds 0.1ml, placed 25 ℃ of water bath heat preservations 20 minutes, pyrogallol solution (preparing) 0.2ml that adds the 3mmol/L of 25 ℃ of pre-temperature then with 10mmol/LHCl, pour in the cuvette after shaking up rapidly, under 319nm, measured light absorption value once, coreaction 9 minutes every 1 minute, with 10mmol/L HCl solution is blank zeroing, and control group replaces for test agent with the equal-volume deionized water.Make the absorbancy regression equation of change curve in time, its slope is the autoxidation speed V of pyrogallol, is calculated as follows sample to O
-2Clearance rate.
Clearance rate SA (%)=[1-V
1/ V
0] * 100%
In the formula: V
1Be the absorbancy of sample sets rate over time, V
0Be blank group group absorbancy rate over time.
3, result and discussion
3.1 concrete experimental result is as shown in table 4:
The resistance of oxidation of table 4 Mactra veneriformis protein polypeptide (concentration 100 μ g/ml,
N=3)
Show that by table 4 experimental result the Mactra veneriformis protein polypeptide that method provided by the invention prepares has the ability of fine removing DPPH free radical, hydroxy radical qiao and ultra-oxygen anion free radical, and has stronger reducing power.Therefore, Mactra veneriformis protein polypeptide provided by the invention is expected to develop the medicine or the protective foods of antioxidant and anti-aging that becomes a new generation, can be used for preventing and treating diseases such as aging and aging relevant disorders such as cancers, cardiovascular and autoimmunity.
The above only is a preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (8)
1. Mactra veneriformis protein polypeptide with anti-oxidant activity, it is characterized in that, it prepares by the following method: at first get the Mactra veneriformis software and clean and to blend homogenate, the trypsinase that adds suitable homogenate weight 0.05%～0.5%, in pH7.5～8.5, enzymolysis is 30～90 minutes under 45～55 ℃ of conditions of temperature, then 90～100 ℃ of following enzyme-deactivatings 10 minutes, centrifugal, get supernatant liquor and carry out ultrafiltration, molecular weight cut-off is the ultrafiltrated of 10～50kDa, concentrating under reduced pressure, and spraying drying is promptly.
2. the described preparation method with Mactra veneriformis protein polypeptide of anti-oxidant activity of claim 1 is characterized in that, may further comprise the steps:
Getting the Mactra veneriformis software cleans, blend homogenate, get homogenate, add the trypsinase of suitable homogenate weight 0.05%～0.5%, at pH7.5 to 8.5, enzyme digestion reaction is 30 to 90 minutes under 45～55 ℃ of conditions of temperature, and enzymolysis finishes the back at go out enzyme 10 minutes of 90～100 ℃ of water-baths, and is centrifugal, get supernatant liquor and carry out ultrafiltration, molecular weight cut-off is the ultrafiltrated of 10～50kDa, and concentrating under reduced pressure promptly gets the Mactra veneriformis protein polypeptide after the spraying drying.
3. the preparation method with Mactra veneriformis protein polypeptide of anti-oxidant activity according to claim 2 is characterized in that, described tryptic add-on is 0.25%～0.5% of a Mactra veneriformis homogenate weight.
4. the preparation method with Mactra veneriformis protein polypeptide of anti-oxidant activity according to claim 2 is characterized in that the pH value of enzyme digestion reaction is 8.5.
5. the preparation method with Mactra veneriformis protein polypeptide of anti-oxidant activity according to claim 2 is characterized in that, the temperature of enzyme digestion reaction is 45 degree, and the time of enzyme digestion reaction is 70～90 minutes.
6. the preparation method with Mactra veneriformis protein polypeptide of anti-oxidant activity according to claim 2 is characterized in that, the centrifugal gained supernatant liquor of enzymolysis solution carries out ultrafiltration, holds back and obtains the ultrafiltrated that molecular weight is 10～30kDa.
7. the described application of Mactra veneriformis protein polypeptide in preparation antioxidant and anti-aging medicine of claim 1 with anti-oxidant activity.
8. the described application of Mactra veneriformis protein polypeptide in preparation antioxidant and anti-aging healthcare products or foodstuff additive of claim 1 with anti-oxidant activity.
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| CN103989135A (en) * | 2014-04-29 | 2014-08-20 | 江苏恒顺醋业股份有限公司 | Clam flavor enhancer and making method thereof |
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| CN106636271A (en) * | 2016-11-21 | 2017-05-10 | 浙江海洋大学 | Method for preparing clam angiotensin converting enzyme inhibitory peptide by mixed enzymolysis |
| CN106916868A (en) * | 2015-12-25 | 2017-07-04 | 无限极（中国）有限公司 | A kind of white clam polypeptide with anti-oxidant in vivo and neuroprotective function and preparation method and application |
| CN107604033A (en) * | 2017-10-31 | 2018-01-19 | 浙江海洋大学 | The method that female cuttlefish spawn tangled gland enzymolysis prepares high anti-oxidation active peptides |
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