CN104789626A - Preparation method and application of Cyclina sinensis enzymolysis polypeptide - Google Patents
Preparation method and application of Cyclina sinensis enzymolysis polypeptide Download PDFInfo
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- CN104789626A CN104789626A CN201510099953.2A CN201510099953A CN104789626A CN 104789626 A CN104789626 A CN 104789626A CN 201510099953 A CN201510099953 A CN 201510099953A CN 104789626 A CN104789626 A CN 104789626A
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Abstract
The invention relates to a preparation method of Cyclina sinensis enzymolysis polypeptide. The method includes: subjecting fresh Cyclina sinensis to evisceration and homogenization, taking the homogenate, adding alkaline protease to perform enzymolysis, centrifuging the enzymolysis solution and taking the supernatant, carrying out rotary evaporation and concentration, and freeze drying so as to obtain Cyclina sinensis enzymolysis polypeptide with a molecular weight of 15-25kDa. The Cyclina sinensis enzymolysis polypeptide has good antioxidant activity, has a DPPH free radical clearance rate up to 91.24%, a hydroxyl free radical clearance rate up to 62.83%, chelating power to Fe<2+> of 61.13%, and reducing power of 0.331, and also has certain protective effect on hydroxyl free radical induced damaged pBR322 DNA, and can be widely used in antioxidation active products.
Description
Technical field
The present invention relates to field of marine biotechnology, particularly relate to a kind of preparation method and application of clam enzymolysis polypeptide.
Background technology
Clam (Cyclina sinensis) belongs to Mollusca, lamellibranchiata, curtain clam order, Veneridae, and being commonly called as black clam, circle clam, iron clam etc., being distributed in the silt particle beach of China coast tideland, is the common a kind of shellfish in China's Coastal Areas.Clam delicious meat, nutritious, apart from outside higher edibleness or a kind of important marine drug, clam is all embodied in wherein as a kind of common drug by Shennong's Herbal, Compendium of Material Medica and supplementary Amplifications of the Compendium of Materia Medica.
More to the enzymolysis polypeptide research of sea-food in recent years, as: the patent No. Chinese invention patent " preparation method of Ruditapes philippinarum enzymatic polypeptide " that is ZL201010253745.0 (Authorization Notice No. is CN 102372765 B) discloses the preparation method of a kind of Ruditapes philippinarum enzymatic polypeptide (Asp Trp Pro His), also disclose the application of this enzymolysis polypeptide simultaneously.Current research shows, clam meat extract can increase the macrophage activity in lymphocyte, macrophage activity and the lymphoglandula in senile rat spleen, has important effect to raising cellullar immunologic response and body normal immunity etc.; Thick clam polysaccharide has anti-oxidant activity in vitro, has important provide protection, but have no report at present about the research of clam enzymolysis polypeptide to the Mouse Liver toxic model of tetrachloro-methane induction, the especially effect of clam enzymolysis polypeptide in anti-oxidant activity.
Summary of the invention
First technical problem to be solved by this invention is the preparation method providing a kind of clam enzymolysis polypeptide for prior art.
Second technical problem to be solved by this invention provides the application of above-mentioned clam enzymolysis polypeptide in anti-oxidant activity.
The present invention solves the technical scheme that first technical problem adopt: a kind of preparation method of clam enzymolysis polypeptide,
It is characterized in that comprising the following steps:
(1) get fresh clam, shell and get internal organ, broken with refiner, obtain clam internal organ homogenate;
(2) ratio in 1:3 ~ 4 in above-mentioned homogenate adds distilled water, pH value to 9 ~ 11 of homogenate are regulated with certain density hydrochloric acid and sodium hydroxide solution, add 1000 ~ 2000u/g Sumizyme MP, 40 ~ 50 DEG C of enzymolysis 4 ~ 8h, boiling water deactivation 10 ~ 15min;
(3) enzymolysis solution after deactivation is cooled to room temperature, the centrifugal 10 ~ 15min of 9000 ~ 10000r/min, get supernatant liquor rotary evaporation and concentrate, lyophilize obtains clam enzymolysis polypeptide.
As preferably, in described step (2), hydrolysis temperature is 50 DEG C, and enzymolysis time is 4h, and Sumizyme MP addition is 2000u/g, and enzymolysis pH is 9.
As preferably, the molecular weight of described clam enzymolysis polypeptide is 15 ~ 25KDa.
The present invention solves the technical scheme that second technical problem adopt: the application of above-mentioned clam enzymolysis polypeptide in anti-oxidant activity product.
Further, described anti-oxidant activity be scavenging(action) to free radical, to Fe
2+the sequestering action of ion, reducing power and the provide protection to pBR322DNA damage.
Further again, described free radical is hydroxyl radical free radical and DPPH free radical, and described pBR322DNA damage is induced by hydroxyl radical free radical.
Further again, when described clam enzymolysis polypeptide concentration is 1.57mg/mL, 50% is reached to the clearance rate of DPPH free radical, when described clam enzymolysis polypeptide concentration is 5mg/mL, 91.24% is reached to the clearance rate of DPPH free radical; When described clam enzymolysis polypeptide concentration is 2.1mg/mL, 50% is reached to the clearance rate of DPPH free radical, when described clam enzymolysis polypeptide concentration is 5mg/mL, 62.83% is reached to the clearance rate of DPPH free radical; Clam enzymolysis polypeptide is to Fe
2+the EC50 of the sequestering power of ion is 2.75mg/mL, and when clam enzymolysis polypeptide concentration is 5mg/mL, chelating ability is 61.13%; Reducing power and its concentration of described clam enzymolysis polypeptide are proportionate, and reducing power is 0.331.
Anti-oxidant activity product in such scheme can be food, medicine, healthcare products and makeup.
Compared with prior art, the invention has the advantages that: clam is a kind of common marine shellfish, with fresh clam for raw material enzymolysis and extraction clam enzymolysis polypeptide, raw material sources is wide, and cost is low, easy to operate, and the clam enzymolysis polypeptide extracted has good anti-oxidant activity, wherein up to 91.24%, can reach 62.83%, to Fe to the clearance rate of hydroxy radical qiao to the clearance rate of DPPH free radical
2+chelating ability be 61.13%, reducing power is 0.331, and has certain provide protection to the pBR322DNA by hydroxy radical qiao induced damage, can be widely used in anti-oxidant activity product.
Accompanying drawing explanation
Fig. 1 be in embodiment 2 clam enzymolysis polypeptide to the clearance rate schematic diagram of DPPH free radical;
Fig. 2 be in embodiment 3 clam enzymolysis polypeptide to the clearance rate schematic diagram of hydroxy radical qiao;
Fig. 3 be in embodiment 4 clam enzymolysis polypeptide to Fe
2+the sequestering action schematic diagram of ion;
Fig. 4 is the reducing power schematic diagram of clam enzymolysis polypeptide in embodiment 5;
Fig. 5 is the agarose gel electrophoresis figure of clam enzymolysis polypeptide anti-hydroxy radical qiao induction pBR322DNA lesion capability in embodiment 6;
Fig. 6 is the SDS-PAGE electrophorogram of clam enzymolysis polypeptide in embodiment 7.
Embodiment
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.
The raw material used in following each embodiment and reagent as follows:
Fresh clam is purchased from Nan Zhen market, Zhoushan, shells and gets internal organ, and-20 DEG C of freezen protective are for subsequent use; Sumizyme MP is purchased from Heng Xin bio tech ltd, Asia-Pacific, Beijing; Phenanthroline, DPPH, the Tripotassium iron hexacyanide are purchased from the brilliant pure reagent company limited in Shanghai; All the other reagent are that analytical pure all purchases Chemical Reagent Co., Ltd., Sinopharm Group.
Instrument is as follows:
BSA1245 type electronic balance (Sai Duolisi scientific instrument company limited); CF16RX high speed freezing centrifuge (Hitachi, Ltd); UV-1600PC type ultraviolet spectrophotometer (Shanghai Mei Puda Instrument Ltd.); SSW type Microcomputerized electric Water Tank with Temp.-controlled (Shanghai Medical Equipment Plant of Bo Xun Industrial Co., Ltd.); DYCZ-28C type electrophoresis apparatus (Liuyi Instruments Plant, Beijing).
Embodiment 1: enzyme-squash techniqued clam enzymolysis polypeptide
Get above-mentioned clam internal organ as raw material, raw material is broken with refiner, obtain clam internal organ homogenate.Ratio in 1:3 ~ 4 in above-mentioned homogenate adds distilled water, and regulate the pH value of homogenate with certain density hydrochloric acid and sodium hydroxide solution, add Sumizyme MP and carry out enzymolysis, enzymolysis terminates rear boiling water deactivation 10 ~ 15min.Enzymolysis solution after deactivation is cooled to room temperature, the centrifugal 10 ~ 15min of 9000 ~ 10000r/min, get supernatant liquor rotary evaporation and concentrate, lyophilize obtains clam enzymolysis polypeptide.
As shown in table 1, consider four factors in enzymolysis process: 1 temperature, 2 times, 3 enzyme concentrations, 4pH, on the impact of hydrolysis result, each factor is chosen three levels and is carried out orthogonal experiment, the relatively DPPH free radical scavenging activity of Sumizyme MP under different hydrolysising condition (detailed process is see embodiment 2), thus determine the optimum hydrolysising condition of Sumizyme MP.
Table 1 Sumizyme MP factor and level
This experiment for index with DPPH clearance rate, is carried out the orthogonal experiment of the horizontal L9 of four factor three (34), be the results are shown in Table 2.From in table 2, each factorial arrangement order affecting hydrolysis by novo polypeptide DPPH clearance rate is D > A > C > B, and namely pH value has the greatest impact, and hydrolysis time impact is minimum.Therefore hydrolysis by novo clam obtains the optimum hydrolysising condition of polypeptide is A3B1C3D1.The temperature of selection is 50 DEG C, time 4h, enzyme live addition 2000u/g, pH 9 as optimum hydrolysising condition.
Table 2 Sumizyme MP enzymolysis clam internal organ Orthogonal experiment results
Embodiment 2: clam enzymolysis polypeptide is to the clearance rate of DPPH free radical
By the hydrolyzate lyophilize obtained under the optimum enzymolysis condition obtained in embodiment 1, carry out dissolving with distilled water and obtain detection sample, the mass concentration detecting sample is 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, 5mg/mL.DPPH concentration 0.1mmol/L (anhydrous alcohol solution) keeps in Dark Place, and method is 1mL sample+1mL DPPH, and after mixing, centrifugal 10min, the 517nm place of keep in Dark Place 30min, 4000r/min surveys absorbance A i.Survey the survey absorbance A j of 1mL dehydrated alcohol+1mL sample mix, mix with 1mL DPPH+1mL water and survey absorbance A c, calculation formula is: DPPH clearance rate (%)=[1-(Ai-Aj)/Ac] × 100% simultaneously.
As shown in Figure 1, the clearance rate of visible clam hydrolysis by novo thing to DPPH free radical increases along with the rising of concentration result.Particularly, when concentration is 5mg/mL, clearance rate reaches 91.24%; When the Scavenging activity of clam polypeptide to DPPH reaches 50%, required peptide masses concentration is 1.57mg/mL.
Embodiment 3: clam enzymolysis polypeptide is to the clearance rate of hydroxy radical qiao
Get 0.75mmol/L phenanthroline 1mL in test tube, add the PBS 2mL that pH value is 7.4 successively, sample liquid 1mL, add the ferrous sulfate 1mL that concentration is 0.75mol/L after vibration mixing, fully mix, then add 0.03% hydrogen peroxide 1mL, 37 DEG C of water-bath 1h, survey its absorbance A s at 536nm place; Blank group water replaces hydrogen peroxide, is Ab; Damage group water replaces sample, is Ap, and clearance rate (%)=[(As-Ap)/(Ab-Ap)] × 100%, sample preparation is with embodiment 2.
As shown in Figure 2, the hydrolysis by novo thing of visible different concns all has scavenging(action) to a certain degree to hydroxy radical qiao to result, and raises along with concentration, clearance rate increases, but enzymolysis polypeptide is to the clearance rate of hydroxy radical qiao lower than DPPH, and when concentration is 5mg/mL, clearance rate is 62.83%; When the clearance rate of clam enzymolysis polypeptide to hydroxy radical qiao reaches 50%, required peptide masses concentration is 2.1mg/mL.
Embodiment 4: clam enzymolysis polypeptide is to the sequestering action of Fe2+
Given the test agent (1mg/mL ~ 5mg/mL) 0.5mL of different mass concentration is added successively, 20 μMs of FeCl in clean tool plug test tube
2solution 1mL, adds 0.5mM ferrozine 1mL after mixing, measure absorbance A b after 25 DEG C of reaction 20min in 562nm place; Blank group replaces sample with water, and be Ac, chelating ability (%)=(1-Ab/Ac) × 100%, sample preparation is with embodiment 2.
As shown in Figure 3, as seen along with the rising of hydrolyzate concentration, chelating ability increases result, and when concentration is 5mg/mL, chelating ability is 61.13%, Fe
2+the EC50 of sequestering power be 2.75mg/mL.
Embodiment 5: the reducing power of clam enzymolysis polypeptide measures
Sample 1mL is added successively respectively by clean tool plug test tube, separately add 1mL Tris-HCl (0.2mol/L, pH are 6.6) and 1% Tripotassium iron hexacyanide 2.5mL, in 50 DEG C of water-bath 20min after mixing, then 10% trichoroacetic acid(TCA) 1.5mL is added, the centrifugal 10min of 3000r/min after mixing.Accurately pipette supernatant liquor 2.0mL, add distilled water 2.0mL and 0.1% iron trichloride 0.5mL, leave standstill 10min after mixing, measure absorbance A in 700nm place, absorbance shows that more greatly reducing power is stronger, and sample preparation is with embodiment 2.
According to the measuring method of reducing power, larger in the absorbancy at 700nm place, the reducing power of hydrolyzate is stronger.As can be seen from Figure 4, in set concentration range, along with concentration increases, reducing power strengthens, and namely presents concentration dependent.
Embodiment 6: clam enzymolysis polypeptide is to the pBR322DNA provide protection of hydroxy radical qiao induced damage
The pBR322DNA of 0.5 μ L being dissolved in 3 μ L concentration is in the PBS liquid of 50mmol/L, and transfers in Eppendorf tube, adds the freshly prepared FeSO of 2mmol/L simultaneously
4the clam enzymolysis polypeptide solution 2 μ L of solution 3 μ L and different concns, then add the H of 4 μ L massfractions 30%
2o
2solution, after mixture reacts 30min at 37 DEG C, adds DNA sample-loading buffer (fluorescence) and terminates reaction.Blank group is only pBR322DNA and sample-loading buffer, and respectively group point sample is in 1% sepharose, electrophoresis 70min under 100V voltage conditions.Utilize gel imaging system to observe electrophoresis result, and take pictures, sample preparation is with embodiment 2.
Three kinds of form-superhelix (supercoil DNA are cracked into when DNA is exposed in hydroxy radical qiao, SC-DNA), open loop structure (open cycle DNA, and linear structure form OC-DNA), open loop structure represents single-strand DNA breaks, and linear structure represents double-strand break.Clam enzymolysis polypeptide to the provide protection of the DNA oxidative damage that hydroxy radical qiao is induced as shown in Figure 5; as can be seen from the figure; not by former pBR322 DNA (Blank) DNA almost all in superhelix of injured by hydroxyl free radicals, and control group (control) namely adds Fe
2+and H
2o
2after reaction, DNA is almost all in open loop situations, show that hydroxy radical qiao has damage pBR322DNA, but after adding clam enzymolysis polypeptide, the DNA of superhelix increases gradually, and the DNA of open loop structure reduces gradually, and increase gradually along with sample concentration, the DNA showed increased existed with supercoiled form.And gel there is not linear strip, this may be that the hydroxy radical qiao effect degree produced in this experiment is not enough to make plasmid DNA double-strand break.These results show, the DNA oxidative damage that this polypeptide can suppress hydroxy radical qiao to be induced significantly, may can stop Fe with this polypeptide
2+and H
2o
2reaction, and by providing hydrogen atom and electronics and direct scavenging hydroxyl, and then protection plasmid DNA is not damaged relevant.
Embodiment 7: the SDS-PAGE of clam enzymolysis polypeptide analyzes
Separation gel with 15%, 5% concentrated glue record SDS-PAGE gel, the separation gel of 15% to be added in electrophoresis sheet glass and with water, separation gel surface to be flattened, after 20min, water being outwelled, pour into the concentrated glue of 5%.Applied sample amount is 0.01mL, and the voltage that switches on power is adjusted to 90V, enters after separation gel be adjusted to 130V until sample.Powered-down when sample arrives 0.5cm bottom distance separation gel, take out gel and to dye in staining fluid 1h, finally decolour 4h, takes pictures, and sample preparation is with embodiment 2.
As shown in Figure 6, visible zymolyte component, through SDS-PAGE electrophoresis, be shown as single band, and band molecular weight shows the mensuration of clam enzymolysis polypeptide molecular weight between 15-25KDa after dyeing, decolouring.
Claims (8)
1. a preparation method for clam enzymolysis polypeptide, is characterized in that comprising the following steps:
(1) get fresh clam, shell and get internal organ, broken with refiner, obtain clam internal organ homogenate;
(2) ratio in 1:3 ~ 4 in above-mentioned homogenate adds distilled water, pH value to 9 ~ 11 of homogenate are regulated with certain density hydrochloric acid and sodium hydroxide solution, add 1000 ~ 2000u/g Sumizyme MP, 40 ~ 50 DEG C of enzymolysis 4 ~ 8h, boiling water deactivation 10 ~ 15min;
(3) enzymolysis solution after deactivation is cooled to room temperature, the centrifugal 10 ~ 15min of 9000 ~ 10000r/min, get supernatant liquor rotary evaporation and concentrate, lyophilize obtains clam enzymolysis polypeptide.
2. the preparation method of clam enzymolysis polypeptide as claimed in claim 1, is characterized in that, it is characterized in that, in described step (2), hydrolysis temperature is 50 DEG C, and enzymolysis time is 4h, and Sumizyme MP addition is 2000u/g, and enzymolysis pH is 9.
3. the preparation method of clam enzymolysis polypeptide as claimed in claim 1 or 2, it is characterized in that, the molecular weight of described clam enzymolysis polypeptide is 15 ~ 25KDa.
4. the application of a clam enzymolysis polypeptide as claimed in claim 1 in anti-oxidant activity product.
5. the application of clam enzymolysis polypeptide in anti-oxidant activity product as claimed in claim 4, is characterized in that, described anti-oxidant activity is scavenging(action) to free radical, to Fe
2+the sequestering action of ion, reducing power and the provide protection to pBR322DNA damage.
6. the application of clam enzymolysis polypeptide in anti-oxidant activity product as claimed in claim 5, it is characterized in that, described free radical is hydroxyl radical free radical and DPPH free radical, and described pBR322DNA damage is induced by hydroxyl radical free radical.
7. the application of clam enzymolysis polypeptide in anti-oxidant activity product as claimed in claim 6, it is characterized in that, when described clam enzymolysis polypeptide concentration is 1.57mg/mL, 50% is reached to the clearance rate of DPPH free radical, when described clam enzymolysis polypeptide concentration is 5mg/mL, 91.24% is reached to the clearance rate of DPPH free radical;
When described clam enzymolysis polypeptide concentration is 2.1mg/mL, 50% is reached to the clearance rate of DPPH free radical, when described clam enzymolysis polypeptide concentration is 5mg/mL, 62.83% is reached to the clearance rate of DPPH free radical; Clam enzymolysis polypeptide is to Fe
2+the EC50 of the sequestering power of ion is 2.75mg/mL, and when clam enzymolysis polypeptide concentration is 5mg/mL, chelating ability is 61.13%;
Reducing power and its concentration of described clam enzymolysis polypeptide are proportionate, and reducing power is 0.331.
8. the application of clam enzymolysis polypeptide in anti-oxidant activity product as described in claim arbitrary in claim 4 ~ 7, is characterized in that, described anti-oxidant activity product is food, medicine, healthcare products and makeup.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434811A (en) * | 2016-11-21 | 2017-02-22 | 浙江海洋大学 | Preparation method and application of cyclina sinensis immune-enhancing peptide |
| CN106467569A (en) * | 2016-05-17 | 2017-03-01 | 浙江海洋学院 | A kind of extracting method of shellfish anti-tumor activity thing |
| CN106636270A (en) * | 2016-11-21 | 2017-05-10 | 浙江海洋大学 | Preparation method of antibacterial peptide of cyclina sinensis |
| CN107007831A (en) * | 2017-06-16 | 2017-08-04 | 南京佰泰克生物技术有限公司 | The immunologic adjuvant of hepatitis B DNA vaccine |
| CN108277249A (en) * | 2018-01-25 | 2018-07-13 | 浙江海洋大学 | A kind of clam polypeptide production methods to nonalcoholic fatty liver model with repair |
| CN108339109A (en) * | 2018-01-17 | 2018-07-31 | 浙江海洋大学 | A kind of clam immunoloregulation polypeptide tablet and preparation method |
| CN110687287A (en) * | 2019-11-12 | 2020-01-14 | 吉林鑫水科技开发有限公司 | Analysis method of deer fetus oligopeptide for immunoregulation of macrophage |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106467569A (en) * | 2016-05-17 | 2017-03-01 | 浙江海洋学院 | A kind of extracting method of shellfish anti-tumor activity thing |
| CN106434811A (en) * | 2016-11-21 | 2017-02-22 | 浙江海洋大学 | Preparation method and application of cyclina sinensis immune-enhancing peptide |
| CN106636270A (en) * | 2016-11-21 | 2017-05-10 | 浙江海洋大学 | Preparation method of antibacterial peptide of cyclina sinensis |
| CN107007831A (en) * | 2017-06-16 | 2017-08-04 | 南京佰泰克生物技术有限公司 | The immunologic adjuvant of hepatitis B DNA vaccine |
| CN108339109A (en) * | 2018-01-17 | 2018-07-31 | 浙江海洋大学 | A kind of clam immunoloregulation polypeptide tablet and preparation method |
| CN108277249A (en) * | 2018-01-25 | 2018-07-13 | 浙江海洋大学 | A kind of clam polypeptide production methods to nonalcoholic fatty liver model with repair |
| CN110687287A (en) * | 2019-11-12 | 2020-01-14 | 吉林鑫水科技开发有限公司 | Analysis method of deer fetus oligopeptide for immunoregulation of macrophage |
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Application publication date: 20150722 |